猪传染性胃肠炎病毒荧光定量RT-PCR检测方法的建立

摘要

【Objective】 The study was to provide technical support for clinic diagnosis,prevention and cure of TGE by establishing RT-PCR method for transmissible gastroenteritis virus.【Method】 One pair of specific primers based on the conservative region of TGEV TH98 N gene sequences was designed,then target region was amplified and cloned into vector to construct the plasmid standards.The PCR assay was developed by optimizing the varity of conditions,and then 30 clinic samples were detected with this method.【Result】 Plasmid standards and the PCR assay of detecting TGEV were developed successfully.The data showed high specificity and good repeatability,and demonstrated that the method was of high repeatability,which was 100 times higher than general PCR,and the linear range was from 1.0×107 to 1.0×101 copies/μL.30 clinic samples were tested by two PCR methods,and the result showed that four samples were positive,and the PCR results were 100% positive coincident with general PCR.【Conclusion】 The PCR assay for detecting TGEV has high sensitivity and specificity as well as short time,which provides technical support for the diagnosis,prevention and cure of TGE.%【目的】建立一种能检测猪传染性胃肠炎病毒（TGEV）含量的荧光定量RT-PCR方法,为猪传染性胃肠炎（TGE）的诊断和防治提供技术支持。【方法】根据TGEV TH98株的N基因序列设计1对特异性引物,扩增出目的片段,连接克隆载体后构建质粒标准品;对荧光定量的循环条件进行优化,建立猪传染性胃肠炎病毒荧光定量RT-PCR检测方法,对其重复性、特异性进行检测,并与普通PCR检测方法进行比较,最后应用该方法对采自陕西杨凌周边的30份临床样品进行检测。【结果】成功构建了质粒标准品,建立了检测猪传染性胃肠炎病毒的荧光定量RT-PCR方法,该方法特异性高、重复性好、敏感性比普通PCR检测方法高2个数量级,对质粒标准品的线性检测范围为1.0×107～1.0×101拷贝/μL。用建立的检测方法对从陕西杨凌周边猪场采集的30份样品进行检测,检出4份阳性,与普通PCR检测方法符合率为100%。【结论】建立的TGEV荧光定量RT-PCR方法敏感性高、特异性好、省时省力,可以对猪传染性胃肠炎病毒进行快速检测。