新型冠状病毒N亚基因组荧光定量PCR检测方法的建立及初步应用

Establishment and preliminary application of quantitative real-time PCR assay for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA

目的建立一种快速检测新型冠状病毒N亚基因组(SgN)的方法,探讨其在新型冠状病毒肺炎病例及环境样本中的应用价值。方法根据全球共享流感数据库(GISAID)公布的新型冠状病毒全基因组序列设计SgN特异性引物和探针,优化退火温度、引物与探针浓度等反应条件,建立新型冠状病毒SgN荧光定量PCR检测方法,绘制标准曲线,评价该方法的灵敏性、重复性和特异性。采用建立的病毒SgN荧光定量PCR检测方法检测21份环境和351份临床样本,并选取25份SgN阳性样本进行病毒分离以评估该检测方法的应用效果。结果 SgN的引物和探针对新型冠状病毒具有良好特异性。初步建立的病毒SgN荧光定量PCR检测方法最低检测限为1.5×102copies/ml,变异系数小于1%。372份样本的gRNA阳性率为97.04%(361/372),gRNA阳性样本中阳性环境样本和阳性临床样本的SgN阳性率分别为36.84%(7/19)和49.42%(169/342)。其中野生型毒株样本的SgN阳性率及拷贝数均比德尔塔(Delta)毒株低。在25份SgN阳性样本中,采样时间在5 d内的12份样本均分离到病毒;13份采样时间在12 d以上的样本未发生细胞病变。结论成功建立了一种检测新型冠状病毒SgN的荧光定量PCR方法,该方法的灵敏度、特异性和重复性均较好。

Objective To establish a rapid and specific quantitative real-time PCR(qPCR)method for the detection of SARS-CoV-2 subgenomic nucleocapsid RNA(SgN)in patients with COVID-19 or environmental samples.Methods The qPCR assay was established by designing specific primers and TaqMan probe based on the SARS-CoV-2 genomic sequence in Global Initiative of Sharing All Influenza Data(GISAID)database.The reaction conditions were optimized by using different annealing temperature,different primers and probe concentrations and the standard curve was established.Further,the specificity,sensitivity and repeatability were also assessed.The established SgN and genomic RNA(gRNA)qPCR assays were both applied to detect 21 environmental samples and 351 clinical samples containing 48 recovered patients.In the specimens with both positive gRNA and positive SgN,25 specimens were inoculated on cells.Results The primers and probes of SgN had good specificity for SARS-CoV-2.The minimum detection limit of the preliminarily established qPCR detection method for SgN was 1.5×102 copies/ml,with a coefficient of variation less than 1%.The positive rate of gRNA in 372 samples was 97.04%(361/372).The positive rates of SgN in positive environmental samples and positive clinical samples were 36.84%(7/19)and 49.42%(169/342),respectively.The positive rate and copy number of SgN in Wild strain were lower than those of SARS-CoV-2 Delta strain.Among the 25 SgN positive samples,12 samples within 5 days of sampling time were all isolated with virus;13 samples sampled for more than 12 days had no cytopathic effect.Conclusion A qPCR method for the detection of SARS-CoV-2 SgN has been successfully established.The sensitivity,specificity and repeatability of this method are good.