key: cord-0862831-h7zi82a8 authors: Wang, Xueliang; Yao, Hangping; Xu, Xing; Zhang, Pengyin; Zhang, Minmin; Shao, Junbin; Xiao, Yanqun; Wang, Hualiang title: Limits of Detection of Six Approved RT–PCR Kits for the Novel SARS-coronavirus-2 (SARS-CoV-2) date: 2020-04-13 journal: Clin Chem DOI: 10.1093/clinchem/hvaa099 sha: e8ded24129d04e714c3bf8098127b28124d373b2 doc_id: 862831 cord_uid: h7zi82a8 nan The novel SARS coronavirus 2 (SARS-CoV-2, previously 2019-nCoV) can cause lethal coronavirus disease 2019 (COVID-19) (1) . Since its outbreak in December 2019, COVID-19 has posed a great threat to human health and life in China and the world (2) . Nucleic acid testing is the gold standard method for confirming infection (3) . Many real-time reverse transcription (RT)-PCR kits have been developed and used by the World Health Organization, the United States Centers for Disease Control and Prevention, the Chinese Center for Disease Control and Prevention, and private companies. However, in China, false-negative results have been reported at rates as high as 20 to 40 percent in cases for which both clinical symptoms and imaging evidence raised strong suspicions of disease (4) . False negatives may be caused by various factors, including the specimen source, timing of sampling, personnel operation, and the test kit quality. The limit of detection (LoD), the lowest analyte concentration that a kit can detect, is an important performance parameter in evaluating kit quality. To cope with the COVID-19 epidemic, the China National Medical Products Administration (NMPA) approved six RT-PCR kits for SARS-CoV-2, and some of which subsequently received CE marking. However, because the research and development time was short, the approved kits were not verified or optimized with appropriate numbers of clinical samples, which may have affected their LoDs. To examine whether LoD is a factor contributing to the observed false-negative results, we evaluated and compared the LoDs of these six kits using real viral RNA. Viral RNA was extracted from cultured SARS-CoV-2 (SARS-CoV-2/ZJU-01/Human/2020) with the QIAamp Viral RNA Mini Kit (Qiagen). To verify its applicability, the viral RNA was tested with the six kits provided by Shanghai Liferiver Bio-tech Co., Ltd, Wuhan Huada Bio-tech Co., Ltd, Shanghai GeneoDx Biotech Co., Ltd, DAAN Gene Co., Ltd of Sun Yat-sen University, Sansure Biotech Inc., and Shanghai BioGerm Medical Co., Ltd. The different target genes ( Table 1 ) produced typical S-shaped amplification curves, indicating that the RNA could be used in the six kits to evaluate their LoDs. The viral RNA concentration was determined with RT-droplet digital PCR (RT-ddPCR), which allows the absolute quantification of viral RNA by counting single molecules, without reference to an external standard curve. The RT-ddPCR primers and probes were designed to target ORF1ab, Table 1) . The poor LoD of the latter may be attributable to technical deficiencies in the product's manufacture, including unreasonable primer design, primer or probe impurities, reagent instability, or inappropriate reagent ratios. The low sensitivity of the kit implies that it may fail to identify many COVID-19 patients who consequently would be unlikely to receive appropriate Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia A novel coronavirus emerging in China -key questions for impact assessment General Office of National Health Committee. Notice on the issuance of a program for the diagnosis and treatment of novel coronavirus (SARS-CoV-2) infected pneumonia Correlation of chest CT and RT-PCR testing in coronavirus disease 2019 (COVID-19) in China: A report of 1014 cases Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR We acknowledge the six manufacturers for providing the SARS-CoV-2 RT-PCR detection kits.