伪狂犬病病毒野毒荧光定量PCR检测方法的建立

根据伪狂犬病病毒gE基因的序列,设计和合成了一对特异的可用于检测伪狂犬病病毒野毒的PCR引物和一条Taqman荧光探针,采用Li ght Cycl e 480荧光定量PCR仪,建立了一种可实时定量检测伪狂犬病病毒野毒的荧光定量PCR技术。该方法的线性范围为1.0×102～1.0×1010拷贝,灵敏度可达4拷贝。检测速度快,仪器的运行时间仅为1 h。对13株猪伪狂犬病病毒野毒进行了检测,结果均为阳性;与伪狂犬病gE基因缺失疫苗、猪细小病毒和鸭瘟病毒无非特异性反应。与病毒分离培养比较,该方法具有快速、灵敏、特异、定量、重复性好等优点,可望用于临床上伪狂犬病病毒野毒与疫苗毒的区分,伪狂犬病病毒野毒的检测和病毒分布的研究等。

Development of the fluorescent quantitative PCR method for detecting Pseudorabies virus wild strains

According to the sequences of gE gene of pseudorabies virus(PRV),one pair of primers and one Taqman probe were designed and synthesized for differentiating the wild strains of pseudorabies virus from the field samples by using LightCycle 480 real-time PCR machine.The detectable linear range of this novel qPCR method was from 1.0×102 to 1.0×1010 copies with the lowest detectable DNA limit of 4 copies.This rapid and sensitive qPCR test could be run in one hour to acquire the results.Its specificity by detecting 13 wild strains of pseudorabies viruses isolated in our laboratory was confirmed in which no any positive amplification for swine parvovirus and duck plague virus(duck enteritis virus) were produced.This method was more highly rapid,sensitive,specific and repeatable than the classical virus isolation and culture methods,and could be used for the detection of wild type of PRV in the field samples.