Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2

doi:10.7717/peerj.9278

. 2020 Jun 3:8:e9278.

doi: 10.7717/peerj.9278. eCollection 2020.

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2

Affiliations

¹ Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
² Department of Pathology, Hospital Sungai Buloh, Selangor, Malaysia.
³ Clinical Research Centre, Hospital Sungai Buloh, Selangor, Malaysia.
⁴ Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
⁵ Institute for Clinical Research (ICR), National Institutes of Health (NIH), Ministry of Health Malaysia, Putrajaya, Malaysia.

PMID: 32547882
PMCID: PMC7275676
DOI: 10.7717/peerj.9278

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2

Yee Ling Lau et al. PeerJ. 2020.

. 2020 Jun 3:8:e9278.

doi: 10.7717/peerj.9278. eCollection 2020.

Authors

Affiliations

¹ Department of Parasitology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
² Department of Pathology, Hospital Sungai Buloh, Selangor, Malaysia.
³ Clinical Research Centre, Hospital Sungai Buloh, Selangor, Malaysia.
⁴ Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia.
⁵ Institute for Clinical Research (ICR), National Institutes of Health (NIH), Ministry of Health Malaysia, Putrajaya, Malaysia.

PMID: 32547882
PMCID: PMC7275676
DOI: 10.7717/peerj.9278

Abstract

Background: Highly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid.

Methods: A rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection.

Results: This assay detected one copy/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.

Keywords: COVID-19; Diagnosis; RT-LAMP; Rapid detection.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

**Figure 1. Assessment of RT-LAMP results based on hydroxynaphthol blue visualization of color change.**
A positive reaction indicated by sky blue color is seen in tubes 1–2 and the positive control (PC), while a violet color indicates a negative reaction in tubes 3–4 and the non-template negative control (NC).

**Figure 2. RT-qPCR results for SARS-CoV-2 detection.**
The chart was generated by plotting relative fluorescence (RFU) vs. cycle number, with each colored line representing one sample. Seven positive samples and a positive control (PC) are shown with cycle threshold levels between 21 and 28 cycles.

See this image and copyright information in PMC

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References

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[1] Cardoso TC, Ferrari HF, Bregano LC, Silva-Frade C, Rosa ACG, Andrade AL. Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye. Molecular and Cellular Probes. 2010;24(6):415–417. doi: 10.1016/j.mcp.2010.08.003. - DOI - PMC - PubMed

[2] Cardoso TC, Ferrari HF, Bregano LC, Silva-Frade C, Rosa ACG, Andrade AL. Visual detection of turkey coronavirus RNA in tissues and feces by reverse-transcription loop-mediated isothermal amplification (RT-LAMP) with hydroxynaphthol blue dye. Molecular and Cellular Probes. 2010;24(6):415–417. doi: 10.1016/j.mcp.2010.08.003. - DOI - PMC - PubMed

[3] Centers for Disease Control Real-time RT-PCR panel for detection 2019-novel Coronavirus. 2020. https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-for-det.... [22 March 2020]. https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-for-det...

[4] Centers for Disease Control Real-time RT-PCR panel for detection 2019-novel Coronavirus. 2020. https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-for-det.... [22 March 2020]. https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-for-det...

[5] Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, Bleicker T, Brünink S, Schneider J, Schmidt ML, Mulders DG, Haagmans BL, Van Der Veer B, Van Den Brink S, Wijsman L, Goderski G, Romette JL, Ellis J, Zambon M, Peiris M, Goossens H, Reusken C, Koopmans MP, Drosten C. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance. 2020;25(3):2000045. doi: 10.2807/1560-7917.ES.2020.25.3.2000045. - DOI - PMC - PubMed

[6] Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, Bleicker T, Brünink S, Schneider J, Schmidt ML, Mulders DG, Haagmans BL, Van Der Veer B, Van Den Brink S, Wijsman L, Goderski G, Romette JL, Ellis J, Zambon M, Peiris M, Goossens H, Reusken C, Koopmans MP, Drosten C. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Eurosurveillance. 2020;25(3):2000045. doi: 10.2807/1560-7917.ES.2020.25.3.2000045. - DOI - PMC - PubMed

[7] Hu S-F, Li M, Zhong L-L, Lu S-M, Liu Z-X, Pu J-Y, Wen J-S, Huang X. Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection and differentiation of dengue virus serotypes 1–4. BMC Microbiology. 2015;15(1):265. doi: 10.1186/s12866-015-0595-1. - DOI - PMC - PubMed

[8] Hu S-F, Li M, Zhong L-L, Lu S-M, Liu Z-X, Pu J-Y, Wen J-S, Huang X. Development of reverse-transcription loop-mediated isothermal amplification assay for rapid detection and differentiation of dengue virus serotypes 1–4. BMC Microbiology. 2015;15(1):265. doi: 10.1186/s12866-015-0595-1. - DOI - PMC - PubMed

[9] Kwallah A, Inoue S, Muigai AW, Kubo T, Sang R, Morita K, Mwau M. A real-time reverse transcription loop-mediated isothermal amplification assay for the rapid detection of yellow fever virus. Journal of Virological Methods. 2013;193(1):23–27. doi: 10.1016/j.jviromet.2013.05.004. - DOI - PubMed

[10] Kwallah A, Inoue S, Muigai AW, Kubo T, Sang R, Morita K, Mwau M. A real-time reverse transcription loop-mediated isothermal amplification assay for the rapid detection of yellow fever virus. Journal of Virological Methods. 2013;193(1):23–27. doi: 10.1016/j.jviromet.2013.05.004. - DOI - PubMed

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Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2

Affiliations

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Similar articles

Cited by

References

Grants and funding

LinkOut - more resources

Full Text Sources

Miscellaneous