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. 2019 Nov 11;13(4):341–351. doi: 10.1007/s13206-019-3404-3

A Simple and Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of SARS-CoV

Jin Hwa Kim 1,#, Minhee Kang 1,2,✉,#, Eunkyoung Park 1,2, Doo Ryeon Chung 3,4,5,✉, Jiyeon Kim 6,7, Eung Soo Hwang 6,7
PMCID: PMC7097549  PMID: 32226589

Abstract

The current diagnosis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) relies on laboratory-based tests since its clinical features are nonspecific, unlike other respiratory pathogens. Therefore, the development of a rapid and simple method for on-site detection of SARS-CoV is crucial for the identification and prevention of future SARS outbreaks. In this study, a simple colorimetric and multiplex loop-mediated isothermal amplification (LAMP) assay was developed to rapid screening of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). It can be visually detected based on color change and monitored in real-time with fluorescent signals. The performance of this assay, based on six primers targeting open reading frame (ORF1b) and nucleocapsid (N) genes located in different regions of the SARS-CoV, was compared with real-time RT-PCR assay using various concentrations of target genes. The detection limit of the LAMP assay was comparable to that of real-time RT-PCR assay and therefore a few target RNA to 104-105 copies could be detected within a short period of time (20–25 min). In addition, we established a multiplex real-time LAMP assay to simultaneously detect two target regions within the SARS-CoV genome. Two target sequences were amplified by specific primers in the same reaction tube and revealed that it was able to detect down to 105 copies. The standard curve had a linear relationship with similar amplification efficiencies. The LAMP assay results in shorter “sample-to-answer” time than conventional PCR method. Therefore, it is suitable not only for diagnosis of clinical test, but also for surveillance of SARS virus in developing countries.

Electronic Supplementary Material

Supplementary material is available for this article at 10.1007/s13206-019-3404-3 and is accessible for authorized users.

Keywords: SARS-CoV, Loop-mediated isothermal amplification, Colorimetric detection, Point-of-care test

Electronic supplementary material

13206_2019_3404_MOESM1_ESM.pdf (1.7MB, pdf)

Supplementary Table 1. The target sequences for amplification.

Supplementary Figure 1S. Color change in LAMP Assay. Primer set II (six primers) for N genes was used for amplification. A, Before amplification; B, After amplification. An orange color indicates a positive and a neutral pink color indicates a negative reaction. C, Gel electrophoresis of LAMP products (POS: positive; NEG: negative; C: control).

Supplementary Figure 2S. LAMP primer set optimization with two LAMP primer set. A, four primers (F3, B3, FIP, BIP); and B, six primers (added Loop F and Loop B primers to A primer set). Detection of LAMP products by (a) naked eye with color change and (b) agarose gel electrophoresis (Top: negative control (non-template control, NTC); Bottom: positive control with 50 ng of genomic DNA). An orange color indicates a positive and a neutral pink color indicates a negative reaction. (b) Agarose electrophoresis results of LAMP assay (temperature gradient from 50°C (lane 2) to 72°C (lane 12), 1kb DNA ladder; lane 1). Optimal temperature was determined to be 65°C for both targets.

Acknowledgements

This research was supported by the government-wide R&D Fund for the research of infectious diseases in Republic of Korea (HG18C 0062), and the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science (2016M3 A9B6919187, 2016M3A9B6919189).

Footnotes

Conflict of Interests The authors declare no competing financial interests.

These authors contrilbuted equally.

Contributor Information

Minhee Kang, Email: minhee.kang@samsung.com.

Doo Ryeon Chung, Email: minikang@skku.edu.

