Abstract: The purpose of this study was to establish a highly sensitive 3D digital PCR (3D-dPCR) method for the detection of equine herpesvirus 1 (EHV-1),which could accurately and quantitatively detect the samples with low EHV-1 content and realize the early diagnosis and prevention of equine rhinopneumonia.According to the conserved region of EHV-1 glycoprotein B gene,we designed specific primers and probes,optimized the concentration and annealing temperature of primers in the 3D-dPCR reaction system,analyzed the sensitivity,specificity and repeatability of this method,and established the 3D-dPCR method of EHV-1.In this study,the best concentration of primer and probe of 3D-dPCR was 0.4 and 0.4 μmol/L respectively,the best annealing temperature was 60 ℃,R² of the absolute quantitative curve of the method was 0.998,the linear relationship was good,the sensitivity was about 10 times higher than that of Real-time PCR,and the minimum detection limit was 5.83 copies/μL.There was no cross reaction with EHV-4,Theileria equi and the nucleic acid of equine arteritis.The results showed that the positive rate of 3D-dPCR was 66.7%,which was higher than that of Real-time PCR for EHV-1 in OIE (64.2%).The results of 3D-dPCR were consistent with those of Real-time PCR,and the sensitivity of 3D-dPCR to the samples with low virus content was higher,which could effectively detect suspicious samples.The results showed that the established 3D-dPCR method was more sensitive,specific and reproducible for the detection of clinical samples with low copy number,and could be used for the accurate and quantitative detection of EHV-1.

Key words: equine herpesvirus type 1 (EHV-1); 3D digital PCR (3D-dPCR); absolute quantification