H3亚型犬流感病毒荧光定量RT-PCR检测方法的建立

Establishment of fluorescence quantitative RT-PCR for detection of H3 subtype canine influenza virus

为建立H3亚型犬流感病毒(CIV)荧光定量PCR检测方法,本研究根据H3亚型CIV HA基因的保守区序列,设计合成一对特异性引物和Taq Man MGB探针,经条件优化后初步建立了H3亚型CIV荧光定量RT-PCR检测方法。结果显示,构建的重组质粒标准品在109拷贝/μL~10^(2)拷贝/μL浓度范围内有良好的线性关系,标准曲线的相关系数为0.999。该方法仅能特异性扩增H3N2亚型CIV核酸,与H3N2亚型禽流感病毒、人流感病毒和猪流感病毒及H9N2亚型CIV、H1N1亚型猪流感病毒、犬腺病毒、犬瘟热病毒、犬细小病毒、犬冠状病毒等病毒核酸均无交叉反应。该方法最低可检测到的质粒标准品浓度为19拷贝/μL,敏感性高于普通PCR方法。组内与组间重复性试验的变异系数均小于1%。利用该方法对83份临床样品的核酸进行检测,检测结果与病毒分离结果总符合率为97.59%。本研究建立的荧光定量PCR检测方法特异性强、敏感性高、重复性好、操作简便,为临床H3亚型CIV的快速检测提供可靠方法。

In order to establish a fluorescence quantitative PCR method for detection of canine influenza H3 viruses,a pair of specific primers and Taq Man MGB probe were designed and synthesized according to the conserved region of HA genes of canine influenza H3 viruses.After optimization of the conditions,a fluorescent quantitative RT-PCR method for detecting the canine influenza H3 viruses was established.The results showed that the constructed recombinant plasmid standard had a good linear relationship in the concentration range of 109copies/μL-10^(2)copies/μL,and the correlation coefficient of the standard curve was0.999.The results showed that only the nucleic acid of canine influenza H3N2 virus could be amplified by using this method,but not any of H3N2 viruses from avian,human or swine origins,H9N2 canine influenza virus,H1N1 swine influenza virus,canine adenovirus,canine distemper virus,canine parvovirus,canine coronavirus or other viruses.The minimum detectable concentration of a plamid in this method was 19 copies/μL,which was more sensitive than ordinary PCR methods.The variant coefficient of intra-assay and inter-assay in repeatability tests were less than 1%.Nucleic acid detection of 83 clinical samples were compared by using this method and virus isolation,and the coincidence rate between the two methods was 97.59%.The established assay in this study has high specificity,sensitivity,repeatability and operability,which could provide a reliable method for the rapid detection of clinical canine influenza H3 viruses.