Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets

• Published in: Emerging microbes & infections 2020-01, Vol.9 (1), p.1175-1179
• Main Authors: Liu, Xinjin, Feng, Jiangpeng, Zhang, Qiuhan, Guo, Dong, Zhang, Lu, Suo, Tao, Hu, Wenjia, Guo, Ming, Wang, Xin, Huang, Zhixiang, Xiong, Yong, Chen, Guozhong, Chen, Yu, Lan, Ke
• Format: Article
• Language: English
• Subjects: Betacoronavirus - genetics; Coronavirus Infections - diagnosis; Coronavirus Infections - virology; COVID-19; diagnosis; digital PCR; DNA Primers; DNA Probes; false negative; false positive; Humans; Letter; Multiplex Polymerase Chain Reaction - methods; Pandemics; Pneumonia, Viral - diagnosis; Pneumonia, Viral - virology; real time PCR; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; SARS-CoV-2; Sensitivity and Specificity; Severe acute respiratory syndrome coronavirus 2; Viral Load
• ISSN: 2222-1751; 2222-1751
• DOI: 10.1080/22221751.2020.1772679

Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10 −4 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.