牛呼吸道病毒四重实时荧光定量PCR检测方法的建立与应用OA

Bovine alphaherpesvirus type 1(BoHV-1),bovine parainfluenza virus type 3(BPIV3),bovine adenovirus type 3(BAdV3),and bovine respiratory syncytial virus(BRSV)are the primary pathogens causing bovine respiratory disease complex(BRDC).This study aimed to develop a quadruplex real-time fluorescent quantitative polymerase chain reaction(qPCR)method capable of simultaneously detecting and quantifying these four pathogens to meet the demand for rapid clinical diagnosis of BRDC.Specific primers and probes were designed based on gene sequences from GenBank.Reaction conditions were optimized,standard curves were established,and the method was evaluated for sensitivity,specificity,repeatability,and accuracy.The method was further validated using clinical samples and compared with conventional PCR.The results showed that the amplification efficiency for the target pathogens ranged from 93%to 103%,correlation coefficients were all 1.00,and slopes ranged from-3.497 4 to-3.256 7,demonstrating suitability for multiplex quantification.The limits of detection for BoHV-1,BPIV3,BAdV3,and BRSV were 10¹,10²,10¹,and 10² copies/μL,respectively,with no cross-reaction with other pathogens,indicating high sensitivity and specificity.The coefficient of variation(CV)of quantification cycle(Cq)values ranged from 0.14%to 1.41%,indicating good inter-assay repeatability.The CVs for samples containing single or mixed pathogens ranged from 1.03%to 2.75%,showing good accuracy.Application of the method to 217 clinical samples detected 7 BoHV-1-positive,1 BPIV3-positive,12 BAdV3-positive,and 0 BRSV-positive samples,with higher positive detection rates than conventional PCR.In conclusion,a highly sensitive,specific,repeatable,and accurate quadruplex qPCR method was successfully established,suitable for rapid clinical differentiation and diagnosis of BoHV-1,BPIV3,BAdV3,and BRSV.