犬细环病毒SYBR GreenⅠ荧光定量PCR检测方法的建立  

Development of a SYBR GreenⅠreal-time PCR method for detection of Torque tenocanis virus

作　　者：解长占 孙文超[2,3] 张萍[1,2] 崔卓栋 赵冠宇[2,4] 曹亮[1,2] 南福龙 张金勇[1,2] 马海彬[2] 韩继承[2] 柴丹 鲁会军[2] 金宁一[2] 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130118 [2]军事医学科学院军事兽医研究所,吉林长春130122 [3]广西大学动物科技学院,广西南宁530004 [4]吉林大学动物科技学院,吉林长春130062

出　　处：《中国预防兽医学报》2017年第6期471-474,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家高技术研究与中国发展计划(863计划)(2011AA10A208)

摘　　要：为建立犬细环病毒(TTCV)的快速检测方法,本研究根据GenBank中登录的TTCV ORF7基因设计合成一对特异性引物,并对反应条件和反应体系进行优化,建立了检测TTCV的SYBR Green I荧光定量PCR方法。结果显示:建立的荧光定量PCR方法 Ct值与标准品模板在5.82×10~9拷贝/μL^5.82×10~3拷贝/μL范围内呈良好的线性关系。该检测方法仅对TTCV检测为阳性,而对狂犬病病毒、犬冠状病毒、犬细小病毒、犬瘟热病毒和犬副流感病毒均无特异扩增;该检测方法灵敏度可达5.82×103拷贝/μL。对27份犬血清样品检测结果显示,建立的荧光定量PCR阳性检测率为11.11%,而普通PCR方法检测阳性率为3.7%。本实验建立了TTCV SYBR Green Ⅰ荧光定量PCR检测方法,对TTCV诊断及致病机制的研究提供了高通量的定量检测方法。In order to rapidly detect Torque tenocanis virus （TTCV）, the SYBR Green I real-time PCR method was proposed to detect TTCV with a pair of specific primers were synthesized according to the TTCV ORF7 gene, and the reaction conditions and reaction system were optimized. The results showed that the Ct value of the proposed assay linearly related to the standard template in the range of 5.82×109 copies/μL to 5.82×103 copies/μL. This assay was only positive for TTCV detection, while showed no specific amplification for RABV, CCV, CPV, CDV, RV; the sensitivity of the assay was 5.82×103 copies/μL. The detection for 27 canine serum samples showed that the positive rate of the proposed real-time PCR method was 11.11%. While, the positive rate of conventional PCR was 3.7%. In this study, the developed TTCV SYBR Green I real-time PCR could provide a high-throughput quantitative detection for the research of diagnosis and pathogenesis of TTCV.

关 键 词：犬细环病毒 SYBR Green Ⅰ 荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]