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

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

doi:10.1016/j.jcvp.2023.100134

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

J Clin Virol Plus. 2023 Jun;3(2):100134. doi: 10.1016/j.jcvp.2023.100134. Epub 2023 Jan 27.

Authors

Affiliations

¹ Laboratório de Pesquisa em Bioquímica e Biologia Molecular de Micro-organismos, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.
² Laboratório de Genômica Estrutural, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.
³ Laboratório de Biotecnologia do Câncer, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.
⁴ Laboratório de Vacinologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.
⁵ Laboratório de Virologia e Imunologia, Faculdade de Veterinária, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brazil.
⁶ Laboratório de Lipidômica e Bio-Orgânica, Universidade Federal de Pelotas, Rio Grande do Sul, Brazil.
⁷ Laboratório de Microbiologia Molecular, Universidade FEEVALE, Novo Hamburgo, Rio Grande do Sul, Brazil.

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.

Keywords: COVID-19; Healthcare workers; RT-LAMP; SARS-CoV-2.