猪流行性腹泻病毒SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立  

Development of SYBR Green Ⅰ based real-time PCR assay for detection of porcine epidemic diarrhea virus

作　　者：曹贝贝[1,2] 韩丽[1,2] 于朋飞[1,2] 兰培英[1,2] 程慧芳[1,2] 宋月[1,2] 胡慧[2] 

机构地区：[1]河南省动物性食品安全重点实验室,河南郑州450002 [2]河南农业大学牧医工程学院,河南郑州450002

出　　处：《中国预防兽医学报》2015年第6期461-464,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(30500165)

摘　　要：为建立一种快捷、特异、敏感检测猪流行性腹泻病毒(PEDV)的方法,本研究根据Gen Bank中登录的PEDV ZKHFQ株的N基因序列的保守区设计一对特异性引物,经过对反应条件进行优化,建立了检测PEDV的SYBR Green I荧光定量RT-PCR方法。该方法对常见的病毒均未检测到荧光信号,而仅对PEDV检测为阳性,其灵敏度为57拷贝/μL;组内和组间变异系数均小于1%。利用该方法对26份疑似PEDV感染的病料样品进行检测,结果显示本研究所建立的方法对PED的检出率比常规PCR高23.07%。本实验建立的荧光定量RT-PCR方法具有特异、敏感、快速、定量、重复性好等优点,可以用于临床PEDV的检测。To establish a sensitive assay for porcine epidemic diarrhea virus （PEDV） detection, a SYBR Green I based real-time RT-PCR was developed with a pair of primers designed according to the conserved regions of the N gene sequences of PEDV ZKHFQ strain. By optimization of reacting conditions, the assay was specific to detect PEDV, but had no cross-reaction to other swine viruses. In addition, the sensitivity of the assay was 57 copies/μL which was 10 time higher than that of the traditional RT-PCR detection. The coefficient of variations in intra- and inter-assay were both less than 1%. These results indicated the established real-time PCR had the potential to be used for the detection of clinical PEDV infection.

关 键 词：猪流行性腹泻病毒 SYBR Green I 荧光定量RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]