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![Isothermal nucleic acid amplification methods that are based on polymerase extension plus a single-strand cutting event. In SDA (Becton Dickinson) [42,43], NEMA (Ustar) [45] and ICA (RapleGene) [46], an intermediate target is generated using strand-displacing amplification via sacrificial outer bumper primers. NEAR (Ionian Technologies) [44] does not use bumper primers and involves shorter amplicons than the other methods. In SDA, phosphothioates are incorporated into the amplicon during polymerization so that a restriction endonuclease only cuts one strand. NEAR and NEMA both use nicking endonucleases that are inherently single strand cutting. In ICA, a single strand cut is facilitated through RNase H and DNA-RNA-DNA chimeric primers. After the single-strand cutting event, amplicons are generated through strand-displacing amplification, for short amplicons further facilitated by thermal denaturation. These amplicons re-prime, and lead to exponential feedback amplification. EXPAR [47] amplifies short trigger oligonucleotides, which can be generated via the so-called ''fingerprinting'' reaction [48] from adjacent nicking enzyme recognition sites in genomic DNA. This is followed by rapid exponential amplification, mediated by a template sequence that contains two copies of the trigger complement that are separated by the nicking enzyme recognition site complement. BAD-AMP [49] uses a molecular beacon for signal generation and as a template for single-strand nicking and re-priming.](profile/Tanya-Ferguson-2/publication/50288941/figure/fig2/AS:667194107109384@1536082879698/Isothermal-nucleic-acid-amplification-methods-that-are-based-on-polymerase-extension-plus.png)
Isothermal nucleic acid amplification methods that are based on polymerase extension plus a single-strand cutting event. In SDA (Becton Dickinson) [42,43], NEMA (Ustar) [45] and ICA (RapleGene) [46], an intermediate target is generated using strand-displacing amplification via sacrificial outer bumper primers. NEAR (Ionian Technologies) [44] does not use bumper primers and involves shorter amplicons than the other methods. In SDA, phosphothioates are incorporated into the amplicon during polymerization so that a restriction endonuclease only cuts one strand. NEAR and NEMA both use nicking endonucleases that are inherently single strand cutting. In ICA, a single strand cut is facilitated through RNase H and DNA-RNA-DNA chimeric primers. After the single-strand cutting event, amplicons are generated through strand-displacing amplification, for short amplicons further facilitated by thermal denaturation. These amplicons re-prime, and lead to exponential feedback amplification. EXPAR [47] amplifies short trigger oligonucleotides, which can be generated via the so-called ''fingerprinting'' reaction [48] from adjacent nicking enzyme recognition sites in genomic DNA. This is followed by rapid exponential amplification, mediated by a template sequence that contains two copies of the trigger complement that are separated by the nicking enzyme recognition site complement. BAD-AMP [49] uses a molecular beacon for signal generation and as a template for single-strand nicking and re-priming.
Source publication
Nucleic acid testing for infectious diseases at the point of care is beginning to enter clinical practice in developed and developing countries; especially for applications requiring fast turnaround times, and in settings where a centralized laboratory approach faces limitations. Current systems for clinical diagnostic applications are mainly PCR-b...
Context in source publication
Context 1
... in Biotechnology May 2011, Vol. 29, No. 5 and primer annealing ( Figure 2b) [32][33][34][35]; strand displace- ment using polymerases only, with multiple linear primer sets ( Figure 2c) [36][37][38][39]; and strand displacement from a circular or circularized target (Figure 2d) [40,41]. Many isothermal amplification methods use polymerase exten- sion in conjunction with a single-strand cutting event ( Figure 3). [42][43][44][45][46][47][48][49][50]. ...
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Background
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Citations
... Meeting precision in biological sample detection is critical, especially in scenarios where only trace amounts of material are available. This demand of precision and accuracy is facilitated by sensitive and specific detection methods like loop-mediated isothermal amplification (LAMP) [1]. LAMP offers several advantages, including high sensitivity and specificity, rapid results, and simplicity of operation [2]. ...
Our research has developed a highly sensitive and simple assay to detect small amounts of animal and human biological material in less than 40 min. The handheld SaLux19 device developed at the Max Planck Institute of Experimental Medicine in Göttingen, Germany, was used to validate our concept. The proposed system uses isothermal amplification of DNA in a rapid assay format. Our results show that the assay can detect Sus scrofa nucleic acids with very high sensitivity and specificity. This detection system has potential for forensic scenarios.
