Barboza Victor Dos Santos, Domingues William Borges, de Souza Thobias Toniolo, Collares Tiago Veiras, Seixas Fabiana Kommling, Pacheco Bruna Silveira, Sousa Fernanda Severo Sabedra, Oliveira Thaís Larré, de Lima Marcelo, de Pereira Claúdio Martin Pereira, Spilki Fernando Rosado, Giongo Janice Luehring, Vaucher Rodrigo de Almeida
Laboratório de Pesquisa em Bioquímica e Biologia Molecular de Micro-organismos, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.
Laboratório de Genômica Estrutural, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.
J Clin Virol Plus. 2023 Jun;3(2):100134. doi: 10.1016/j.jcvp.2023.100134. Epub 2023 Jan 27.
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 ( = 0.957 and = 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.
2019年12月，中国疾病预防控制中心报告了中国湖北省武汉市的一场肺炎疫情，这场疫情令全球陷入恐慌，引发了全球大流行。此次疫情由一种名为严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的β冠状病毒引起。在医院工作并身处疫情一线提供护理的医护人员中发现了多例感染病例。在本研究中，使用医护人员的鼻咽拭子样本评估逆转录环介导等温扩增（RT-LAMP）检测方法的性能，并随后与实时逆转录定量PCR（RT-qPCR）方法进行比较。因此，在本研究中，我们验证了一种基于RT-LAMP检测SARS-CoV-2的方法，该方法可用于诊断这些医护人员。所采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Bland-Altman方法设计的散点图来估计一致性。在SARS-CoV-2疫情期间，从巴西佩洛塔斯有症状和无症状的医护人员中总共采集了100份鼻咽拭子临床标本。使用RT-LAMP检测方法，使用E基因和N基因引物大约可以在96.7%接受检测的医护人员中检测到SARS-CoV-2，而对于人类β-肌动蛋白基因，检测率约为100%。观察到的一致性对于E基因和N基因的引物组分别被认为是极好的（分别为 = 0.957和 = 0.896）。RT-LAMP检测方法对于E基因引物组的灵敏度为阳性，每个反应大约可检测到2个拷贝。对于N基因引物组，该检测方法大约可验证每个反应253个拷贝的检测限。在执行RT-LAMP检测后，对所检测的任何呼吸道病毒均未观察到阳性反应。因此，我们得出结论，RT-LAMP在COVID-19疫情期间对医护人员进行快速分子诊断是有效的。
