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建立逆转录重组酶聚合酶扩增检测方法,快速直接目视检测严重急性呼吸综合征冠状病毒 2(SARS-CoV-2)。

Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

机构信息

Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.

Department of Pathology, Hospital Sungai Buloh, Selangor, Malaysia.

出版信息

PLoS One. 2021 Jan 6;16(1):e0245164. doi: 10.1371/journal.pone.0245164. eCollection 2021.


DOI:10.1371/journal.pone.0245164
PMID:33406112
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7787525/
Abstract

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

摘要

快速诊断是管理严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 爆发的重要干预措施。实时逆转录聚合酶链反应 (RT-qPCR) 仍然是诊断新病毒株的主要手段,但它既耗时又昂贵。重组酶聚合酶扩增 (RPA) 是一种不需要 PCR 机器的等温扩增检测方法。它是一种经济实惠、快速且简单的检测方法。在这项研究中,我们使用 SYBR Green I 和/或侧流 (LF) 条带开发并优化了一种用于快速检测 SARS-CoV-2 的灵敏 RT-RPA 检测方法。通过使用 10 倍系列稀释的合成 RNA 和类似病毒的基因组 RNA 分别测试了 RT-RPA 检测方法的分析灵敏度和特异性。使用 78 个阳性和 35 个阴性鼻咽样本进行了 RT-RPA 检测方法的临床灵敏度和特异性检测。RPA 和 RT-qPCR 检测方法的检测限分别为 7.659 和 5 拷贝/μL RNA,与其他病毒无交叉反应。RT-RPA 的临床灵敏度和特异性分别为 98%和 100%。我们的研究表明,RT-RPA 是 RT-qPCR 检测 SARS-CoV-2 的一种可行替代方法,尤其是在基础设施有限的地区。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b16c/7787525/10dd8ba71e8d/pone.0245164.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b16c/7787525/ea6c3da390a6/pone.0245164.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b16c/7787525/10dd8ba71e8d/pone.0245164.g002.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b16c/7787525/ea6c3da390a6/pone.0245164.g001.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b16c/7787525/10dd8ba71e8d/pone.0245164.g002.jpg

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[3]
SARS-CoV-2 recombinase polymerase amplification assay with lateral flow readout and duplexed full process internal control.

Sens Diagn. 2024-1-12

[4]
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Diagnostics (Basel). 2024-2-29

[5]
The Diagnostic Performance of Various Clinical Specimens for the Detection of COVID-19: A Meta-Analysis of RT-PCR Studies.

Diagnostics (Basel). 2023-9-26

[6]
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Egypt J Med Hum Genet. 2022

[7]
Rapid isothermal point-of-care test for screening of SARS-CoV-2 (COVID-19).

Asp Mol Med. 2023

[8]
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本文引用的文献

[1]
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medRxiv. 2020-10-25

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Clin Chem. 2020-8-1

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