SYBR GreenⅠ荧光定量RT-PCR检测犬瘟热病毒方法的建立及应用

Development of a SYBR GreenⅠ Real-time RT-PCR assay for detection of canine distemper virus

根据GenBank发表的犬瘟热病毒(CDV)核衣壳蛋白(NP)基因序列,设计合成一对引物,建立了基于SYBR GreenⅠReal-time RT-PCR检测CDV核酸。以含有83bp扩增产物的pGM-T载体质粒为参考,构建了标准曲线。对该方法的特异性、敏感性、可重复性进行了评价,与常规RT-PCR及胶体金免疫检测技术(GIA)进行了敏感性比较。结果表明,该方法能特异性检测到CDV的扩增信号;检测范围在5.2×102～5.2×108拷贝之间有良好的线性关系,相关系数为0.999,扩增效率为96.80%;敏感度高,最低检测限为52拷贝,比常规RT-PCR高103倍,比GIA高105倍;组内变异系数为0.51%～1.90%,组间变异系数为1.96%～2.63%。用建立的Real-time RT-PCR检测11例临床病例,CDV阳性率为100%。对犬瘟热弱毒活疫苗中的CDV进行定量,其含量在1.24×105～4.88×105拷贝/头份之间。该方法的建立为CDV的早期快速诊断、分子流行病学调查及疫苗质量监测等提供了一种新的快速定量检测手段。

A SYBR Green Ⅰ based Real-time RT-PCR assay was developed for detection of canine distemper virus （CDV） using a pair of primers derived from the nucleocapsid protein （NP） gene sequence. The specificity, sensitivity and reproducibility of the assay were evaluated and compared with conventional RT-PCR and Colloidal gold immunoassay （GIA）. The result showed that the Real-time RT-PCR assay was highly specific and had a broad linear detecting range （5.2 × 10^2-5.2 × 10^7 copies, R=0.999） with 96.8 % PCR amplification efficiency. It had a detection threshold of 52 copies of plasmid DNA and was 103 and l05 folds more sensitive than the conventional RT-PCR and GIA, respectively. The assay was highly reproducible and had an intra-assay and inter assay coefficient of variations （CVs） of 0.51%-1.90 % and 1.96 %-2.63 %, respectively. Test on positive clinical samples showed 100 % consistency. The real time PCR assay was used to quantify CDV in the multivalent live attenuated vaccines and showed that those vaccines contained 1.24 × 10^5-4.88 × 10^5 CDV viruses per dose. The assay provided an effective means for rapid early diagnosis, vaccine quality control and molecular epidemiological investigation of CDV.