基于gB基因的IBRV微滴式数字PCR检测方法的建立及初步应用

Development and application of a droplet digital PCR assay for detection of infectious bovine rhinotracheitis virus based on gB gene

为实现牛传染性鼻气管炎病毒(infectious bovine rhinotracheitis virus,IBRV)精准的定量检测,以IBRV gB基因为靶基因,建立IBRV微滴式数字PCR(droplet digital PCR,ddPCR)定量检测方法,进行了ddPCR退火温度、引物和探针浓度的优化、特异性和灵敏性试验。结果显示,建立的ddPCR方法最佳退火温度为56.5℃;反应体系中最佳引物、探针浓度分别为900和250 nmol/L;能够实现IBRV的特异性检测,与其他常见的牛呼吸系统疫病病原无交叉反应;以重组质粒pMD19-T-gB为模板,ddPCR方法的检出限为1.8 copies/μL,是实时荧光PCR方法敏感性的10倍。对119份具有呼吸道症状的牛鼻拭子样本分别进行ddPCR方法和实时荧光PCR方法进行检测,结果显示,ddPCR方法和实时荧光PCR方法的检出率分别为(15.13%,18/119)和(12.60%,15/119)。本研究建立的ddPCR方法特异性强、灵敏度高,可作为牛呼吸道临床样品中IBRV精准定量检测的有效手段。

In order to make the accurate quantitative analysis of the infectious bovine rhinotracheitis virus(IBRV),a droplet digital PCR(ddPCR)assay was developed based on gB gene in this study,and optimization of ddPCR annealing temperature,primer and probe concentrations,and specificity and sensitivity tests were performed.The results showed that the optimal annealing temperature was 56.5℃,and the optimal primers and probe concentration in the reaction system was 900 nmol/L and 250 nmol/L.The developed ddPCR assay could achieve the specific detection of IBRV,and there were no cross-reactions with other common viruses causing bovine respiratory diseases.Using recombinant plasmid PMD19-T-gB as template,the detection limit of ddPCR was 1.8 copies/μL,which was 10 times more sensitive than real-time PCR.A total of 119 bovine nasal swab samples with respiratory symptoms were detected by ddPCR and real-time PCR.The detection rates of ddPCR and real-time PCR respectively were 15.13%(18/119)and 12.60%(15/119).The ddPCR assay developed in this study was demonstrated to have high specificity and good sensitivity,which could be used as an effective method for accurate quantitative detection of IBRV in clinical bovine respiratory samples.