Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients’ viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.

中文翻译：

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快速检测SARS-CoV-2感染避免RNA提取的简化方法：工作流验证

从2020年初开始，严重的急性呼吸综合症冠状病毒2（SARS-CoV-2）感染已在世界范围内迅速传播。通过核酸（NA）分子分析在样品中存在病毒RNA是诊断COVID-19疾病的唯一方法并评估患者的病毒载量。由于对实验室试剂的需求增加，因此全球范围内都缺乏RNA提取试剂盒。因此，我们开发了一种快速且经济高效的病毒基因组分离方法，该方法与定量RT-PCR分析相结合，可检测患者样品中的SARS-CoV-2 RNA。该方法依赖于蛋白酶K的添加，然后进行受控的热激孵育，然后再进行E基因通过RT-qPCR进行评估。已针对灵敏度，特异性，线性，可重复性和精密度进行了验证。它可以检测到低至10个病毒拷贝/样品，而​​且速度很快，并且已在60例感染COVID-19的患者中鉴定出特征。与自动提取方法相比，我们的预处理可确保相同的阳性率，并具有缩短分析时间并降低成本的优势。这是一个快速的工作流程，旨在帮助医疗保健系统快速识别受感染的患者，例如在病原体相关的暴发期间。由于其固有特性，该工作流程适合于大规模筛选。