伪狂犬病病毒实时荧光PCR的建立和应用

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。

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

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 de...