一步法多重荧光定量RT-PCR检测人杯状病毒的建立及应用

Establishment and application of one-step multiplex real-time RT-PCR assay for the detection of human calicivirus

目的建立检测人杯状病毒(human calicivirus,HuCVs,主要包括SV、NoV-GⅠ和NoV-GⅡ)的一步法多重荧光定量RTPCR(rRT-PCR)法。方法用Primer Express 3.0软件设计特异性引物和探针,通过合成的对应病毒基因的质粒来评价该方法的灵敏度、特异性和重复性,检测临床标本并对阳性结果进行基因测序验证。结果建立的rRT-PCR法检测SV、NoV-GⅠ和NoV-GⅡ的灵敏度均达到103copies/mL,特异性为100%,且变异系数(CV)均≤1.275%。HuCVs检出率为21.43%(75/350),其中SV、NoV-GⅠ和NoV-GⅡ分别为14.67%(11/75)、21.33%(16/75)和64.00%(48/75)。随机选择检测阳性结果进行测序,结果均与该法检测结果一致。结论建立的rRT-PCR法可同时检测并区分SV、NoV-GⅠ和NoV-GⅡ病毒,可用于HuCVs引起的急性腹泻散发和暴发疫情的监测。

Objective To establish a one-step multiplex real-time RT-PCR（ rRT-PCR） method for detecting human calicivirus（ HuCVs）,such as sapovirus（ SV）,norovirus（ NoV）-GⅠ and NoV-GⅡ. Methods The specific primers and probes of SV,NoV-GⅠ and NoV-GⅡ were designed by the Primer Express 3. 0 software,and the one-step multiplex rRT-PCR method was established. Then,the sensitivity,specificity and repeatability of the method were evaluated by the specific plasmids of these viruses. In addition,the stool samples from acute diarrhea patients were detected by the method,and the positive results were further verified by sequencing assay.Results The sensitivity,specificity and coefficients of variation（ CV） of the established multiplex rRT-PCR method for the detection of SV,NoV-GⅠ and NoV-GⅡ were 103 copies / mL,100% and ≤1. 275%,respectively. Of the 350 stool samples,the positive rate of HuCVs was 21. 43%（ 75 /350）,including 14. 67%（ 11 /75） of SV,21. 33%（ 16 /75） of NoV-GⅠ,and 64. 0%（ 48 /75） of NoV-GⅡ. The positive samples were selected randomly for sequencing assay,and there were consistent results between them. Conclusion The established one-step multiplex rRT-PCR assay may simultaneously detect and discriminate SV,NoV-GⅠ and NoV-GⅡ viruses,which may be used to monitor epidemic situation of acute diarrhea caused by HuCVs.