A Rapid, Simple, Inexpensive, and Mobile Colorimetric Assay COVID-19-LAMP for Mass On-Site Screening of COVID-19

Franklin Wang-Ngai Chow, Tony Tat-Yin Chan, Anthony Raymond Tam, Suhui Zhao, Weiming Yao, Joshua Fung, Flora Ka-Kei Cheng, George Chi-Shing Lo, Stella Chu, Kam Leng Aw-Yong, James Yat-Man Tang, Chi-Ching Tsang, Hayes Kam-Hei Luk, Antonio Cheuk-Pui Wong, Kenneth Sze-Ming Li, Longchao Zhu, Zirong He, Emily Wan Ting Tam, Tom Wai-Hin Chung, Sally Cheuk Ying Wong, Tak-Lun Que, Kitty Sau-Chun Fung, David Christopher Lung, Alan Ka-Lun Wu, Ivan Fan-Ngai Hung, Patrick Chiu-Yat Woo, Susanna Kar-Pui Lau, Franklin Wang-Ngai Chow, Tony Tat-Yin Chan, Anthony Raymond Tam, Suhui Zhao, Weiming Yao, Joshua Fung, Flora Ka-Kei Cheng, George Chi-Shing Lo, Stella Chu, Kam Leng Aw-Yong, James Yat-Man Tang, Chi-Ching Tsang, Hayes Kam-Hei Luk, Antonio Cheuk-Pui Wong, Kenneth Sze-Ming Li, Longchao Zhu, Zirong He, Emily Wan Ting Tam, Tom Wai-Hin Chung, Sally Cheuk Ying Wong, Tak-Lun Que, Kitty Sau-Chun Fung, David Christopher Lung, Alan Ka-Lun Wu, Ivan Fan-Ngai Hung, Patrick Chiu-Yat Woo, Susanna Kar-Pui Lau

Abstract

To control the COVID-19 pandemic and prevent its resurgence in areas preparing for a return of economic activities, a method for a rapid, simple, and inexpensive point-of-care diagnosis and mass screening is urgently needed. We developed and evaluated a one-step colorimetric reverse-transcriptional loop-mediated isothermal amplification assay (COVID-19-LAMP) for detection of SARS-CoV-2, using SARS-CoV-2 isolate and respiratory samples from patients with COVID-19 (n = 223) and other respiratory virus infections (n = 143). The assay involves simple equipment and techniques and low cost, without the need for expensive qPCR machines, and the result, indicated by color change, is easily interpreted by naked eyes. COVID-19-LAMP can detect SARS-CoV-2 RNA with detection limit of 42 copies/reaction. Of 223 respiratory samples positive for SARS-CoV-2 by qRT-PCR, 212 and 219 were positive by COVID-19-LAMP at 60 and 90 min (sensitivities of 95.07% and 98.21%) respectively, with the highest sensitivities among nasopharyngeal swabs (96.88% and 98.96%), compared to sputum/deep throat saliva samples (94.03% and 97.02%), and throat swab samples (93.33% and 98.33%). None of the 143 samples with other respiratory viruses were positive by COVID-19-LAMP, showing 100% specificity. Samples with higher viral load showed shorter detection time, some as early as 30 min. This inexpensive, highly sensitive and specific COVID-19-LAMP assay can be useful for rapid deployment as mobile diagnostic units to resource-limiting areas for point-of-care diagnosis, and for unlimited high-throughput mass screening at borders to reduce cross-regional transmission.

Keywords: COVID-19; RT-LAMP; SARS-CoV-2; diagnosis; mass screening; mobile Diagnostic; on-site screening.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Illustration of a small van-sized mobile COVID-19-LAMP diagnostic unit. A drawn-to-scale layout of a van-sized mobile COVID-19-LAMP diagnostic unit, with sample processing and LAMP reactions compartments have been illustrated. A cargo van/lorry can be modified quickly to become a mobile diagnostic unit for rapid deployment in any region.
Figure 2
Figure 2
Box and whisker plot of COVID-19-LAMP results. The COVID-19-LAMP results of each SARS-CoV-2 qRT-PCR positive samples have been illustrated in the box and whisker plot at (A): 30 min, (B): 60 min, (C): 90 min with corresponding qRT-PCR Ct values of samples.
Figure 3
Figure 3
COVID-19-LAMP evaluation profile. SARS-CoV-2 qRT-PCR positive and negative respiratory samples are used for evaluation of COVID-19-LAMP.

