犬细小病毒的新型原液荧光定量PCR方法的建立

发布时间：2018-12-15 17:11

【摘要】：为建立一种不提取病毒DNA,直接采用样本上清液检测犬细小病毒(CPV)的TaqMan荧光定量PCR方法,通过扩增CPV NS基因部分片段构建重组质粒,建立TaqMan荧光定量PCR方法;对建立的方法进行反应条件、特异性、敏感性的检测;通过建立的方法对核酸荧光定量PCR和原液荧光定量PCR进行对比;最后对临床中疑似的CPV病例进行检测。结果显示,建立的方法特异性好,对犬常见病毒无扩增。该方法检测的灵敏度达101copies/L,该方法的批内变异系数为0.09%~1.30%,批间变异系数为0.57%~0.94%,重复性良好。核酸荧光定量PCR和原液荧光定量PCR的最低检测浓度均可以达到101 copies/L,且原液荧光定量PCR的产物浓度约为核酸荧光定量PCR的7倍,提示核酸在提取过程中有大量的损耗。对187份临床病料进行检测,建立的方法共检出128份阳性,与普通PCR和胶体金快速检测板的阳性符合率均为100%,核酸法与原液法的符合率为100%。因此,本试验建立的CPV病料原液TaqMan荧光定量PCR方法与普通PCR和胶体金快速检测板相比,具有阳性检出率更高、准确率更好的特点。
[Abstract]:In order to establish a TaqMan fluorescence quantitative PCR method for detecting canine parvovirus (CPV) by sample supernatant, the recombinant plasmid was constructed by amplifying part of CPV NS gene, and the TaqMan fluorescence quantitative PCR method was established. The reaction conditions, specificity and sensitivity of the established method were detected. The fluorescence quantitative PCR of nucleic acid was compared with the fluorescence quantitative PCR of the original solution by the established method. Finally, the suspected cases of CPV in clinic were detected. The results showed that the method was specific and could not amplify common viruses in dogs. The sensitivity of this method is 101copias / L, the coefficient of variation within the assay is 0.09 and the coefficient of variation between batches is 0.570.94 and the repeatability is good. The detection concentration of nucleic acid fluorescence quantitative PCR and original fluorescence quantitative PCR can reach 101 copies/L, and the product concentration of fluorescence quantitative PCR in the original solution is about 7 times of that of nucleic acid fluorescence quantitative PCR, which indicates that nucleic acid has a large loss in the extraction process. A total of 128 positive samples were detected from 187 clinical samples. The positive coincidence rate with PCR and colloidal gold rapid detection plate was 100, and the coincidence rate between nucleic acid method and original solution method was 100. Therefore, compared with ordinary PCR and colloidal gold rapid detection plate, the TaqMan quantitative PCR method of CPV raw material has higher positive detection rate and better accuracy than that of ordinary PCR and colloidal gold rapid detection plate.
【作者单位】：成都军区疾病预防控制中心;四川农业大学动物医学院动物疫病与人类健康四川省重点实验室;
【基金】：国家公益性行业(农业)科研重大专项(201303042) 全军医学科技青年培育项目(14QNP057) 成都大熊猫繁育研究基金会项目(CPF2015-4)
【分类号】：S852.655

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