塞内卡病毒和口蹄疫病毒二重实时荧光RT-PCR鉴别检测方法的建立

Seneca Valley virus and foot-and-mouth disease virus by MGB based real-time RT-PCR

为了建立一种快速、敏感、特异的能够鉴别诊断口蹄疫病毒(FMDV)和塞内卡病毒(SVV)二重实时荧光RTPCR方法,根据FMDV及SVV的保守基因序列,设计了2套特异性引物和不同荧光素标记的MGB探针。通过对PCR反应体系和反应条件的优化筛选,建立了FMDV和SVV二重实时荧光RT-PCR检测方法,并对二重实时荧光RT-PCR检测方法进行了特异性、敏感性、重复性试验;利用所建立的方法对98份疑似FMDV和SVV感染的临床样品进行了检测。结果显示,成功建立的FMDV及SVV二重实时荧光RT-PCR检测方法,模板在101~107拷贝/μL有很好的线性关系;对pGEM-T/FMDV和pGEM-T/SVV重组质粒出现阳性扩增信号,但对正常细胞培养物对照和其他7种病原对照未扩增出特异性曲线,方法特异性较好;最低检测模板浓度为10拷贝/μL;自98份疑似FMDV和SVV感染样品中检出9份FMDV阳性,10份SVV阳性,2份FMDV和SVV双阳性,并且和克隆测序结果一致。本研究建立的FMDV及SVV二重实时荧光RT-PCR检测方法,可用于FMDV和SVV的快速鉴别检测,为FMDV和SVV的鉴别诊断提供特异、敏感和高通量的方法。

In order to establish a rapid,sensitive and specific method for differential diagnosis of foot-and-mouth disease virus(FMDV)and Seneca Valley virus(SVV)through a double real-time fluorescent RT-PCR method,two sets of specific primers and different fluorescent-labeled MGB probes were designed based on the conserved gene sequences of FMDV and SVV.The FMDV and SVV double real-time fluorescent RT-PCR detection methods were established by optimizing the PCR reaction system and reaction conditions,and the specific,sensitive and reproducible tests for the double-real-time fluorescent RT-PCR detection method were carried out.And 98clinical samples suspected of FMDV and SVV infection were tested using the established method.The results showed that the successfully established double-real-time RT-PCR detection method of FMDV and SVV had a good linear relationship within the template range of 101-107 copies/μL;positive amplification signals were generated for the pGEM-T/FMDV and pGEM-T/SVV recombinant plasmids,but the specificity curves were not amplified for the normal cell culture control and the other 7pathogen controls,proving that the method was better in specificity;The minimum detection template concentration was 10copies/μL;9FMDV-positive and 10SVV-positive and 2pairs of FMDV/SVV samples were detected from 98suspected FMDV and SVV infected samples,and the results were consistent with the results of cloning sequencing.The double-real-time fluorescence RT-PCR detection method of FMDV and SVV established in this study can be used for rapid identification detection of FMDV and SVV,and provides a specific,sensitive and high-throughput method for the differential diagnosis of FMDV and SVV.