References

    1. Coronaviridae Study Group of the International Committee on Taxonomy of Viruses The species Severe acute respiratory syndrome-related coronavirus: Classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 2020;5:536–544. doi: 10.1038/s41564-020-0695-z.
    1. Verity R., Okell L.C., Dorigatti I., Winskill P., Whittaker C., Imai N., Cuomo-Dannenburg G., Thompson H., Walker P.G.T., Fu H., et al. Estimates of the severity of coronavirus disease 2019: A model-based analysis. Lancet Infect. Dis. 2020 doi: 10.1016/S1473-3099(20)30243-7.
    1. Woo P.C., Lau S.K., Tsoi H.W., Chan K.H., Wong B.H., Che X.Y., Tam V.K., Tam S.C., Cheng V.C., Hung I.F., et al. Relative rates of non-pneumonic SARS coronavirus infection and SARS coronavirus pneumonia. Lancet. 2004;363:841–845. doi: 10.1016/S0140-6736(04)15729-2.
    1. Lau S.K., Woo P.C., Li K.S., Huang Y., Tsoi H.W., Wong B.H., Wong S.S., Leung S.Y., Chan K.H., Yuen K.Y. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc. Natl. Acad. Sci. USA. 2005;102:14040–14045. doi: 10.1073/pnas.0506735102.
    1. Lau S.K., Feng Y., Chen H., Luk H.K., Yang W.H., Li K.S., Zhang Y.Z., Huang Y., Song Z.Z., Chow W.N., et al. Severe Acute Respiratory Syndrome (SARS) Coronavirus ORF8 Protein Is Acquired from SARS-Related Coronavirus from Greater Horseshoe Bats through Recombination. J. Virol. 2015;89:10532–10547. doi: 10.1128/JVI.01048-15.
    1. Luk H.K.H., Li X., Fung J., Lau S.K.P., Woo P.C.Y. Molecular epidemiology, evolution and phylogeny of SARS coronavirus. Infect. Genet. Evol. 2019;71:21–30. doi: 10.1016/j.meegid.2019.03.001.
    1. Woo P.C., Lau S.K., Wernery U., Wong E.Y., Tsang A.K., Johnson B., Yip C.C., Lau C.C., Sivakumar S., Cai J.P., et al. Novel betacoronavirus in dromedaries of the Middle East, 2013. Emerg. Infect. Dis. 2014;20:560–572. doi: 10.3201/eid2004.131769.
    1. Andersen K.G., Rambaut A., Lipkin W.I., Holmes E.C., Garry R.F. The proximal origin of SARS-CoV-2. Nat. Med. 2020;26:450–452. doi: 10.1038/s41591-020-0820-9.
    1. Sheridan C. Fast, portable tests come online to curb coronavirus pandemic. Nat. Biotechnol. 2020 doi: 10.1038/d41587-020-00010-2.
    1. Xu Y., Li X., Zhu B., Liang H., Fang C., Gong Y., Guo Q., Sun X., Zhao D., Shen J., et al. Characteristics of pediatric SARS-CoV-2 infection and potential evidence for persistent fecal viral shedding. Nat. Med. 2020;26:502–505. doi: 10.1038/s41591-020-0817-4.
    1. To K.K., Tsang O.T., Leung W.S., Tam A.R., Wu T.C., Lung D.C., Yip C.C., Cai J.P., Chan J.M., Chik T.S., et al. Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: An observational cohort study. Lancet Infect. Dis. 2020 doi: 10.1016/S1473-3099(20)30196-1.
    1. Udugama B., Kadhiresan P., Kozlowski H.N., Malekjahani A., Osborne M., Li V.Y.C., Chen H., Mubareka S., Gubbay J.B., Chan W.C.W. Diagnosing COVID-19: The Disease and Tools for Detection. ACS Nano. 2020 doi: 10.1021/acsnano.0c02624.
    1. Li X., Geng M., Peng Y., Meng L., Lu S. Molecular immune pathogenesis and diagnosis of COVID-19. J. Pharm. Anal. 2020 doi: 10.1016/j.jpha.2020.03.001.
