Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay
Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay
复制标题
通过逆转录环介导的等温扩增测定快速直观地检测 2019 新型冠状病毒 (SARS-CoV-2)
DOI：
10.1016/j.cmi.2020.04.001
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发表时间：
2020-06-01
影响因子：
14.2
通讯作者：
Yuan, J.
中科院分区：
文献类型：
--
作者：
Yan, C.;Cui, J.;Yuan, J.
目的：目的：建立检测SARS冠状病毒2型（SARS-CoV-2）的逆转录环介导等温扩增（RT-LAMP）方法，并与RT-PCR方法进行比较。使用QIAamp Viral RNA Mini Kit提取总病毒RNA。我们优化了RT-LAMP检测方法，并通过实时浊度监测和目视观察来评估其检测的灵敏度和特异性。引物组orf 1ab-4和S-123在最短时间内扩增基因，平均（+/- SD）时间分别为18 +/- 1.32 min和20 +/- 1.80 min，63 ℃为最佳反应温度。使用引物组orf 1ab-4和S-123，灵敏度分别为每次反应2 x 10（1）个拷贝和2 x 10（2）个拷贝。该检测试剂盒与其他60种呼吸道病原体无交叉反应性。为了描述该方法在临床诊断中的可用性，我们收集了130例临床疑似SARS-CoV-2感染患者的标本。其中58份经RT-LAMP检测为阳性，72份为阴性。敏感性为100%（95% CI 92.3%~ 100%），特异性为100%（95% CI 93.7% e100%）。结论：建立了一种快速、简便、特异、灵敏的RT-LAMP方法，可用于临床标本中SARS-CoV-2的检测。它将成为SARS-CoV-2识别以及监测疑似患者、密切接触者和高危人群的有力工具。(C)2020年欧洲临床微生物学和传染病学会。由爱思唯尔有限公司出版。保留所有权利。
Objective: To evaluate a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and compare it with RTPCR.Methods: We designed primers specific to the orf1ab and S genes of SARS-CoV-2. Total viral RNA was extracted using the QIAamp Viral RNA Mini Kit. We optimized the RT-LAMP assay, and evaluated it for its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation.Results: The primer sets orf1ab-4 and S-123 amplified the genes in the shortest times, the mean (+/- SD) times were 18 +/- 1.32 min and 20 +/- 1.80 min, respectively, and 63 degrees C was the optimum reaction temperature. The sensitivities were 2 x 10(1) copies and 2 x 10(2) copies per reaction with primer sets orf1ab-4 and S-123, respectively. This assay showed no cross-reactivity with 60 other respiratory pathogens. To describe the availability of this method in clinical diagnosis, we collected 130 specimens from patients with clinically suspected SARS-CoV-2 infection. Among them, 58 were confirmed to be positive and 72 were negative by RT-LAMP. The sensitivity was 100% (95% CI 92.3%-100%), specificity 100% (95% CI 93.7% e100%). This assay detected SARS-CoV-2 in a mean (+/- SD) time of 26.28 +/- 4.48 min and the results can be identified with visual observation.Conclusion: These results demonstrate that we developed a rapid, simple, specific and sensitive RT-LAMP assay for SARS-CoV-2 detection among clinical samples. It will be a powerful tool for SARS-CoV-2 identification, and for monitoring suspected patients, close contacts and high-risk groups. (C) 2020 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.