牛病毒性腹泻病毒内标双重RT-PCR检测方法的建立及初步应用  

Establishment and Application of Duplex RT-PCR for Detection of Bovine Viral Diarrhea Virus with Internal Control

作　　者：季新成[1] 段琛[2] 王克栋[3] 史茜[1] 于学辉[1] 冉多良[3] 

机构地区：[1]新疆出入境检验检疫局,新疆乌鲁木齐830063 [2]国际关系学院,北京100091 [3]新疆农业大学动物医学院,新疆乌鲁木齐832052

出　　处：《动物医学进展》2014年第1期17-21,共5页Progress In Veterinary Medicine

基　　金：新疆自治区高新技术项目(201010101;201141147);国家质量监督检验检疫总局科研项目(2011IK017)

摘　　要：为了对BVDV-1和BVDV-2进行同步检测,针对不同基因型牛病毒性腹泻病毒(BVDV)基因组高度保守的5′-UTR核苷酸序列设计特异性引物(扩增长度为288bp)。为了指示假阴性结果,以牛GAPDH基因为内标基因,设计特异性引物(扩增序列长度为152bp)。通过反应条件的优化,建立了BVDV通用型内标双重RT-PCR检测方法,该方法可以监测RNA提取与RT-PCR反应过程,指示假阴性结果的发生。该方法的检测灵敏度约为100TCID50/mL,且具有较好的特异性和可重复性。通过对566份临床样品检验,检测到阳性样品19份,阳性率3.36%。该方法可用于临床样品中BVDV检测,并可对检测结果进行质量控制。To simultaneously detect gene of BVDV-1 and BVDV-2, we designed specific primers to amplify a 288 bp highly conserved sequence of 5r-UTR. Furthermore, we obtained a 152 bp sequence of bovine GAPDH as an internal control with specific primers to supervise false-nagative results. After optimization of reaction conditions, we established a universal and duplex PCR method for detection of BVDV. The method can eliminate false negative results. This duplex PCR sensitivity of detection is 100 TCID50/mL and has a high specificity and repeatability. We used this method to detect 566 clinical samples and the pos- itive rate is 3.36% （19/566）. The results showed that this duplex PCR can be applied to detect BVDV from clinical samples and monitor the test results with internal control.

关 键 词：牛病毒性腹泻病毒 内标 双重反转录-聚合酶链反应 共扩增 

分 类 号：S852.659.5[农业科学—基础兽医学] Q789[农业科学—兽医学]