摘要
根据犬瘟热病毒核蛋白基因的保守序列分别设计一对引物及其相应的Taqman探针建立荧光定量PCR。构建FQ-PCR绝对定量的标准品,并对反应体系进行优化,建立绝对定量的标准曲线。利用十倍稀释法检验方法的灵敏度并与普通RT-PCR法进行比较;特异性检验后进行临床标本的检验。结果显示:实验成功构建了绝对定量的参照质粒,标准曲线相关系数为0.994。犬瘟热病毒FQ-PCR检测反应的灵敏度为10TCID50;5种非犬瘟热病毒病原体检测均为阴性;说明实验建立的FQ-PCR具有快速、灵敏和稳定的特点,适用于临床样品的检验。
According to conservative sequence of canine distemper virus nuclear proteins gene, a pair primers and TaqMan probe were designed, respectively. The standard recombinant plasmids for N gene were constructed as reference standard used in absolute quantification assay. The reaction conditions of FQ-PCR were optimized. The sensitivity of assay was tested with ten fold serial dilution of CDV and compared with ordinary RT-PCR. The quantitative curve was established using reference standard plasmids. The clinic samples were detected after specificity assay. The results demonstrated that the plasmid for FQPCR was favorable. The regression coefficient of the quantitative curve was 0.994. The detection sensitivity of FQ-PCR was 10 TCID50 and the specificity was determined by testing five other specimens, all of which yielded negative results. The detection system based on real-time RT-PCR was rapid, sensitive and steady, which could be used to detect the clinic samples.
出处
《中国农学通报》
CSCD
2007年第11期33-37,共5页
Chinese Agricultural Science Bulletin
基金
中国人民解放军总后勤部科技攻关项目(06G138)资助
