Figure - available via license: Creative Commons Attribution 4.0 International
Content may be subject to copyright.
Source publication
Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus disease 2019 (COVID-19), a respiratory tract infection. The standard molecular diagnostic test is a multistep process involving viral RNA extraction and real-time quantitative reverse transcriptase PCR (qRT-PCR). Laboratories across the globe face constraints on equipme...
Context in source publication
Similar publications
Background: At the beginning of the outbreak of coronavirus infected disease 2019 (COVID-19), children in Wuhan were experiencing an extremely strong influenza A epidemic. This study is aimed to explore the epidemic dynamics characteristics of children with influenza A and its correlation with the early stage spread of COVID-19 in Wuhan.
Methods: T...
The outbreak of SARS-CoV-2 coronavirus rapidly altered radiotherapy service delivery around the world.
Aim:
The main objective of this study was to assess the impact of precautionary measures implemented in response to the COVID-19 pandemic on the performance of a radiation oncology departments and on mitigation the risk of COVID-19 contagion bet...
Until vaccines and effective therapeutics become available, the practical way to transit safely out of the current lockdown may include the implementation of an effective testing, tracing and tracking system. However, this requires a reliable and clinically validated diagnostic platform for the sensitive and specific identification of SARS-CoV-2. H...
Efficient management of an epidemic requires identification and isolation of infected individuals from healthy
population to curb community spread of the pathogen. Therefore, a faster and efficient diagnostic method
for population screening is desirable to flatten the curve of daily raise of cases. This will also help medical
professional to treat...
The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organ...
Citations
... The novel coronavirus SARS-CoV-2 was reported in Wuhan, China, in December of 2019. SARS-CoV-2 cause COVID-19 challenged the health public system worldwide and genetic sequencing of the virus suggest that SARS-CoV-2 closely linked to SARS-CoV-1, affecting more than 180 countries [8][9][10] . The most widely used test for detection of SARS-CoV-2 fall into nucleic-acid test, as a multistep that involves, nasopharyngeal swab sample collection, isolation of viral genetic material and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) [10][11][12][13] . ...
... SARS-CoV-2 cause COVID-19 challenged the health public system worldwide and genetic sequencing of the virus suggest that SARS-CoV-2 closely linked to SARS-CoV-1, affecting more than 180 countries [8][9][10] . The most widely used test for detection of SARS-CoV-2 fall into nucleic-acid test, as a multistep that involves, nasopharyngeal swab sample collection, isolation of viral genetic material and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) [10][11][12][13] . ...
... During the first few weeks of the COVID-19 pandemic, the global demand for nucleic acid extraction kits and required reagents had already in short supply, making them a limiting source for SARS-CoV-2 testing due to those kits are mainly produced in industrialized countries, which means a disadvantage in the access to COVID-19 testing. Consequently, being a challenge for middle and low-income countries in need of improving SARS-CoV-2 testing fueling the development of alternative SARS-CoV-2 RNA isolation methods and protocols 10,[12][13][14][15][16] . Most European countries and the United States have to deal with the accelerated growth of infections and enormous pressure on their health systems, where cases started to grow exponentially 17 . ...
In late 2019 a new virus reported in Wuhan, China, identified as SARS-CoV-2 spread rapidly challenging the healthcare system around the world. The need for rapid, timely and accurate detection was critical to the prevention of community outbreaks of the virus. However, the high global demand for reagents during the years 2020 and 2021 generated a bottleneck in kits used for detection, greatly affecting developing countries, lagging their ability to diagnose and control the virus in the population. The difficulty in importing reagents, high costs and limited public access to the SARS-CoV-2 detection test led to the search for alternative methods. In this framework, different commercial nucleic acid extraction methodologies were evaluated and compared against heat shock as an alternative method for SARS-CoV-2 detection by RT-PCR, in order to determine the diagnostic yield and its possible low-cost compared to other methodologies. Nasopharyngeal samples were used where the diagnostic efficiency of the alternative method was 70 to 73%. The evaluation of the discriminatory efficacy of the method took the sensitivity and specificity to establish its cut-off point, being 0.73 to 0.817, which allows discriminating between COVID-19 positives and negatives. As for the diagnostic effectiveness expressed as the proportion of subjects correctly classified, it is between 80 and 84%. On the other hand, in terms of the costs necessary to carry out the detection, the alternative method is more economical and accessible in terms of direct cost close to 47 and 49 USD, and indirect cost around 35 and 50 USD compared to the commercial methods available in this comparison and evaluation, being possible its implementation in developing countries with high infection rates, allowing access to the diagnostic test with a reliable and low-cost method. Keywords: COVID-19, RT-PCR, Viral RNA.
