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Viral diagnostics in the era of digital polymerase chain reaction
Abstract
Unlike quantitative polymerase chain reaction (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR. Here we review current literature that demonstrates dPCR's potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection.
... Methods to rapidly detect and absolutely quantify adventitious agents such as viruses are needed to monitor and halt the spread of infectious diseases, to accelerate the virus-related research, and critical in biosafety release testing in biopharmaceutical and cell manufacturing process [1][2][3]. Regulatory guidelines from the United States Food and Drug Administration (FDA) often mandate the identification of live adventitious agents, i.e. their replication competency [4] that can benefit from the rapid diagnostic methods that allow their absolute quantification. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is considered a gold standard for viral detection [5]. ...
... However, quantification by RT-qPCR relies on the use of accurate and stable external standards or references, with a 20-30% variability reported even within trained laboratories or testing sites [2,6,7]. Thus, an absolute quantification method with improved speed is needed for establishing reference benchmarks, and for allowing reliable cross-site standardized comparisons in international research collaborations, global health surveillance, and food and drug safety supervision/surveillance [8,9]. ...
... dPCR is increasingly being used as a highly accurate and sensitive method for absolute quantification of nucleic acids [2,8,10,11]. Since thousands of PCR reactions take place in individual partitions independently, absolute quantification by dPCR is more robust than RT-qPCR, less sensitive to inhibitors and poor amplification efficiency [2,7]. ...
Rapid diagnostics of adventitious agents in biopharmaceutical/cell manufacturing release testing and the fight against viral infection have become critical. Quantitative real-time PCR and CRISPR-based methods rapidly detect DNA/RNA in one hour (1h) but suffer from inter-site variability. Absolute quantification of DNA/RNA by methods such as digital PCR reduce this variability but are currently too slow for wider application. Here, we report a RApid DIgital Crispr Approach (RADICA) for absolute quantification of nucleic acids within 1h. Using SARS-CoV-2 as a proof-of-concept target, RADICA allows for absolute quantification with a linear dynamic range of 0.6 to 2027 copies/μL (R² value > 0.99), high accuracy and low variability, no cross-reactivity to similar targets, and high tolerance to human background DNA. RADICA’s versatility is validated against other targets such as Epstein-Barr virus (EBV) from human B cells and patients’ serum. RADICA can accurately detect and absolutely quantify EBV DNA with similar dynamic range of 0.5 to 2100 copies/μL (R² value > 0.98) in 1h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based detection. RADICA therefore enables rapid and sensitive absolute quantification of DNA/RNA which can be widely applied across clinical, research, and biomanufacturing areas.
... Methods to rapidly detect and absolutely quantify adventitious agents such as viruses are needed to monitor and halt the spread of infectious diseases, to accelerate the virus-related research, and critical in biosafety release testing in biopharmaceutical and cell manufacturing process [1][2][3]. Regulatory guidelines from the United States Food and Drug Administration (FDA) often mandate the identification of live adventitious agents, i.e. their replication competency [4] that can benefit from the rapid diagnostic methods that allow their absolute quantification. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is considered a gold standard for viral detection [5]. ...
... However, quantification by RT-qPCR relies on the use of accurate and stable external standards or references, with a 20-30% variability reported even within trained laboratories or testing sites [2,6,7]. Thus, an absolute quantification method with improved speed is needed for establishing reference benchmarks, and for allowing reliable cross-site standardized comparisons in international research collaborations, global health surveillance, and food and drug safety supervision/surveillance [8,9]. ...
... dPCR is increasingly being used as a highly accurate and sensitive method for absolute quantification of nucleic acids [2,8,10,11]. Since thousands of PCR reactions take place in individual partitions independently, absolute quantification by dPCR is more robust than RT-qPCR, less sensitive to inhibitors and poor amplification efficiency [2,7]. ...
... Methods to rapidly detect and absolutely quantify adventitious agents such as viruses are needed to monitor and halt the spread of infectious diseases, to accelerate the virus-related research, and critical in biosafety release testing in biopharmaceutical and cell manufacturing process [1][2][3]. Regulatory guidelines from the United States Food and Drug Administration (FDA) often mandate the identification of live adventitious agents, i.e. their replication competency [4] that can benefit from the rapid diagnostic methods that allow their absolute quantification. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is considered a gold standard for viral detection [5]. ...
... However, quantification by RT-qPCR relies on the use of accurate and stable external standards or references, with a 20-30% variability reported even within trained laboratories or testing sites [2,6,7]. Thus, an absolute quantification method with improved speed is needed for establishing reference benchmarks, and for allowing reliable cross-site standardized comparisons in international research collaborations, global health surveillance, and food and drug safety supervision/surveillance [8,9]. ...
... dPCR is increasingly being used as a highly accurate and sensitive method for absolute quantification of nucleic acids [2,8,10,11]. Since thousands of PCR reactions take place in individual partitions independently, absolute quantification by dPCR is more robust than RT-qPCR, less sensitive to inhibitors and poor amplification efficiency [2,7]. ...
Quantitative real-time PCR and CRISPR-based methods detect SARS-CoV-2 in 1 hour but do not allow for the absolute quantification of virus particles, which could reduce inter-lab variability and accelerate research. The 4-hour reaction time of the existing digital PCR-based method for absolute virus quantification is too long for widespread application. We report a RApid DIgital Crispr Approach (RADICA) for the absolute quantification of SARS-CoV-2 DNA and Epstein–Barr virus DNA in human samples that yields results within 1 hour. For validation, we compared RADICA to digital PCR for quantifying synthetic SARS-CoV-2 DNA and Epstein–Barr viral DNA. RADICA allows absolute quantification of DNA with a dynamic range from 0.6 to 2027 copies/µL (R ² value > 0.98), without cross-reactivity on similar virus or human background DNA. Thus, RADICA can accurately detect and quantify nucleic acid in 1h without thermal cycling, providing a 4-fold faster alternative to digital PCR-based virus detection.
... dPCR is a highly sensitive and accurate method for absolute quantification of DNA samples, eliminating the need for standard curves. This technique involves distributing DNA across multiple replicate reactions, enabling the use of Poisson statistics for precise quantification (Sedlak & Jerome 2013). By directly calculating the DNA molecule number from positive and negative reactions, dPCR provides absolute quantification and can determine the number of DNA copies per ml, particularly for low viral loads (Sedlak & Jerome 2013). ...
... This technique involves distributing DNA across multiple replicate reactions, enabling the use of Poisson statistics for precise quantification (Sedlak & Jerome 2013). By directly calculating the DNA molecule number from positive and negative reactions, dPCR provides absolute quantification and can determine the number of DNA copies per ml, particularly for low viral loads (Sedlak & Jerome 2013). Moreover, dPCR exhibits increased sensitivity and precision compared to traditional PCR assays or even multiplex PCR, making it capable of detecting mutant sequences that may be undetected by sequencing methods. ...
Climate change and the associated environmental temperature fluctuations are contributing to increases in the frequency and severity of disease outbreaks in both wild and farmed aquatic species. This has a significant impact on biodiversity and also puts global food production systems, such as aquaculture, at risk. Most infections are the result of complex interactions between multiple pathogens, and understanding these interactions and their co-evolutionary mechanisms is crucial for developing effective diagnosis and control strategies. In this review, we discuss current knowledge on bacteria-bacteria, virus-virus, and bacterial and viral co-infections in aquaculture as well as their co-evolution in the context of global warming. We also propose a framework and different novel methods (e.g. advanced molecular tools such as digital PCR and next-generation sequencing) to (1) precisely identify overlooked co-infections, (2) gain an understanding of the co-infection dynamics and mechanisms by knowing species interactions, and (3) facilitate the development multi-pathogen preventive measures such as polyvalent vaccines. As aquaculture disease outbreaks are forecasted to increase both due to the intensification of practices to meet the protein demand of the increasing global population and as a result of global warming, understanding and treating co-infections in aquatic species has important implications for global food security and the economy.
... While quantitative real-time PCR (qPCR) has been the standard quantification method in molecular diagnostics ever since its development [2,3], dPCR has got more and more attention during the last years [4,5] facilitated by technological advances in microfluidic systems [6]. Especially its superior precision makes dPCR an attractive alternative to qPCR in various molecular diagnostic applications like the monitoring of low pathogenic loads [4,7], monitoring of minimal residual disease [8][9][10], the analysis of copy number variations [11] and the detection of rare mutants [12]. Over the last years dPCR systems have been extensively compared to qPCR systems for such use cases [13][14][15][16] confirming the enormous potential of this technology. ...
... The shown data was generated by employing Python 3.7 on a Lenovo ThinkPad T470 (Intel1 Core™ i5-6300U CPU @ 2.40 GHz, 8 GB RAM) to perform the calculations. Formula (7) allows to check whether a certain number of target copies C is covered by the confidence interval, but it does not provide a direct calculation of the upper and lower border of the confidence interval, C U and C L , respectively. Therefore, a successive approximation using multiple iterations was performed for the determination of the borders. ...
The precision of compartment-based quantification methods is subject to multiple effects, of which partitioning and subsampling play a major role. Partitioning is the process of aliquoting the sample liquid and consequently the contained target molecules, whereas subsampling denotes the fact that usually only a portion of a sample is analyzed. In this work, we present a detailed statistical description comprising the effects of partitioning and subsampling on the relative uncertainty of the test result. We show that the state-of-the-art binomial model does not provide accurate results for the level of subsampling present when analyzing the nucleic acid content of single specific cells. Hence, in this work we address partitioning and subsampling effects separately and subsequently combine them to derive the relative uncertainty of a test system and compare it for single cell content analysis and body fluid analysis. In point-of-care test systems the area for partitioning and detection is usually limited, which means that a trade-off between the number of partitions (related to a partitioning uncertainty) and the amount of analyzed volume (related to a subsampling uncertainty) might be inevitable. In case of low target concentration, the subsampling uncertainty is dominant whereas for high target concentration, the partitioning uncertainty increases, and a larger number of partitions is beneficial to minimize the combined uncertainty. We show, that by minimizing the subsampling uncertainty in the test system, the quantification uncertainty of low target concentrations in single cell content analysis is much smaller than in body fluid analysis. In summary, the work provides the methodological basis for a profound statistical evaluation of partitioning and subsampling effects in compartment-based quantification methods and paves the way towards an improved design of future digital quantification devices for highly accurate molecular diagnostic analysis at the point-of-care.
... Molecular techniques, such as real-time reverse transcription-polymerase chain reaction (RT-PCR), yield the cycle threshold (Ct) which is a rough estimate of the number of the viral genomes in a virus preparation [11]. At present, several advanced molecular techniques can enumerate the copy numbers of the viral genomes with high precision, such as quantitative real-time PCR [12] and digital droplet PCR [13]. Even though these molecular techniques are highly sensitive, they cannot differentiate between the genomes derived from infectious and dead viruses. ...
... Actual copy numbers of the target genes can be determined if the run is conducted along with the standard viral RNA and a calibration curve. In contrast, digital RT-PCR (based on measurement of a large number of positive micro-reactions in oil droplets) can determine the copy numbers of target genes without viral RNA standard and calibration curve [13,24]. ...
Zika virus (ZIKV) was isolated from the archival urine, serum, and autopsy specimens by intrathoracic inoculation of Toxorhynchitis splendens and followed by three blind sub-passaging in C6/36 mosquito cells. The virus isolates were identified using an immunofluorescence assay and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). This study analyzed 11 ZIKV isolates. One isolate (0.6%) was obtained from 171 urine samples, eight (8.7%) from 92 serum samples and two from tissues of an abortive fetus. After propagation in C6/36 cells, ZIKV was titrated by plaque and focus forming unit (FFU) assays in Vero cell monolayers, and viral genomes were determined via real-time and digital RT-PCR. Plaque and FFU assay quantitations were comparable, with the amount of infectious viruses averaging 10 ⁶ −10 ⁷ PFU or FFU/ml. Real-time RT-PCR semi-quantified the viral genome numbers, with Ct values varying from 12 to 14. Digital RT-PCR, which precisely determines the numbers of the viral genomes, consistently averaged 10–100 times higher than the number of infectious units. There was good correlation between the results of these titration methods. Therefore, the selection of a method should be based on the objectives of each research studies.
