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

【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 1.47×102copies/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.