References

  • 1.Peiris JSM, Lai ST, Poon LLM, Guan Y, Yam LYC, Lim W, Nicholls J, Yee WKS, Yan WW, Cheung MT, Cheng VCC, Chan KH, Tsang DNC, Yung RWH, Ng TK, Yuen KY. Coronavirus as a possible cause of severe acute respiratory syndrome. The Lancet. 2003;361(9366):1319–1325. doi: 10.1016/S0140-6736(03)13077-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Tsang Kenneth W., Ho Pak L., Ooi Gaik C., Yee Wilson K., Wang Teresa, Chan-Yeung Moira, Lam Wah K., Seto Wing H., Yam Loretta Y., Cheung Thomas M., Wong Poon C., Lam Bing, Ip Mary S., Chan Jane, Yuen Kwok Y., Lai Kar N. A Cluster of Cases of Severe Acute Respiratory Syndrome in Hong Kong. New England Journal of Medicine. 2003;348(20):1977–1985. doi: 10.1056/NEJMoa030666. [DOI] [PubMed] [Google Scholar]
  • 3.Kuiken Thijs, Fouchier Ron AM, Schutten Martin, Rimmelzwaan Guus F, van Amerongen Geert, van Riel Debby, Laman Jon D, de Jong Ton, van Doornum Gerard, Lim Wilina, Ling Ai Ee, Chan Paul KS, Tam John S, Zambon Maria C, Gopal Robin, Drosten Christian, van der Werf Sylvie, Escriou Nicolas, Manuguerra Jean-Claude, Stöhr Klaus, Peiris J S Malik, Osterhaus Albert DME. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. The Lancet. 2003;362(9380):263–270. doi: 10.1016/S0140-6736(03)13967-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Schlagenhauf Patricia, Ashra Haroon. Severe acute respiratory syndrome spreads worldwide. The Lancet. 2003;361(9362):1017. doi: 10.1016/S0140-6736(03)12843-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Kenneth J. Severe Acute Respiratory Syndrome (SARS): The new epidemic. Natl. Med. J. India. 2003;16:115–116. [PubMed] [Google Scholar]
  • 6.Ksiazek Thomas G., Erdman Dean, Goldsmith Cynthia S., Zaki Sherif R., Peret Teresa, Emery Shannon, Tong Suxiang, Urbani Carlo, Comer James A., Lim Wilina, Rollin Pierre E., Dowell Scott F., Ling Ai-Ee, Humphrey Charles D., Shieh Wun-Ju, Guarner Jeannette, Paddock Christopher D., Rota Paul, Fields Barry, DeRisi Joseph, Yang Jyh-Yuan, Cox Nancy, Hughes James M., LeDuc James W., Bellini William J., Anderson Larry J. A Novel Coronavirus Associated with Severe Acute Respiratory Syndrome. New England Journal of Medicine. 2003;348(20):1953–1966. doi: 10.1056/NEJMoa030781. [DOI] [PubMed] [Google Scholar]
  • 7.Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003, https://doi.org/www.who.int/csr/sars/country/table2004_04_21/en/
  • 8.SARS (Severe Acute Respiratory Syndrome), https://doi.org/www.who.int/ith/diseases/sars/en/
  • 9.Chan Jasper F. W., Lau Susanna K. P., To Kelvin K. W., Cheng Vincent C. C., Woo Patrick C. Y., Yuen Kwok-Yung. Middle East Respiratory Syndrome Coronavirus: Another Zoonotic Betacoronavirus Causing SARS-Like Disease. Clinical Microbiology Reviews. 2015;28(2):465–522. doi: 10.1128/CMR.00102-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Gong Shu-ran, Bao Lin-lin. The battle against SARS and MERS coronaviruses: Reservoirs and Animal Models. Animal Models and Experimental Medicine. 2018;1(2):125–133. doi: 10.1002/ame2.12017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Cheng V. C. C., Lau S. K. P., Woo P. C. Y., Yuen K. Y. Severe Acute Respiratory Syndrome Coronavirus as an Agent of Emerging and Reemerging Infection. Clinical Microbiology Reviews. 2007;20(4):660–694. doi: 10.1128/CMR.00023-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Wong S C C. Development of a quantitative assay for SARS coronavirus and correlation of GAPDH mRNA with SARS coronavirus in clinical specimens. Journal of Clinical Pathology. 