... Indeed, performing every single PCR in an isolated droplet provides higher resistance to a variety of inhibitors that could be contained within biological samples and consequently significantly increases reproducibility of the obtained data and comparability of the results between different laboratories 15 . The increased tolerance of ddPCR to PCR inhibitory molecules makes it an attractive alternative to qPCR for medical applications including viral diagnostics 16 . Recently, ddPCR was used to quantify SARS-CoV-2 RNA in lower respiratory A c c e p t e d M a n u s c r i p t 6 tract samples of COVID-19 patients, and showed higher sensitivity than RT-PCR, especially with low viral loads 17 . ...
... In the present study, SARS-CoV- within biological samples 15 . This increased tolerance of ddPCR to PCR inhibitory molecules makes it an attractive alternative to quantitative PCR or RT-PCR for medical applications including viral diagnostics 16 and cancer research 13 . Finally, digital RT-PCR was described as presenting high potentiality over conventional real time RT-qPCR 21 . ...
Background:
Coronavirus disease 2019 (COVID-19) is a global public health problem that has already caused more than 662,000 deaths worldwide. Although the clinical manifestations of COVID-19 are dominated by respiratory symptoms, some patients present other severe damage such as cardiovascular, renal and liver injury or/and multiple organ failure, suggesting a spread of the SARS-CoV-2 in blood. Recent ultrasensitive polymerase chain reaction (PCR) technology now allows absolute quantification of nucleic acids in plasma. We herein intended to use the droplet-based digital PCR technology to obtain sensitive detection and precise quantification of plasma SARS-CoV-2 viral load (SARS-CoV-2 RNAaemia) in hospitalized COVID-19 patients.
Methods:
Fifty-eight consecutive COVID-19 patients with pneumonia 8 to 12 days after onset of symptoms and 12 healthy controls were analyzed. Disease severity was categorized as mild-to-moderate in 17 patients, severe in 16 patients and critical in 26 patients. Plasma SARS-CoV-2 RNAaemia was quantified by droplet digital Crystal Digital PCR™ next-generation technology (Stilla Technologies, Villejuif, France).
Results:
Overall, SARS-CoV-2 RNAaemia was detected in 43 (74.1%) patients. Prevalence of positive SARS-CoV-2 RNAaemia correlated with disease severity, ranging from 53% in mild-to-moderate patients to 88% in critically ill patients (p=0.036). Levels of SARS-CoV-2 RNAaemia were associated with severity (p=0.035). Among nine patients who experienced clinical deterioration during follow-up, eight had positive SARS-CoV-2 RNAaemia at baseline while only one critical patient with undetectable SARS-CoV-2 RNAaemia at the time of analysis died at day 27.
Conclusion:
SARS-CoV-2 RNAaemia measured by droplet-based digital PCR constitutes a promising prognosis biomarker in COVID-19 patients.
... Digital PCR (dPCR) was first reported in the 1990s and has since been used in a wide range of areas, particularly in bacterial and viral diagnostics [1][2][3][4][5][6][7]. Droplet digital PCR (ddPCR) is performed by partitioning the nucleic acid in thousands or millions of droplets at a concentration that will ensure that a proportion of the droplets contain no nucleic acid template [8,9]. ...
... The advantages of RT-ddPCR, compared with RT-qPCR, include absolute quantification without the need of a standard curve, and increased specificity with comparable sensitivity [7]. This could offer cost and time-savings with an assay that is less labor-intense compared with RT-qPCR since there is no reference standard to manage, prepare and qualify. ...
During vaccine production, RNA from chimeric yellow fever-dengue (CYD) vaccine viruses (CYD1, CYD2, CYD3 and CYD4) is currently quantified using separate serotype-specific RT-qPCR assays. Here we describe the results from a proof-of-concept study on the development of a multiplex reverse transcriptase droplet digital PCR (RT-ddPCR) assay for simultaneous quantification of RNA for all four viruses. Serotype-specific simplex RT-ddPCRs were developed using the serotype-specific PCR systems (forward and reverse primers and FAM (fluorescent chromophores 6-carboxyfluorescein) and YY (Yakima Yellow)-labelled probes), used in the routine RT-qPCR. The PCR systems were specific and gave similar quantification results to those from the RT-qPCR assay. Linear regression analyses were used to select relative probe concentrations to obtain distinct clusters for each target RNA in a 2-D cluster plot in a multiplex RT-ddPCR assay. We showed the clusters were positioned as predicted in the model for each CYD RNA and were well separated. The multiplex RT-ddPCR gave similar quantification results to those obtained by the serotype-specific RT-qPCR assays for triplicate samples containing 7, 8 or 9 Log10 Geq/mL. In conclusion, these results demonstrate that it is possible to quantify RNA from four CYD serotypes with a multiplex RT-ddPCR assay in a single assay.
... The disadvantage of the method is the high purchasing cost of the system (QIAcuity Digital PCR System, Qiagen, Germany), as well as of the plates, assays, and kits required for determination. Previous research has used the dPCR method in molecular diagnostics [73], personalized medicine [62], and virology [74,75] and has mainly been limited to the quantitative determination of viruses [75][76][77][78][79][80][81][82]. For some time, this method has also been used in miR studies related to the quantification of this molecule in various types of cancer [53,54], bacterial diseases [55], and neurological [56], cardiovascular [57], and neuromuscular disorders [58]. ...
MicroRNAs (miRNAs, miRs) are a group of small, 17–25 nucleotide, non-coding RNA sequences that, in their mature form, regulate gene expression at the post-transcriptional level. They participate in many physiological and pathological processes in both humans and animals. One such process is viral infection, in which miR-155 participates in innate and adaptive immune responses to a broad range of inflammatory mediators. Recently, the study of microRNA has become an interesting field of research as a potential candidate for biomarkers for various processes and disease. To use miRNAs as potential biomarkers of inflammation in viral diseases of animals and humans, it is necessary to improve their detection and quantification. In a previous study, using reverse transcription real-time quantitative PCR (RT-qPCR), we showed that the expression of ocu-miR-155-5p in liver tissue was significantly higher in rabbits infected with Lagovirus europaeus/Rabbit Hemorrhagic Disease Virus (RHDV) compared to healthy rabbits. The results indicated a role for ocu-miR-155-5p in Lagovirus europaeus/RHDV infection and reflected hepatitis and the impairment/dysfunction of this organ during RHD. MiR-155-5p was, therefore, hypothesized as a potential candidate for a tissue biomarker of inflammation and examined in tissues in Lagovirus europaeus/RHDV infection by dPCR. The objective of the study is the absolute quantification of ocu-miR-155-5p in four tissues (liver, lung, kidney, and spleen) of rabbits infected with Lagovirus europaeus/RHDV by digital PCR, a robust technique for the precise and direct quantification of small amounts of nucleic acids, including miRNAs, without standard curves and external references. The average copy number/µL (copies/µL) of ocu-miRNA-155-5p in rabbits infected with Lagovirus europaeus GI.1a/Rossi in the liver tissue was 12.26 ± 0.14, that in the lung tissue was 48.90 ± 9.23, that in the kidney tissue was 16.92 ± 2.89, and that in the spleen was 25.10 ± 0.90. In contrast, in the tissues of healthy control rabbits, the average number of copies/µL of ocu-miRNA-155-5p was 5.07 ± 1.10 for the liver, 23.52 ± 2.77 for lungs, 8.10 ± 0.86 for kidneys, and 42.12 ± 3.68 for the spleen. The increased expression of ocu-miRNA-155-5p in infected rabbits was demonstrated in the liver (a fold-change of 2.4, p-value = 0.0003), lung (a fold-change of 2.1, p-value = 0.03), and kidneys (a fold-change of 2.1, p-value = 0.01), with a decrease in the spleen (a fold-change of 0.6, p-value = 0.002). In the study of Lagovirus europaeus/RHDV infection and in the context of viral infections, this is the first report that shows the potential use of dPCR for the sensitive and absolute quantification of microRNA-155-5p in tissues during viral infection. We think miR-155-5p may be a potential candidate for a tissue biomarker of inflammation with Lagovirus europaeus/RHDV infection. Our report presents a new path in discovering potential candidates for the tissue biomarkers of inflammation.
... Using Poisson binomial distributions, the absolute copy number of a template can be calculated because the number of templates is positively correlated with the number of positive droplets. ddPCR has been widely applied in many laboratories and for diagnosis of various clinical infectious diseases because of its high sensitivity and accuracy (14)(15)(16). ...
This study aimed to develop a rapid and sensitive droplet digital PCR (ddPCR) assay for the specific detection of Klebsiella pneumoniae in fecal samples, and to evaluate its application in the clinic by comparison with real-time PCR assay and conventional microbial culture. Specific primers and a probe targeting the K. pneumoniae hemolysin (khe) gene were designed. Thirteen other pathogens were used to evaluate the specificity of the primers and probe. A recombinant plasmid containing the khe gene was constructed and used to assess the sensitivity, repeatability, and reproducibility of the ddPCR. Clinical fecal samples (n = 103) were collected and tested by the ddPCR, real-time PCR, and conventional microbial culture methods. The detection limit of ddPCR for K. pneumoniae was 1.1 copies/μL, about a 10-fold increase in sensitivity compared with real-time PCR. The ddPCR was negative for the 13 pathogens other than K. pneumoniae, confirming its high specificity. Clinical fecal samples gave a higher rate of positivity in the K. pneumoniae ddPCR assay than in analysis by real-time PCR or conventional culture. ddPCR also showed less inhibition by the inhibitor in fecal sample than real-time PCR. Thus, we established a sensitive and effective ddPCR-based assay method for K. pneumoniae. It could be a useful tool for K. pneumoniae detection in feces and may serve as a reliable method to identify causal pathogens and help guide treatment decisions.
IMPORTANCE Klebsiella pneumoniae can cause a range of illnesses and has a high colonization rate in the human gut, making it crucial to develop an efficient method for detecting K. pneumoniae in fecal samples.
... In general, methods of quantifying viruses can be divided into two categories: molecular assays that detect the presence of viral nucleic acid (DNA or RNA) and assays that measure virus infectivity in vitro 12 . Quantitative polymerase chain reaction (qPCR, for DNA) or quantitative reverse transcription polymerase chain reaction (qRT-PCR, for RNA) 13 and digital droplet PCR 14 are examples of common molecular techniques used to quantitate the viral nucleic acid in a given sample 15 . However, these highly sensitive molecular techniques cannot differentiate between viable and non-viable viruses 15 . ...
... Outbreaks and epidemics of viral diseases in recent years have raised the active search for new methods for differential diagnostics and virus detection. Modern methods of virus detection, such as polymerase chain reactions (PCR) [1] and enzyme-linked immunosorbent assays (ELISA) [2], have high sensitivity in determining the presence of influenza viruses in a sample [3,4] but have a number of disadvantages: laboriousness, time duration of the assays, low versatility, and a high percentage of false positive results [5,6]. ...
We demonstrate the possibility of applying surface-enhanced Raman spectroscopy (SERS) combined with machine learning technology to detect and differentiate influenza type A and B viruses in a buffer environment. The SERS spectra of the influenza viruses do not possess specific peaks that allow for their straight classification and detection. Machine learning technologies (particularly, the support vector machine method) enabled the differentiation of samples containing influenza A and B viruses using SERS with an accuracy of 93% at a concentration of 200 μg/mL. The minimum detectable concentration of the virus in the sample using the proposed approach was ~0.05 μg/mL of protein (according to the Lowry protein assay), and the detection accuracy of a sample with this pathogen concentration was 84%.
... Viral enfeksiyonların teşhisinde birçok yöntem bulunmasına rağmen bu yöntemlerin pratikte kullanılmasının zaman alıcı olması, duyarlılıklarının ve özgünlüklerinin düşük olması ve pahalı yöntemler olması nedeniyle kullanılması zor olmaktadır. Bu yüzden PCR gibi hızlı moleküler tekniklerin duyarlılığının yüksek olması, düşük maliyetli olması nedeniyle viral enfeksiyonların tanısında önem arz etmektedir (36)(37). CCoV' un tespitine yönelik birçok serolojik ve virolojik metodlar bulunmaktadır. ...
