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There are more than 350 realtime polymerase chain reaction (RTPCR) coronavirus disease (COVID19) testing kits commercially available but these kits have not been evaluated for pooled sample testing. Thus, this study was planned to compare and evaluate seven commercially available kits for pooled samples testing. Diagnostic accuracy of (1) TRUPCR SARSCoV2 Kit (Black Bio), (2) TaqPath RTPCR COVID19 Kit (Thermo Fisher Scientific), (3) Allplex nCOV Assay (Seegene), (4) Patho detect COVID19 PCR kit (My Lab), (5) LabGun COVID19 RTPCR Kit (Lab Genomics, Korea), (6) Fosun COVID19 RTPCR detection kit (Fosun Ltd.), (7) Realtime Fluorescent RTPCR kit for SARS CoV2 (BGI) was evaluated on precharacterised 40 positive and 10 negative COVID19 sample pools. All seven kits detected all sample pools with low C t values (<30); while testing weak positive pooled samples with high C t value (>30); the TRUPCR Kit, TaqPath Kit, Allplex Assay, and BGI RTPCR kit showed 100% sensitivity, specificity, and accuracy. However, the Fosun kit, LabGun Kit, and Patho detect kit could detect only 90%, 85%, and 75% of weakly positive samples, respectively. We conclude that all seven commercially available RTPCR kits included in this study can be used for routine molecular diagnosis of COVID19. However, regarding performing pooled sample testing, it might be advisable to use those kits that performed best regarding positive identification in samples' pool, that is TRUPCR SARSCoV2 Kit, TaqPath RTPCR COVID19 Kit, Allplex nCOV Assay, and BGI Realtime RTPCR kit for detecting SARS CoV2.
Keywords: commercial kits, COVID19, realtime PCR, sample pooling, World Health Organization
The ongoing coronavirus disease (COVID19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARSCoV2) started in December from Wuhan, China. 1 World Health Organization (WHO) declared a pandemic on March 11, 2 and urged that the most effective way to prevent infections and save lives is breaking the chains of transmission, and to do that escalation of COVID19 testing is urgently required. 3
The test available for laboratory diagnosis of COVID19 includes point of care antigen detection, Conventional realtime polymerase chain reaction (RTPCR), cartridgebased test molecular test like GeneXpert (Cepheid) and Truenat (Molbio), and automated high throughput molecular assays like Roche Cobas, Abbot molecular SARS CoV2 assay, Glenmark ePlex assay, and so forth. Amongst them, conventional RTPCR is the preferred and most widely used test for COVID19 diagnosis. 4 , 5
There are more than 350 Conventional RTPCR COVID19 testing kits available commercially, of which 29 kits have been approved by the United States food and drug administration (USFDA). 6 RTPCR is an expensive test and requires a well equipped molecular laboratory with trained manpower. Many countries are experiencing acute shortages of diagnostic kits and manufacturers of molecular testing kits and consumables are also struggling to keep with the demand. It has become important to come up with novel ideas to conserve the reagents used for molecular tests. However, at the same time, the disease is new it is important to validate modifications to the testing protocol before universal adoption.
Several researchers are advocating that it is time to reintroduce the Dorfman theory 7 of sample pooling in the era of molecular testing. 8 , 9 In a recent study from the University of Nebraska Medical Centre, Omaha, the authors have used a webbased application and determined the most efficient pool size to be five samples when the incidence rate of SARSCoV2 infection is 10% or less and concluded that group testing will result in saving of reagents and increase in the testing capability of at least 69%. 10 Several countries are performing pooled sample testing for COVID19, however, none of the available RTPCR kits has been tested for pooled sample either by kit manufacturer or research groups. Thus, this study was planned to compare and evaluate seven commercially available COVID19 RTPCR kits for pooled samples' testing (Table 1).
Overview of RTPCR kits evaluated in the study
S No Name of Kit Manufacturer Regulatory clearance Target genes Limit of detection Kit interpretation 1 Allplex nCoV assay See gene USFDA E, N, RdRP copy/mlC t<40
positive
2 Patho Detect RTPCR kit Mylab ICMR, India E, RdRP 3 FOSUN COVID19 RT PCR Kit Fosun USFDA E, N, ORF1ab 300 copy/mlC t<36
positive
4 TRUPCR SARSCoV2 RTqPCR kit Black Biotech USFDA E, N, RdRP 10 copy/μlC t<35
positive
5 TaqPath COVID19 Combo Kit Thermo Fisher Scientific USFDA S, N ORF1ab 2 copy/μlC t<40
positive
6 Lab Gun RealTime PCR Kit Lab Genomics USFDA E, RdRP 20 copy/μlC t<40
positive
7 RealTime Fluorescent RTPCR Kit for nCoV BGI Genomics USFDA CEIVD ORF1ab 150 copy/μlC t<37
positive
Open in a new tabThis prospective observational study was designed and conducted at the Department of Microbiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences (SGPGIMS), Lucknow, India. The swabs were collected by healthcare workers at Rajdhani Corona Hospital, SGPGIMS in a 3ml viral transport media (VTM) and transported to the COVID19 laboratory in a cold chain.