2005;58(3):276–280. doi: 10.1136/jcp.2004.016592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.He Zhongping, Zhuang Hui, Zhao Chunhui, Dong Qingming, Peng Guoai, Dwyer Dominic E. Using patient-collected clinical samples and sera to detect and quantify the severe acute respiratory syndrome coronavirus (SARS-CoV) Virology Journal. 2007;4(1):32. doi: 10.1186/1743-422X-4-32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Wu Ho-Sheng, Chiu Shu-Chun, Tseng Tsan-Chang, Lin Szu-Fong, Lin Jih-Hui, Hsu Yu-Fen, Wang Mei-Ching, Lin Tsuey-Li, Yang Wen-Zieh, Ferng Tian-Lin, Huang Kai-Hung, Hsu Li-Ching, Lee Li-Li, Yang Jyh-Yuan, Chen Hour-Young, Su Shun-Pi, Yang Shih-Yan, Lin Ting-Hsiang, Su Ih-Jen. Serologic and Molecular Biologic Methods for SARS-associated Coronavirus Infection, Taiwan. Emerging Infectious Diseases. 2004;10(2):305–310. doi: 10.3201/eid1002.030731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Wang Yongzhong, Yu Li, Kong Xiaowei, Sun Leming. Application of nanodiagnostics in point-of-care tests for infectious diseases. International Journal of Nanomedicine. 2017;Volume 12:4789–4803. doi: 10.2147/IJN.S137338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Loopmediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28:E63. doi: 10.1093/nar/28.12.e63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Tomlinson J.A., Dickinson M.J., Boonham N. Detection of Botrytis cinerea by loop-mediated isothermal amplification. Letters in Applied Microbiology. 2010;51(6):650–657. doi: 10.1111/j.1472-765X.2010.02949.x. [DOI] [PubMed] [Google Scholar]
  • 18.Kubota R., Vine B. G., Alvarez A. M., Jenkins D. M. Detection of Ralstonia solanacearum by Loop-Mediated Isothermal Amplification. Phytopathology. 2008;98(9):1045–1051. doi: 10.1094/PHYTO-98-9-1045. [DOI] [PubMed] [Google Scholar]
  • 19.Mori Yasuyoshi, Nagamine Kentaro, Tomita Norihiro, Notomi Tsugunori. Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation. Biochemical and Biophysical Research Communications. 2001;289(1):150–154. doi: 10.1006/bbrc.2001.5921. [DOI] [PubMed] [Google Scholar]
  • 20.Iwamoto T., Sonobe T., Hayashi K. Loop-Mediated Isothermal Amplification for Direct Detection of Mycobacterium tuberculosis Complex, M. avium, and M. intracellulare in Sputum Samples. Journal of Clinical Microbiology. 2003;41(6):2616–2622. doi: 10.1128/JCM.41.6.2616-2622.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Hill J., Beriwal S., Chandra I., Paul V. K., Kapil A., Singh T., Wadowsky R. M., Singh V., Goyal A., Jahnukainen T., Johnson J. R., Tarr P. I., Vats A. Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Common Strains of Escherichia coli. Journal of Clinical Microbiology. 2008;46(8):2800–2804. doi: 10.1128/JCM.00152-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Zhou Qian-Jin, Wang Lei, Chen Jiong, Wang Rui-Na, Shi Yu-Hong, Li Chang-Hong, Zhang De-Min, Yan Xiao-Jun, Zhang Yan-Jun. Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals. Journal of Microbiological Methods. 2014;104:26–35. doi: 10.1016/j.mimet.2014.06.008. [DOI] [PubMed] [Google Scholar]
  • 23.Kong Qing-Ming, Lu Shao-Hong, Tong Qun-Bo, Lou Di, Chen Rui, Zheng Bin, Kumagai Takashi, Wen Li-Yong, Ohta Nobuo, Zhou Xiao-Nong. Loop-mediated isothermal amplification (LAMP): Early detection of Toxoplasma gondii infection in mice. Parasites & Vectors. 2012;5(1):2. doi: 10.1186/1756-3305-5-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Kargar Mohammad, Askari Ahzam, Doosti Abbas, Ghorbani-Dalini Sadegh. Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hepatitis C virus. Indian Journal of Virology. 2012;23(1):18–23. doi: 10.1007/s13337-012-0067-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Yoneyama Tetsuo, Kiyohara Tomoko, Shimasaki Noriko, Kobayashi Gen, Ota Yoshinori, Notomi Tsugunori, Totsuka Atsuko, Wakita Takaji. Rapid and real-time detection of hepatitis A virus by reverse transcription loop-mediated isothermal amplification assay. Journal of Virological Methods. 2007;145(2):162–168. doi: 10.1016/j.jviromet.2007.05.023. [DOI] [PubMed] [Google Scholar]
