Yang Zhang1, Chunyang Dai1, Huiyan Wang1, Yong Gao2, Tuantuan Li2, Yan Fang2, Zuojun Shen1, Lichang Chen2, Zhaowu Chen1, Xuejun Ma3,4, Ming Li5. 1. Department of Clinical Laboratory, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230031, China. 2. Department of Clinical Laboratory, Fuyang Second People's Hospital, Fuyang Infectious Disease Clinical College, Anhui Medical University, Fuyang, 236015, China. 3. Chinese Center for Disease Control and Prevention, National Institute for Viral Disease Control and Prevention, Beijing, 102206, China. maxj@ivdc.chinacdc.cn. 4. Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China. maxj@ivdc.chinacdc.cn. 5. Department of Clinical Laboratory, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230031, China. liming19831002@163.com.
Abstract
BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. METHOD: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. RESULT: The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23-1145.69) for ORF1ab and 528.1 (95% CI: 347.7-1248.7) for N, 401.8 (95% CI: 284.8-938.3) for ORF1ab and 336.8 (95% CI: 244.6-792.5) for N, and 194.74 (95% CI: 139.7-430.9) for ORF1ab and 189.1 (95% CI: 130.9-433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. CONCLUSION: In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.
BACKGROUND:Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. METHOD: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19patients. RESULT: The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23-1145.69) for ORF1ab and 528.1 (95% CI: 347.7-1248.7) for N, 401.8 (95% CI: 284.8-938.3) for ORF1ab and 336.8 (95% CI: 244.6-792.5) for N, and 194.74 (95% CI: 139.7-430.9) for ORF1ab and 189.1 (95% CI: 130.9-433.9) for N, respectively. Of the 34 clinical samples from COVID-19patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. CONCLUSION: In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.
Authors: Gian Paolo Caviglia; Maria Lorena Abate; Francesco Tandoi; Alessia Ciancio; Antonio Amoroso; Mauro Salizzoni; Giorgio Maria Saracco; Mario Rizzetto; Renato Romagnoli; Antonina Smedile Journal: J Hepatol Date: 2018-04-03 Impact factor: 25.083
Authors: Giacomo Pirovano; Alvaro A Ordonez; Sanjay K Jain; Thomas Reiner; Laurence S Carroll; Naga Vara Kishore Pillarsetty Journal: Nucl Med Biol Date: 2021-05-25 Impact factor: 2.947