... In this scenario, the standard test adopted to perform the diagnosis based on the extraction of viral RNA is the Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) [3]. However, the work presented in [4] found a false-negative rate of about 26.7% and 27% for critical and moderate cases, respectively. ...
Background
In December 2019, the first case of COVID-19 was described in Wuhan, China, and by July 2022, there were already 540 million confirmed cases. Due to the rapid spread of the virus, the scientific community has made efforts to develop techniques for the viral classification of SARS-CoV-2.
Results
In this context, we developed a new proposal for gene sequence representation with Genomic Signal Processing techniques for the work presented in this paper. First, we applied the mapping approach to samples of six viral species of the Coronaviridae family, which belongs SARS-CoV-2 Virus. We then used the sequence downsized obtained by the method proposed in a deep learning architecture for viral classification, achieving an accuracy of 98.35%, 99.08%, and 99.69% for the 64, 128, and 256 sizes of the viral signatures, respectively, and obtaining 99.95% precision for the vectors with size 256.
Conclusions
The classification results obtained, in comparison to the results produced using other state-of-the-art representation techniques, demonstrate that the proposed mapping can provide a satisfactory performance result with low computational memory and processing time costs.
... [40,41] The clinical and epidemiological elements associated with the likelihood of infection should be the deciding factor in conducting a test repeatedly for individual patients. [42] The importance of rapid development of asymptomatic testing, [43] random access, [44] and POC [45] biosensor and rapid molecular testing [46,47] need to be implemented to control the disease. Patients on suspicion undergo the test for the virus with NA amplification tests, such as RT-PCR, reverse transcriptionloop-mediated isothermal amplification (RT-LAMP), and reverse transcription-rolling circle amplification. ...
... In this study, we utilized common chemicals to decrease costs as much as possible. Previous studies have indicated that adding VTM samples directly as templates to PCR reactions can be an inexpensive technique for detecting virus genomes (10,11). However, there are two problems with this technique; first, the results are affected by the quality of different materials obtained from different manufacturers. ...
... The second is the sample's purity. As a result of the use of direct samples in PCR reactions, the authors could only use 2l of VTM samples in a 20l PCR reaction to achieve the best results (10,11,14). To achieve a higher level of purity of samples, a simple and cost-benefit method was used to eliminate the inhibitors. ...
Background: The COVID-19 pandemic affected different aspects of human life seriously, including health issues. Unfortunately, the process of RNA extraction using commercial kits is highly expensive. Replacement of this technique with a cheaper one may help us catch a more affordable approach. Objectives: This study aims to introduce a simple and cost-benefit procedure to extract nucleic acid from swab samples of patients infected with SARS-COV-2. Methods: All 41 positive extracted samples were extracted with three methods separately. The first method was based on the commercial kit using a silica filter column. The second method was made of ammonium acetate, sodium acetate, and alcohol as an extraction solution, and the last method was applied using only the sodium acetate and alcohol solution. Results: All samples extracted with a commercial kit based on a silica column were positive (100%) with Cts 21 ± 4.9, 21.4 ± 4.8, and 28.1 ± 1.8 for RNA-dependent RNA polymerase (RdRp), N, and RNase P genes, respectively. In the precipitation method using ammonium acetate, 40 samples were detected positive (97.5%), and the Cts were 26.3 ± 4.5, 23.6 ± 5.3, and 25.7 ± 3.5 for the above three genes, respectively. Similar to the conventional extraction method, the third method also showed positive results (97.5%) significantly. The mean CTs were 26 ± 4.3, 23 ± 5.4, and 23.7 ± 2.3, respectively. Conclusions: Our results indicated that the precipitation method using ammonium acetate, sodium acetate, and ethanol could be an alternative extraction method instead of the column-based method for SARS-COV-2 by swab samples.
... Some of these compounds can inhibit or interfere with NAATs if left unprocessed or untreated. Grant, Wee, Lee, Morecchiato, and colleagues overcame the issues associated with using crude samples by either reducing the input volume to 4-5% of the total reaction mixture or by diluting the sample by a factor of 4-5 prior to its addition to the NAAT master mix [14][15][16][17]. ...
... [11] Swab in viral transport medium RT-PCR Reduced input volume of swabs eluted in viral transport medium * Very easy to implement as no sample treatment is required. [14] Nasopharyngeal swab in UTM Fluorescence RT-LAMP, RT-PCR Dilution of sample in RNase-free water * Very easy to implement as no sample treatment is required. [16,17] Nasopharyngeal swabs and saliva RT-PCR and RT-ddPCR Elution of swabs into Chelex-TED buffer (50% Chelex-100, TE buffer, DMSO) or addition to saliva, followed by heat treatment at 98 • C for 5 min and centrifugation. ...
