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A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2.
Front Cell Infect Microbiol. 2021; 11:653616.FC

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.

Authors+Show Affiliations

Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Peru.Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Peru.Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Peru.Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Peru.Laboratorio de Infecciones Respiratorias Agudas, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Peru.Laboratorio de Virus Respiratorios, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Peru.Laboratorio de Virus Respiratorios, Centro Nacional de Salud Pública, Instituto Nacional de Salud, Lima, Peru.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

34268131

Citation

Juscamayta-López, Eduardo, et al. "A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2." Frontiers in Cellular and Infection Microbiology, vol. 11, 2021, p. 653616.
Juscamayta-López E, Valdivia F, Horna H, et al. A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2. Front Cell Infect Microbiol. 2021;11:653616.
Juscamayta-López, E., Valdivia, F., Horna, H., Tarazona, D., Linares, L., Rojas, N., & Huaringa, M. (2021). A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2. Frontiers in Cellular and Infection Microbiology, 11, 653616. https://doi.org/10.3389/fcimb.2021.653616
Juscamayta-López E, et al. A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2. Front Cell Infect Microbiol. 2021;11:653616. PubMed PMID: 34268131.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A Multiplex and Colorimetric Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of Novel SARS-CoV-2. AU - Juscamayta-López,Eduardo, AU - Valdivia,Faviola, AU - Horna,Helen, AU - Tarazona,David, AU - Linares,Liza, AU - Rojas,Nancy, AU - Huaringa,Maribel, Y1 - 2021/06/29/ PY - 2021/01/14/received PY - 2021/06/11/accepted PY - 2021/7/16/entrez PY - 2021/7/17/pubmed PY - 2021/7/21/medline KW - COVID-19 KW - Multiplex RT-LAMP KW - SARS-CoV-2 KW - diagnostic accuracy KW - primary health care KW - rapid molecular tests KW - resource-limited settings KW - timely diagnosis SP - 653616 EP - 653616 JF - Frontiers in cellular and infection microbiology JO - Front Cell Infect Microbiol VL - 11 N2 - 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. SN - 2235-2988 UR - https://www.unboundmedicine.com/medline/citation/34268131/A_Multiplex_and_Colorimetric_Reverse_Transcription_Loop_Mediated_Isothermal_Amplification_Assay_for_Sensitive_and_Rapid_Detection_of_Novel_SARS_CoV_2_ L2 - https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/34268131/ DB - PRIME DP - Unbound Medicine ER -