Coronaviridae familyasında yer alan coronaviruslar insan ve hayvanlarda sindirim ve solunum sistemi enfeksiyonlarına neden olmaktadır. Alfacoronavirus içerisinde yer alan canine coronaviruslar (CCoV)’ın CCoV I ve CCoV II olmak üzere iki alt tipi bulunmaktadır. CCoV-II ise CCoV- IIa ve IIb olmak üzere iki genotipe ayrılmaktadır. CCoV her yaştaki ve her türlü beslenme şekline sahip köpekleri etkilemesine rağmen özellikle yeni doğan yavrular daha duyarlı ve ciddi şekilde etkilenebilmektedir. Yapılan literatür araştırmalarına göre ülkemizde özellikle alt solunum yolu enfeksiyonlarındaki canine coronavirus varlığının tespitine yönelik moleküler çalışmalara rastlanılmamıştır. Bu çalışmada alt solunum yolu enfeksiyonu tanımlanan barınak köpeklerinde CCoV’un tespiti ve moleküler karakterizasyonunun yapılması amaçlandı. Bu amaçla alt solunum yolu enfeksiyonu tespit edilen 40 adet barınak köpeğinden alınan Bronkoalveolar Lavaj (BAL) sıvıları incelendi. Test edilen 40 köpeğe ait BAL sıvılarından 3 tanesinde CCoV tespit edildi. Yapılan dizin analizi sonrasında elde edilen dizinler ile filogenetik ağaç yapıldı. Filogenetik ağaçta pozitif bulunan 3 örnekten 2 sinin CCoVI, bir örneğin ise CCoV-II olduğu tespit edildi. Sonuç olarak bu çalışma ile barınak köpeklerinin alt solunum yolu rahatsızlıklarında CCoV-I ve CCoV-II’ nin rol oynayabileceği ortaya konulmuştur. Ayrıca aynı barınakta farklı hayvanlarda iki farklı CCoV’nun tespiti önemli bir veri olarak değerlendirilmiş olup özellikle barınak koşulları gibi kalabalık ortamda barındırılan köpeklerdeki her iki tipin tespiti gelecekte oluşabilecek yeni varyantların ya da alt tiplerin oluşabilme ihtimalinin de göz ardı edilmemesi gerektiğini göstermektedir.
... Several publications have reviewed the application of dPCR technology (Baker, 2012;Morley, 2014), including clinical application (Sedlak and Jerome, 2013;Kuypers and Jerome, 2017;Mao et al., 2019;Pomari et al., 2019;Salipante and Jerome, 2020;Lei et al., 2021;Tan et al., 2021), as well as environmental applications for biodegradation (Cao et al., 2020), and genetically modified organisms (Lievens et al., 2016;Pecoraro et al., 2019). However, the application of dPCR remains limited for environmental monitoring of pathogens and other clinically relevant microorganisms (Rothrock et al., 2013;Rački et al., 2014aRački et al., , 2014bWang et al., 2016;Staley et al., 2018;Bivins et al., 2021a;Graham et al., 2021;Heijnen et al., 2021;Shi et al., 2021;Simpson et al., 2021;Ahmed et al., 2022b;Ahmed et al., 2022c). ...
Digital polymerase chain reaction (dPCR) is emerging as a reliable platform for quantifying microorganisms in the field of health-related water microbiology. This paper reviews the fundamental principles of dPCR and its applications for health-related water microbiology. The relevant literature indicates increasing adoption of dPCR for measuring fecal indicator bacteria, microbial source tracking marker genes, and pathogens in various aquatic environments. The adoption of dPCR has accelerated recently due to increasing use for wastewater surveillance of SARS-CoV-2 the virus that causes COVID-19. The collective experience in the scientific literature indicates that well-optimized dPCR assays can quantify genetic fragments of microorganisms without the need for a calibration curve and often with superior analytical performance (i.e., greater sensitivity, precision, and reproducibility) than quantitative polymerase chain reaction (qPCR). Nonetheless, dPCR should not be viewed as a panacea for the fundamental uncertainties and limitations associated with measuring microorganisms in health-related water microbiology. With dPCR platforms, the sample analysis cost and processing time typically are greater than qPCR. However, if improved analytical performance (i.e., sensitivity and accuracy) is required, dPCR can be an alternative option for quantifying microorganisms, including pathogens, in aquatic environments.
... Droplet digital PCR (ddPCR), as a new and accurate quantitative technique, is an absolute quantitative method for the detection of genes (10,11), especially for trace specimens (12)(13)(14). Compared with quantitative PCR (qPCR), ddPCR has higher sensitivity and precision, so it can be used for early detection and subsequent monitoring of treatment of low-load pathogenic microorganisms, providing quantitative thresholds for judgment (15)(16)(17)(18). ddPCR has been successfully used to detect Mycobacterium tuberculosis in plasma specimens of pulmonary tuberculosis patients (19). ...
Background: Pneumocystis jirovecii is a human-specific opportunistic fungus that causes Pneumocystis pneumonia (PCP), a life-threatening opportunistic lung infection that affects immunocompromised patients. P. jirovecii colonization may be linked to the transmission of the infection. The detection of P. jirovecii in immunocompromised patients is thus especially important. The low fungal load and the presence of PCR inhibitors limit the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of P. jirovecii in specimens. Droplet digital PCR (ddPCR), however, presents a methodology that allows higher sensitivity and accuracy. Here, we developed a ddPCR method for detecting P. jirovecii DNA in respiratory specimens, and evaluated its sensitivity against qPCR.
Materials and Methods: One bronchoalveolar fluid (BALF) sample each was collected from 82 patients with potential PCP to test the presence of P. jirovecii DNA using both ddPCR and qPCR, and samples with inconsistent results between the two methods were further tested by metagenomic next generation sequencing (mNGS). In addition, 37 sputum samples from 16 patients diagnosed with PCP, as well as continuous respiratory tract specimens from nine patients with PCP and treated with sulfonamides, were also collected for P. jirovecii DNA testing using both ddPCR and qPCR.
Results: ddPCR and qPCR gave the same results for 95.12% (78/82) of the BALF samples. The remaining four specimens tested positive using ddPCR but negative using qPCR, and they were found to be positive by mNGS. Detection results of 78.37% (29/37) sputum samples were consistent between ddPCR and qPCR, while the other eight samples tested positive using ddPCR but negative using qPCR. The P. jirovecii load of patients with PCP decreased to undetectable levels after treatment according to qPCR, but P. jirovecii was still detectable using ddPCR.
Conclusions: ddPCR was more sensitive than qPCR, especially at detecting low-pathogen-load P. jirovecii . Thus, ddPCR represents a useful, viable, and reliable alternative to qPCR in P. jirovecii testing in patients with immunodeficiency.
... In addition to characteristic clinical presentations, most infectious disease diagnosis is based upon the identification of pathogen-specific molecular signatures (via culture, PCR/ RT-PCR or sequencing for DNA or RNA, or immunocapture assays for antigen or antibody) in a relevant biological fluid (Evans and Kaslow, 1997;Ksiazek et al., 1999;Bausch et al., 2000;Drosten et al., 2002Drosten et al., , 2003Mahony, 2008;Muldrew, 2009;Kortepeter et al., 2011;Sedlak and Jerome, 2013;Liu et al., 2014b). Exciting new approaches enabled by high-throughput sequencing have shown the promise of pre-symptomatic detection using genomic (Zaas et al., 2009;Woods et al., 2013) or transcriptional (Malhotra et al., 2013;Caballero et al., 2014;Connor et al., 2015;Speranza et al., 2018) expression profiles in the host (Shurtleff et al., 2015). ...
Background and Objectives: Early warning of bacterial and viral infection, prior to the development of overt clinical symptoms, allows not only for improved patient care and outcomes but also enables faster implementation of public health measures (patient isolation and contact tracing). Our primary objectives in this effort are 3-fold. First, we seek to determine the upper limits of early warning detection through physiological measurements. Second, we investigate whether the detected physiological response is specific to the pathogen. Third, we explore the feasibility of extending early warning detection with wearable devices.
Research Methods: For the first objective, we developed a supervised random forest algorithm to detect pathogen exposure in the asymptomatic period prior to overt symptoms (fever). We used high-resolution physiological telemetry data (aortic blood pressure, intrathoracic pressure, electrocardiograms, and core temperature) from non-human primate animal models exposed to two viral pathogens: Ebola and Marburg (N = 20). Second, to determine reusability across different pathogens, we evaluated our algorithm against three independent physiological datasets from non-human primate models (N = 13) exposed to three different pathogens: Lassa and Nipah viruses and Y. pestis. For the third objective, we evaluated performance degradation when the algorithm was restricted to features derived from electrocardiogram (ECG) waveforms to emulate data from a non-invasive wearable device.
Results: First, our cross-validated random forest classifier provides a mean early warning of 51 ± 12 h, with an area under the receiver-operating characteristic curve (AUC) of 0.93 ± 0.01. Second, our algorithm achieved comparable performance when applied to datasets from different pathogen exposures – a mean early warning of 51 ± 14 h and AUC of 0.95 ± 0.01. Last, with a degraded feature set derived solely from ECG, we observed minimal degradation – a mean early warning of 46 ± 14 h and AUC of 0.91 ± 0.001.
Conclusion: Under controlled experimental conditions, physiological measurements can provide over 2 days of early warning with high AUC. Deviations in physiological signals following exposure to a pathogen are due to the underlying host’s immunological response and are not specific to the pathogen. Pre-symptomatic detection is strong even when features are limited to ECG-derivatives, suggesting that this approach may translate to non-invasive wearable devices.
... A newer iteration of PCR testing is digital PCR (dPCR), whereby samples are diluted and thousands of PCR reactions are run in parallel in separate wells. This process works using Poisson statistical theory, assuming that for each micro-PCR amplification well there are either 0 or 1 copies of the nucleic acid of interest, allowing absolute quantification of nucleic acid present in a sample [14]. ...
This review discusses the current testing methodologies for COVID-19 diagnosis and explores next-generation sequencing (NGS) technology for the detection of SARS-CoV-2 and monitoring phylogenetic evolution in the current COVID-19 pandemic. The review addresses the development, fundamentals, assay quality control and bioinformatics processing of the NGS data. This article provides a comprehensive review of the obstacles and opportunities facing the application of NGS technologies for the diagnosis, surveillance, and study of SARS-CoV-2 and other infectious diseases. Further, we have contemplated the opportunities and challenges inherent in the adoption of NGS technology as a diagnostic test with real-world examples of its utility in the fight against COVID-19.
... In recent years, we have seen the advance of digital PCR (dPCR) as a complementary approach for measuring nucleic acids, a technique that is highly accurate and reproducible when targeting the viral genes of SARS-CoV-2 [114][115][116][117]. The advantages of digital PCR compared to quantitative PCR include quantification without the need for calibration curves, higher accuracy, and sensitivity that may arise from sub-optimal amplification efficacy because dPCR can detect low amounts of nucleic acid [118,119]. ...
The coronavirus disease 2019 (COVID-19) has caused a large global outbreak. It is accordingly important to develop accurate and rapid diagnostic methods. The polymerase chain reaction (PCR)-based method including reverse transcription-polymerase chain reaction (RT-PCR) is the most widely used assay for the detection of SARS-CoV-2 RNA. Along with the RT-PCR method, digital PCR has emerged as a powerful tool to quantify nucleic acid of the virus with high accuracy and sensitivity. Non-PCR based techniques such as reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) are considered to be rapid and simple nucleic acid detection methods and were reviewed in this paper. Non-conventional molecular diagnostic methods including next-generation sequencing (NGS), CRISPR-based assays and nanotechnology are improving the accuracy and sensitivity of COVID-19 diagnosis. In this review, we also focus on standardization of SARS-CoV-2 nucleic acid testing and the activity of the National Metrology Institutes (NMIs) and highlight resources such as reference materials (RM) that provide the values of specified properties. Finally, we summarize the useful resources for convenient COVID-19 molecular diagnostics.
... In IWW samples with low RNA concentrations, qPCR could potentially be affected by inhibitors present in the sample, resulting in poor amplification efficiency, less precision and the need for relative quantification, which might lead to the occurrence of false-negative results (Klein, 2002;Kuypers and Jerome, 2017;Suo et al., 2020). It is hypothesized that dPCR could potentially be more tolerant to PCR inhibitors compared to qPCR (Baker, 2012;Dingle et al., 2013;Sedlak and Jerome, 2013;Suo et al., 2020). These PCR inhibitors are potentially highly present in IWW and could be co-concentrated during sample preparation (Ahmed et al., 2020d;Gibson et al., 2012). ...
