Discrimination of False Negative Results in RT-PCR Detection of SARS-CoV-2 RNAs in Clinical Specimens by Using an Intern
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RESEARCH ARTICLE
Discrimination of False Negative Results in RT-PCR Detection of SARSCoV-2 RNAs in Clinical Specimens by Using an Internal Reference Yafei Zhang1,2 • Changtai Wang1,2 • Mingfeng Han3 • Jun Ye1,2 • Yong Gao3 • Zhongping Liu1,2 Tengfei He1,2 • Tuantuan Li3 • Mengyuan Xu1,2 • Luping Zhou1,2 • Guizhou Zou1,2 • Mengji Lu4 Zhenhua Zhang1,2
• •
Received: 25 May 2020 / Accepted: 15 July 2020 Ó Wuhan Institute of Virology, CAS 2020
Abstract Reverse transcription-polymerase chain reaction (RT-PCR) is an essential method for specific diagnosis of SARS-CoV-2 infection. Unfortunately, false negative test results are often reported. In this study, we attempted to determine the principal causes leading to false negative results of RT-PCR detection of SARS-CoV-2 RNAs in respiratory tract specimens. Multiple sputum and throat swab specimens from 161 confirmed COVID-19 patients were tested with a commercial fluorescent RT-PCR kit targeting the ORF1ab and N regions of SARS-CoV-2 genome. The RNA level of a cellular housekeeping gene ribonuclease P/MRP subunit p30 (RPP30) in these specimens was also assessed by RT-PCR. Data for a total of 1052 samples were retrospectively re-analyzed and a strong association between positive results in SARS-CoV-2 RNA tests and high level of RPP30 RNA in respiratory tract specimens was revealed. By using the ROC-AUC analysis, we identified Ct cutoff values for RPP30 RT-PCR which predicted false negative results for SARS-CoV-2 RT-PCR with high sensitivity (95.03%–95.26%) and specificity (83.72%–98.55%) for respective combination of specimen type and amplification reaction. Using these Ct cutoff values, false negative results could be reliably identified. Therefore, the presence of cellular materials, likely infected host cells, are essential for correct SARS-CoV-2 RNA detection by RT-PCR in patient specimens. RPP30 could serve as an indicator for cellular content, or a surrogate indicator for specimen quality. In addition, our results demonstrated that false negativity accounted for a vast majority of contradicting results in SARS-CoV-2 RNA test by RT-PCR. Keywords COVID-19 SARS-CoV-2 RT-PCR False negative results Internal reference
Yafei Zhang, Changtai Wang, Mingfeng Han, Jun Ye and Yong Gao have contributed equally to this work.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12250-020-00273-8) contains supplementary material, which is available to authorized users. & Zhenhua Zhang [email protected] & Mengji Lu [email protected] 1
Department of Infectious Diseases, The Second Hospital of Anhui Medical University, Hefei 230601, China
2
Institute of Clinical Virology, The Second Hospital of Anhui Medical University, Hefei 230601, China
3
Department of Internal Medicine, The Second Hospital of Fuyang, Fuyang 236015, China
4
Institute for Virology, University Hospital of Essen, University of Duisburg-Essen, 45147 Essen, Germany
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