猪伪狂犬病病毒野毒株与疫苗株荧光定量PCR检测方法的建立和应用

Establishment and application of a real-time fluorescent quantitative PCR for pseudorabies wild-type virus and vaccine virus

根据伪狂犬病病毒gD、gE基因序列设计2对引物及其对应的探针,通过对引物、探针浓度的优化,建立了猪伪狂犬病病毒野毒和减毒活疫苗株荧光定量PCR检测鉴别方法,并评价其特异性、敏感性和重复性。结果显示,猪圆环病毒、猪瘟病毒、猪流行性乙型脑炎病毒、猪细小病毒、猪繁殖与呼吸综合征病毒、猪博卡病毒、猪流行性腹泻病毒和猪传染性胃肠炎病毒均无扩增曲线。野毒株均出现gD和gE基因扩增曲线,而疫苗株只出现了gD基因扩增曲线,表明该检测方法具有良好的特异性。建立的gD和gE基因标准曲线Ct值和模板终浓度在1×10^8~1×10 copies/μL范围内均具有良好的线性关系,灵敏度均可达1×10 copies/μL。对39份猪组织样品进行检测,荧光定量PCR检出5份样品猪伪狂犬病病毒gD、gE阳性,表明这5份猪组织样品为猪伪狂犬病病毒野毒感染。常规PCR检出5份猪伪狂犬病病毒阳性,经测序为野毒感染,2种方法符合率为100%。本研究建立的荧光定量PCR可用于猪伪狂犬病病毒的快速检测和流行病学调查。

According to gD and gE genes of pseudorabies virus,two pairs of primers and their corresponding probes were designed to establish a real-time fluorescent quantitative PCR for detection of pseudorabies virus by optimizing the concentration of primers and probes.Specificity,sensitivity and repeatability of the method were evaluated.The results showed this method had no cross reaction with other diarrhea virus(porcine circovirus,classical swine fever virus,Japanese encephalitis virus,footand-mouth disease virus,porcine parvovirus,porcine reproductive and respiratory syndrome virus,porcine bocavirus,porcine epidemic diarrhea virus,and transmissible gastroenteritis virus).The Ct value of standard curve had a linear relationship to the final concentration of template in the range of 1×108-1×10 copies/μL.The detection limit was 1×10 copies/μL.Among the 39 samples from porcine,5 samples were positive based on the real-time PCR for both gD and gE genes,which indicated that these 5 samples were infected by wild-type virus.In addition,5 positive samples were diagnosed by the conventional PCR methods,and was confirmed wild virus infection after sequencing.These positive samples were the same samples based on real-time PCR and conventional PCR.The concordance of the results based on two methods was 100%.In conclusion,the real-time PCR could be used for the detection and epidemiological investigation for pseudorabies virus.