马鼻肺炎病毒双重PCR分型检测方法的建立

Establishment of a duplex PCR assay for typing and detection of equine herpesviruses type 1 and 4

根据GenBank中登录的马鼻肺炎病毒1、4型(EHV1、EHV4)糖蛋白B(gB)基因特异保守序列(登录号分别为AY665713、AF030027.1),各设计1对引物,建立了同时分型检测EHV1、EHV4的双重PCR方法;确定其最佳工作条件,并对该方法进行了特异性和敏感性试验。结果显示,在EHV1、EHV4中分别扩增出265bp和537bp的特异性目的条带,而马流感病毒cDNA、马动脉炎病毒cDNA、马乙型脑炎病毒cDNA及马传染性贫血弱毒疫苗株cDNA均未扩增出任何条带。EHV1的DNA最低检出限为2.1pg,EHV4的最低检出限为15pg。表明本试验建立的方法具备很高的应用价值。

According to the conserved sequences of glycoprotein B（gB） gene of equine herpesviruses（EHVs） type 1 and 4 in GenBank,a set of primers was designed to establish a duplex PCR method for detecting the two kinds of viruses.And the optimal conditions,specificity and sensitivity test were determined.In result,the PCR products for EHV1 and EHV4 were 265 bp（EHV1） and 537 bp（EHV4） with detection limit of 2.1 pg and 15 pg of DNA copies,respectively,while no product was found from closely realted virus,including equine arteritis virus,Japanese encephalitis virus and inactivated vaccine strain of equine infectious anemia,which indicated that the duplex PCR had potential application value.