Wastewater-based epidemiology of SARS-CoV-2 could play a role in monitoring the spread of the virus in the population and controlling possible outbreaks. However, sensitive sample preparation and detection methods are necessary to detect trace levels of SARS-CoV-2 RNA in influent wastewater (IWW).
Unlike predecessors, method optimization of a SARS-CoV-2 RNA concentration and detection procedure was performed with IWW samples with high viral SARS-CoV-2 loads. This is of importance since the SARS-CoV-2 genome in IWW might have already been subject to in-sewer degradation into smaller genome fragments or might be present in a different form (e.g. cell debris,…). Centricon Plus-70 (100 kDa) centrifugal filter devices resulted in the lowest and most reproducible Ct-values for SARS-CoV-2 RNA. Lowering the molecular weight cut-off did not improve our limit of detection and quantification (approximately 10⁰ copies/μL for all genes). Quantitative polymerase chain reaction (qPCR) was employed for the amplification of the N1, N2, N3 and E -gene fragments.
This is one of the first studies to apply digital polymerase chain reaction (dPCR) for the detection of SARS-CoV-2 RNA in IWW. dPCR showed high variability at low concentration levels (10⁰ copies/μL), indicating that variability in bioanalytical methods for wastewater-based epidemiology of SARS-CoV-2 might be substantial. dPCR results in IWW were in line with the results found with qPCR.
On average, the N2-gene fragment showed high in-sample stability in IWW for 10 days of storage at 4 °C. Between-sample variability was substantial due to the low native concentrations in IWW. Additionally, the E-gene fragment proved to be less stable compared to the N2-gene fragment and showed higher variability. Freezing the IWW samples resulted in a 10-fold decay of loads of the N2- and E-gene fragment in IWW.
... Digital PCR is a new approach for nucleic acid detection and quantification that offers an alternative method for absolute quantification. This technology enables the detection and quantification of low-abundance pathogens with higher precision, accuracy, sensitivity, and reproducibility than quantitative Real-Time PCR [67,68]. Life Technologies have successfully applied dPCR technology for the detection of WNV [69]. ...
Introduction
Molecular technology has played an important role in arboviruses diagnostics. PCR-based methods stand out in terms of sensitivity, specificity, cost, robustness, and accessibility, and especially the isothermal amplification (IA) method is ideal for field-adaptable diagnostics in resource-limited settings (RLS).
Areas covered
In this review, we provide an overview of the various molecular methods for West Nile, Zika, Dengue and Chikungunya. We summarize literature works reporting the assessment and use of in house and commercial assays. We describe limitations and challenges in the usage of methods and opportunities for novel approaches such as Next Generation Sequencing (NGS).
Expert opinion
The rapidity and accuracy of differential diagnosis is essential for a successful clinical management, particularly in co-circulation area of arboviruses. Several commercial diagnostic molecular assays are available, but many are not affordable by RLS and not usable as Point-of-care/Point-of-need (POC/PON) such as Real Time RT-PCR, Array-based methods and NGS. In contrast, the IA-based system fits better for POC/PON but it is still not ideal for the multiplexing detection system. Improvement in the characterization and validation of current molecular assays is needed to optimize their translation to the point of care.
... Many of the advantages of ddPCR are derived from its use of end point quantification rather than quantification by real-time amplification used by qPCR. 11 This qualitative measurement for each droplet confers increased resistance to inhibitors, 12 reduced susceptibility to poor amplification efficiency, 13 and compartmentalization of the individual droplets for a higher confidence of low positive results 14 because of lower competition for reaction resources with higher-frequency targets. 15 To ensure optimal detection, a minimum number of accepted droplets is required for analysis. ...
Objectives:
The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR-in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results.
Methods:
We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer's specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription-polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated.
Results:
The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate.
Conclusions:
The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.
... Digital PCR (dPCR) is a method that can be performed as an SItraceable reference measurement procedure when measuring DNA in buffered solution [13] and can perform with high reproducibility when incorporating extraction protocol to measure DNA from whole bacteria [14] and viruses [15]. In combination with reverse transcription (RT), dPCR was proposed as method to provide accurate and robust quantification of HIV RNA in plasma samples in the clinically relevant low concentration range [16]. Previous dPCR studies reported to date have quantified HIV-1 DNA as well as cell associated and synthetic RNA [7]. ...
Viral load monitoring in human immunodeficiency virus type 1 (HIV-1) infection is often performed using reverse transcription quantitative PCR (RT-qPCR) to observe response to treatment and identify the development of resistance. Traceability is achieved using a calibration hierarchy traceable to the International Unit (IU). IU values are determined using consensus agreement derived from estimations by different laboratories. Such a consensus approach is necessary due to the fact that there are currently no reference measurement procedures available that can independently assign a reference value to viral reference materials for molecular in vitro diagnostic tests. Digital PCR (dPCR) is a technique that has the potential to be used for this purpose. In this paper, we investigate the ability of reverse transcriptase dPCR (RT-dPCR) to quantify HIV-1 genomic RNA without calibration. Criteria investigated included the performance of HIV-1 RNA extraction steps, choice of reverse transcription approach and selection of target gene with assays performed in both single and duplex format. We developed a protocol which was subsequently applied by two independent laboratories as part of an external quality assurance (EQA) scheme for HIV-1 genome detection. Our findings suggest that RT-dPCR could be used as reference measurement procedure to aid the value assignment of HIV-1 reference materials to support routine calibration of HIV-1 viral load testing by RT-qPCR.
... Droplet digital PCR is not dependent on amplification efficiency and, therefore, may provide more accurate measurements than qPCR. Droplet digital PCR is used widely in clinical research (Kiselinova et al. 2014;Strain et al. 2013;Palmer 2013;Hall Sedlak and Jerome 2014;Sedlak and Jerome 2013;Baker 2012), and it has recently been applied in very few studies to detect and quantify enteric viruses in wastewater (Racki et al. 2014b;Kishida et al. 2014;Coudray-Meunier et al. 2015;Ishii et al. 2014). ...
The quantification and trends in concentrations for naturally occurring rotaviruses (RV) and enteroviruses (EV) in untreated sewage in various wastewater systems have not often been compared. There is now greater interest in monitoring the infections in the community including live vaccine efficacy by evaluating untreated sewage. The goals of this study were to 1) survey the concentrations of naturally occurring RV and EV in untreated sewage using a reverse transcription—droplet digital polymerase chain reaction (RT-ddPCR) and 2) investigate the use of a new adsorption elution (bag-mediated filtration system (BMFS) using ViroCap filters) against more traditional polyethylene glycol (PEG) precipitation for virus concentration. Sewage samples were collected from lagoons in Kenya and Michigan (MI), the United States (USA) and from wastewater treatment plants (WWTPs) in the USA. RVs were detected at geometric mean concentrations in various locations, California (CA) 1.31 × 10⁵ genome copies/L (gc/L), Kenya (KE) 2.71 × 10⁴ gc/L and Virginia (VA) 1.48 × 10⁵ gc/L, and EVs geometric means were 3.72 × 10⁶ gc/L (CA), 1.18 × 10⁴ gc/L (Kenya), and 6.18 × 10³ gc/L (VA). The mean RV concentrations using BMFS-ViroCap in split samples compared to PEG precipitation methods demonstrated that the levels were only 9% (#s BMFS/PEG) in the Michigan lagoons which was significantly different (p < 0.01). This suggests that RV concentrations in Kenya are around 1.69 × 10⁶ gc/L. Overall, there was no difference in concentrations for the other sampling locations across the methods of virus recovery (i.e., PEG precipitation and HA filters) using one-way ANOVA (F = 1.7, p = 0.2739) or Tukey–Kramer pairwise comparisons (p > 0.05). This study provides useful data on RV and EV concentrations in untreated sewage in Kenya and the USA. It also highlights on the usefulness of the RT-ddPCR for absolute quantification of RV and EV in sewage samples. The BMFS using ViroCap filters while less efficient compared to the more traditional PEG precipitation method was able to recover RVs and EVs in untreated sewage and may be useful in poor resource settings while underestimating viruses by 1 to 1.5 logs.
... Interest is growing in the application of dPCR for direct quantification of HIV-1 DNA (Bosman et al., 2015, Eriksson et al., 2013, Henrich et al., 2012, Jones et al., 2014. In contrast to qPCR, dPCR can provide sensitive, absolute measurements without the need for a standard curve (Sedlak and Jerome, 2013). dPCR has been reported to have enhanced precision and better tolerance to primer-probe mismatches compared to qPCR; a desirable attribute for measuring highly variable HIV-1 sequences , Rutsaert et al., 2018a. ...
Metrology, the study of measurement, is an emerging concept within molecular diagnosis of infection. Metrology promotes high-quality, reproducible data to be used in clinical management of infection, through characterisation of technical error and measurement harmonisation. This influences measurement accuracy, which has implications for setting thresholds between healthy and disease states, monitoring disease progression, and establishing cures. This thesis examines the placing of metrology in molecular diagnosis of infectious diseases. Sources of experimental error in advanced methodologies – dPCR and MALDI-TOF MS – that can influence measurement accuracy for RNA, DNA and protein biomarkers were investigated for HIV-1, methicillin-resistant Staphylococcus spp and organisms associated with hospital transmission. Measurement error introduced at different stages of a method can directly impact upon clinical results. A 30% bias was introduced between dPCR and qPCR quantification of HIV-1 DNA in clinical samples, owing to instability in the qPCR calibration material. In addition, experimental variability was found to influence classification of protein profiles which can limit the resolution of MALDI-TOF MS for strain typing bacteria. This thesis also addresses the prospective role of these advanced methods in supporting accurate clinical measurements. dPCR offers precise measurements of RNA and DNA targets and could be used to support qPCR, or for value assignment of reference materials to harmonise inter-laboratory results. MALDI-TOF MS demonstrated potential for strain typing Acinetobacter baumannii; results correlated with epidemiological data and WGS, although were not consistent with reference typing. Further work should examine the extent to which MALDI-TOF MS can support or replace contemporary strain typing methods for identifying nosocomial outbreaks. Molecular approaches possess a crucial role in the detection, quantification and characterisation of pathogens, and are invaluable tools for managing emerging diseases. Supporting accuracy and reproducibility in molecular measurements could help to strengthen diagnostic efforts, streamline clinical pathways and provide overall benefit to patient care.
... The pros of dPCR compared to a conventional quantitative polymerase chain reaction (qPCR) are that there is no requirement for a standard curve for analysis, it is able to tolerate any PCR inhibitors [128], able to analyze the presence of uncommon targets in large sample mixture, and capable of identifying minute fold changes [129]. In addition, sample segregation and absolute quantification in dPCR allow it to be suitable detection candidates of rare allele [130,131], genomic mutations such as variation, DNA deletions or replication [132][133][134], viral load, and next-generation sequencing libraries quantification [129,[135][136][137][138][139][140]. In general, digital PCR is utilized for circulating tumor DNA and miRNA identification in cancer patients [66,141,142]. ...
Triple-negative breast cancer (TNBC) is an aggressive breast type of cancer with no expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2). It is a highly metastasized, heterogeneous disease that accounts for 10–15% of total breast cancer cases with a poor prognosis and high relapse rate within five years after treatment compared to non-TNBC cases. The diagnostic and subtyping of TNBC tumors are essential to determine the treatment alternatives and establish personalized, targeted medications for every TNBC individual. Currently, TNBC is diagnosed via a two-step procedure of imaging and immunohistochemistry (IHC), which are operator-dependent and potentially time-consuming. Therefore, there is a crucial need for the development of rapid and advanced technologies to enhance the diagnostic efficiency of TNBC. This review discusses the overview of breast cancer with emphasis on TNBC subtypes and the current diagnostic approaches of TNBC along with its challenges. Most importantly, we have presented several promising strategies that can be utilized as future TNBC diagnostic modalities and simultaneously enhance the efficacy of TNBC diagnostic.