... Molecular nucleic acid testing (NAT) has revolutionized the landscape of diagnostics, offering rapid and precise detection of nucleic acids from various biological samples (Mackay et al., 2002;Niemz et al., 2011;Zhu et al., 2020). NAT involves a series of intricate steps, starting from sample collection to detection assays. ...
... The extracted nucleic acids are then amplified, typically using Polymerase Chain Reaction (PCR) or Reverse Transcription-PCR (RT-PCR), to increase the quantity of the target to detectable levels (Liu and Tsutsui, 2023). Following amplification, detection and interpretation are performed, which involves electrophoresis, real-time PCR, or next-generation sequencing (Esbin et al., 2020;Niemz et al., 2011). ...
... There has been a significant shift in molecular diagnostics towards the development and implementation of point-of-care testing (POCT) NAT devices (Niemz et al., 2011). This transition is driven by the growing need for rapid, on-site diagnostic tools that can provide immediate results, thereby facilitating timely clinical decisions (Bissonnette and Bergeron, 2012). ...
Nucleic acid testing has changed the diagnostic landscape, offering precise, rapid, and scalable detection platforms for various biological samples. The growing need for on-site diagnostics has driven the development of these technologies for point-of-care applications. Despite the recent advances, there are still significant challenges, the largest being sample preparation. Poor sample preparation can significantly diminish the sensitivity of downstream assays. There is a need for universal and portable sample preparation strategies to alleviate the bottle neck on NAT. In this work we present a semi-automated, easy-to-use, and portable centrifuge kit and device for the extraction of nucleic acids. Extraction reagents show stability out to 6-months of storage and device extractions on battery power remain viable out to 30 trials. Portable spin column procedures show an increased drying ability that does not affect PCR performance. Characterization of our portable centrifuge (varying rotor size and spin time) showed a maximum ΔC q of 0.45 and 0.71, respectively. Extractions using the portable centrifuge showed efficiencies greater than 84% for high concentrated samples with linearity (R 2 = 0.811) between C q time and RNA concentrations. The portable centrifuge showed a strong correlation (r > 0.96) and strong linearity (R 2 > 0.92) when compared to the benchtop centrifuge when processing all three sample types: HIV RNA in buffer, HIV RNA in plasma, and SARS-CoV-2 RNA in saliva. Therefore, we present an easy-to-use, semi-automated, and portable centrifuge kit and device for the universal preparation of nucleic acids in low resource areas.
... Rapid, inexpensive, and sensitive nucleic acid detection methods are desired for early and pointof-care (POC) diagnostics. Currently, widely applied nucleic acid detection methods, including polymerase chain reaction (PCR) assays [1][2][3][4] and DNA sequencing 5 , are time-consuming, inaccessible for field use, and dependent on expensive instruments and skilled technical staff 6 . ...
Most CRISPR-based biosensors rely on labeled reporter molecules and expensive equipment for signal readout. A recent alternative approach quantifies analyte concentration by sizing native λ DNA reporters, providing a simple and label-free solution for ultrasensitive detection. However, this method faces challenges in accurately quantifying size reduction of long DNA reporters via gel electrophoresis due to the sensitivity of DNA band shift to other interferences such as gel distortion. To overcome these limitations, here we developed a simple and robust ratiometric signaling strategy using CRISPR-Cas12a-induced supercoil relaxation of dsDNA plasmid reporters. In the presence of target, we observed that the fraction of supercoiled plasmid DNA decreased, and the amount of relaxed conformation (circular) increased over time. The relative percentage of supercoiled DNA to the relaxed circular DNA was analyzed by gel electrophoresis to generate an intensity-based ratiometric signal for more accurate target concentration quantification. This simple and inexpensive method is ∼100 times more sensitive when compared with the typical fluorescent reporter system. This self-referenced strategy solves the potential application limitations of previously demonstrated DNA sizing-based CRISPR-Dx without compromising the sensitivity. Finally, we demonstrated the applicability of ratiometric sensing strategy using model DNA targets such as AAV and HPV 16, highlighting its feasibility for point-of-use CRISPR-Dx applications.
