双淬灭TaqMan探针应用于新型冠状病毒核酸检测的初步研究

Preliminary Study on A Dual⁃quencher TaqMan Probe Applied for Detection of the Nucleic Acids of SARS⁃CoV⁃2

核酸检测是新型冠状病毒肺炎(COVID⁃19)诊断的金标准。目前,临床上主要采用基于TaqMan探针的荧光定量PCR(qPCR)来检测新型冠状病毒(SARS⁃CoV⁃2)的N基因和ORF1a/b序列。在实际的核酸检测中,因采样和qPCR灵敏度问题导致的假阴性结果时有发生。为了优化SARS⁃CoV⁃2的核酸检测,我们设计了含有两个淬灭基团的双淬灭TaqMan探针,与单淬灭TaqMan探针相比,双淬灭TaqMan探针的背景荧光信号更低(161±9 vs.290±18)。同时,我们以N基因质粒和假病毒作为核酸检测的模板,发现双淬灭TaqMan探针检测体系具有更好的检测精密度(CV 1.4%vs 2.3%;0.6%vs 2.1%)以及更低的检测下限。

Detection of the nucleic acids(NAs)of severe acute respiratory disease⁃coronavirus⁃2(SARS⁃CoV⁃2)is the“gold standard”for the diagnosis of coronavirus disease⁃2019(COVID⁃19).TaqMan probe⁃based fluorescence quantitative polymerase chain reaction(qPCR)was used to detect the N gene and ORF1a/b sequence of SARS⁃CoV⁃2.In practical NA detection,false⁃negative results often occur due to sampling errors and a deficiency in qPCR sensitivity.In order to optimize the NA detection of SARS⁃COV⁃2,a dual⁃quencher TaqMan probe containing two quenching groups was designed.The background fluorescence signal of the dual⁃quencher TaqMan probe was lower than that of the single⁃quencher TaqMan probe(161±9 vs.290±18).Meanwhile,using an N gene plasmid and pseudovirus as a template for NA detection,we found that the dual⁃quencher TaqMan probe had greater detection precision(coefficient of variation of 1.4%vs.2.3%and 0.6%vs.2.1%,respectively)and a lower limit of detection.