A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2 Juscamayta-López, E. es_PE Valdivia, F. es_PE Horna, H. es_PE Tarazona, D. es_PE Linares, L. es_PE Rojas, N. es_PE Huaringa, M. es_PE 2024-05-30T23:13:38Z 2024-05-30T23:13:38Z 2021
dc.description This work was supported by the CONCYTEC-FONDECYT Program of Proyectos Especiales: Respuesta al COVID-19 2020-01-01 [grant number 034-2020-FONDECYT] and the National Institute of Health of Peru.
dc.description.abstract Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4–100.0%) and 98.6% (95% CI: 94.9–99.8%), respectively, showing high consistency (Cohen’s kappa, 0.986; 95% CI: 0.966–1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device. © Copyright © 2021 Juscamayta-López, Valdivia, Horna, Tarazona, Linares, Rojas and Huaringa.
dc.description.sponsorship Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica - Concytec
dc.identifier.scopus 2-s2.0-85110180024
dc.language.iso eng
dc.publisher Frontiers Media S.A.
dc.relation.ispartof Frontiers in Cellular and Infection Microbiology
dc.rights info:eu-repo/semantics/openAccess
dc.subject timely diagnosis
dc.subject COVID-19 es_PE
dc.subject diagnostic accuracy es_PE
dc.subject Multiplex RT-LAMP es_PE
dc.subject primary health care es_PE
dc.subject rapid molecular tests es_PE
dc.subject resource-limited settings es_PE
dc.subject SARS-CoV-2 es_PE
dc.title A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2
dc.type info:eu-repo/semantics/article
dspace.entity.type Publication