马鼻肺炎双重荧光定量PCR检测方法的建立与应用

Establishment and Application of Duplex Quantitative Real-Time PCR Method for Detection of Equine Rhinopneumonitis

【目的】建立快速、准确检测马疱疹病毒1型(EHV1)和4型(EHV4)的双重荧光定量PCR检测方法。【方法】以EHV1、EHV4的全基因组序列为基础,针对糖蛋白B(gB)基因的保守序列,分别设计EHV1与EHV4特异性引物和探针,筛选引物,优化反应条件,分型检测马疱疹病毒(EHV1、EHV4)。【结果】检测到EHV1、EHV4、EIV、EAV、EVJ和EIAV标准毒株,EHV1、EHV4为阳性结果,其余病毒为阴性结果;EHV1、EHV4DNA模板的最低检出限为1.47×10^2copies/μL;检测64份临床样本,阳性检出率为14.06%,病毒分离证实100%符合。【结论】双重荧光定量PCR检测方法可直接应用于检测并区分EHV1、EHV4,并可在短时间内获得对EHV1/EHV4特异性及敏感性的检测结果。

【Objective】The study was to establish of a duplex quantitative real-time PCR method for rapid and accurate detection of equine herpesvirus 1(EHV1)and equine herpesvirus 4(EHV4).【Method】Based on the complete genome sequence of EHV1 and EHV4,according to the conserved sequences of glycoprotein B(gB)gene in GenBank,a series of specific primers and probes were designed.By selecting suitable primers,optimizing reaction conditions,two kinds of equine herpesviruses(EHV1,EHV4)were detected.【Result】The standard strains such as EHV1,EHV4,EIV,EAV,EVJ and EIAV were detected,among which EHV1 and EHV4 was positive and other stains were negative.The minimum detection limit was 1.47×10^2copies/μL.The positive detection rate in 64 clinical samples was 14.06%,which was 100%consistent with virus isolation.【Conclusion】The duplex quantitative real-time PCR detection method can be directly applied to detect and distinguish EHV1 and EHV4,and the test results of specificity and sensitivity of EHV1/EHV4 can be obtained in a short time.