Non-specific Primers Reveal False-negative Risk in Detection of COVID-19 Infections

BackgroundA novel coronavirus disease 2019 (COVID-19) broke out in Wuhan of Hubei province and had spread throughout the world since December 2019. Because the clinically diagnosed cases in Hubei province were reported for the first time on February 13, 2020, a very high peak of new cases in China was observed. The reason why so many clinically diagnosed cases appeared was not clear. MethodsAll data of new cases in China were acquired from WHO situation reports. Linear fitting was used to infer the ability to detect COVID-19 infections. Primer-BLAST and nucleotide blast were applied to check the specificity of primers. Expression data of human mRNA in different tissues was obtained from Human Protein Atlas. FindingsBased on the data and analysis of changes of new laboratory-confirmed cases and new clinically diagnosed cases, it was inferred that there were many false-negative results in all clinically diagnosed cases in Hubei province. There were eight non-specific primers in dozens of primers used in clinical or research detection of COVID-19. Among them, a pair of primer for the ORF1ab regions of SARS-CoV-2 genome well matched some human mRNAs such as Cathepsin C transcripts. Compared to other transcripts, Cathepsin C mRNA had a high abundance in tonsil, lung and small intestine. InterpretationSome non-specific RT-PCR primers could cause the serious interference during RT-PCR amplification so as to increase the risk of false-negative diagnoses for COVID-19 infections. FundingKey Research Project of the Higher Education of Henan Province Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThe author searched PubMed on April 15, 2020, for papers that describe false-negative RT-PCR detection of COVID-19 by using the search terms "COVID-19", "false-negative" and "RT-PCR", with no language or time restrictions. Eleven investigations only presented the rate of false-negative detection or the importance of positive chest CT finding. There were no reports referring the primer problems of false-negative detection in COVID-19 infections. Added value of this studyThe author had found that some primers could amplify the human mRNA in specimens, which mixed SARS-CoV-2 viral particles and other tissue cells. A pair of primer provided by China CDC could vastly match the sequences of human CTSC transcripts with high abundance. That could lead to false-negative results in detection of COVID-19 infections. Implications of all the available evidenceAlthough there were so many false-negative results in detection of COVID-19 infections in China, the exact reason was not clear. Problems in sampling and test condition were discussed thoroughly, but conclusions were usually contradictory. Therefore, the work could promote the verification of the false-negative detection of COVID-19 infections in China.