中国兽医杂志2026，Vol.62Issue(6)：45-53,9.DOI:10.20157/j.cnki.zgsyzz.2026.06.006
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
Development and Application of a Quadruplex Real-Time Fluorescent Quantitative PCR Assay for Bovine Respiratory Viruses
摘要
Abstract
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.关键词
牛疱疹病毒1型(BoHV-1)/牛副流感病毒3型(BPIV3)/牛腺病毒3型(BAdV3)/牛呼吸道合胞体病毒(BRSV)/四重实时荧光定量聚合酶链式反应(qPCR)/牛呼吸道疾病综合征(BRDC)分类
农业科技引用本文复制引用
王朋朋,焦晓宇,马文阁,牛康,于越洋,宋佰芬,彭辰,吴文学..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[J].中国兽医杂志,2026,62(6):45-53,9.基金项目
国家重点研发计划(2022YFD1800700) （2022YFD1800700）
国家现代农业产业技术体系(CARS36) （CARS36）
