O型口蹄疫病毒和塞内卡病毒双重荧光定量RT-PCR检测方法的建立

Establishment of a duplex fluorescent quantitative RT-PCR method for detecting foot-and-mouth disease type-O virus and Seneca Valley virus

为建立O型口蹄疫病毒(FMDV-O)和塞内卡病毒(SVV)快速鉴别检测方法,本实验根据FMDV-O VP2基因序列和SVV P3基因序列,采用Primer Express3.0软件分别设计引物与探针,并经各反应条件的优化初步建立了可同时鉴别检测FMDV-O和SVV的双重荧光定量RT-PCR方法。利用该方法检测临床常见猪病毒,结果显示可同时检测出FMDV-O和SVV核酸,且与猪水泡性口炎病毒(VSV)、猪水疱病毒(SVDV)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)、A型口蹄疫病毒(FMDV-A)、A-1型口蹄疫病毒(FMDVA-1)核酸均无交叉反应,并可检测到FMDV-O PanAsia株、cathay株、Ind-2001株和Mya-98株等主要流行株,特异性强;该方法对含FMDV-O和SVV拼接质粒标准品的最低检测限为7.17×10^(2)拷贝/μL,敏感性高;利用该方法检测了同一时间和不同时间提取的3种不同浓度的质粒标准品,结果显示批内和批间重复性试验Ct值的变异系数均小于3%,重复性好。利用本实验所建立的方法对30份临床样品进行检测,结果与各病毒单一荧光定量RT-PCR检测结果一致。本研究建立的FMDV-O和SVV双重荧光定量RT-PCR方法能够快速准确的鉴别检测FMDV-O和SVV,为口岸检疫的快速通关提供了技术手段。

To establish a rapid detection and identification method for foot-and-mouth disease virus type-O(FMDV-O)and Seneca Valley virus(SVV),primers and probes were designed based on the VP2 gene of FMDV-O and the P3 gene of SVV.After optimizing the reaction conditions,a duplex real-time RT-PCR method was established for simultaneous detection and identification of FMDV-O and SVV.The results showed that this method has strong specificity and can detect the VP2 gene of FMDV-O and P3 gene of SVV simultaneously without cross-reaction to other viral nucleic acids,including vesicular stomatitis virus(VSV),swine vesicular disease virus(SVDV),classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV),porcine parvovirus(PPV),foot-and-mouth disease virus type-A(FMDV-A)and foot-and-mouth disease virus type-A-1(FMDV-A-1)nucleic acids.The method exhibited broad-spectrum coverage of epidemic strains of foot-and-mouth disease virus type-O.The method demonstrated good sensitivity,and the detection limit for the plasmid standard could reach about 7.17×10^(2)copies/μL in this study.It also exhibited good repeatability with a variation coefficient of Ct values of less than 3%.The method established in this experiment was used to detect 30 clinical samples,and the results were consistent with those of the reference method.In conclusion,this method enables rapid and accurate identification of FMDV-O and SVV,creating technical conditions for efficient quarantine at ports of entry.