Department of Virology, Wageningen Bioveterinary Research (WBVR), Lelystad, the Netherlands.
Department of Virology, Wageningen Bioveterinary Research (WBVR), Lelystad, the Netherlands; Department of Biochemistry, North West University, Potchefstroom, South Africa.
J Virol Methods. 2019 Aug;270:106-112. doi: 10.1016/j.jviromet.2019.05.002. Epub 2019 May 13.
PCR diagnostics are often the first line of laboratory diagnostics and are regularly designed to either differentiate between or detect all pathogen variants of a family, genus or species. The ideal PCR test detects all variants of the target pathogen, including newly discovered and emerging variants, while closely related pathogens and their variants should not be detected. This is challenging as pathogens show a high degree of genetic variation due to genetic drift, adaptation and evolution. Therefore, frequent re-evaluation of PCR diagnostics is needed to monitor its usefulness. Validation of PCR diagnostics recognizes three stages, in silico, in vitro and in vivo validation. In vitro and in vivo testing are usually costly, labour intensive and imply a risk of handling dangerous pathogens. In silico validation reduces this burden. In silico validation checks primers and probes by comparing their sequences with available nucleotide sequences. In recent years the amount of available sequences has dramatically increased by high throughput and deep sequencing projects. This makes in silico validation more informative, but also more computing intensive. To facilitate validation of PCR tests, a software tool named PCRv was developed. PCRv consists of a user friendly graphical user interface and coordinates the use of the software programs ClustalW and SSEARCH in order to perform in silico validation of PCR tests of different formats. Use of internal control sequences makes the analysis compliant to laboratory quality control systems. Finally, PCRv generates a validation report that includes an overview as well as a list of detailed results. In-house developed, published and OIE-recommended PCR tests were easily (re-) evaluated by use of PCRv. To demonstrate the power of PCRv, in silico validation of several PCR tests are shown and discussed.
PCR 诊断通常是实验室诊断的第一线，通常旨在区分或检测一个家族、属或种的所有病原体变体。理想的 PCR 检测应能检测到目标病原体的所有变体，包括新发现和新兴的变体，而不应检测到密切相关的病原体及其变体。这是具有挑战性的，因为病原体由于遗传漂变、适应和进化而表现出高度的遗传变异。因此，需要频繁重新评估 PCR 诊断以监测其有用性。PCR 诊断的验证认可三个阶段，即计算机模拟、体外和体内验证。体外和体内测试通常成本高、劳动强度大，并涉及处理危险病原体的风险。计算机模拟验证减轻了这种负担。计算机模拟验证通过将引物和探针的序列与可用的核苷酸序列进行比较来检查它们。近年来，高通量和深度测序项目使可用序列的数量急剧增加，这使得计算机模拟验证更具信息性，但也更具计算密集性。为了方便 PCR 测试的验证，开发了一种名为 PCRv 的软件工具。PCRv 具有用户友好的图形用户界面，并协调使用 ClustalW 和 SSEARCH 软件程序，以对不同格式的 PCR 测试进行计算机模拟验证。使用内部控制序列使分析符合实验室质量控制系统。最后，PCRv 生成一份验证报告，其中包括概述以及详细结果列表。使用 PCRv 可以轻松（重新）评估内部开发、已发表和 OIE 推荐的 PCR 测试。为了展示 PCRv 的强大功能，展示并讨论了几个 PCR 测试的计算机模拟验证。
