猪嵴病毒荧光定量RT-PCR检测方法的建立和应用

Establishment and application of the real-time reverse transcription polymerase chain reaction for detection of porcine kobuvirus

根据猪嵴病毒3D基因保守序列设计引物和TaqMan探计．通过优化反应体系．建立了猪嵴病毒荧光定量RT—PCR检测方法，并对该方法进行特异性、敏感性和重复性评价；同时以10倍梯度稀释的质粒标准品建立荧光定量RT—PCR的标准曲线，构建定量分析模型。结果显示．建立的方法能特异性检测猪嵴病毒，与其他引起猪腹泻的病毒无交叉反应，对标准品的检测灵敏度达10copies／pL；标准曲线CI值和模板终浓度在107～10copies／μL范围内有良好的线性关系；重复性试验变异系数均小于2．5％。对139份猪粪便样品进行检测，荧光定量RT-PCR阳性检出率为30．94％（43／139），常规RT—PCR为29．50％（41／139）。本研究建立的荧光定量RT-PCR可用于猪嵴病毒的快速检测和流行病学调查。

To develop a rapid real time PCR assay for detection of porcine kobuvirus,primer and probe were designed based on the conservative region of 3D of porcine kobuvirus. Specificity,sensitiv- ity and repeatability of the method were evaluated. The standard curve and nucleic acid quantitative analysis were constructed using the standard plasmid in series dilution. The result showed this method had no cross reaction with other diarrhea virus（porcine epidemic diarrhea virus、transmissible gastro- enteritis virus、porcine astrovirus、group A porcine rotavirus、porcine bocavirus、porcine torovirus、 porcine pseudorabies virus）. The lowest detection limit was 10 copies/μL. The Ct value of the standard curve had a linear relationship to the final concentration of template in the range of 10^7-10 copies/μL. The coefficient of variation were less than 2.5%.Tested on the 139 fecal samples from porcine,the positive rate of the real-time PCR assay was 30.94%（43/139）,the conventional RT-PCRwas 29.50% （41/139）.In conclusion,the real-time RT-PCR could be used for the detection and the epidemiological investigation of porcine kobuvirus.