Table of Contents (TOC)
... Centrifuges are critical equipment in medical diagnostics, employed for separating plasma from blood, extracting pathogens and parasites from biological fluids such as urine and feces, and for use in immunoassay, blood analysis, and concentration determination experiments [1][2][3]. Centrifugation enables the separation and discarding of unnecessary substances, reducing interference and thereby enhancing the accuracy and reliability of rapid diagnoses crucial in detecting maladies like malaria, HIV, and tuberculosis [4][5][6][7]. Traditional centrifuges, commonly used and driven by motors to achieve high speeds and generate centrifugal force, are capable of layering samples based on density, size, and weight. However, due to their high cost, substantial size, and dependence on electricity, their use is limited under certain conditions. ...
Traditional centrifuges, extensively employed in biology, chemistry, medicine, and other domains for tasks such as blood separation and pathogen extraction, have certain limitations. Their high cost, substantial size, and reliance on electricity restrict their range of application. Contemporary centrifuges, inspired by everyday items like paper trays and egg beaters, boast characteristics such as ease of operation, independence from electricity, and portability. These features offer unique advantages in specific situations, such as electricity shortages, inadequate infrastructure, and challenging medical conditions. Consequently, we designed a hand-powered portable centrifuge driven by pulling a rope. Our experiments revealed significant performance factors, including load capacity, rope length, and frequency of rope pulling. The results demonstrated that the revolutions per minute (RPM) of a hand-powered portable centrifuge were directly proportional to the length of the rope and the frequency of pulling, up to a certain limit, while inversely proportional to the load. When used for separating and washing polystyrene microspheres, the portable centrifuge’s performance equaled that of traditional centrifuges. According to relevant calculations, this centrifuge could be capable of meeting the application of blood separation. Therefore, we believe this portable centrifuge will find meaningful applications in similar areas, particularly in resource-poor settings.
... Antibody-dependent LF for NA detection is similar to LF immunoassays, with the exception that antigenic labels must be included in the amplicon via primers or labeled dNTPs, and a second labeled probe is frequently required. Wax printing and PDMS are widely used methods in the manufacturing of LF devices [119]. Microfluidic paper-based analytical devices (μPADs) have recently been very popular for developing POCT. ...
Timely and accurate detection of the causal agent of a disease is crucial to restrict suffering and save lives. Mere symptoms are often not enough to detect the root cause of the disease. Better diagnostics applied for screening at a population level and sensitive detection assays remain the crucial component of disease surveillance which may include clinical, plant, and environmental samples, including wastewater. The recent advances in genome sequencing, nucleic acid amplification, and detection methods have revolutionized nucleic acid-based testing (NABing) and screening assays. A typical NABing assay consists of three modules: isolation of the nucleic acid from the collected sample, identification of the target sequence, and final reading the target with the help of a signal, which may be in the form of color, fluorescence, etc. Here, we review current NABing assays covering the different aspects of all three modules. We also describe the frequently used target amplification or signal amplification procedures along with the variety of applications of this fast-evolving technology and challenges in implementation of NABing in the context of disease management especially in low-resource settings.
Graphical Abstract
... Point-of-care (POC) testing which is defined as nearpatient testing performed at home, clinic, doctor's office, or a hospital has a fast turnaround time [5]; this may range from several minutes to more than an hour. Current HIV testing algorithms in the United States (U.S.) rely on laboratory-based Ag/Ab tests as the first step to detect HIV infection [6]. ...
Background
Integration of a sensitive point-of-care (POC) HIV viral load (VL) test into screening algorithms may help detect acute HIV infection earlier, identify people with HIV (PWH) who are not virally suppressed, and facilitate earlier referral to antiretroviral therapy (ART), or evaluation for pre-exposure prophylaxis (PrEP). This report describes a randomized clinical trial sponsored by the Centers for Disease Control and Prevention (CDC): “Ending the HIV Epidemic Through Point-of-Care Technologies” (EHPOC). The study’s primary aim is to evaluate the use of a POC HIV VL test as part of a testing approach and assess the impact on time to linkage to ART or PrEP. The study will recruit people in Baltimore, Maryland, including patients attending a hospital emergency department, patients attending an infectious disease clinic, and people recruited via community outreach. The secondary aim is to evaluate the performance characteristics of two rapid HIV antibody tests approved by the United States Food and Drug Administration (FDA).
