猪捷申病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒的多重RT-PCR检测方法的建立及应用  

Establishment of the mutiplex RT-PCR detecting of Porcine teschovirus,Classical swine fever virus and Porcine reproductive and respiratory syndrome virus

作　　者：刘杉杉[1] 赵亚荣 胡峰[1] 吕超超 胡泉博 王贵华 崔尚金[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪传染病研究室,黑龙江哈尔滨150001 [2]大北农动物医学研究中心,北京100097

出　　处：《中国预防兽医学报》2011年第9期704-708,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然基金(31001069);兽医生物技术国家重点实验室自主课题项目(NKLVBP2001002);中央级公益性科研院所基本科研业务费专项(2009ZJKJ01)

摘　　要：为建立同时检测猪捷申病毒(PTV)、猪瘟病毒(CSFV)和猪繁殖与呼吸综合征病毒(PRRSV)的多重RT-PCR(mRT-PCR)检测方法,本研究以PRRSV ORF6基因、CSFV Erns基因和PTV 5'-UTR基因为对象,建立检测3种病毒的mRT-PCR方法。特异性试验表明,该方法可以特异扩增3种病毒的基因,对大肠杆菌、猪圆环病毒Ⅱ型、猪细小病毒、猪伪狂犬病毒和猪流感病毒呈阴性反应;敏感性试验表明,PRRSV、CSFV和PTV的最低核酸检出量分别为7.11×102拷贝、7.81×103拷贝和8.43×102拷贝,略低于单一RT-PCR敏感性;应用该方法对69份送检样品进行检测,其中PRRSV、CSFV和PTV单纯感染检出率分别为28.99%、27.54%和56.52%,PRRSV和CSFV混合感染检出率为4.35%,PRRSV和PTV的混合感染检出率为11.59%,CSFV和PTV的混合感染检出率为13.04%,3种病毒的混合感染检出率为8.70%,与单一RT-PCR检测结果符合率为98.6%。本研究建立的检测方法快速简便,可以用于疾病的初步筛选,对临床早期诊断和流行病学调查具有重要意义。To develop a mutiplex RT-PCR for detection of Porcine reproductive and respiratory syndrome virus（PRRSV）,Classical swine fever virus（CSFV） and Porcine teschovirus（PTV）,three pairs of primers were designed and synthesized for amplification of PRRSV ORF6,CSFV Erns,and PTV 5＇-UTR sequences.This method had a detection limits of 7.11×10^2（PRRSV）,7.81×10^3（CSFV） and 8.43×10^2（PTV） copies,respectively,and no reaction with other porcine virus.A total of 69 clinic samples were tested by this method,the results indicated that the single infection was 28.99%,27.54% and 56.52% for PRRSV,CSFV and PTV,respectively,the dual infections was 4.35% for PRRSV and CSFV,11.59% for PRRSV and PTV,13.04% for PTV and CSFV,respectively,and the co-infenction was 8.70% for the 3 viruses.The consistency rate between mutiplex RT-PCR and single RT-PCR was 98.60%.This method was a rapid and sensitive tool for early diagnosis and the routine surveillance of the viral infections in pigs.

关 键 词：猪繁殖与呼吸综合征病毒 猪瘟病毒 猪捷申病毒 mRT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]