猪伪狂犬病毒环介导等温扩增技术检测方法的建立

Establishment of Detection Method for Loop-mediated Isothermal Amplification of Porcine Pseudorabies Virus

为建立通用性猪伪狂犬病毒(PRV)环介导等温扩增快速检测方法,本实验根据PRV gB基因序列的保守区段设计四套特异性引物,基于环介导等温扩增技术引入环引物,筛选出特异性引物PRV-1,对其特异性、灵敏度进行评估,进行临床样本和人工干扰样本检测试验,并与实时荧光定量PCR(Quantitative real-time PCR,qPCR)检测结果进行比较分析。结果表明该检测方法能够特异性的检出PRV的存在,具有良好的特异性;灵敏度检测下限为10 fg/μL;且本文建立的检测方法其结果与临床样品和人工干扰样本qPCR检测结果具有高度的一致性。因此,本实验建立的PRV环介导等温扩增检测方法特异性强、灵敏度高,具有较好的稳定性,并且操作安全、简便、高效,该方法的建立为猪伪狂犬病的快速诊断和基层检疫提供了新的选择。

To establish a universal rapid detection method for porcine pseudorabies virus(PRV)loop-mediated isothermal amplification,four sets of specific primers were designed based on the conserved segments of the PRV gB gene sequence,and loop primers were introduced based on loop-mediated isothermal amplification.The specific primer PRV-1 was screened,and its specificity and sensitivity were evaluated.Clinical samples and artificial interference samples were tested and compared with real-time quantitative real-time PCR(qPCR).The results showed that the detection method could detect the presence of PRV specifically,and had good specificity.The limit of sensitivity detection is 10 fg/μL.The results of the detection method established in this work were highly correlated with the results of qPCR detection of clinical samples and artificial interference samples.Therefore,the PRV loop-mediated isothermal amplification detection method established in this work is specific,sensitive,and has good stability.The operation is safe,simple and efficient.It is potential to be a rapid diagnosis and grassroots of pseudorabies.