Establishment and Preliminary Application of Droplet Digital PCR Method for Detection of Bovine viral diarrhea virus
Bovine viral diarrhea virus(BVDV)is one of the main pathogens causing viral diarrhea of calves(Bos sp.),which leads to decreased immunity and increase of morbidity and mortality.At present,the commonly used detection methods for BVDV have low sensitivity and are prone to false positives,This research is based on droplet digital PCR(ddPCR)technology to establish the accurate detection technology of BVDV.Firstly,primers and probes were designed for the BVDV 5'UTR gene conserved sequence region,and the BVDV 5'UTR gene was synthesized and the plasmid standard was constructed.Real-time quantitative PCR(qPCR)and ddPCR methods were established using BVDV plasmid standards,and the reaction parameters and conditions were optimized to analyze their sensitivity,specificity and repeatability,and the clinical application was carried out.The results showed that the optimal primers and probe concentrations were 300 and 500 nmol/L,respectively.The standard curve showed a good linear relationship,and the minimum detection limit was 1 × 104 copies/μL,with good specificity and repeatability.The minimum detection limit of ddPCR was 0.64 copies/μL,and the sensitivity of ddPCR was 15 625 times higher than that of qPCR.The method had good specificity and repeatability.The detection rate of ddPCR and qPCR was 15.58％and 14.63％respectively in 82 samples.The ddPCR method established in this study was more sensitive,efficient,specific and reproducible than the qPCR method,which provides technical support for the differential diagnosis of BVDV.
Bovine viral diarrhea virus(BVDV)Droplet digital PCR(ddPCR)Real-time quantitative PCR(qPCR)SensitivitySpecificityRepeatability
国家科技期刊平台
NSTL
万方数据
