猪瘟和猪蓝耳病病毒多重RT-PCR检测方法的建立及初步应用  

Development and preliminary application of multiplex RT-PCR for classical swine fever virus and porcine reproductive and respiratory syndrome virus

作　　者：陈甜甜[1] 程福亮[1] 梁瑾[1] 宿志瑞[1] 范群平[1] 

机构地区：[1]山东宝来利来生物工程股份有限公司,山东泰安271000

出　　处：《中国生物制品学杂志》2013年第3期425-429,共5页Chinese Journal of Biologicals

摘　　要：目的建立猪瘟病毒（Classical swine fever virus,CSFV）和猪蓝耳病病毒（Porcine respiratory and reproductivesyndrome virus,PRRSV）多重RT-PCR检测方法,并进行验证及初步应用。方法根据GenBank中登录的CSFV和PRRSV疫苗株基因组序列,分别设计针对CSFV和PRRSV的两对引物,建立多重RT-PCR检测方法,并进行敏感性及特异性验证。用建立的多重RT-PCR法检测20份疑似猪瘟、猪蓝耳病或混合感染病料,并与市售RT-PCR试剂盒的检测结果进行比较;检测病料的部分PCR产物测序后,与9株具有代表性的CSFV毒株和10株具有代表性的PRRSV毒株进行核苷酸序列同源性比对。结果以CSFV和PRRSV混合引物扩增,分别可扩增出286和664 bp的特异性条带。建立的多重RT-PCR检测方法可检出100倍稀释的病毒RNA;该方法可检出CSFV、PRRSV及CSFV与PRRSV混合病毒,而猪流行性腹泻病毒（Porcine epidemic diarrhea virus,PEDV）、猪传染性胃肠炎病毒（Trans-missible gastro-enteritis virus,TGEV）、猪圆环病毒（Porcine circovirus,PCV）、猪细小病毒（Porcine parvovirus,PPV）和猪伪狂犬病病毒（Porcine pseudorabies virus,PRV）的检测结果均为阴性;该方法检测20份疑似猪瘟、猪蓝耳病或混合感染病料的结果与市售RT-PCR试剂盒比较,灵敏度达100%;选取的两份CSFV扩增片段与9株具有代表性的CSFV毒株的核苷酸序列同源性在92.3%~98.2%之间,扩增的PRRSV与10株具有代表性的PRRSV毒株的核苷酸序列同源性在94.1%~97.9%之间。结论成功建立了CSFV和PRRSV多重RT-PCR检测方法,为猪瘟和猪蓝耳病的早期诊断及流行病学调查奠定了基础。Objective A multiplex RT-PCR method for detection of classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus（PRRSV） was developed, verified and preliminarily applied. Methods According to the genomic sequences of CSFV and PRRSV reported in GenBank, two specific pairs of primers were designed, based on which a multiplex RT-PCR method was developed and verified for sensitivity and specificity. Twenty suspected samples with CSFV, PRRSV or mixed infection were detected by the developed method, of which the results were compared with those by commercial RT-PCR kit. The PCR products of partial samples were sequenced, and analyzed for homologies of nucleotides to those of representative nine CSFV strains and ten PRRSV strains. Results Specific gene fragments at lengths of 664 and 286 bp respectively were amplified by RT-PCR using the mixed primers for CSFV and PRRSV. The viral RNA at a dilution of 1 ： 100 was detected by the developed method. The detection results of CSFV, PPRSV and mixture of the two viruses were positive, while those of porcine epidemic diarrhea virus （PEDV）, transmissible gastro-en- teritis virus （TGEV）, porcine circovirus （PCV）, porcine parvovirus （PPV） and porcine pseudorabies virus （PRV） were negative. Compared with that of commercial RT-PCR kit, the sensitivity of the developed method for 20 suspected samples was 100%. The homologies of nucleotides amplified CSFV to those of nine representative CSFV strains were 92. 3% -98. 2%, while those of amplified PRRSV to ten representative PRRSV strains were 94. 1% - 97. 9%. Conclusion A multiplex RT-PCR method for detection of CSFV and PRRSV was successfully developed, which laid a foundation of early diagnosis and epidemiological investigation of classical swine fever and porcine respiratory and reproductive syndrome.

关 键 词：猪瘟病毒 猪蓝耳病病毒 多重逆转录聚合酶链反应 

分 类 号：S852.651[农业科学—基础兽医学] S851.347.1[农业科学—兽医学]