A Streamlined Approach to Rapidly Detect SARS-CoV-2 Infection Avoiding RNA Extraction: Workflow Validation.
A Streamlined Approach to Rapidly Detect SARS-CoV-2 Infection Avoiding RNA Extraction: Workflow Validation.
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DOI：
10.1155/2020/8869424
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Curcio F
中科院分区：
文献类型：
--
作者：
Mio C;Cifù A;Marzinotto S;Bergamin N;Caldana C;Cattarossi S;Cmet S;Cussigh A;Martinella R;Zucco J;Verardo R;Schneider C;Marcon B;Zampieri S;Pipan C;Curcio F
自2020年初以来，严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）感染在全球迅速蔓延。核酸（NA）分子分析样品中病毒RNA的存在是诊断COVID-19疾病和评估患者病毒载量的唯一方法。由于对实验室试剂的需求增加，世界范围内一直存在RNA提取试剂盒的短缺。因此，我们开发了一种快速且具有成本效益的病毒基因组分离方法，结合定量RT-PCR检测患者样本中的SARS-CoV-2 RNA。该方法依赖于添加蛋白酶K，然后进行受控热休克孵育，然后通过RT-qPCR对E基因进行评估。验证了该方法的灵敏度、特异性、线性度、重现性和精密度。它检测到低至10个病毒拷贝/样本，速度快，并已在60名感染covid -19的患者中得到证实。与自动化提取方法相比，我们的预处理保证了相同的阳性率，缩短了分析时间，降低了成本。这是一种快速的工作流程，旨在帮助医疗保健系统快速识别受感染的患者，例如在与病原体有关的爆发期间。由于其固有的特点，该工作流程适合于大规模筛选。
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.
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