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Before conducting the study, a survey was done on commercially available RTPCR kits regarding necessary approvals (USFDA/CE/ICMR, India), the lower limit of detection, usage, and availability in India, compatibility with different PCR platforms, cost, and so forth. Based on the survey results, the following kits were selected and procured: (1) TRUPCR SARSCoV2 Kit (Black Bio Biotech), (2) TaqPath RTPCR COVID19 Kit (Thermo Fisher Scientific), (3) Allplex nCOV Assay (Seegene), (4) Patho detect COVID19 qualitative PCR kit (My Lab), (5) LabGun COVID19 RTPCR Kit (Lab Genomics), (6) Fosun COVID19 RTPCR detection kit (Fosun Ltd.), and (7) Realtime Fluorescent RTPCR kit for detecting SARS CoV2 (BGI Genomics). None of the manufacturers were involved in the assessment and interpretation of the study results.
The samples (VTM) were opened in biosafety cabinet classII and 300μl of the VTM was further processed for viral nucleic acid extraction by a chemagic Viral DNA/RNA Kit on a chemagic 360 instrument (Perkin Elmar) as per the manufacturer's protocol. To avoid RNA degradation, the study was planned in such a manner that the entire experiment was completed in 24h.
A 25μl reaction was prepared for qualitative detection of SARS Corona virus2 by RTqPCR utilizing 5μl of extracted RNA, 12.5μl of 2× PCR buffer and 1μl of AgPath OneStep RTPCR Reagents (Thermo Fisher Scientific) and primer and probe sequences targeting E genes, RdRP and RnaseP as per WHO protocol. 11 All oligonucleotides were synthesized and provided by Thermo Fisher Scientific and thermal cycling was performed at 55°C for 10 min for reverse transcription, followed by 95°C for 3min and then 40 cycles of 95°C for 15s, 58°C for 30s using an Applied biosystem RealTime PCR system (Thermo Fisher Scientific). All samples that were initially screened for the E gene and positive samples were confirmed by detection of specific RdRP gene. The cutoff threshold (C t value) for each sample was recorded and samples with C t value<40 were considered as positive. All the samples were tested in triplicate before including them in the study. Once the positive and negative samples were identified by WHO protocol; we tested 40 positive samples and 10 negative samples by all seven commercial kits selected for study following kits protocol on an Applied biosystem RealTime PCR system.
Positive pools were created using 60μl VTM from an RTPCR confirmed COVID19positive patient specimen added to 60μl VTM from each of four negative patient samples to prepare a final volume of 300μl. Similarly, negative sample pools were also created. The pooled 300μl VTM was used as starting material for RNA extraction and nucleic acid extraction was performed on each pool using viral nucleic acid extraction by a chemagic Viral DNA/RNA Kit on a chemagic 360 instrument (Perkin Elmar). Finally, 100μl of RNA was eluted and the same RNA was used in further experiments. The study protocol was designed in such a way that the positive sample with a low cutoff threshold (C t value<30) and high C t value (>30) were included. All RTPCR tests were performed using RNA extracted from pooled samples on an Applied biosystem RealTime PCR system and thermocycling settings and results interpretation was performed as per manufacturer's instructions.
A total of 500 samples were tested for SARS Cov2. Sixty samples were positive and the rest negative; we retested all positive and 10 negative samples in triplicate and finally 40 positive samples and 10 negative samples were selected for the study. Twenty positive samples had high viral loads with a low cutoff threshold (<30) and twenty samples had low viral loads with a high C t value (>30). The selected 50 samples were tested by RTPCR for SARS CoV2 by using seven commercial kits. All seven commercially RTPCR kits could correctly identify 40 positive and 10 negative samples, as a weak positive sample with a cut of threshold in the range of 3538 was also detected by these kits, it can be safely concluded that they can be used for routine diagnostics of COVID19.