The COVID-19 pandemic has resulted in an unprecedented global demand for in vitro diagnostic reagents. Supply shortages and hoarding have impacted testing capacity which has led to inefficient COVID-19 case identification and transmission control, predominantly in developing countries. Traditionally, RNA extraction is a prerequisite for conducting SARS-CoV-2 nucleic acid amplification tests (NAAT); however, simplified methods of sample processing have been successful at bypassing typical nucleic acid extraction steps, enabling extraction-free SARS-CoV-2 NAAT workflows. These methods involve chemical and physical approaches that are inexpensive and easily accessible alternatives to overcome extraction kit supply shortages, while offering acceptable test performance. Here we provide an overview of three main sample preparation strategies that have been shown to facilitate extraction-free SARS-CoV-2 NAATs.
... In addition, a range of Cq values were included. Although these samples were not routinely screened to eliminate the presence of other respiratory pathogens, the real-time RT-PCR assay has previously been shown not to cross-react [30]. ...
... All samples were initially screened at University College London Hospitals by realtime RT-PCR targeting the N gene and using the extraction-free SARS-CoV-2 diagnosis method [30]. In brief, 2 µL of sample in VTM was added to the RT-PCR mix comprised of 5 µL of 4X TaqMan Fast Virus 1-step Master Mix (Applied Biosystems, Waltham, MA, USA), SARS-CoV-2 N gene primers, RNase P primers and probes each at a final concentration of 250 nM and the reaction volume made up to 20 µL with nuclease-free water. ...
The COVID-19 pandemic has unveiled a pressing need to expand the diagnostic landscape to permit high-volume testing in peak demand. Rapid nucleic acid testing based on isothermal amplification is a viable alternative to real-time reverse transcription polymerase chain reaction (RT-PCR) and can help close this gap. With the emergence of SARS-CoV-2 variants of concern, clinical validation of rapid molecular tests needs to demonstrate their ability to detect known variants, an essential requirement for a robust pan-SARS-CoV-2 assay. To date, there has been no clinical validation of reverse transcription recombinase polymerase amplification (RT-RPA) assays for SARS-CoV-2 variants. We performed a clinical validation of a one-pot multi-gene RT-RPA assay with the E and RdRP genes of SARS-CoV-2 as targets. The assay was validated with 91 nasopharyngeal samples, with a full range of viral loads, collected at University College London Hospitals. Moreover, the assay was tested with previously sequenced clinical samples, including eleven lineages of SARS-CoV-2. The rapid (20 min) RT-RPA assay showed high sensitivity and specificity, equal to 96% and 97%, respectively, compared to gold standard real-time RT-PCR. The assay did not show cross-reactivity with the panel of respiratory pathogens tested. We also report on a semi-quantitative analysis of the RT-RPA results with correlation to viral load equivalents. Furthermore, the assay could detect all eleven SARS-CoV-2 lineages tested, including four variants of concern (Alpha, Beta, Delta, and Omicron). This variant-proof SARS-CoV-2 assay offers a significantly faster and simpler alternative to RT-PCR, delivering sensitive and specific results with clinical samples.
... Our findings are consistent with previous studies showing that 0.1% SDS with 0.1% NP-40 [10] and 0.3% tri(n-butyl)phosphate (TNBP) with 1.0% Triton X-100 [8] could inactivate SARS-CoV. Recent studies on SARS-CoV-2 showed that several lysis buffers from extraction kits like ATL (1%-10% SDS) and VXL (30%-50% guanidine hydrochloride and 1%-10% Triton X-100) from Qiagen [14] and others containing guanidine hydrochloride [18] and guanidinium [19] inactivated the virus. Several RNA extraction kits contain a lysis buffer effective at inactivating SARS-CoV-2 [20]. ...
Inactivation methods allow for hazard group 3 (HG3) pathogens to be disposed of and used safely in downstream experiments and assays to be carried out at lower containment levels. Commonly used viral inactivation methods include heat inactivation, fixation methods, ultraviolet (UV) light and detergent inactivation. Here we describe known methods used to inactivate SARS-CoV-2 for safe downstream biological assays.
... It has also been suggested that the absence of the preliminary heat inactivation step in direct RT-PCR may lead to insufficient release of RNA from the virion and a decrease in the efficiency of RNA detection by direct RT-PCR [10]. On the other hand, the heat treatment step (95 • C for 5 min), a prior direct RT-PCR aimed to lyse epithelial cells, may, however, reduce the detection rate of the samples with low viral load due to the inverse effect of thermolysis on RNA integrity as was noted by others [11][12][13]. ...
... Several direct RT-PCR protocols have been described and validated using a pre-heat step at high temperatures above 95 • C for direct RT-qPCR without RNA extraction [8][9][10]30]. The heat pretreatment procedure introduced in order to lyse epithelial cells may, however, reduce sensitivity and increase false negative rate as it was noted for temperature regimen 95 • C [12,13,31] as well as 70-75 • C [9]. In our study, the direct RT-qPCR was in complete agreement with the standard RT-PCR method with all four types of upper respiratory tract samples. ...