... Droplet digital PCR is an absolute quantification method without the requirement for a standard curve and has superior sensitivity and is highly replicable, which is especially suited to esophageal tissue, which has a low viral load. [76][77][78] Furthermore, HPV viral load measurement should be standardized. ...
The causal link between high‐risk human papillomavirus (hr‐HPV) infection and cervical, anogenital, and some oropharyngeal malignancies has been established by both molecular and epidemiological data. The association between HPV and esophageal squamous cell carcinoma (ESCC) remains controversial, as is the true prevalence of HPV infection in ESCC. The wide range in reported rates reflects variability in the primary literature, with some larger scale case–control studies suggesting the infection rates range from 0% to 78%. Interactions between HPV and the Barrett's metaplasia–dysplasia–carcinoma sequence have been explored, and these studies have shown some conflicting data. Overall, systematic reviews have reported the prevalence of HPV‐positive DNA in esophageal adenocarcinoma patients of between 13% and 35%. Postulated reasons for discrepancies in HPV prevalence rates in esophageal cancer include variations in testing methodology and assay sensitivities; technical issues, including the lack of a gold‐standard primer; types of specimens utilized (fresh‐frozen versus formalin‐fixed tissue); geographical variation; cross‐contamination; and small sample sizes. Thus, efforts must be undertaken to (1) standardize HPV testing, ideally in a central laboratory and utilizing tests that detect viral transcriptional activity; (2) avoid cross‐contamination; and (3) recruit large numbers of patients to accurately ascertain HPV rates in esophageal malignancy.
... The use of sensitive processes for Salmonella detection brings along the application using real-time PCR, which is widely used for the diagnosis of foodborne pathogens. Considering the fact that Real-Time PCR stands as quite sensitive against inhibitory agents, a comparatively new technology, ddPCR is now proposed to be used for this purpose, with its ability to detect even one copy of DNA in a sample [36]. In this study, it is ought to increase the sensitivity of ddPCR by adding a very short pre-enrichment step, which will not set back the aim of having the same-day results. ...
With its ability to easily infect each step of food production chain, Salmonella spp. stands as one of the most important foodborne pathogens which impose threats against economy and human health by creating high morbidity and mortality, worldwide. Although sensitive screening and detection methodologies based on common conventional techniques have been developed for Salmonella spp., their long turnaround time and strict legislative demands directs the researchers to develop more sensitive and rapid methods. Digital PCR has many potential advantages over its closest competitor, Real-Time PCR. It gives more accurate, sensitive, and precise results, especially for samples that contain low pathogen concentrations and shows more resistance against PCR inhibitors. In this study, we developed a droplet digital PCR screening methodology with a short pre-enrichment step. For this purpose, 25 g raw minced meat was subjected to Salmonella contamination, and following a very short 3 h enrichment step, bacterial isolation from 5 mL of sample was achieved. For the specific detection of the Salmonella spp., bipA gene region was selected as a better candidate. Subsequently, droplet digital PCR setup was performed and detection sensitivity of the test was determined as 1.39 cfu/mL. Furthermore, parallel experiments were also conducted using Real-Time PCR, for comparative and validative purposes. Conclusively, data gathered in this study have shown that droplet digital PCR with our proposed setup can sensitively detect Salmonella spp. in raw minced meat samples while generating the same-day results.
... The fraction of amplification positive partitions is used to estimate the concentration of the initial target sequence using binomial Poisson statistics (4,6). Nowadays, different dPCR commercial platforms are available as a useful tool for precise quantification of nucleic acids in a variety of basic research and clinical applications (4,(7)(8)(9). ...
Bluetongue (BT) is a major Office International des Epizooties (OIE)-listed disease of wild and domestic ruminants caused by several serotypes of Bluetongue virus (BTV), a virus with a segmented dsRNA genome belonging to the family Reoviridae, genus Orbivirus. BTV is transmitted through the bites of Culicoides midges. The aim of this study was to develop a new method for quantification of BTV Seg-10 by droplet digital RT-PCR (RTdd-PCR), using nucleic acids purified from complex matrices such as blood, tissues, and midges, that notoriously contain strong PCR inhibitors. First, RTdd-PCR was optimized by using RNAs purified from serially 10-fold dilutions of a BTV-1 isolate (105.43TCID50/ml up to 10−0.57 TCID50/ml) and from the same dilutions spiked into fresh ovine EDTA-blood and spleen homogenate. The method showed a good degree of linearity (R² ≥ 0.995). The limit of detection (LoD) and the limit of quantification (LoQ) established were 10−0.67TCID50/ml (0.72 copies/μl) and 100.03TCID50/ml (3.05 copies/μl) of BTV-1, respectively. Second, the newly developed test was compared, using the same set of biological samples, to the quantitative RT-PCR (RT-qPCR) detecting Seg-10 assay widely used for the molecular diagnosis of BTV from field samples. Results showed a difference mean of 0.30 log between the two assays with these samples (p < 0.05). Anyway, the analysis of correlation demonstrated that both assays provided similar measurements with a very close agreement between the systems.
Digital PCR (dPCR) is a technique for absolute quantification of nucleic acid molecules. To develop a dPCR technique that enables more accurate nucleic acid detection and quantification, we established a novel dPCR apparatus known as centrifugal force real-time dPCR (crdPCR). This system is efficient than other systems with only 2.14% liquid loss by dispensing samples using centrifugal force. Moreover, we applied a technique for analyzing the real-time graph of the each micro-wells and distinguishing true/false positives using artificial intelligence to mitigate the rain, a persistent issue with dPCR. The limits of detection and quantification were 1.38 and 4.19 copies/μL, respectively, showing a two-fold higher sensitivity than that of other comparable devices. With the integration of this new technology, crdPCR will significantly contribute to research on next-generation PCR targeting absolute micro-analysis.
Nowadays digital PCR (dPCR) is a nucleic acid quantification method widely used in genetic analysis. One of the most significant advantages of dPCR over other methods is the possibility for absolute quantitative determination of genetic material without construction of calibration curves, which allows one to detect even single molecules of nucleic acids, and, hence, early diagnosis of diseases. A specific characteristic of dPCR is the detection of the analyzed biological object in each microreaction, followed by the presentation of the analysis results in a binary system, thereby giving the method name. The key aspects of developing the dPCR method, i.e. from the first devices based on microfluidic chip technology to modern systems capable of measuring a target at a concentration of up to 1 in 100 000 copies were shown in the current work. We analyzed the data on the detection of various pathogens using dPCR, as well as summarized various study results demonstrating the innovativeness of this method “point-of-care”. Both the possibilities of multiplex dPCR analysis and its potential in clinical practice were presented. The review also addresses the issue of the dPCR role in the development of non-invasive methods for oncological diseases to be analyzed. Possible ways of developing dPCR technology were emphasized, including the use as a “point-of-care” systems.
This study was conducted to design primer set and to map restriction site targeting synonymous mutation c.297A>G of the POU domain class 1 transcription factor 1 gene ( POU1F1 ) of Friesian Holstein in silico . Primer set was designed to target exon 3 of the POU1F1 based on nucleotide sequence at GenBank database with accession number NC_037328.1 using primer3. Simulations of amplification targeting POU1F1 fragment and restriction fragment length polymorphism (RFLP) were conducted in silico using serial cloner version 2.6 and NEBcutter version 3.0, respectively. This study used a primer pair (5′-CCT TTT AGA ACT GAG ACT GGC TG-3′ and 5′-CCC ACA GCT GTT AAC AAG CA-3′) that produce 356 bp of estimated product size. Moreover, a synonymous mutation, c.297A>G of the POU1F1 , could be detected using BsII restriction enzyme in silico . The BsII did not have restriction site for AA genotype. On the other hand, it could cut the PCR product size into two fragments (167 and 189 bp) for GG genotype. It can be concluded that in silico analysis successfully amplified target region using primer designed in this study and RFLP simulation using BsII could detect synonymous mutation c.297A>G of the POU1F1 . Further in vitro study should be conducted to identify c.297A>G in Friesian Holstein population.
The COVID-19 pandemic is to escalate globally and acquire new mutations quickly, so accurate diagnostic technologies play a vital role in controlling and understanding the epidemiology of the disease. A plethora of technologies acquires diagnosis of individuals and informs clinical management of COVID. Some important biochemical parameters for COVID diagnosis are the elevation of liver enzymes, creatinine, and nonspecific inflammatory markers such as C-reactive protein (CRP) and Interleukin 6 (IL-6). The main progression predictors are lymphopenia, elevated D-dimer, and hyperferritinemia, although it is also necessary to consider LDH, CPK, and troponin in the marker panel of diagnosis. Owing to the greater sensitivity and accuracy, molecular technologies such as conventional polymerase chain reaction (PCR), reverse transcription (RT)-PCR, nested PCR, loop-mediated isothermal amplification (LAMP), and xMAP technology have been extensively used for COVID diagnosis for some time now. To make so many diagnostics accessible to general people, many techniques may be exploited, including point of care (POC), also called bedside testing, which is developing as a portable promising tool in pathogen identification. Some other lateral flow assay (LFA)-centered techniques like SHERLOCK, CRISPR-Cas12a (AIOD-CRISPR), and FNCAS9 editor limited uniform detection assay (FELUDA), etc. have shown auspicious results in the rapid detection of pathogens. More recently, low-cost sequencing and advancements in big data management have resulted in a slow but steady rise of next-generation sequencing (NGS)-based approaches for diagnosis that have potential relevance for clinical purposes and may pave the way toward a better future. Due to the COVID-19 pandemic, various institutions provided free, specialized websites and tools to promote research and access to critically needed advanced solutions by alleviating research and analysis of data within a substantial body of scientific and patent literature regarding biochemical and molecular diagnosis published since January 2020. This circumstance is unquestionably unique and difficult for anyone using patent information to find pertinent disclosures at a specific date in a trustworthy manner.
In Vitro Diagnosis (IVD) technology is able to accurately detect pathogens or biomarkers at an initial stage of disease, which works as an important toolbox for disease diagnosis. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system, as an emerging IVD method, plays a crucial role in the field of infectious disease detection due to its superior sensitivity and specificity. Recently, an increasing number of scientists have been devoted to improving the performance of CRISPR-based detection and on-site point-of-care testing (POCT) from extraction-free detection, amplification-free, modified Cas/crRNA complexes, quantitative assays, one-pot detection and multiplexed platform. In this review, we describe the potential roles of these novel approaches and platforms in one-pot methods, quantitative molecular diagnostics as well as multiplexed detection. This review will not only help guide the full use of the CRISPR-Cas tools for quantification, multiplexed detection, POCT and as next-generation diagnostic biosensing platforms but also inspire new ideas, technological advances and engineering strategies to address real-world challenges like the ongoing COVID-19 pandemic.
Berries are important vehicles for norovirus (NoV) and hepatitis A virus (HAV) foodborne outbreaks. Sensitive and quantitative detection of these viruses in food samples currently relies on RT-qPCR, but remains challenging due to their low concentration and the presence of RT-qPCR inhibitors. Moreover, quantification requires a standard curve. In this study, crystal digital RT-PCR (RT-cdPCR) assays were adapted from RT-qPCR sets of primers and probe currently used in our diagnostic laboratory for the detection and precise quantification of norovirus genogroups I and II (NoV GI, GII) and hepatitis A virus (HAV) RNA in frozen raspberry samples. We selected assay conditions based on optimal separation of positive and negative droplets, and peak resolution. Using virus-specific in vitro RNA transcripts diluted in raspberry RNA extracts, we showed that all three RT-cdPCR assays were sensitive, and we estimated the 95 % detection limit at 9 copies per RT-cdPCR reaction for NoV GI, 3 for NoV GII, and 14 for HAV. Serial dilutions of the RNA transcripts showed excellent linearity over a range of four orders of magnitude. We achieved precise quantification (CV ≤ 35 %) of the RNA transcripts between runs down to 15–145 copies per reaction for NoV GI, <20 for NoV GII, and < 15 for HAV. The three RT-cdPCR assays also proved to be tolerant to inhibitors from frozen raspberries, although not as tolerant as the RT-qPCR assays in the case of NoV GI and HAV. We further evaluated the assays with inoculated frozen raspberry samples and compared their performance to that of the RT-qPCR assays. As compared to the corresponding RT-qPCR assays, the NoV GI and HAV RT-cdPCR assays showed a decreased qualitative sensitivity, while the NoV GII RT-cdPCR assay had an increased sensitivity. As for quantification, the NoV GI and NoV GII RT-cdPCR assays produced similar estimates of RNA copy number than their respective RT-qPCR assays, whereas for HAV, the RT-cdPCR assay produced lower estimates than the RT-qPCR assay. However, all the RT-cdPCR assays provided more precise quantitative measurements at low levels of contamination than the RT-qPCR assays. In conclusion, the potential of the RT-cdPCR assays in this study to detect viral RNA from frozen raspberries varied according to assay, but these RT-cdPCR assays should be considered for precise absolute quantification in difficult matrices such as frozen raspberries.
