Preliminary discussion on results of droplet digital PCR and real-time fluorescence quantitative PCR in nucleic acid detection of 2019-nCoV
ZHOU Shuai-feng1, LI Shi-kang1, CHEN Yu1, ZHANG Fan1, PAN Jia3, HU Shi-xiong1, HUANG Yi-wei1, DAI Zhi-hui1, ZHAN Zhi-fei1, MA Xue-jun2
1. Hunan Provincial Key Laboratory of Microbial Molecular Biology, Hunan Provincial Center for Disease Control and Prevention, Changsha, Hunan 410005, China; 2. National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; 3. The First Hospital of Changsha City, Changsha, Hunan 410005, China
Abstract：Objective To analyze the 2019 novel coronavirus (2019-nCoV) nucleic acid results detected by droplet digital PCR (ddPCR) and real-time fluorescence quantitative PCR (qPCR), to compare the differences in detecting various specimens by the two assays, and to provide data support for improving detection scheme of 2019-nCoV nucleic acid. Methods ddPCR and qPCR were used to detect 2019-nCoV nucleic acid in 22 blood, urine and feces specimens collected at different times in 3 clinically-confirmed COVID-19 patients. Results The gene amplification results of a conserved region from the human cells were consistent with the two methods: whole blood samples had the strongest signal, followed by urine, and the least feces. The positive microdrops of ORF-1ab and N genes were detected in 1 whole blood specimen, 1 urine specimen and 5 feces specimens, respectively by ddPCR, while only 3 feces specimens were detected positive by qPCR. The average concentration of 2019-nCoV genes in the 3 missed samples was 128 copies/ml. ddPCR could screen the positive specimens less than 5 days, within 5-15 days and more than 15 days after the onset of the disease, whilepositive specimens from COVID-19 patients at middle-late stage were mainly detected by qPCR.Positive droplets were detected in three kinds of specimens of severe cases by ddPCR, but all specimens were negative by qPCR. Only fecal specimens of mild cases were detected 2019-nCoV positive, and the positive rate of fecal detection by ddPCR in mild cases was higher than that of qPCR. Conclusions ddPCR can effectively overcome the lack of sensitivity of qPCR, and is a useful supplement to qPCR, especially for the blood, urine and suspected fecal or anal swab samples with low viral load, which is suitable for the early and discharge diagnosis of 2019-nCoV.
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2020, Vol. 27 