Nasopharyngeal swab (NPS) and oropharyngeal swab (OPS) are the most widely used upper respiratory tract specimens for diagnosis of SARS-CoV-2 using RT-qPCR. In contrast, nasal swab (NS) and saliva (SS), recently recommended by the WHO, are rarely used, and their test accuracy is limited. The method for direct RT-PCR detection of SARS-CoV-2 does not require an RNA extraction and is faster and easier than standard RT-PCR tests with RNA extraction. This study aimed to compare the diagnostic performance of upper respiratory tract samples for SARS-CoV-2 detection using the direct RT-PCR without preliminary heat inactivation. Here we report the application and validation of direct RT-PCR SARS-CoV-2 RNA on 165 clinical specimens of NPS/OP, and 36 samples of NS/NPS and 37 saliva samples (for the latter with prior deproteinization). The overall sensitivity estimates were 95.9%, 94.2%, 88.9%, and 94.6% for NPS/OPS/NS/SS samples, respectively, and the specificity was 100% against standard RT-PCR with RNA extraction. Overall, NS and SS testing by direct RT-PCR had sufficient sensitivity to detect SARS-CoV-2. They can be acceptable alternative to NPS/OPS for rapid detection of SARS-CoV-2 infections in future.
... In our study, before RNA extraction, samples in VTM were pooled, which helped in saving reagents both for extraction and PCR. For reagent use and time required, the RNA extraction step remains one of the most rate limiting stages for SARS-CoV-2 RT-PCR (Grant et al., 2020). At this point in time, number of studies have been published on conventional pooling strategies for COVID-19 RT-PCR testing (Praharaj et al., 2020;Barak et al., 2021, Prakash et al., 2021, Sawicki et al., 2021. ...
In the on-going COVID-19 pandemic, pooled testing of samples by RT-PCR has been recommended at certain scenarios to increase labs’ testing capacity and reduce cost of testing. This paper describes the evaluation of bi-directional matrix pooling strategies with clinical samples in a 5×5 and 10×10 matrix. Nasopharyngeal swab samples in viral transport medium (VTM) previously tested (positive or negative) by real time RT-PCR for SARS-CoV-2 were used for these experiments. Ten sets of 5×5 (250 samples) and ten sets of 10×10 (1000 samples) pooling of samples in both directions was done with known positive samples introduced at random positions. Extracted nucleic acid was tested for SARS-CoV-2 E-gene by RT-PCR. Sensitivity or concordance and feasibility of matrix pooling were assessed in comparison to direct RT-PCR testing. In comparison to direct testing, the overall concordance was 86.6% for 5×5 pooling, 73.3% for 10×10 with 200 µL extraction volume and 86.6% for 10×10 with 400 µL extraction volume. Bi-directional matrix pooling can be adopted with advantage over conventional direct or pool testing for COVID-19 by RT-PCR under the following conditions: i) sample positivity rate of ≤5%, ii) matrix pool size of 8-10 samples, iii) use of min. 40 µL VTM from each sample and iv) utilization of automated liquid handling equipment, if available, for sample addition to avoid human errors.
... With or without addition of commercial buffers or detergents, a Ct difference between four and seven cycles was observed compared with extracted vRNA [8][9][10][11][12][13][14][15][16]. Other methods include the addition of proteinase K to patient swab samples, showing 4-6 cycle differences, but no proof of virus inactivation was shown for these samples [17,18]. ...
Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify the host factors involved and treatments to combat infection. Quantification of released virions often requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable to clinical isolates. Viral RNA purification is expensive in terms of time and resources, and is often unsuitable for high-throughput screening. Direct lysis protocols were explored for patient swab samples, but the lack of virus inactivation, cost, sensitivity, and accuracy is hampering their application and usefulness for in vitro studies. Here, we show a highly sensitive, accurate, fast, and cheap direct lysis RT-qPCR method for quantification of SARS-CoV-2 in culture supernatant. This method inactivates the virus and permits detection limits of 0.043 TCID50 virus and <1.89 copy RNA template per reaction. Comparing direct lysis with RNA extraction, a mean difference of +0.69 ± 0.56 cycles was observed. Application of the method to established qPCR methods for RSV (-ve RNA), IAV (segmented -ve RNA), and BHV (dsDNA) showed wider applicability to other enveloped viruses, whereby IAV showed poorer sensitivity. This shows that accurate quantification of SARS-CoV-2 and other enveloped viruses can be achieved using direct lysis protocols, facilitating a wide range of high- and low-throughput applications.
