牛病毒性腹泻病毒SYBR Green I实时定量RT-PCR检测方法的建立及应用

Establishment and Application of SYBR Green I RealI Real-Time Quantitative RT-PCR Assay Ffor Thethe Detection Of of Bovine Viral Diarrhea Virus

[目的]在建立一种快速定量的用于检测牛病毒性腹泻病毒（BVDV）的实时荧光定量RT-PCR检测方法.[方法]根据GenBank公布的BVDV 5′端非编码区（5′ UTR）核苷酸序列,软件分析后设计特异性扩增引物,建立SYBR GreenⅠ实时荧光定量PCR方法.[结果]建立的方法只能检测到BVDV,而与猪瘟病毒、牛传染性鼻气管炎病毒、牛轮状病毒及牛冠状病毒没有交叉反应,具有高度的特异性;在102～108copies/μL模板范围内具有良好的线性关系,所制作的标准曲线相关系数为0.998,最低可检测下限可达到102 copies/μL,具有灵敏度高的特点;不同情况下3次重复实验,变异系数均小于1.5％,具有重复性好的特点.[结果]成功建立了一种用于检测BVDV的实时荧光定量RT-PCR技术,并可用于临床样品的检测,适用于BVDV的快速诊断和流行病学调查.

[Objective] The research was aimeds to develop a SYBR Green Ⅰ real-time quantitative RT -PCR assay for rapid quantitative detection of bovine viral diarrhea virus （BVDV）.[Method] According to the nucleotide sequence of BVDV 5＇-untranslated region （5＇-UTR） available in GenBank,the specific primers were designed to perform in the real-time fluorescent quantitative RT-PCR assay.[Result]The results showed that the specificity of this assay was high without any cross-reaction with classical swine fever virus,bovine rhinotracheitis virus,bovine rotavirus and bovine coronavirus.A good linear correlation was demonstrated in the standard curve for the real-time PCR assay within the range of 102 ～ 108 copies/μL,with a correlation coefficient of O.998.The detection limit of the real-time PCR assay was 102 copies of plasmids,indicating a good sensitivity.And the intra-assay and the inter-assay coefficient of variation values were maintained at less than 1.5％.[Conclusion] The established SYBR Green Ⅰ real-time quantitative RTPCR can be used for rapid detection,diagnosis and epidemiological investigation for of BVDV.