猪繁殖与呼吸综合征病毒一步多重RT-PCR检测方法的建立及应用

摘要

A multiplex reverse transcription polymerase chain reaction (multiplex RT-PCR) was developed for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Three pairs of primer selected from four pairs were designed based on the sequence of high conservative region of PRRSV. And PCR product was 228, 345 and 490 bp. Diagnostic accuracy of the multiplex RT-PCR shown to be 10-fold more sensitive than the conventional single RT-PCR (conventional RT-PCR) method, and the testing time is shorten greatly. Simultaneously, the specificity and sensitivity of this method were evaluated. The result indicated that this assay could reliably differentiate between PRRSV and other swine viral disease, such as classical swine fever virus (CSFV), porcine rabies virus (PRV). The developed multiplex RT-PCR in this study may provide a new avenue to the rapid detection of this important pathogen in one reaction, which possess the good character in specificity, sensibility,simple and convenient.%本研究旨在建立猪繁殖与呼吸综合征病毒(PRRSV)的一步多重RT-PCR检测方法.通过多序列比对以及反应条件的优化,从4对引物中精选了3对特异性和敏感性较好、扩增片段分别为228、345和490 bp的引物,并应用所建立的方法对4种分离株的细胞毒和组织毒、田间样品及临床症状相似的疾病猪瘟和猪伪狂犬病毒做了检测；并对田间及实验室样品进行符合率试验,对阳性样品进行重复性试验.与常规RT-PCR法进行比较,证明该方法比常规RT-PCR法敏感性提高了10倍；而且利用该方法可使检测时间大大缩短；特异性试验证实该方法无交叉反应；阳性和阴性样本的符合率均为100％；3次阳性样品重复性检测结果完全相同；稳定性试验显示该试剂盒至少可保存1年以上.本研究所建立的PRRSV一步多重RT-PCR检测方法的敏感性高、特异性强、操作简便、省时、结果重复性好,不仅适用于临床病例尤其低微含量样品的诊断,而且在筛查隐形带毒动物及兽医检疫方面具有重要的应用价值.