Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay as a rapid molecular diagnostic tool for COVID-19 in healthcare workers

IF 1.6 Q4 INFECTIOUS DISEASES
Victor dos Santos Barboza , William Borges Domingues , Thobias Toniolo de Souza , Tiago Veiras Collares , Fabiana Kommling Seixas , Bruna Silveira Pacheco , Fernanda Severo Sabedra Sousa , Thaís Larré Oliveira , Marcelo de Lima , Claúdio Martin Pereira de Pereira , Fernando Rosado Spilki , Janice Luehring Giongo , Rodrigo de Almeida Vaucher
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引用次数: 4

Abstract

In December 2019, the Chinese Center for Disease Control (CDC of China) reported an outbreak of pneumonia in the city of Wuhan (Hubei province, China) that haunted the world, resulting in a global pandemic. This outbreak was caused by a betacoronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Several of these cases have been observed in healthcare professionals working in hospitals and providing care on the pandemic's frontline. In the present study, nasopharyngeal swab samples of healthcare workers were used to assess the performance of the reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay and subsequently compared with the real‐time reverse-transcription quantitative PCR (RT-qPCR) method. Thus, in this study, we validated a method for detecting SARS-CoV-2 based on RT-LAMP that can be used to diagnose these workers. The methodology used was based on analyzing the sensitivity, specificity, evaluation of the detection limit, and cross-reaction with other respiratory viruses. The agreement was estimated using a dispersion diagram designed using the Bland-Altman method. A total of 100 clinical specimens of nasopharyngeal swabs were collected from symptomatic and asymptomatic healthcare workers in Pelotas, Brazil, during the SARS-CoV-2 outbreak. RT-LAMP assay, it was possible to detect SARS-CoV-2 in 96.7% of the healthcare professionals tested using the E gene and N gene primers approximately and 100% for the gene of human β-actin. The observed agreement was considered excellent for the primer set of the E and N genes (k = 0.957 and k = 0.896), respectively. The sensitivity of the RT-LAMP assay was positive for the primer set of the E gene, detected to approximately 2 copies per reaction. For the primer set of the N gene, the assay was possible to verify an LoD of approximately 253 copies per reaction. After executing the RT-LAMP assay, no positive reactions were observed for any of the virus respiratory tested. Therefore, we conclude that RT-LAMP is effective for rapid molecular diagnosis during the COVID-19 outbreak period in healthcare professionals.

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逆转录环介导等温扩增(RT-LAMP)检测作为医护人员新冠肺炎的快速分子诊断工具
2019年12月,中国疾病控制中心(中国疾控中心)报告称,武汉市(中国湖北省)爆发了一场困扰世界的肺炎疫情,导致了一场全球大流行。这次疫情是由一种名为严重急性呼吸综合征冠状病毒2型(严重急性呼吸系统综合征冠状病毒-2)的β冠状病毒引起的。在医院工作并在疫情前线提供护理的医护人员中观察到了其中一些病例。在本研究中,医护人员的鼻咽拭子样本用于评估逆转录环介导的等温扩增(RT-LAMP)测定的性能,随后与实时逆转录定量PCR(RT-qPCR)方法进行比较。因此,在这项研究中,我们验证了一种基于RT-LAMP的检测严重急性呼吸系统综合征冠状病毒2型的方法,该方法可用于诊断这些工作人员。所使用的方法基于分析灵敏度、特异性、检测限评估以及与其他呼吸道病毒的交叉反应。使用布兰德-奥特曼方法设计的色散图来估计一致性。在严重急性呼吸系统综合征冠状病毒2型爆发期间,共从巴西佩洛塔斯有症状和无症状的医护人员身上采集了100份鼻咽拭子临床标本。RT-LAMP检测,使用E基因和N基因引物检测的96.7%的医护人员中有可能检测到严重急性呼吸系统综合征冠状病毒2型,人类β-肌动蛋白基因的检测率为100%。观察到的一致性被认为对于E和N基因的引物组是极好的(分别为k=0.957和k=0.896)。RT-LAMP测定的灵敏度对E基因的引物组是阳性的,每个反应检测到大约2个拷贝。对于N基因的引物组,该测定可以验证每个反应大约253个拷贝的LoD。在进行RT-LAMP测定后,未观察到任何呼吸道测试病毒的阳性反应。因此,我们得出结论,RT-LAMP在新冠肺炎爆发期间对医护人员的快速分子诊断是有效的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Journal of clinical virology plus
Journal of clinical virology plus Infectious Diseases
CiteScore
2.20
自引率
0.00%
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0
审稿时长
66 days
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