猪瘟病毒和猪繁殖与呼吸综合征病毒双重RT-PCR检测方法的建立

Establishment of Duplex RT-PCR Method for Detecting Classical Swine Fever Virus and Porcine Reproductive and Respiratory Syndrome Virus

为建立一种猪瘟病毒和猪繁殖与呼吸综合征病毒的快速、鉴别诊断方法,根据GenBank中已发表的2种病毒的基因组序列,选择病毒的保守基因各设计了一对特异性检测引物,建立了可以同时鉴别检测猪瘟病毒和猪繁殖与呼吸综合征病毒的双重RT-PCR方法。该方法能从猪瘟病毒和猪繁殖与呼吸综合征病毒分别扩增出310bp和161bp的特异性片段,对2种病毒混合基因组同时扩增出310bp和161bp的特异性片段;该方法对猪乙型脑炎病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪细小病毒、猪圆环病毒2型、猪伪狂犬病毒的扩增结果均为阴性;该方法最低可以检出0.2pg病毒RNA含量。本研究建立的RTPCR方法具有良好的敏感性、特异性、重复性,在一个PCR反应体系中就可以准确、快速鉴别检测出猪瘟病毒和猪繁殖与呼吸综合征病毒,可以用于临床病料的快速检测。

In order to establish a rapid, differential diagnosis method for classical swine fever virus（SCFV） and porcine reproductive and respiratory syndrome virus（PRRSV）, we designed a pairs of specfic primers targeting to the conservative genes according to the genomic sequence of CSFV and PRRSV in GenBank, and established a duplex RT-PCR method for simultaneously detecting SCFV and PRRSV. This method can amplify 310 bp and/or 161 bp specific fragments from either SVFV and PRRSV single sample or mixed samples,and the minimum RNA content for detection is 0. 2 pg. This methed could specifically detect SCFV and PRRSV,while amplification results for JEV, PEDV, PGEV, PPV, PCV-2, PRV were nega- tive. The differential diagnosis RT-PCR method in this study had good sensitivity, specificity and repeat- ability. It can accurately, quickly, and differentialy diagnose CSFV and PRRSV in a PCR system, which can be used for rapid detection of clinical samples as well.