Anal Chem
. 2020 May 22.
doi: 10.1021/acs.analchem.0c01884. Online ahead of print.
A Novel One-Step Single-Tube Nested Quantitative Real-Time PCR Assay for Highly Sensitive Detection of SARS-CoV-2
Ji Wang, Kun Cai, Ruiqing Zhang, Xiaozhou He, Xinxin Shen, Jun Liu, Junqiang Xu, Feng Qiu, Wenwen Lei, Jinrong Wang, Xinna Li, Yuan Gao, Yongzhong Jiang, Wenbo Xu, Xuejun Ma
- PMID: 32438806
- DOI: 10.1021/acs.analchem.0c01884
Abstract
Coronavirus disease 2019 (COVID-19) has become a public health emergency. The reverse transcriptase real-time quantitative PCR (qRT-PCR) test is currently considered as a gold standard in the laboratory for the etiological detection of COVID-19. However, qRT-PCR results could be false-negative due to the inadequate sensitivity of qRT-PCR. In this study, we have developed and evaluated a novel one-step single-tube nested quantitative Real-Time PCR (OSN-qRT-PCR) assay for highly sensitive detection of SARS-CoV-2 targeting the ORF1aband N genes. The sensitivity of the OSN-qRT-PCR assay was 1 copy/reaction and 10-fold higher than that of commercial qRT-PCR kit (10 copies/reaction). The clinical performance of the OSN-qRT-PCR assay was evaluated using 181 clinical samples. Among them, 14 qRT-PCR-negative samples (7 had no repetitive results, and 7 had no Ct values) were detected by OSN-qRT-PCR. Moreover, the 7qRT-PCR-positives in the qRT-PCR grey zone (Ct value of ORF1ab ranged from 37.48 to 39.07, and Ct value of N ranged from 37.34 to 38.75) were out of the grey zone thus were deemed to be positive by OSN-qRT-PCR, indicating the positivity of these samples is confirmative. Compared to the qRT-PCR kit, OSN-qRT-PCR assay revealed higher sensitivity and specificity hence better suited to clinical applications for the detection of SARS-CoV-2 in patients with low viral load.