多重PCR技术同时检测牛传染性鼻气管炎和赤羽病方法的建立和应用

Establishment and Primary Application of Multiplex Polymerase Chain Reaction for Detection of Infectious Bovine Rhinotracheitis Virus and Akabane Virus

为满足同时对IBRV和AKAV进行快速诊断的需要,根据牛疱疹病毒1型(BHV1)gB基因序列和赤羽病病毒(AKAV)的S基因序列,设计了2对针对这2种病毒的特异引物,并建立了多重PCR方法,分别对其最佳反应条件、特异性及敏感性进行了测定,结果表明:该方法能同时扩增得到2条与试验设计相符的311 bp(IBRV)和392 bp(AKAV)特异性条带;IBRV的灵敏度为0.13 pg/25μL,AKAV的灵敏度为1.04 pg/25μL。本试验方法的建立对于加强进出口牛IBRV和AKAV的检验检疫具有十分重要的意义。

In order to detect both IBRV and AKAV rapidly, a multiplex polymerase chain reaction（M- PCR） was established and optimized to detect bovine herpesvirus 1 （BHV-1） and akabane virus （AKAV） simultaneously. Two sets of specific primers were designed according to the conservative gB sequence of ]BRV and the S sequence of AKAV in the GenBank. It was showed that all samples which contained IBRV or AKAV could be amplified by the multiplex PCR using these two sets primers. Two specific bands of IBRV 311 bp and AKAV 392 bp were detected using agarose gel electrophoresis in this multiplex PCR and accorded with designed result. The detection limit of the M-PCR assay was 0.13 pg/25μL for IBRV and 1.04 pg/25μL for AKAV, respectively The method could prove very useful for laboratories working with early rapid identification of IBRV or AKAV.