Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

Pedro A Alves; Ellen G de Oliveira; Ana Paula M Franco-Luiz; Letícia T Almeida; Amanda B Gonçalves; Iara A Borges; Flávia de S Rocha; Raissa P Rocha; Matheus F Bezerra; Pâmella Miranda; Flávio D Capanema; Henrique R Martins; Gerald Weber; Santuza M R Teixeira; Gabriel Luz Wallau; Rubens L do Monte-Neto

doi:10.3389/fmicb.2021.713713

Optimization and Clinical Validation of Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification, a Fast, Highly Sensitive and Specific COVID-19 Molecular Diagnostic Tool That Is Robust to Detect SARS-CoV-2 Variants of Concern

Front Microbiol. 2021 Nov 18:12:713713. doi: 10.3389/fmicb.2021.713713. eCollection 2021.

Authors

Pedro A Alves^{1

2}, Ellen G de Oliveira¹, Ana Paula M Franco-Luiz¹, Letícia T Almeida¹, Amanda B Gonçalves¹, Iara A Borges¹, Flávia de S Rocha¹, Raissa P Rocha², Matheus F Bezerra³, Pâmella Miranda⁴, Flávio D Capanema⁵, Henrique R Martins^{6

7}, Gerald Weber⁴, Santuza M R Teixeira², Gabriel Luz Wallau⁸, Rubens L do Monte-Neto¹

Affiliations

¹ Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, Brazil.
² Centro de Tecnologia em Vacinas, UFMG/Fiocruz, Belo Horizonte, Brazil.
³ Departamento de Microbiologia, Instituto Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Brazil.
⁴ Departamento de Física, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
⁵ Núcleo de Inovação Tecnológica, Fundação Hospitalar do Estado de Minas Gerais, Belo Horizonte, Brazil.
⁶ Visuri Equipamentos e Serviços, Belo Horizonte, Brazil.
⁷ Departamento de Engenharia Elétrica, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
⁸ Departamento de Entomologia e Núcleo de Bioinformática, Instituto Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, Brazil.

Abstract

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/μL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3-99.5%] sensitivity and 100% (95% CI = 94.5-100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non-SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction-free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

Keywords: COVID-19; RT-LAMP; SARS-CoV-2; diagnostic test; molecular test; respiratory virus.