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Highly accurate and sensitive diagnostic detection of SARS-CoV-2 by digital PCR

By Lianhua Dong, Junbo Zhou, Chunyan Niu, Quanyi Wang, Yang Pan, Sitong Sheng, Xia Wang, Yongzhuo Zhang, Jiayi Yang, Manqing Liu, Yang Zhao, Xiaoying Zhang, Tao Zhu, Tao Peng, Jie Xie, Yunhua Gao, Di Wang, Yun Zhao, Xinhua Dai, Xiang Fang

Posted 18 Mar 2020
medRxiv DOI: 10.1101/2020.03.14.20036129

BACKGROUNDThe outbreak of COVID-19 caused by a novel Coronavirus (termed SARS-CoV-2) has spread to over 140 countries around the world. Currently, reverse transcription quantitative qPCR (RT-qPCR) is used as the gold standard for diagnostics of SARS-CoV-2. However, the positive rate of RT-qPCR assay of pharyngeal swab samples are reported to vary from 30[~]60%. More accurate and sensitive methods are urgently needed to support the quality assurance of the RT-qPCR or as an alternative diagnostic approach. METHODSWe established a reverse transcription digital PCR (RT-dPCR) protocol to detect SARS-CoV-2 on 194 clinical pharyngeal swab samples, including 103 suspected patients, 75 close contacts and 16 supposed convalescents. RESULTSThe limit of blanks (LoBs) of the RT-dPCR assays were [~]1.6, [~]1.6 and [~]0.8 copies/reaction for ORF 1ab, N and E genes, respectively. The limit of detection (LoD) was 2 copies/reaction. For the 103 fever suspected patients, the sensitivity of SARS-CoV-2 detection was significantly improved from 28.2% by RT-qPCR to 87.4% by RT-dPCR. For close contacts, the suspect rate was greatly decreased from 21% down to 1%. The overall sensitivity, specificity and diagnostic accuracy of RT-dPCR were 90%, 100% and 93 %, respectively. In addition, quantification of the viral load for convalescents by RT-dPCR showed that a longer observation period was needed in the hospital for elderly patients. CONCLUSIONRT-dPCR could be a confirmatory method for suspected patients diagnosed by RT-qPCR. Furthermore, RT-dPCR was more sensitive and suitable for low viral load specimens from the both patients under isolation and those under observation who may not be exhibiting clinical symptoms.

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