猪瘟病毒和猪繁殖与呼吸综合征病毒一步法双重荧光RT-PCR检测方法的建立及应用

Establishment and Application of an One-step Fluorescent RT-PCR Method for Dection of CSFV and PRRSV

为建立猪瘟病毒(CSV)和猪繁殖与呼吸综合征病毒(PRRSV)现场快速鉴别检测方法,参照GenBank中CSV和PRRSV特异性基因序列,设计特异引物和TaqMan探针,通过优化反应条件,建立了检测CSV和PRRSV的一步法荧光RT-PCR方法,并验证了该方法的特异性、敏感性、重复性。结果显示,该方法检测CSV和PRRSV的灵敏度分别可达0.25和1.99 TCID_(50)/100μL,对猪细小病毒(PPV)、猪乙型脑炎病毒(JEV)、猪伪狂犬病毒(PRV)、猪圆环病毒2型(PCV2)的检测结果均为阴性。本试验建立的TaqMan一步法荧光定量RT-PCR检测方法,可对CSV和PRRSV进行快速诊断,适合现场检测。

In order to establish a field rapid indentification method for detection of Classical swine fever virus(CSFV)and Porcine reproductive and respiratory syndrome virus(PRRSV),specific primers and TaqMan probes were designed according to the specific gene sequences of CSFV and PPRSV in GenBank,then an one-step fluorescent RT-PCR method for CSFV and PRRSV detection was established by optimizing the reaction conditions,and the specificity,sensitivity and repeatability of the method were verified.The results showed that the minimum values of CSFV and PPRSV detected by this method were 0.25 and 1.99 TCID50/100μL,respectively,and the detection results of Porcine parvovirus(PPV),Japanese encephalitis virus(JEV),Pseudorabies virus(PRV)and Porcine circovirus type 2(PCV2)were all negative.As a conclusion,the one-step fluorescent RT-PCR method established in this article could be used for rapid diagnosis of CSFV and PRRSV,and it was suitable for field detection.