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一种简化的快速检测 SARS-CoV-2 感染的方法,避免 RNA 提取:工作流程验证。

A Streamlined Approach to Rapidly Detect SARS-CoV-2 Infection Avoiding RNA Extraction: Workflow Validation.

机构信息

Department of Medicine (DAME), University of Udine, Udine, Italy.

Department of Laboratory Medicine, University Teaching Hospital of Udine, Udine, Italy.

出版信息

Dis Markers. 2020 Dec 9;2020:8869424. doi: 10.1155/2020/8869424. eCollection 2020.


DOI:10.1155/2020/8869424
PMID:33343767
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7727018/
Abstract

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, 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.

摘要

严重急性呼吸综合征冠状病毒 2(SARS-CoV-2)自 2020 年初开始在全球范围内迅速传播。通过核酸(NA)分子分析检测样本中病毒 RNA 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,全球范围内出现了 RNA 提取试剂盒短缺的情况。因此,我们开发了一种快速且经济有效的病毒基因组分离方法,结合定量 RT-PCR 检测,可检测患者样本中的 SARS-CoV-2 RNA。该方法依赖于添加蛋白酶 K,然后进行控制热休克孵育,然后通过 RT-qPCR 进行评估。它已针对灵敏度、特异性、线性、重现性和精密度进行了验证。它可以检测低至 10 个病毒拷贝/样本,速度快,已在 60 名 COVID-19 感染患者中得到了表征。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,具有缩短分析时间和降低成本的优势。这是一种快速工作流程,旨在帮助医疗保健系统快速识别感染患者,例如在与病原体相关的爆发期间。由于其固有特性,该工作流程适合大规模筛查。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0de/7727018/ef85e4cd392f/DM2020-8869424.002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0de/7727018/0e948dccc87d/DM2020-8869424.001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0de/7727018/ef85e4cd392f/DM2020-8869424.002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0de/7727018/0e948dccc87d/DM2020-8869424.001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b0de/7727018/ef85e4cd392f/DM2020-8869424.002.jpg

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[5]
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Anal Chem. 2021-11-9

[8]
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[9]
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[10]
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本文引用的文献

[1]
The COVID-19 Pandemic: A Comprehensive Review of Taxonomy, Genetics, Epidemiology, Diagnosis, Treatment, and Control.

J Clin Med. 2020-4-24

[2]
An alternative workflow for molecular detection of SARS-CoV-2 - escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020.

Euro Surveill. 2020-4

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Infez Med. 2020

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Clin Exp Pediatr. 2020-4

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J Med Virol. 2020-3-5

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Combination of RT-qPCR testing and clinical features for diagnosis of COVID-19 facilitates management of SARS-CoV-2 outbreak.

J Med Virol. 2020-6

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges.

Int J Antimicrob Agents. 2020-2-17

[10]
Updated understanding of the outbreak of 2019 novel coronavirus (2019-nCoV) in Wuhan, China.

J Med Virol. 2020-2-12

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