All seven commercial kits were evaluated with 40 positive and 10 negative sample pools in duplicate. Results showed that all kits performed well when strongly positive samples were tested and all seven kits detected all sample pools with low C t value (<30). When testing weak positive pooled samples (C t value>30), TRUPCR Kit, TaqPath Kit, Allplex Assay, and BGI RTPCR kit showed 100% sensitivity, specificity, and accuracy. (Table 2) The Fosun kit, LabGun Kit, and Patho detect kit could detect only 18 (90%), 17 (85%), and 15 (75%) of weakly positive samples respectively. The C t value of all samples that could not be detected on pool testing by RT PCR was more than 37. (Table 3) The C t value of three pool by WHO protocol that was not detected by LabGun kit was 37.8, 38.2, and 38.4, among them the LabGun kit could detect all three with the good sigmoid graph at C t value>40 but was interpreted as negative as per manufacturer recommendations, the LabGun RTPCR kit demonstrated a sensitivity, specificity, and accuracy of 93.0%, 100%, and 94% respectively.
Overall diagnostic efficacy of kits used in the study
Lab Gun Fosun BGI Thermo Fischer Scientific Black Bio My Lab Seegene Sensitivity 93.02% (80.9%8.5%) 95.2% (83.8%99.4%) 100.0% (91.1%100.0%) 100.0% (91.1%100.0%) 100.0% (91.1%100.0%) 88.8% (75.9% 96.2%) 100.0% (91.1%100.0%) Specificity 100% (69.1%100%) 100% (69.1%100%) 100% (69.1%100%) 100% (69.1%100%) 100% (69.1%100%) 100% (69.1%100%) 100% (69.1%100%) Positive predictive value 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% Negative predictive value 76.9% (52.8%90.8%) 83.3% (56.3% 95.0%) 100.0% 100.0% 100.0% 66.6% (46.6%82.0%) 100.0% Accuracy 94.3% 96.15% 100.0% 100.0% 100.0% 90.9% 100.0% Open in a new tabShowing Cut off threshold (C t value) of positive sample pools not detected by few commercial kits
S. No. E gene (In house) Lab Gun Fosun BGI Thermo Fischer Scientific Black Bio My Lab Seegene 1 38.4 Negative (41.4) Negative (38.1) 35.4 37.2 34.2 Not detected 38.0 2 37.8 Negative (40.6) 35.6 36.6 35.8 32.8 Not detected 38.4 3 37.5 38.9 Negative (37.0) 35.2 36.8 35.0 Not detected 38.4 4 38.2 Negative (41.0) 35.8 36.2 37.2 33.6 Not detected 38.8 5 37.8 38.8 34.8 37.0 36.4 34.0 Not detected 38.2 Open in a new tabThe C t value by WHO protocol of two sample pools tested negative by FOSUN kit was 37.5 and 38.4, the FOSUN RTPCR kit detected them at a C t value of 37.0 and 38.1, respectively, but was interpreted negatively as per manufacturer recommendations (C t value>36 Negative). The Patho detect kit by MyLab could not detect five (25%) sample pool and there was no sigmoid shaped graph even at a higher C t value. The sensitivity, specificity, and positive predictive value of the Mylab kit is 88.8%, 100%, and 100.0%, respectively, but documented a low negative predictive value of 66%.
Amidst the ongoing COVID19 pandemic, the World Health Organization has globally emphasized the importance of the molecular diagnosis of SARS CoV2 to limit the spread as well as to appropriately treat those patients who have a serious infection. 12 WHO has further emphasized that an urgent increase in laboratory testing, isolation, and contact tracing should be the backbone of the pandemic control strategy. 4 Accurate and timely results are important for decision making during this current outbreak, both in the inpatient and OPD settings. For patients admitted to the hospital, the results are critical for medical management, patient cohorting and infection control measures. Likewise, the results are also very important in the outpatient setting; as community prevalence data and identification of disease hot spots help the decisionmakers to form the basis for social distancing, the lockdown of infected areas, and a disease combating strategy. 13 A short turnaround time of COVID19 test reports is also critical for judicious use of limited resources, such as the availability of holding area beds, isolation rooms, and realtime cohorting decisions. In addition, timely test results are required to ensure the safety of healthcare workers and minimize their exposure as levels of personal protective equipment required by healthcare professionals also vary depending on whether a patient is COVID19 positive or negative. 14
The recommended test for diagnosis of COVID19 is RTPCR, and to conduct this test, a fully functional molecular laboratory is required equipped with specialized equipment like biosafety cabinets, automated RNA extractors, a Realtime PCR machine, and trained manpower to process the samples while ensuring biosafety and biosecurity. It is difficult to set up a new molecular testing laboratory in the midst of the COVID19 pandemic and increasing the testing capacity of the existing laboratory by sample pooling strategy is a practical solution. 15 The pandemic reached India in March and the testing capacity of the nation was less than samples/day. On 13.04., WHO in collaboration with the Indian Council Of Medical Research, New Delhi issued an advisory recommending pool testing of fivesample pools in an area where COVID19 prevalence is <5%. 16 With time the molecular laboratory network in India has been strengthened and using the sample pooling strategy currently 110,000 samples are tested every day and to date, 60 million samples have been tested with 5 million COVID19 positive cases. 17 Sample polling conserves PCR Kits and consumables and significantly decreases manpower requirement. At our center, we have performed 300,000 COVID19 RTPCR to date and data suggest that fivesample pooling saves 55% reagents and increases the testing capacity 2.5 times using the same infrastructure and manpower (unpublished data).
