非洲猪瘟病毒微滴数字PCR (ddPCR)方法的建立及应用

A novel high-sensitivity droplet digital PCR (ddPCR) for detection of African swine fever virus

摘要:

【目的】非洲猪瘟病毒(African swine fever virus，ASFV)能导致猪群高死亡率，从而造成严重的经济损失。因此，建立严密、高效的防控系统，包括灵敏、准确的诊断方法以及高效的预报预警机制等，以避免ASF传入我国。本文旨在建立一种新型而高灵敏度的ASFV微滴数字PCR (Droplet digital PCR，ddPCR)检测方法。【方法】针对ASFV K205R基因设计一对特异性引物和TaqMan探针，通过反应条件优化，建立了ASFV实时荧光定量PCR (qPCR)和ddPCR检测方法；并对两种方法的线性关系、灵敏性、特异性和重复性进行了评估，利用建立的ASFV检测方法对163份国内和进境的组织样本及血清样本进行检测，血清样本经商品化ASFV ELISA试剂盒复检，评估不同方法检测结果的符合率。【结果】建立的ASFV qPCR和ddPCR检测方法线性关系较好(R2≥0.998)，ddPCR的最低检测限度可达到0.36拷贝，在20 μL反应体系中约为10拷贝/反应，检测灵敏度是qPCR的10倍。ASFV ddPCR检测方法具有很高的特异性和重复性，不与其他猪常见病毒发生交叉反应。【结论】该方法灵敏度高、特异性强，可作为一种有效的分子生物学方法来诊断ASFV，为防止该病传入我国以及ASFV的实时监测提供新的技术储备，同时促进ddPCR技术在我国的发展和应用。

Abstract:

[Objective] African swine fever virus (ASFV) can cause high mortality in swine and lead to huge economic losses. Therefore to establish a strict and efficient control system, including the sensitive and accurate diagnosis methods, effective warning mechanism, which avoid the spread of ASF in China. The aim of this study was to develop a novel and high-sensitive droplet digital PCR (ddPCR) method to detect African swine fever virus (ASFV). [Methods] The methods of ASFV real-time quantitative PCR (qPCR) and ddPCR were established and optimal reaction conditions were confirmed based on K205R gene of ASFV. Each method was evaluated for linearity, limit of detection and specificity. The methods were tested in 163 specimens which were collected from domestic or imported clinical sample or serum samples. [Results] The results indicated that the method both had a high degree of linearity (R2≥0.998). The detection limit of ddPCR reached 0.36 copies and was approximately 10 copies/reaction, which was approximately a 10-fold greater sensitivity than qPCR. The cross-reaction was performed with other porcine pathogens, and negative amplification of the cross-reaction assay demonstrated the high specificity of this method. [Conclusion] This high sensitivity and specificity method could be used as an efficient molecular biology tool to diagnose ASFV, which is a reserve technique and very important for prevention of the spread of diseases across borders, and it promotes the development of ddPCR in China.