摘要
本研究建立了检测牛副流感病毒3型的SYBR Green Ⅰ Q-PCR方法。根据Gen Bank发表的BPIV3序列进行对比分析,选取P基因上保守区域设计特异性引物。优化反应体系,建立Q-PCR检测方法,并对该方法的特异性、敏感性和重复性进行验证。结果显示,构建的标准曲线在10~4~10~8copies.μL^(-1)内具有较好的线性关系,相关系数达到0.998,斜率为-3.370,Q-PCR扩增效率为98%。特异性试验中,利用该方法对BPIV3a及BPIV3c进行检测为阳性,对牛疱疹病毒I型、牛病毒性腹泻病毒进行检测,结果呈阴性。敏感性试验中,该方法对标准品的最小检出量为1.0×10~3 copies.μL^(-1)。重复性试验中,Ct值变异系数均小于1.0%。表明SYBR Green Ⅰ Q-PCR法能够快速地对病原进行诊断,该方法具有较强的特异性、良好的敏感性及重复性,为实验室诊断及定量分析提供了更快速、稳定、可靠的方法。
To establish a SYBR Green I Q-PCR assay for the detection of bovine parainfluenza virus type 3. Based on the sequences of BPIV3 published in GenBank, the numerous sequences of complete genome were analyzed. A pair of specific primers was designed, which specifically targeted P gene of BPIV3. The assay was established and its specificity, sensitivity and reproducibility were assessed upon the optimization of reaction condition of Q-PCR assay. Results showed that the standard curve based on 10^4-10^8 copies. μ L^-1 of BPIV3-P positive plasmid represented a sharp linearity by Q-PCR assay in this study, which the correlation coefficient of standard curve was 0.998 and the slope value of standard curve was -3.370. The efficiency of Q-PCR was 98%. In the specificity, the assay showed highly specificity for amplification BPIV3a and BPIV3c, instead of bovine viral diarrhea virus, and infectious bovine rhinotraeheitis. In the sensitivity test, the minimum detectable count of Q-PCR was 1.0X 103 copies. μL^-1. In the reproducibility test, the Ct value variation coefficient was less than 1.0%. The Q-PCR assay established in this study enabled to detect BPIV3a and BPIV3c and demonstrated high specificity, sensitivity and repeatability, which offered a rapid, stable and reliable method for early diagnosis and quantitative analysis.
出处
《现代畜牧兽医》
2017年第5期8-14,共7页
Modern Journal of Animal Husbandry and Veterinary Medicine
基金
黑龙江省农垦总局攻关课题(HNK125B-11-08A)
黑龙江八一农垦大学研究生创新科研项目(YJSCX2016-Y27)
