ABSTRACT

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.

摘要

在努力减轻 COVID-19 大流行的影响的过程中，已证明早期发现严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 至关重要。过去几个月出现了几种诊断方法，每种方法都有不同的缺点和局限性。当前的黄金标准，使用荧光探针的 RT-qPCR，依赖于苛刻的设备要求以及探针和特定反应混合物的高成本。为了扩大可分配给此任务的试剂和热循环仪的可能性，我们优化了 RT-qPCR 诊断的替代策略。这是基于广泛使用的 DNA 嵌入染料，可以使用几种不同的 qPCR 试剂和仪器来实现。值得注意的是，所提出的 qPCR 方法与广泛使用的基于 TaqMan 的检测相似，在特异性和敏感性方面，因此是一种可靠的工具。我们认为，通过使用广泛的热循环仪模型和实验室设施进行 SARS-CoV-2 诊断，这里提出的替代方案可以显着提高检测能力，尤其是在获得昂贵技术和试剂的国家有限。