鉴别猪瘟强毒和弱毒的反转录-复合套式聚合酶链式反应(RT-nPCR)检测方法的建立  

A Reverse Transcription-Multiplex Nested Polymerase Chain Reaction for Detection and Differentiation of Wild-type and Vaccine Viruses of Classical Swine Fever Virus

作　　者：李艳[1,2] 仇华吉[1] 王秀荣[1] 张守发[3] 朱庆虎[1] 李娜[1,4] 李国新[1,4] 童光志[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 [2]延边大学农学院,龙井133400 [3]延边大学农学院 [4]东北农业大学动物医学院,哈尔滨150030

出　　处：《中国农业科学》2006年第9期1907-1914,共8页Scientia Agricultura Sinica

基　　金：国家"973"计划资助(2005CB523202)

摘　　要：【目的】建立一种能区分猪瘟病毒(classicalswinefever,CSFV)强毒和弱毒的反转录-复合套式聚合酶链式反应(RT-nPCR)检测方法。【方法】根据GenBank上登录的现有CSFV基因组全序列,选择高度保守区设计了一对CSFV通用引物,并在该对引物跨越区域的内部设计了猪瘟兔化弱毒疫苗和强毒特异性引物,建立了一种能区分猪瘟强毒和弱毒的RT-nPCR鉴别诊断方法。【结果】应用该方法从猪瘟兔化弱毒疫苗和石门强毒株基因组中扩增出了大小分别为447和343bp的一条特异性片段,从两种病毒基因组混合物中扩增出了大小为447和343bp的两条特异性片段,但对牛病毒性腹泻病毒和其它常见猪源病毒细胞培养物以及正常细胞基因组进行检测时均为阴性。该方法可以检测出0.04pg的CSFVRNA。对从黑龙江省采集的15份疑似猪瘟病料进行了检测,结果表明,有14份类似猪瘟强毒,1份类似弱毒疫苗。限制性片段长度多态性和种系发生分析证实了RT-nPCR的检测结果。【结论】本研究建立的RT-nPCR可以有效区分猪瘟强毒和弱毒,减少了未感染的免疫猪被误杀的可能性。[ Objective ] A reverse transcription-multiplex nested polymerase chain reaction （RT-nPCR） was developed for the differentiation of wild-type classical swine fever virus （CSFV） from the C-strain vaccine. [ Method ] A palr of primers corresponding to the highly conserved regions ofCSFV genome was used as a common primer pair in the first round of the RT-nPCR. Another two primers specific for CSFV C-strain vaccine or highly virulent Shimen strain, respectively, were used as the forward primer together with the common reverse primer in the second round of the RT-nPCR. [Result] A fragment of 447 or 343 bp was amplified from genomic RNA of C-strain or Shimen, respectively, in the RT-nPCR, and two fragments of 447 and 343 bp were simultaneously amplified from the mixed RNA sample of C-strain and Shimen. No amplification was achieved for mock infected cells, or cells infected with other common viruses of porcine origin. The RT-nPCR could detect 0.04 pg of CSFV RNA. Fourteen of fifteen field samples suspected of CSF from Heilongjiang Province were found to be Shimen-like, and 1 to be C-strain-like by this method.Restrictive fragment length polymorphism and phylogenetic analyses confirmed the RT-nPCR results. [ Conclusion] The RT-nPCR can be used to differentiate pigs infected with wild-type CSFV from those vaccinated with the C-strain vaccine. It thus minimizes the possibility of miss-killed vaccinates during outbreaks.

关 键 词：猪瘟病毒 反转录-复合套式聚合酶链式反应 限制性片段长度多态性分析 

分 类 号：S854.43[农业科学—临床兽医学]