猪伪狂犬病病毒经典毒株与变异毒株PCR鉴别方法的建立

Establishment of PCR assay for differentiation of variant strains from classic strains of porcine pseudorabies virus

猪伪狂犬病是危害世界养猪业的重要疫病之一。近年来我国免疫猪群出现暴发、流行,经证实其抗原性和毒力发生了变异。为了有效区分变异毒株和经典毒株,根据国内外伪狂犬病病毒（PRV）变异毒株和经典毒株基因序列比对结果,针对UL44和UL36区域的基因序列设计了2对引物,建立两步法PCR。第一步PCR针对UL44区域,区分出经典毒株（疫苗株HB98除外）感染与变异毒株感染。第二步PCR针对UL36区域,区分出HB98株与伪狂犬病病毒变异毒株。两步法PCR的敏感度分别达到2~20TCID50和50TCID50病毒量。对猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪瘟病毒、日本脑炎病毒、脑心肌炎病毒和猪流行性腹泻病毒的核酸应用此方法进行扩增,结果均为阴性。该方法与猪伪狂犬病病毒国家标准PCR检测方法的符合率达到91%,可用于实验室快速诊断。

Pseudorabies virus（PRV）is one of the very important pathogens spread internationally in the pig industry across the world, lt was proved that the antigenicity and virulence of the variant PRY strains isolated from pig farms changed greatly in recent years. To distinguish variant PRV strains from the classic,we developed 2-step PCR by designing 2 pairs of primes based on the genome sequences alignment results of PRV variant and classic isolates. Variant PRY strains could be distinguished from classic strains except HB98 vaccine strain by using the primers based on UL44 gene sequences（ist PCR）. PRY variant strain and HB98 could be differentiated further by using primers based on UL36. The detection sensitivity of 2-step PCR was 2-20 TCIDs0 and 50 TCID50 PRY load. Meanwhile,no bands were amplified by this method from the classical swine fever virus,porcine reproductive and respiratory syndrome virus, porcine circovirus type 2,Japanese encephalitis virus,encephalomyocarditis virus and porcine epidem- ic diarrhea virus cultural samples. The coincidence rate between this method and national standard PCR was 91%. This method can be used for rapid diagnosis of PRV in laboratory.