In December 2019, a disease linked to the coronavirus (CoV) was identified in the capital of China’s Wuhan. When seen under an electron microscope, CoVs, which are enveloped positive-sense RNA viruses, appear like crown-shaped viruses. There are four subtypes of CoVs such as (a) alpha, (b) beta, (c) delta, (d) gamma CoV. Coronavirus disease is caused by the extreme acute respiratory syndrome coronavirus 2, which is caused by a beta coronavirus (-CoVs or Beta-CoVs) (SARS-CoV-2). Infected people may have fever of 38°C, cough, and shortness of breath. WHO officially called COVID-19, an abbreviated form of coronavirus disease 2019, on February 12, 2020.
Breast carcinoma is a disease with a high degree of heterogeneity. Every year, around 1 million new instances of breast carcinoma are diagnosed around the world. Triple negative breast cancer (TNBC) is a type of breast cancer that lacks estrogen, progesterone, and ERBB2 receptor expression, making them a particularly difficult therapeutic target because of their invading nature and limited responsiveness to therapies. They account for 15% to 20% of all cases of breast carcinoma. Gene expression analysis shows that most TNBC possesses a basal-like molecular profile. Their pathological and clinical characteristics are similar to inherited BRCA1 breast tumors. TNBC usually affects younger females and is characterized by higher relapse, CNS and visceral metastasis, and early mortality. TNBC is divided into six subtypes, each with its own molecular profile, prognosis, and likely treatment responses. Increased hip-to-waist ratios, higher parity, the shorter period of breastfeeding, young age at first-term pregnancy, and shorter period of breast-feeding have all been linked to an elevated frequency of triple-negative breast cancers among premenopausal African-American women. TNBC has specific imaging characteristics, commonly showing as a mass on mammograms and ultrasonography with generally benign characteristics and more alarming results on magnetic resonance imaging. Epigenetics is a promising area of inquiry in modern cancer research. Improvements in cancer therapy, detection, and prevention are feasible by researching the epigenetic processes driving tumorigenesis–DNA methylation, non-coding RNAs, and histone changes. To summarize, TNBC is a complicated disease that will most likely necessitate many distinct targeted therapies in order to achieve substantial progress in improving results.
Die Polymerasekettenreaktion (Polymerase Chain Reaction, PCR) ist ein Verfahren zur synthetischen Vervielfältigung (Amplifikation) von Nucleinsäuren (Desoxyribonucleinsäure, DNA) bzw. Nucleinsäureabschnitten mithilfe des Enzyms DNA-Polymerase, welches in allen Lebewesen vorkommt und vor der Zellteilung während der Replikation die DNA der Zelle verdoppelt. Das Verfahren wurde 1983 zum ersten Mal wissenschaftlich durch Kary B. Mullis vollständig beschrieben, wofür er 1993 mit dem Nobelpreis für Chemie geehrt wurde.
Droplet digital polymerase chain reaction (ddPCR) is a rapidly developing technology used for accurate, quantitative analysis of rare samples. However, ddPCR has only been implemented in research file but rarely in clinical trials due to its relatively high cost and negative user experiences compared with qPCR. We developed a novel programmable on-demand droplet generator based on a microfluidic adaptive printing system (MAP-ddPCR) to create a cost-effective ddPCR process. This process easily produces low-volume, spot-on-demand droplet dispensing and data analysis using simple equipment and workflow. Compared with the existing droplet generation systems that rely on surface-assistant, the proposed MAP system generates a variety of droplet arrays on regular non-surface-treated glass slides. This system directly processes PCR and performs data analysis without requiring a secondary droplets transfer. The static and independent properties of each droplet dramatically avoid cross-contamination during PCR, provide the opportunity to trace droplets in real-time and simplify the analysis. We demonstrated that the MAP-ddPCR produces reliable data using gradient concentrations of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in human genomic cDNA. These concentrations were further verified by quantitative PCR (qPCR). In addition, a very low viral load of SARS-CoV-2 was precisely detected and quantified by the MAP-ddPCR system. Finally, this system is affordable and simpler to integrate compared to other more expensive commercial digital PCR methods. Therefore, the proposed MAP-ddPCR system is expected to have a significant impact on market applications.
The isothermal molecular diagnosis with CRISPR has attracted particular interest for the sensitive, specific detection of nucleic acids. However, most of the assays with Cas enzymes were performed in bulk assays using multistep approaches and hard to realize quantitative detection. Herein, we report Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA), a digital format of SHERLOCK with enhanced robustness and sensitivity. We first address the macromolecular crowding problems when combining the recombinase polymerase amplification (RPA) and Cas13a detection into a one-pot SHERLOCK assay. After the assay optimization, the enhanced one-pot SHERLOCK (E-SHERLOCK) achieves high robustness and 200-fold increased sensitivity. Leveraging droplet microfluidics, we streamline the E-SHERLOCK to eliminate undesired input targets caused by pre-amplification before partition, enabling background-free absolute quantification. From the real-time monitoring, MEDICA enables qualitative detection within 10 min and absolute quantification within 25 min. For the proof of concept, we applied MEDICA to quantify HPV 16 and 18 viral loads in 44 clinical samples, indicating perfect accordance with qPCR results. MEDICA highlights the CRISPR-based isothermal assays are promising for the next generation of point-of-care diagnostics.
After World Health Organization (WHO) announced COVID-19 outbreak a pandemic, we all again realized the importance of developing rapid diagnostic kits. In this article, we introduced a lightweight and field-portable biosensor employing a plasmonic chip based on nanohole arrays integrated to a lensfree-imaging framework for label-free detection of viruses in field-settings. The platform utilizes a CMOS (complementary metal–oxide–semiconductor) camera with high quantum efficiency in the spectral window of interest to monitor diffraction field patterns of nanohole arrays under the uniform illumination of an LED (light-emitting diode) source which is spectrally tuned to the plasmonic mode supported by the nanohole arrays. As an example for the applicability of our biosensor for virus detection, we could successfully demonstrate the label-free detection of H1N1 viruses, e.g., swine flu, with medically relevant concentrations. We also developed a low-cost and easy-to-use sample preparation kit to prepare the surface of the plasmonic chip for analyte binding, e.g., virus-antibody binding. In order to reveal a complete biosensor technology, we also developed a user friendly PythonTM – based graphical user interface (GUI) that allows direct access to biosensor hardware, taking and processing diffraction field images, and provides direct virus information to the end-user. Employing highly sensitive nanohole arrays and lensfree-imaging framework, our platform could yield an LOD as low as 10³ TCID50/mL. Providing accurate and rapid sensing information in a handheld platform, weighing only 70 g and 12 cm tall, without the need for bulky and expensive instrumentation, our biosensor could be a very strong candidate for diagnostic applications in resource-poor settings. As our detection scheme is based on the use of antibodies, it could quickly adapt to the detection of different viral diseases, e.g., COVID-19 or influenza, by simply coating the plasmonic chip surface with an antibody possessing affinity to the virus type of interest. Possessing this ability, our biosensor could be swiftly deployed to the field in need for rapid diagnosis, which may be an important asset to prevent the spread of diseases before turning into a pandemic by isolating patients from the population.
Viral infections have threatened public health for a long time and have severely impacted socio-economic development. Traditional diagnostic and therapeutic methods are not enough to control the viral contagions that have been realized by numerous pandemics. Thus, it is necessary to develop novel diagnostic methods, as well as therapeutic strategies against the existing and emerging viruses. The advent of nanotechnology has enabled us to think differentially about the management of viral diseases. Magnetic nanoparticles (MNPs) are a class of nanomaterials that have extensively been used in different biological applications. They have also shown their potential as active antimicrobial agents against several bacteria, viruses, and other microorganisms. Here, we would like to apprise the scientific community about the use of MNPs as a novel tool for viral diagnostics and antiviral applications. This review summarizes the advances in the clinical diagnosis, detection, and therapeutic applications of magnetic nanoparticles against viral infections. This journal is
The implementation of genetically modified organism (GMO) labeling policies requires accurate quantitative methods to measure the GMO content in test samples. A Kemingdao/phospholipase D (KMD/PLD) duplex ddPCR method was established with rice genomic DNA (gDNA) of homozygous KMD as template by optimizing the annealing temperature and cycle number. Duplex ddPCR showed a linear response over the dynamic range from 68 to 175,000 copies, covering four orders of magnitude. The limit of detection (LOD) and limit of quantification (LOQ) for duplex ddPCR were determined to be 9 copies and 34 copies of the rice haploid genome, respectively. A very high dilution factor would result in unacceptable bias and coefficients of variation for determining copy number of the gDNA solution, and more than 1000 copies of the DNA template in one reaction is preferred to obtain accurate quantitative results by duplex PCR. Five blinded DNA samples with copy number ratio of 10%, 5%, 1%, 0.1%, and 0.05%, and three blinded real-life matrix samples with mass fraction of 5%, 1%, and 0.5% were quantified by duplex ddPCR, simplex ddPCR, and qPCR. These three methods all gave comparable GMO content and copy numbers within the required precision, but the duplex ddPCR showed the narrowest uncertainty interval and provided the highest precision in comparison to simplex ddPCR and qPCR. The ddPCR is a more appealing and reliable technology for the accurate quantification of GMO content than simplex ddPCR and qPCR considering the uncertainty and precision of quantitative results, the time consumption of generating droplets, and the cost of ddPCR reagents.Graphical abstract
Droplet microfluidics offers tremendous potential as an enabling technology for high-throughput screening. It promises to yield novel techniques for personalised medicine, drug discovery, disease diagnosis, establishing chemical libraries, and the discovery of new materials. Despite the enormous potential to contribute to a broad range of applications, the expected adoption has not yet been seen, partly due to the interdisciplinary nature and the fact that, up until now, information has been scattered across the literature. This book goes a long way to addressing these issues.
Edited by two leaders, this book has drawn together expertise from around the globe to form a unified, cohesive resource for the droplet microfluidics community. Starting with the basic theory of droplet microfluidics before introducing its use as a tool, the reader will be treated to chapters on important techniques, including robust passive and active droplet manipulations and applications such as single cell analysis, which is key for drug discovery. This book is a go-to resource for the community yearning to adopt and promote droplet microfluidics into different applications and will interest researchers and practitioners working across chemistry, biology, physics, materials science, micro- and nano-technology, and engineering.
E. coli serogroups O157, O26, O45, O103, O111, O121 and O145, when carry major virulence genes, Shiga toxin genes stx 1, stx 2, and intimin gene eae , are important foodborne pathogens. They are referred to as the “top-7” Shiga toxin-producing E. coli (STEC) and were declared by USDA as adulterant to human health. Since the top-7 serogroup-positive cattle feces and ground beef can also contain non-adulterant E. coli strains, regular PCR cannot confirm whether the virulence genes are carried by adulterant or non-adulterant E. coli serogroups. Thus the traditional gold standard STEC detection requires bacterial isolation and characterization, which is not compatible with high-throughput settings, and often takes a week to obtain a definitive result. In this study, we demonstrated that the partition-based multi-channel digital PCR (dPCR) system can be used to detect and associate the E. coli serogroup-specific gene with major virulence genes, and developed a single cell-based dPCR approach for rapid (within one day) and accurate detection and confirmation of major STEC serogroups under high throughput settings. Major virulence genes carried by each of the top-7 STECs were detected by dPCR with appropriately diluted intact bacterial cells from pure culture, culture-spiked cattle feces, and culture-spiked ground beef. Furthermore, from 100 randomly collected, naturally shed cattle fecal samples, three O103 strains carrying eae , and two O45 strains carrying stx 1 were identified by this dPCR assay and verified by the traditional isolation method. This novel and rapid dPCR assay is a culture-independent, high-throughput, accurate, and sensitive method for STEC detection and confirmation.