Methods
The study will recruit people 18 years or older who have risk factors for HIV acquisition and are not on PrEP, or PWH who are not taking ART. Participants will be randomly assigned to either the control arm or the intervention arm. Participants randomized to the control arm will only receive the standard-of-care (SOC) HIV screening tests. Intervention arm participants will receive a POC HIV VL test in addition to the SOC HIV diagnostic screening tests. Follow up will consist of an interim phone survey conducted at week-4 and an in-person week-12 visit. Demographic and behavioral information, and oral fluid and blood specimens will be collected at enrollment and at week-12. Survey data will be captured in a Research Electronic Data Capture (REDCap) database. Participants in both arms will be referred for either ART or PrEP based on their HIV test results.
Discussion
The EHPOC trial will explore a novel HIV diagnostic technology that can be performed at the POC and provide viral assessment. The study may help inform HIV testing algorithms and contribute to the evidence to support same day ART and PrEP recommendations.
Trial registration
NIH ClinicalTrials.gov NCT04793750. Date: 11 March 2021.
... Early diagnosis is recognized as an effective strategy for the prevention and control of disease progression [1]. Although traditional diagnostic methods such as liquid biopsy [2][3][4], imaging examinations [5], and nucleic acid testing [6] have played a positive role, they are still facing problems such as long time consumption, high cost, and poor specificity. With the rapid development of detection technology, protein-based biomarkers [7][8][9], which are easier to isolate and can reflect more information about the actual physiological state of cells or tissues, are becoming faster, more convenient, and more cost-effective models for disease detection. ...
The sensitive and rapid detection of microsamples is crucial for early diagnosis of diseases. The short response times and low sample volume requirements of microfluidic chips have shown great potential in early diagnosis, but there are still shortcomings such as complex preparation processes and high costs. We developed a low-cost smartphone-based fluorescence detection device (Smartphone-BFDD) without precision equipment for rapid identification and quantification of biomarkers on glass capillary. The device combines microfluidic technology with RGB image analysis, effectively reducing the sample volume to 20 μL and detection time to only 30 min. For the sensitivity of the device, we constructed a standard sandwich immunoassay (antibody–antigen–antibody) in a glass capillary using the N-protein of SARS-CoV-2 as a biological model, realizing a low limit of detection (LOD, 40 ng mL−1). This device provides potential applications for different biomarkers and offers wide use for rapid biochemical analysis in biomedical research.
... Approaches have been used in order to accelerate diagnostic testing for COVID-19, reverse transcription loop-mediated isothermal amplification (RT-LAMP), detecting RNA virus in a single reaction at only a single temperature, and presenting a rapid alternative to PCR-based methods [9,10]. However, this assay has low specificity, making it difficult to use for the diagnosis of SARS-CoV-2 [11,12]. ...
Background:
The global public health system has been severely tested by the COVID-19 pandemic. Mass testing was essential in controlling the transmission of the SARS-CoV-2; however, its implementation has encountered challenges, particularly in low-income countries. The urgent need for rapid and accurate tests for SARS-CoV-2 has proven to be extremely important. Point-of-care tests using the CRISPR system for COVID-19 have shown promise, with a reported high sensitivity and rapid detection. The performance of a CRISPR-based SARS-CoV-2 testing system was reported in this study.
Methods:
A total of 29 nasopharyngeal samples were evaluated, including 23 samples from individuals suspected of COVID-19, and six samples positive for H3N2 or respiratory syncytial virus. Two reference samples with known concentrations of SARS-CoV-2 RNA (3000 RNA copies/mL) or viral titer determined by plaque assay (105 PFU/mL) were also evaluated. The LAMP technique was employed to amplify the ORF1ab gene and the results were analyzed using a Gemini XPS fluorescence reader.
Results:
The RT-LAMP-CRISPR/Cas12 assay showed 100% concordance compared to RT-PCR. The RT-PCR presented a detection limit of 0.01 PFU/mL and the CRISPR/Cas12 system showed a limit of 15.6 PFU/mL. The RT-PCR sensitivity was approximately 8 RNA copies/µL and CRISPR/Cas12 at 84 RNA copies/µL.
