Friedrich-Loeffler-Institut, Institute of Diagnostic Virology, 17493 Greifswald-Insel Riems, Germany.
Int J Mol Sci. 2021 Feb 25;22(5):2307. doi: 10.3390/ijms22052307.
African swine fever (ASF) is a contagious viral hemorrhagic disease of domestic pigs and wild boars. The disease is notifiable to the World Organisation for Animal Health (OIE) and is responsible for high mortality and serious economic losses. PCR and real-time PCR (qPCR) are the OIE-recommended standard methods for the direct detection of African swine fever virus (ASFV) DNA. The aim of our work was the simplification and standardization of the molecular diagnostic workflow in the lab. For validation of this "easy lab" workflow, different sample materials from animal trials were collected and analyzed (EDTA blood, serum, oral swabs, chewing ropes, and tissue samples) to identify the optimal sample material for diagnostics in live animals. Based on our data, the EDTA blood samples or bloody tissue samples represent the best specimens for ASFV detection in the early and late phases of infection. The application of prefilled ready-to-use reagents for nucleic acid extraction or the use of a Tissue Lysis Reagent (TLR) delivers simple and reliable alternatives for the release of the ASFV nucleic acids. For the qPCR detection of ASFV, different published and commercial kits were compared. Here, a lyophilized commercial kit shows the best results mainly based on the increased template input. The good results of the "easy lab" strategy could be confirmed by the ASFV detection in field samples from wild boars collected from the 2020 ASFV outbreak in Germany. Appropriate internal control systems for extraction and PCR are key features of the "easy lab" concept and reduce the risk of false-negative and false-positive results. In addition, the use of easy-to-handle machines and software reduces training efforts and the misinterpretation of results. The PCR diagnostics based on the "easy lab" strategy can realize a high sensitivity and specificity comparable to the standard PCR methods and should be especially usable for labs with limited experiences and resources.
非洲猪瘟（ASF）是一种传染性病毒性出血性疾病，可感染家猪和野猪。该疾病须向世界动物卫生组织（OIE）报告，其可导致高死亡率和严重的经济损失。聚合酶链式反应（PCR）和实时聚合酶链式反应（qPCR）是 OIE 推荐的直接检测非洲猪瘟病毒（ASFV）DNA 的标准方法。我们的工作旨在简化和标准化实验室分子诊断工作流程。为了验证这个“简易实验室”工作流程，我们收集和分析了来自动物试验的不同样本材料（EDTA 血液、血清、口腔拭子、咀嚼绳和组织样本），以确定用于活体动物诊断的最佳样本材料。根据我们的数据，EDTA 血液样本或带血组织样本代表了在感染的早期和晚期阶段检测 ASFV 的最佳标本。使用预填充即用型核酸提取试剂或组织裂解试剂（TLR）可简单可靠地释放 ASFV 核酸。对于 ASFV 的 qPCR 检测，我们比较了不同已发表和商业试剂盒。在这里，冻干商业试剂盒主要基于增加模板输入而显示出最佳结果。通过对德国 2020 年 ASF 暴发期间从野猪采集的现场样本中进行 ASFV 检测，我们验证了“简易实验室”策略的良好结果。提取和 PCR 的适当内部控制系统是“简易实验室”概念的关键特征，可降低假阴性和假阳性结果的风险。此外，使用易于操作的机器和软件可减少培训工作量和结果的误判。基于“简易实验室”策略的 PCR 诊断可实现与标准 PCR 方法相当的高灵敏度和特异性，并且尤其适用于经验和资源有限的实验室。
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