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

摘要:

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。

Abstract:

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.