The clinical significance of the detection of low copy numbers of cytomegalovirus (CMV) DNA in immune-suppressed patients
remains unclear. In this study, we compared the artus CMV Rotor-Gene PCR, utilizing an automated nucleic acid extraction and assay setup (the artus CMV protocol), with the COBAS Amplicor CMV Monitor test (our reference protocol). We then analyzed the results of all CMV
PCR tests ordered following the implementation of the artus CMV protocol at our institution and followed 91 adult patients with positive test results. The artus CMV protocol had a linear range extending from 2.0 to 7.0 log10 copies/ml and had a lower limit of 95% detection of 57 copies/ml. With archived plasma samples, this protocol demonstrated
100% sensitivity and 94% specificity for the detection of CMV DNA. Following implementation of the artus CMV protocol, 320 of 1,403 (22.8%) plasma samples tested positive (compared with 323/3,579 [9.0%] samples in the preceding
6 months), and 227 (16.2%) samples had copy numbers of <400/ml. Ninety-one adult patients had at least one positive test.
The data were analyzed using a threshold of 200 copies/ml, and in 22 episodes, the viral load increased from <200 copies/ml
to ≥200 copies/ml on sequential tests. In 21 of these 22 episodes, either the viral load continued to increase or antiviral
treatment was initiated in response to the repeat value. In summary, we evaluate the performance characteristics of a protocol
utilizing the artus CMV PCR and identify clinically meaningful changes in CMV DNA copy numbers even when they are initially detected at a low
level.
GB virus C (GBV-C) is a lymphotropic human virus discovered in 1995 that is related to hepatitis C virus (HCV). GBV-C infection has not been convincingly associated with any disease; however, several studies found an association between persistent GBV-C infection and improved survival in HIV-positive individuals. GBV-C infection modestly alters T cell homeostasis in vivo through various mechanisms, including modulation of chemokine and cytokine release and receptor expression, and by diminution of T cell activation, proliferation and apoptosis, all of which may contribute to improved HIV clinical outcomes. In vitro studies confirm these clinical observations and demonstrate an anti-HIV replication effect of GBV-C. This review summarizes existing data on potential mechanisms by which GBV-C interferes with HIV, and the research needed to capitalize on this epidemiological observation.
Plasma human immunodeficiency virus type 1 (HIV-1) RNA suppression <50 copies/mL is regarded as the optimal outcome of highly active antiretroviral therapy (HAART). Current viral load (VL) assays show increased sensitivity, but the significance of RNA detection <50 copies/mL is unclear.
This study investigated the virologic outcomes of 1247 patients with VL <50 copies/mL at an arbitrary time point during HAART (= T0), according to whether the actual, unreported (T0)VL was 40-49 copies/mL, RNA detected <40 copies/mL (RNA(+)), or RNA not detected (RNA(-)), as measured by the Abbott Real Time assay. Predictors of rebound >50 and >400 copies/mL over 12 months following T0 were analyzed with Cox proportional hazards models incorporating the (T0)VL and demographic and clinical data.
Rebound rates >50 copies/mL were 34.2% for (T0)VL 40-49 copies/mL, 11.3% for RNA(+), and 4.0% for RNA(-); rebound rates >400 copies/mL were 13.0%, 3.8%, and 1.2%, respectively. The adjusted hazard ratios for rebound >50 copies/mL were 4.67 (95% confidence interval, 2.91-7.47; P < .0001) and 1.97 (1.25-3.11; P < .0001) with (T0)VL 40-49 copies/mL and RNA(+), respectively, relative to RNA(-), and 6.91 (2.90-16.47; P < .0001) and 2.88 (1.24-6.69; P < .0001), respectively, for rebound >400 copies/mL. The association was independent of adherence levels.
In treated patients monitored by RealTime, a VL of 40-49 copies/mL and, to a lesser extent, RNA detection <40 copies/mL predict rebound >50 and >400 copies/mL independently of other recognized determinants. The goal of HAART may need to be revised to a lower cutoff than 50 copies/mL.
Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.
Viruses may very well be the most abundant biological entities on the planet. Yet neither metagenomic studies nor classical
phage isolation techniques have shed much light on the identity of the hosts of most viruses. We used a microfluidic digital
polymerase chain reaction (PCR) approach to physically link single bacterial cells harvested from a natural environment with
a viral marker gene. When we implemented this technique on the microbial community residing in the termite hindgut, we found
genus-wide infection patterns displaying remarkable intragenus selectivity. Viral marker allelic diversity revealed restricted
mixing of alleles between hosts, indicating limited lateral gene transfer of these alleles despite host proximity. Our approach
does not require culturing hosts or viruses and provides a method for examining virus-bacterium interactions in many environments.
Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.
This paper describes a SlipChip to perform digital PCR in a very simple and inexpensive format. The fluidic path for introducing the sample combined with the PCR mixture was formed using elongated wells in the two plates of the SlipChip designed to overlap during sample loading. This fluidic path was broken up by simple slipping of the two plates that removed the overlap among wells and brought each well in contact with a reservoir preloaded with oil to generate 1280 reaction compartments (2.6 nL each) simultaneously. After thermal cycling, end-point fluorescence intensity was used to detect the presence of nucleic acid. Digital PCR on the SlipChip was tested quantitatively by using Staphylococcus aureus genomic DNA. As the concentration of the template DNA in the reaction mixture was diluted, the fraction of positive wells decreased as expected from the statistical analysis. No cross-contamination was observed during the experiments. At the extremes of the dynamic range of digital PCR the standard confidence interval determined using a normal approximation of the binomial distribution is not satisfactory. Therefore, statistical analysis based on the score method was used to establish these confidence intervals. The SlipChip provides a simple strategy to count nucleic acids by using PCR. It may find applications in research applications such as single cell analysis, prenatal diagnostics, and point-of-care diagnostics. SlipChip would become valuable for diagnostics, including applications in resource-limited areas after integration with isothermal nucleic acid amplification technologies and visual readout.
We aim to develop a digital PCR-based method for the quantitative detection of the two common epidermal growth factor receptor (EGFR) mutations (in-frame deletion at exon 19 and L858R at exon 21) in the plasma and tumor tissues of patients suffering from non-small cell lung cancers. These two mutations account for >85% of clinically important EGFR mutations associated with responsiveness to tyrosine kinase inhibitors.
DNA samples were analyzed using a microfluidics system that simultaneously performed 9,180 PCRs at nanoliter scale. A single-mutant DNA molecule in a clinical specimen could be detected and the quantities of mutant and wild-type sequences were precisely determined.
Exon 19 deletion and L858R mutation were detectable in 6 (17%) and 9 (26%) of 35 pretreatment plasma samples, respectively. When compared with the sequencing results of the tumor samples, the sensitivity and specificity of plasma EGFR mutation analysis were 92% and 100%, respectively. The plasma concentration of the mutant sequences correlated well with the clinical response. Decreased concentration was observed in all patients with partial or complete clinical remission, whereas persistence of mutation was observed in a patient with cancer progression. In one patient, tyrosine kinase inhibitor was stopped after an initial response and the tumor-associated EGFR mutation reemerged 4 weeks after stopping treatment.
The sensitive detection and accurate quantification of low abundance EGFR mutations in tumor tissues and plasma by microfluidics digital PCR would be useful for predicting treatment response, monitoring disease progression and early detection of treatment failure associated with acquired drug resistance.
Limiting dilution PCR has become an increasingly useful technique for the detection and quantification of rare species in a population, but the limit of detection and accuracy of quantification are largely determined by the number of reactions that can be analyzed. Increased throughput may be achieved by reducing the reaction volume and increasing processivity. We have designed a high-throughput microfluidic chip that encapsulates PCR reagents in millions of picoliter droplets in a continuous oil flow. The oil stream conducts the droplets through alternating denaturation and annealing zones, resulting in rapid (55-s cycles) and efficient PCR amplification. Inclusion of fluorescent probes in the PCR reaction mix permits the amplification process to be monitored within individual droplets at specific locations within the microfluidic chip. We show that amplification of a 245-bp adenovirus product can be detected and quantified in 35 min at starting template concentrations as low as 1 template molecule/167 droplets (0.003 pg/muL). The frequencies of positive reactions over a range of template concentrations agree closely with the frequencies predicted by Poisson statistics, demonstrating both the accuracy and sensitivity of this platform for limiting dilution and digital PCR applications.
We describe a general method to quantitate the total number of initial targets present in a sample using limiting dilution, PCR and Poisson statistics. The DNA target for the PCR was the rearranged immunoglobulin heavy chain (IgH) gene derived from a leukemic clone that was quantitated against a background of excess rearranged IgH genes from normal lymphocytes. The PCR was optimized to provide an all-or-none end point at very low DNA target numbers. PCR amplification of the N-ras gene was used as an internal control to quantitate the number of potentially amplifiable genomes present in a sample and hence to measure the extent of DNA degradation. A two-stage PCR was necessary owing to competition between leukemic and non-leukemic templates. Study of eight leukemic samples showed that approximately two potentially amplifiable leukemic IgH targets could be detected in the presence of 160,000 competing non-leukemic genomes. The method presented quantitates the total number of initial DNA targets present in a sample, unlike most other quantitation methods that quantitate PCR products. It has wide application, because it is technically simple, does not require radioactivity, addresses the problem of excess competing targets and estimates the extent of DNA degradation in a sample.
The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.
A LightCycler and two TaqMan real-time PCR assays were evaluated against an older PCR with liquid-phase hybridization method
for the detection of enterovirus RNA in 74 patient samples. The two-step LightCycler and the two-step TaqMan formats correlated
well with each other (r2 = 0.90) and were equally sensitive compared to the liquid-phase hybridization method, whereas the one-step recombinant Tth DNA polymerase format was rather insensitive, detecting enterovirus RNA in only about one-half of those patient samples previously
positive by liquid-phase hybridization. The two-step TaqMan method was optimized utilizing 10 μl of cDNA and demonstrated
the highest degree of analytical sensitivity among the methods evaluated in our study, being able to reproducibly quantify
down to 510 copies of enteroviral RNA/ml of cerebrospinal fluid. This new assay can be performed in 4 h, is much less labor
intensive, and showed less cross-reactivity with rhinovirus than the liquid-phase hybridization assay. Thus, the two-step
TaqMan assay should prove useful in the diagnosis of enteroviral meningitis versus bacterial meningitis, thereby resulting
in timely and appropriate clinical management that can amount to significant cost savings to the patient and health care system.
Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens.
The prevalence and concentration of human herpesvirus 6 (HHV-6) DNA in the cerebrospinal fluid (CSF) of the immunocompetent in primary infection was compared with that in viral chromosomal integration. Samples from 510 individuals with suspected encephalitis, 200 young children and 310 older children and/or adults, and 12 other patients were tested. HHV-6 DNA concentration (log(10) copies/ml) was measured in CSF, serum, and whole blood using PCR. Serum HHV-6 immunoglobulin G antibody was measured by indirect immunofluorescence. Primary infection was defined by antibody seroconversion and/or a low concentration of HHV-6 DNA (<3.0 log(10) copies/ml) in a seronegative serum. Chromosomal integration was defined by a high concentration of viral DNA in serum (>/=3.5 log(10) copies/ml) or whole blood (>/=6.0 log(10) copies/ml). The prevalences of CSF HHV-6 DNA in primary infection and chromosomal integration were 2.5% and 2.0%, respectively, in the young children (<2 years) and 0% and 1.3%, respectively, in the older children and/or adults. The mean concentration of CSF HHV-6 DNA in 9 children with primary infection (2.4 log(10) copies/ml) was significantly lower than that of 21 patients with viral chromosomal integration (4.0 log(10) copies/ml). Only HHV-6B DNA was found in primary infection, whereas in viral integration, 4 patients had HHV-6A and 17 patients HHV-6B. Apart from primary infection, chromosomal integration is the most likely cause of HHV-6 DNA in the CSF of the immunocompetent. Our results show that any diagnosis of HHV-6 encephalitis or other type of active central nervous system infection should not be made without first excluding chromosomal HHV-6 integration by measuring DNA load in CSF, serum, and/or whole blood.
