牛病毒性腹泻病毒Real-timePCR方法的建立及其应用

本研究在牛病毒性腹泻病毒5'UTR基因的保守区设计并合成了1对引物,建立了可以定量检测牛病毒性腹泻病毒核酸的SYBR GreenⅠ荧光定量PCR方法。该方法建立的标准曲线相关系数R2为0.999,扩增效率为95.9%,熔解曲线为单一峰值,可检测到初始模板中4.1×101copies/μL的牛病毒性腹泻病毒核酸,是常规RT-PCR方法敏感性的100倍。该检测方法与猪瘟病毒、传染性鼻气管炎病毒、牛呼吸道合胞体病毒、牛副流感病毒3型病毒等其它牛源病毒均不发生交叉反应;批内与批间重复性试验变异系数均小于2.2%。本试验建立的荧光定量PCR检测方法可用于牛病毒性腹泻病毒的临床诊断。同时,应用本方法对临床病例的各组织器官进行了病毒RNA定量检测结果表明,胸腺等淋巴组织的病毒含量最高,证实病毒主要侵害淋巴器官,此研究可为临床病例的病毒分离提供借鉴。

Development of a Real-time PCR Assay for Detection of Bovine Viral Diarrhea Virus

In this study,a SYBR Green I Real- time PCR assay for detecting bovine viral diarrhea vims（BVDV） was established and tested the tissues from clinical diarrhea catfles. A pair of primers was designed according to the conserved region of the BVDV 5′UTR gene and a plasmid containing the target gene was constructed as a standard control. The assay had a detection limit of 4.1 × 10^1 copies/μL viral RNA, which was 100 times more sensitive than the conventional PCR. It was highly specific and no cross - reactions were found with CSFV and other bovine pathogens including IBRV, BRSV, BPDV. This assay was used to detect the BVDV distribution in the tissue samples of the cattle infected BVDV. The results showed that the thymus had the highest virus load. The high sensitivity and specificity of the assay, coupled with a relatively rapid and simple procedure,indicated that the Real- time PCR could be used as a routine assay.