伪狂犬病病毒实时荧光PCR的建立和应用

Development and Application of a Real-time Fluorescence PCR Assay for Pseudorabies Virus

伪狂犬病是世界范围内严重影响养猪业发展的重要动物疫病,快速检测是有效防控该病的重要措施之一。为了建立该病的快速分子生物学检测方法,根据GenBank中登录的伪狂犬病病毒gD基因序列,选择高度保守区域设计引物和TaqMan荧光探针,通过对实时荧光PCR反应条件进行优化,建立了用于检测伪狂犬病病毒的实时荧光PCR方法。特异性试验结果表明,该检测方法只能检测到目的病毒,表明其具有良好的特异性。灵敏性试验发现,其最低检测限可达1.42pg/μL的总DNA,与PCR方法的灵敏度对比试验表明,其敏感度比PCR至少高10倍。对同一样品进行重复性检测,在组内及组间检测的荧光扩增曲线基本重叠,证实其重复性好。对180份临床样品进行伪狂犬病病毒核酸检测,结果发现有52份阳性样品。结果表明,所建立的实时荧光PCR方法可对伪狂犬病病毒进行准确、快速的检测,具有特异性好、灵敏度高、重复性好的优点,是开展伪狂犬病的临床检测和疫情监测工作的有力工具。

Pseudorabies（PR）has already become an important animal disease all over the world,which affects badly on pig production.Rapid,accurate detection is one of main measure to prevent the disease.In order to develop a rapid molecular detection method,this study designed a pair of primers and a TaqMan fluorescence probe based on the gD gene of pseudorabies virus（PRV）.The sequence was reported in GenBank.After optimizing reaction condition of the real-time fluorescence PCR,a real-time fluorescence PCR assay was developed to detect PRV.The results indicated that specificity,sensitivity and reproducibility of the developed method were all high.180 clinical samples were detected by real-time fluorescence PCR assay for pseudorabies virus.52 samples were positive,and others were negative.The study demonstrated that the real-time fluorescence PCR assay developed for pseudorabies virus could be used for clinical test and inspection as a powerful tool.