    1. Konrad R., Eberle U., Dangel A., Treis B., Berger A., Bengs K., Fingerle V., Liebl B., Ackermann N., Sing A. Rapid establishment of laboratory diagnostics for the novel coronavirus SARS-CoV-2 in Bavaria, Germany, February 2020. Eurosurveillance. 2020;25:2000173. doi: 10.2807/1560-7917.ES.2020.25.9.2000173.
    1. Zhai P., Ding Y., Wu X., Long J., Zhong Y., Li Y. The epidemiology, diagnosis and treatment of COVID-19. Int. J. Antimicrob. Agents. 2020:105955. doi: 10.1016/j.ijantimicag.2020.105955.
    1. Pfefferle S., Reucher S., Norz D., Lutgehetmann M. Evaluation of a quantitative RT-PCR assay for the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system. Eurosurveillance. 2020;25:2000152. doi: 10.2807/1560-7917.ES.2020.25.9.2000152.
    1. Kang S., Peng W., Zhu Y., Lu S., Zhou M., Lin W., Wu W., Huang S., Jiang L., Luo X., et al. Recent progress in understanding 2019 novel coronavirus (SARS-CoV-2) associated with human respiratory disease: Detection, mechanisms and treatment. Int. J. Antimicrob. Agents. 2020:105950. doi: 10.1016/j.ijantimicag.2020.105950.
    1. Lee S.H., Baek Y.H., Kim Y.H., Choi Y.K., Song M.S., Ahn J.Y. One-Pot Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP) for Detecting MERS-CoV. Front. Microbiol. 2016;7:2166. doi: 10.3389/fmicb.2016.02166.
    1. 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 Microbiol. 2015;15:265. doi: 10.1186/s12866-015-0595-1.
    1. Rajko-Nenow P., Flannery J., Arnold H., Howson E.L.A., Darpel K., Stedman A., Corla A., Batten C. A rapid RT-LAMP assay for the detection of all four lineages of Peste des Petits Ruminants Virus. J. Virol. Methods. 2019;274:113730. doi: 10.1016/j.jviromet.2019.113730.
    1. Lopez-Jimena B., Bekaert M., Bakheit M., Frischmann S., Patel P., Simon-Loriere E., Lambrechts L., Duong V., Dussart P., Harold G., et al. Development and validation of four one-step real-time RT-LAMP assays for specific detection of each dengue virus serotype. PLoS Negl. Trop. Dis. 2018;12:e0006381. doi: 10.1371/journal.pntd.0006381.
    1. Ashraf W., Unger H., Haris S., Mobeen A., Farooq M., Asif M., Khan Q.M. Genetic detection of peste des petits ruminants virus under field conditions: A step forward towards disease eradication. BMC Vet. Res. 2017;13:34. doi: 10.1186/s12917-016-0940-0.
    1. Shirato K., Semba S., El-Kafrawy S.A., Hassan A.M., Tolah A.M., Takayama I., Kageyama T., Notomi T., Kamitani W., Matsuyama S., et al. Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus. J. Virol. Methods. 2018;258:41–48. doi: 10.1016/j.jviromet.2018.05.006.
    1. Li H., Li K., Bi Z., Gu J., Song D., Lei D., Luo S., Huang D., Wu Q., Ding Z., et al. Development of a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of porcine pegivirus. J. Virol. Methods. 2019;270:59–65. doi: 10.1016/j.jviromet.2019.04.019.
    1. Notomi T., Okayama H., Masubuchi H., Yonekawa T., Watanabe K., Amino N., Hase T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28:E63. doi: 10.1093/nar/28.12.e63.

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