2021-11-23

Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China.; Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China.; Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China.; Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China.; Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China.; Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China.; Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China. Electronic address: gaoyh@nim.ac.cn.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has become a public health emergency. Although many reverse-transcription PCR (RT-PCR) assays have been developed, their performance, especially sensitivity assessment, has been insufficiently tested. In this study, a preliminary comparison of the analytical sensitivity of nine RT-qPCR kits from different manufacturers was first conducted using a certified reference material derived from the genomic RNA of SARS-CoV-2 as the template. Subsequently, three of the nine kits, comprising two highly sensitive kits (DAAN, Huirui) and one less sensitive kit (Geneodx), were selected for further sensitivity and specificity validation. The results revealed variations in the performance between kits of the two groups. For the two highly sensitive kits, the limits of detection at 95 % probability (LOD95%) were 5.6 copies of the N gene and 3.5 copies of the ORF 1ab per reaction (DAAN), and 6.4 (N) and 4.6 (ORF 1ab) copies per reaction (Huirui

2021-06-03

Department of Molecular Biology, Shanghai Centre for Clinical Laboratory, Shanghai, China.; Department of Molecular Biology, Shanghai Centre for Clinical Laboratory, Shanghai, China.; Department of Molecular Biology, Shanghai Centre for Clinical Laboratory, Shanghai, China.; Department of Molecular Biology, Shanghai Centre for Clinical Laboratory, Shanghai, China.; Department of Molecular Biology, Shanghai Centre for Clinical Laboratory, Shanghai, China. Electronic address: xlwang12@126.com.

Recent reports have compared the analytical sensitivities of some SARS-CoV-2 RT-PCR assays, but differences in the viral materials used for these evaluations made comprehensive conclusions difficult. We carried out a direct comparison of the analytical sensitivities of 14 conventional and three rapid RT-PCR assays for the detection of SARS-CoV-2. The comparison was performed utilizing a certified reference material for SARS-CoV-2 RNA that was serially two-fold diluted in RNA storage solution. Our results show that the analytical sensitivities of the 17 assays varied within an 8-fold range (100-800 copies/mL). Moreover, a trend with some rapid assays yielding slightly higher analytical sensitivities (2- to 4-fold) compared with conventional assays was observed. We conclude that most of the RT-PCR assays can be used for routine COVID-19 diagnosis, but some assays with the poorest analytical sensitivities may lead to false-negative results when used to identify asymptomatic individuals

2021-07-22

Animal Production and Health Laboratory, Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Friedenstrasse 1, A-2444, Seibersdorf, Austria. Electronic address: w.dundon@iaea.org.; Animal Production and Health Laboratory, Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Friedenstrasse 1, A-2444, Seibersdorf, Austria.; Department for Molecular Biology, Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety (AGES), Robert Koch Gasse 17, A-2340, Mödling, Austria.; Department for Molecular Biology, Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety (AGES), Robert Koch Gasse 17, A-2340, Mödling, Austria.; Department for Molecular Biology, Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety (AGES), Robert Koch Gasse 17, A-2340, Mödling, Austria.; Department for Molecular Biology, Institute for Veterinary Disease Control, Austrian Agency for Health and Food Safety (AGES), Robert Koch Gasse 17, A-2340, Mödling, Austria.; Animal Production and Health Laboratory, Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Friedenstrasse 1, A-2444, Seibersdorf, Austria.; Animal Production and Health Laboratory, Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Friedenstrasse 1, A-2444, Seibersdorf, Austria.; Animal Production and Health Laboratory, Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Friedenstrasse 1, A-2444, Seibersdorf, Austria.

Transmission mitigation of SARS-CoV-2 requires the availability of accurate and sensitive detection methods. There are several commercial ad hoc molecular diagnostic kits currently on the market, many of which have been evaluated by different groups. However, in low resource settings the availability and cost of these commercial kits can be a limiting factor for many diagnostic laboratories. In such cases alternatives need to be identified. With this in mind, eight commercial reverse transcription quantitative real-time PCR (RT-qPCR) master mixes from Applied Biosystems (Thermo Fisher Scientific), Bio-Rad, Biotech Rabbit, Promega, Qiagen, QuantaBio, Invitrogen (Thermo Fisher Scientific) and Takara using the same commercial primer and probe mix [LightMix® Modular SARS and Wuhan CoV E-gene mix (TIB MolBiol, Germany)] were evaluated. Three ad hoc molecular diagnostic kits [GeneFinder™ COVID-19 Plus RealAmp kit (Osang Healthcare); genesig® Real-Time PCR Coronavirus COVID-19 (Primerdesign

2021-01-17

National Health Commission Key Laboratory of Systems Biology of Pathogens and Christophe Mérieux Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, PR China.; Key Laboratory of Medical Molecular Cell Biology of Shanxi Province, Institutes of Biomedical Sciences, Shanxi University, Taiyuan, 030006, PR China.; National Health Commission Key Laboratory of Systems Biology of Pathogens and Christophe Mérieux Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, PR China.; National Health Commission Key Laboratory of Systems Biology of Pathogens and Christophe Mérieux Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, PR China.; National Health Commission Key Laboratory of Systems Biology of Pathogens and Christophe Mérieux Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, PR China.; National Health Commission Key Laboratory of Systems Biology of Pathogens and Christophe Mérieux Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, PR China.; National Health Commission Key Laboratory of Systems Biology of Pathogens and Christophe Mérieux Laboratory, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100730, PR China; Key Laboratory of Respiratory Disease Pathogenomics, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100730, PR China. Electronic address: renliliipb@163.com.; Key Laboratory of Medical Molecular Cell Biology of Shanxi Province, Institutes of Biomedical Sciences, Shanxi University, Taiyuan, 030006, PR China; The Provincial Key Laboratories for Prevention and Treatment of Major Infectious Diseases, Shanxi, Taiyuan, 030006, PR China. Electronic address: lijg@sxu.edu.cn.

Quick and accurate detection of SARS-CoV-2 is critical for COVID-19 control. Dozens of real-time reverse transcription PCR (qRT-PCR) assays have been developed to meet the urgent need of COVID-19 control. However, methodological comparisons among the developed qRT-PCR assays are limited. In the present study, we evaluated the sensitivity, specificity, amplification efficiency, and linear detection ranges of three qRT-PCR assays, including the assays developed by our group (IPBCAMS), and the assays recommended by WHO and China CDC (CCDC). The three qRT-PCR assays exhibited similar sensitivities, with the limit of detection (LoD) at about 10 copies per reaction (except the ORF 1b gene assay in CCDC assays with a LoD at about 100 copies per reaction). No cross reaction with other respiratory viruses were observed in all of the three qRT-PCR assays. Wide linear detection ranges from 10(6) to 10(1) copies per reaction and acceptable reproducibility were obtained. By using 25 clinical