The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted
as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research
laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility
and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR
product during real-time PCR. These are the DNA binding fluorophores, the 5′ endonuclease, adjacent linear and hairpin oligoprobes
and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development
of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware
and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this
review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations
of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order
to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits
and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as
studies of gene expression and genetic disease.
Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets.
As the less familiar cousin of quantitative PCR moves mainstream, researchers have more options to choose from.
The source, significance and optimal management of low-level viraemia during highly active antiretroviral therapy (HAART) are poorly defined. This review highlights recent observations that have implications for clinical practice.
The definition of low-level viraemia remains elusive. Whereas evidence obtained with second-generation viral load assays indicates that confirmed detection of plasma HIV-1 RNA above 50 copies/ml predicts negative outcomes, third-generation assays detect HIV-1 RNA below this threshold. In patients monitored with the Abbott RealTime assay, the cutoff that should trigger confirmation of viraemia and clinical review can be revised to 40 copies/ml. Further data are needed on the cost-effectiveness of intervening when RNA detection is observed below this cutoff. Discrepancies among viral load assays prevent generalization of these observations. To further compound the issue, most patients on stably suppressive HAART show residual viraemia at around 1-10 copies/ml using research-based ultrasensitive assays. The source of residual viraemia remains controversial, but neither short nor long-term HAART intensification with antiretrovirals such as raltegravir reduces the viraemia. A transient effect of intravenous immunoglobulin has been reported, and different regimens may vary in their propensity to allow HIV-1 RNA persistence. Further studies are required to clarify the relationship between low-level viraemia and the size of proviral DNA reservoirs, and the contribution of cellular and tissue compartments and cell-to-cell spread to ongoing virus replication during HAART.
Understanding the source and clinical significance of HIV-1 RNA persistence in plasma during HAART is required to guide patient care and inform the design of HIV eradication strategies.
In this paper, we are working toward a problem of great importance to global health: determination of viral HIV and hepatitis C (HCV) loads under point-of-care and resource limited settings. While antiretroviral treatments are becoming widely available, viral load must be evaluated at regular intervals to prevent the spread of drug resistance and requires a quantitative measurement of RNA concentration over a wide dynamic range (from 50 up to 10(6) molecules/mL for HIV and up to 10(8) molecules/mL for HCV). "Digital" single molecule measurements are attractive for quantification, but the dynamic range of such systems is typically limited or requires excessive numbers of compartments. Here we designed and tested two microfluidic rotational SlipChips to perform multivolume digital RT-PCR (MV digital RT-PCR) experiments with large and tunable dynamic range. These designs were characterized using synthetic control RNA and validated with HIV viral RNA and HCV control viral RNA. The first design contained 160 wells of each of four volumes (125 nL, 25 nL, 5 nL, and 1 nL) to achieve a dynamic range of 5.2 × 10(2) to 4.0 × 10(6) molecules/mL at 3-fold resolution. The second design tested the flexibility of this approach, and further expanded it to allow for multiplexing while maintaining a large dynamic range by adding additional wells with volumes of 0.2 nL and 625 nL and dividing the SlipChip into five regions to analyze five samples each at a dynamic range of 1.8 × 10(3) to 1.2 × 10(7) molecules/mL at 3-fold resolution. No evidence of cross-contamination was observed. The multiplexed SlipChip can be used to analyze a single sample at a dynamic range of 1.7 × 10(2) to 2.0 × 10(7) molecules/mL at 3-fold resolution with limit of detection of 40 molecules/mL. HIV viral RNA purified from clinical samples were tested on the SlipChip, and viral load results were self-consistent and in good agreement with results determined using the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 Test. With further validation, this SlipChip should become useful to precisely quantify viral HIV and HCV RNA for high-performance diagnostics in resource-limited settings. These microfluidic designs should also be valuable for other diagnostic and research applications, including detecting rare cells and rare mutations, prenatal diagnostics, monitoring residual disease, and quantifying copy number variation and gene expression patterns. The theory for the design and analysis of multivolume digital PCR experiments is presented in other work by Kreutz et al.
Using a multiplexed LNA-based Taqman assay, RT-digital PCR (RT-dPCR) was performed in a prefabricated microfluidic device that monitored absolute viral load in native and immortalized cell lines, overall precision of detection, and the absolute detection limit of an occult RNA virus GB Virus Type C (GBV-C). RT-dPCR had on average a 10% lower overall coefficient of variation (CV, a measurement of precision) for viral load testing than RT-qPCR and had a higher overall detection limit, able to quantify as low as three 5'-UTR molecules of GBV-C genome. Two commercial high-yield in vitro transcription kits (T7 Ribomax Express by Promega and Ampliscribe T7 Flash by Epicentre) were compared to amplify GBV-C RNA genome with T7-mediated amplification. The Ampliscribe T7 Flash outperformed the T7 Ribomax Express in yield of full-length GBV-C RNA genome. THP-1 cells (a model of monocytic derived cells) were transfected with GBV-C, yielding infectious virions that replicated over a 120h time course and could be infected directly. This study provides the first evidence of GBV-C replication in monocytic derived clonal cells. Thus far, it is the only study using a microfluidic device that measures directly viral load of mammalian RNA virus in a digital format without need for a standard curve.
Viral load (VL) measurement is critical for monitoring the effectiveness of highly active antiretroviral therapy (HAART). HAART aims to maintain plasma HIV RNA at undetectable levels. A VL <50 copies/ml was previously considered undetectable. After introducing more sensitive assays many patients with VL <50 copies/ml were found to have very low-level viraemia (VLLV), defined as a detectable VL <40 copies/ml. This study aimed to determine the significance of VLLV.
This retrospective case-control study included 69 individuals on HAART with VLLV. Immunological and virological outcomes over 36 months were compared to those of 70 well-matched controls with persistently undetectable VL.
We detected no significant association between VLLV and the development of virological failure or inferior immunological outcomes. However, individuals with VLLV were significantly less likely to achieve subsequent sustained virological suppression (VL <50 copies/ml, p<0.001), including completely undetectable suppression (undetectable VL <40 copies/ml, p=0.002).
The significance of VLLV has been uncertain. Our results clearly suggest that VLLV is predictive of future suboptimal virological control, particularly a reduced likelihood of achieving virological suppression. Further work should confirm our findings and evaluate strategies for managing VLLV in HAART-treated patients.
The EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] gene is known to harbor genomic alterations in advanced lung cancer involving gene amplification and kinase mutations that predict the clinical response to EGFR-targeted inhibitors. Methods for detecting such molecular changes in lung cancer tumors are desirable.
We used a nanofluidic digital PCR array platform and 16 cell lines and 20 samples of genomic DNA from resected tumors (stages I-III) to quantify the relative numbers of copies of the EGFR gene and to detect mutated EGFR alleles in lung cancer. We assessed the relative number of EGFR gene copies by calculating the ratio of the number of EGFR molecules (measured with a 6-carboxyfluorescein-labeled Scorpion assay) to the number of molecules of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit) (measured with a 6-carboxy-X-rhodamine-labeled TaqMan assay) in each panel. To assay for the EGFR L858R (exon 21) mutation and exon 19 in-frame deletions, we used the ARMS and Scorpion technologies in a DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array.
The digital array detected and quantified rare gefitinib/erlotinib-sensitizing EGFR mutations (0.02%-9.26% abundance) that were present in formalin-fixed, paraffin-embedded samples of early-stage resectable lung tumors without an associated increase in gene copy number. Our results also demonstrated the presence of intratumor molecular heterogeneity for the clinically relevant EGFR mutated alleles in these early-stage lung tumors.
The digital PCR array platform allows characterization and quantification of oncogenes, such as EGFR, at the single-molecule level. Use of this nanofluidics platform may provide deeper insight into the specific roles of clinically relevant kinase mutations during different stages of lung tumor progression and may be useful in predicting the clinical response to EGFR-targeted inhibitors.
Limiting dilution PCR has become an increasingly useful technique for the detection and quantification of rare species in a population, but the limit of detection and accuracy of quantification are largely determined by the number of reactions that can be analyzed. Increased throughput may be achieved by reducing the reaction volume and increasing processivity. We have designed a high-throughput microfluidic chip that encapsulates PCR reagents in millions of picoliter droplets in a continuous oil flow. The oil stream conducts the droplets through alternating denaturation and annealing zones, resulting in rapid (55-s cycles) and efficient PCR amplification. Inclusion of fluorescent probes in the PCR reaction mix permits the amplification process to be monitored within individual droplets at specific locations within the microfluidic chip. We show that amplification of a 245-bp adenovirus product can be detected and quantified in 35 min at starting template concentrations as low as 1 template molecule/167 droplets (0.003 pg/microL). The frequencies of positive reactions over a range of template concentrations agree closely with the frequencies predicted by Poisson statistics, demonstrating both the accuracy and sensitivity of this platform for limiting dilution and digital PCR applications.
Mutations in the epidermal growth factor receptor (EGFR) kinase domain can predict tumor response to tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancer (NSCLC). However, obtaining tumor tissues for mutation analysis is challenging. We hypothesized that plasma-based EGFR mutation analysis is feasible and has value in predicting tumor response in patients with NSCLC.
Plasma DNA samples and matched tumors from 230 patients with stages IIIB to IV NSCLC were analyzed for EGFR mutations in exons 19 and 21 by using denaturing high-performance liquid chromatography. We compared the mutations in the plasma samples and the matched tumors and determined an association between EGFR mutation status and the patients' clinical outcomes prospectively.
In 230 patients, we detected 81 EGFR mutations in 79 (34.3%) of the patients' plasma samples. We detected the same mutations in 63 (79.7%) of the matched tumors. Sixteen plasma (7.0%) and fourteen tumor (6.1%) samples showed unique mutations. The mutation frequencies were significantly higher in never-smokers and in patients with adenocarcinomas (P = .012 and P = .009, respectively). In the 102 patients who failed platinum-based treatment and who were treated with gefitinib, 22 (59.5%) of the 37 with EGFR mutations in the plasma samples, whereas only 15 (23.1%) of the 65 without EGFR mutations, achieved an objective response (P = .002). Patients with EGFR mutations had a significantly longer progression-free survival time than those without mutations (P = .044) in plasma.
EGFR mutations can be reliably detected in plasma DNA of patients with stages IIIB to IV NSCLC and can be used as a biomarker to predict tumor response to TKIs.
The accuracy and precision of quantitative polymerase chain reaction (PCR) results depend not only on the PCR reaction but also on the extraction of viral nucleic acid. Although the extraction of viral nucleic acid from fluids has been extensively studied, less data are available regarding extractions from cellular specimens. We therefore evaluated several commercially available kits for the extraction of nucleic acid from cellular specimens. Although the kits generally performed well, there were some differences in extraction efficiency, especially at low numbers of input cells. Inclusion of a multivirus positive control allowed careful monitoring of extraction efficiency during routine clinical use. Laboratories are encouraged to validate extraction methods carefully in the context of the proposed viral testing.
Colorectal (CR) tumors are usually curable if detected before metastasis. Because genetic alterations are associated with
the development of these tumors, mutant genes may be found in the stool of individuals with CR neoplasms. The stools of nine
patients whose tumors contained mutations of K-ras were analyzed. In eight of the nine cases, the ras mutations were detectable
in DNA purified from the stool. These patients included those with benign and malignant neoplasms from proximal and distal
colonic epithelium. Thus, colorectal tumors can be detected by a noninvasive method based on the molecular pathogenesis of
the disease.
The identification of predefined mutations expected to be present in a minor fraction of a cell population is important for a variety of basic research and clinical applications. Here, we describe an approach for transforming the exponential, analog nature of the PCR into a linear, digital signal suitable for this purpose. Single molecules are isolated by dilution and individually amplified by PCR; each product is then analyzed separately for the presence of mutations by using fluorescent probes. The feasibility of the approach is demonstrated through the detection of a mutant ras oncogene in the stool of patients with colorectal cancer. The process provides a reliable and quantitative measure of the proportion of variant sequences within a DNA sample.
Advocating the Concept of GB Virus C
- D Gretch
Gretch D. Advocating the Concept of GB Virus C Biotherapy Against AIDS. Clin Infect Dis. 2012