Medical Technology Research Centre, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University, Chelmsford, Essex, CM1 1SQ, UK.
Mid and South Essex NHS Foundation Trust, Broomfield Hospital, Chelmsford, Essex, CM1 7ET, UK.
Sci Rep. 2020 Dec 17;10(1):22214. doi: 10.1038/s41598-020-79233-x.
Accurate, reliable and rapid detection of SARS-CoV-2 is essential not only for correct diagnosis of individual COVID-19 disease but also for the development of a rational strategy aimed at lifting confinement restrictions and preparing for possible recurrent waves of viral infections. We have used the MIQE guidelines to develop two versions of a unique five plex RT-qPCR test, termed CoV2-ID, that allows the detection of three viral target genes, a human internal control for confirming the presence of human cells in a sample and a control artificial RNA for quality assessment and potential quantification. Viral targets can be detected either individually with separate fluorophores or jointly using the same fluorophore, thus increasing the test's reliability and sensitivity. It is robust, can consistently detect two copies of viral RNA, with a limit of detection of a single copy and can be completed in around 15 min. It was 100% sensitive and 100% specific when tested on 23 RNA samples extracted from COVID-19 positive patients and five COVID-19 negative patients. We also propose using multiple cycle fluorescence detection, rather than real-time PCR to reduce significantly the time taken to complete the assay as well as assuage the misunderstandings underlying the use of quantification cycles (Cq). Finally, we have designed an assay for the detection of the D614G mutation and show that all of the samples isolated in the Chelmsford, Essex area between mid-April and June 2020, have the mutant genotype whereas a sample originating in Australia was infected with the wild type genotype.
准确、可靠和快速检测 SARS-CoV-2 不仅对于正确诊断个体 COVID-19 疾病至关重要，而且对于制定合理的策略以解除限制和为可能的病毒感染复发波做准备也至关重要。我们使用 MIQE 指南开发了两种独特的五重 RT-qPCR 测试版本，称为 CoV2-ID，该测试可检测三个病毒靶基因、一个用于确认样本中存在人类细胞的人类内参以及一个用于质量评估和潜在定量的人工控制 RNA。病毒靶标可以单独使用不同的荧光团进行检测，也可以使用相同的荧光团进行联合检测，从而提高测试的可靠性和灵敏度。它具有稳健性，可以一致地检测两个拷贝的病毒 RNA，检测限为单个拷贝，并且可以在大约 15 分钟内完成。当在从 23 名 COVID-19 阳性患者和 5 名 COVID-19 阴性患者提取的 RNA 样本上进行测试时，它的灵敏度为 100%，特异性为 100%。我们还建议使用多循环荧光检测，而不是实时 PCR，以显著缩短完成检测所需的时间，并消除对定量循环 (Cq) 使用的误解。最后，我们设计了一种用于检测 D614G 突变的检测方法，并表明在 2020 年 4 月中旬至 6 月期间在埃塞克斯郡切姆斯福德分离的所有样本均具有突变基因型，而源自澳大利亚的样本则感染了野生型基因型。