Currently, there is no literature on the evaluation of the efficacy of various commercially available RealTime PCR kits for pool sample testing, thus this study was planned to compare and evaluate seven commercially available RTPCR kits for the detection of SARSCoV2 in clinical sample pools. We compared the kits by performing testing using the same quantified RNA; thus, allowing parallel evaluation.
All seven RTPCR kits evaluated in this study could correctly identify all 40 positives and 10 negative samples, including a weak positive sample. A similar study evaluated the clinical performance of selected RTPCR kits from seven different manufacturersAltona Diagnostics, BGI, CerTest Biotec, KH Medical, Primer Design, R Biopharm AG, and Seegene and PCR efficiency was found to be 96% for all assays and the authors concluded that all tested RTPCR in the study may be used for routine diagnostics of COVID19 in patients by experienced molecular diagnostic laboratories. 18 In another study evaluating the diagnostic performance of RTPCR kits provided with emergency use authorization by USFDA, the New York SARSCoV2 Realtime PCR Diagnostic Panel (modified CDC) assay, the Simplexa COVID19 Direct (Diasorin Molecular) assay, GenMark ePlex SARSCoV2 (GenMark) assay, and the Hologic Panther Fusion SARSCoV2 (Hologic) assay were tested and the study results suggested that all 4 kits performed similarly. 19 Thus, based on these studies' results and existing literature, it can be concluded that USFDA/CE kits can be used for laboratory diagnosis of COVID19.
To date, to the best of our knowledge, no study has compared the diagnostic performance of commercially available RTPCR kits for pooled sample testing; although there are few studies that have evaluated pool testing efficacy using a single RTPCR kit. In a study on evaluation of RTPCR for pool testing using the Seegene Allplex nCoV assay, the authors pooled up to 32 samples and reported that the kit could detect all positive samples and concluded that using standard protocols sample pooling can be applied immediately in current clinical testing laboratories. 15 In another study on pool sample testing, the authors performed pool testing using a Real Star SARSCoV2 RTPCR Kit (Altona Diagnostics) and suggested that pooling of up to 30 samples per pool can increase test capacity with existing equipment and test kits and detects positive samples with sufficient diagnostic accuracy. 20
This study results show higher analytical sensitivities, specificity, and accuracy of the TRUPCR SARSCoV2 Kit, TaqPath RTPCR COVID19 Kit, Allplex nCOV Assay, and Realtime Fluorescent RTPCR kit for detecting SARS CoV2 (BGI) assays when compared to the FOSUN COVID19 RTPCR detection kit, LabGun COVID19 RTPCR Kit and Patho detect COVID19 qualitative PCR kit assays. The C t value of samples that could not be detected by three RTPCR was more than 37. The Lab Gun kit and Fosun kit could detect all samples with a good sigmoid graph at high C t value but were interpreted as negative as per manufacturer recommendations. Thus, in pooled samples, RTPCR graphs should be analyzed for sigmoid curve even beyond the manufacturerrecommended cutoff threshold, and in case of the appearance of any graph, the RTPCR should be repeated with deconvoluted samples. Further unpublished data from our centre suggest that less then <2% of our RTPCRpositive samples had C t value>37, and by following the abovementioned precautions, we picked the maximum possible positive cases.
We conclude that all seven commercially available RTPCR kits included in this study can be used for the molecular diagnosis of COVID19. When performing pool sample testing, it might be advisable to use those kits that performed best regarding positive identification in the samples pool that is the TRUPCR SARSCoV2 Kit, TaqPath RTPCR COVID19 Kit, Allplex nCOV Assay, and Realtime Fluorescent RTPCR kit for detecting SARS CoV2 (BGI).
The authors declare that there are no conflict of interests.
Atul Garg designed the study, Ujjala Ghoshal prepared and edited the manuscript, Sangram S. Patel, D.V. Singh, and Akshay K. Arya performed the molecular lab work and Shruthi Vasanth performed data analysis.
We would like to thank Mr. VK Mishra and Mr. Hemant Verma for their technical help in molecular work.
Garg A, Ghoshal U, Patel SS, et al. Evaluation of seven commercial RTPCR kits for COVID19 testing in pooled clinical specimens. J Med Virol. ;93:. 10./jmv.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
This section collects any data citations, data availability statements, or supplementary materials included in this article.
The data that support the findings of this study are available from the corresponding author upon reasonable request.