Conclusion:
The RT-LAMP-CRISPR/Cas12a assay offered a promising alternative for the detection of SARS-CoV-2 and reinforces that CRISPR-based diagnostic techniques can be an alternative for fast and accurate assays.
... The isothermal amplification techniques can be classified based on DNA replication, enzyme-based DNA digestion or reaction kinetics. IATs are expanded to detect different targets, including proteins, cells, small molecules and ions (Niemz et al. 2011). The combination of isothermal amplification and nanotechnology has also become a point of interest. ...
Nucleic acids are prominent biomarkers for diagnosing infectious pathogens using nucleic acid amplification techniques (NAATs). PCR, a gold standard technique for amplifying nucleic acids, is widely used in scientific research and diagnosis. Efficient pathogen detection is a key to adequate food safety and hygiene. However, using bulky thermal cyclers and costly laboratory setup limits its uses in developing countries, including India. The isothermal amplification methods are exploited to develop miniaturized sensors against viruses, bacteria, fungi and other pathogenic organisms and have been applied for in situ diagnosis. Isothermal amplification techniques have been found suitable for POC techniques and follow WHO’s ASSURED criteria. LAMP, NASBA, SDA, RCA and RPA are some of the isothermal amplification techniques which are preferable for POC diagnostics. Furthermore, methods such as WGA, CPA, HDA, EXPAR, SMART, SPIA and DAMP were introduced for even more accuracy and robustness. Using recombinant polymerases and other nucleic acid-modifying enzymes has dramatically broadened the detection range of target pathogens under the scanner. The coupling of isothermal amplification methods with advanced technologies such as CRISPR/Cas systems, fluorescence-based chemistries, microfluidics and paper-based sensors has significantly influenced the biosensing and diagnosis field. This review comprehensively analyzed isothermal nucleic acid amplification methods, emphasizing their advantages, disadvantages and limitations.
... The clinical diagnosis of pathogens is generally based on the detection of pathogen-specific genes and proteins using techniques, such as reverse transcription (RT-)polymerase chain reaction (PCR), RT-quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), next-generation sequencing (NGS), f luorescence in situ hybridization (FISH), and chemiluminescence. These tools are very effective and have facilitated the evolution of diagnostics; however, traditional platforms, including serologic methods, which are only workable after antibodies have been produced [18,19], are time-consuming and can identify non-contagious cases as contagious (false-positive results), thus limiting the application of these methods in point-of-care testing (POCT). Pathogen detection tools based on antigen-antibody binding include ELISA [20], which is high in cost due to the heavy use of antibodies. ...
... This novel platform is highly dependent on nucleic acids isolated from pathogens and detection based on nucleic acid amplification. Isothermal amplification methods, such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and RT-LAMP, are effective for pathogen detection in low-resource settings [19,21,[26][27][28]. The CRISPR-Cas-based diagnostic platforms utilize isothermal amplification techniques to detect pathogens for high-speed POCT with improved sensitivity. ...
Pathogenic infections cause severe clinical illnesses in humans and animals. Increased encounters between humans and animals and constant environmental changes exacerbate the transmission of zoonotic infectious diseases. Recently, the World Health Organization has declared some zoonotic epidemics as public health emergencies of international concern. Hence, rapid and accurate detection of the causative pathogen is particularly essential in combating emerging and re-emerging infectious diseases. Traditional pathogen detection tools are time-consuming, costly, and require skilled personnel, which greatly hinder the development of rapid diagnostic tests, particularly in resource-constrained regions. Clustered regularly interspaced short palindromic repeats (CRISPR-)-Cas- and aptamer-based platforms have replaced traditional pathogen detection methods. Herein we review two novel next-generation core pathogen detection platforms that are utilized for clinical and foodborne pathogenic microorganisms: CRISPR-Cas-based systems, including dCas9, Cas12a/b, Cas13, and Cas14; and aptamer-based biosensor detection tools. We highlight CRISPR-Cas- and aptamer-based techniques and compare the strengths and weaknesses. CRISPR-Cas-based tools require cumbersome procedures, such as nucleic acid amplification and extraction, while aptamer-based tools require improved sensitivity. We review the combination of CRISPR-Cas- and aptamer-based techniques as a promising approach to overcome these deficiencies. Finally, we discuss Cas14-based tools as functionally stronger platforms for the detection of non-nucleic acid targets.