  • 26.Thai H. T. C., Le M. Q., Vuong C. D., Parida M., Minekawa H., Notomi T., Hasebe F., Morita K. Development and Evaluation of a Novel Loop-Mediated Isothermal Amplification Method for Rapid Detection of Severe Acute Respiratory Syndrome Coronavirus. Journal of Clinical Microbiology. 2004;42(5):1956–1961. doi: 10.1128/JCM.42.5.1956-1961.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Poon L. L.M. Rapid Detection of the Severe Acute Respiratory Syndrome (SARS) Coronavirus by a Loop-Mediated Isothermal Amplification Assay. Clinical Chemistry. 2004;50(6):1050–1052. doi: 10.1373/clinchem.2004.032011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Njiru Zablon Kithinji. Loop-Mediated Isothermal Amplification Technology: Towards Point of Care Diagnostics. PLoS Neglected Tropical Diseases. 2012;6(6):e1572. doi: 10.1371/journal.pntd.0001572. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Poole Catherine B., Ettwiller Laurence, Tanner Nathan A., Evans Thomas C., Wanji Samuel, Carlow Clotilde K. S. Genome Filtering for New DNA Biomarkers of Loa loa Infection Suitable for Loop-Mediated Isothermal Amplification. PLOS ONE. 2015;10(9):e0139286. doi: 10.1371/journal.pone.0139286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Shirato Kazuya, Semba Shohei, El-Kafrawy Sherif A., Hassan Ahmed M., Tolah Ahmed M., Takayama Ikuyo, Kageyama Tsutomu, Notomi Tsugunori, Kamitani Wataru, Matsuyama Shutoku, Azhar Esam Ibraheem. Development of fluorescent reverse transcription loop-mediated isothermal amplification (RT-LAMP) using quenching probes for the detection of the Middle East respiratory syndrome coronavirus. Journal of Virological Methods. 2018;258:41–48. doi: 10.1016/j.jviromet.2018.05.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Viana Giselle Maria Rachid, Silva-Flannery Luciana, Lima Barbosa Danielle Regina, Lucchi Naomi, do Valle Suiane Costa Negreiros, Farias Samela, Barbalho Nayara, Marchesini Paola, Rossi Juliana Chedid Nogaredi, Udhayakumar Venkatachalam, Póvoa Marinete Marins, de Oliveira Alexandre Macedo. Field evaluation of a real time loop-mediated isothermal amplification assay (RealAmp) for malaria diagnosis in Cruzeiro do Sul, Acre, Brazil. PLOS ONE. 2018;13(7):e0200492. doi: 10.1371/journal.pone.0200492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Sun Miaomiao, Liu Hao, Huang Junbin, Peng Jinbo, Fei Fuhua, Zhang Ya, Hsiang Tom, Zheng Lu. A Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Pectobacterium aroidearum that Causes Soft Rot in Konjac. International Journal of Molecular Sciences. 2019;20(8):1937. doi: 10.3390/ijms20081937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Rahman S. M. Mazidur, Song Hyun Beom, Jin Yan, Oh Jin-Kyoung, Lim Min Kyung, Hong Sung-Tae, Choi Min-Ho. Application of a loop-mediated isothermal amplification (LAMP) assay targeting cox1 gene for the detection of Clonorchis sinensis in human fecal samples. PLOS Neglected Tropical Diseases. 2017;11(10):e0005995. doi: 10.1371/journal.pntd.0005995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Gadkar, V.J., Goldfarb, D.M., Gantt, S. & Tilley, P.A.G. Real-time Detection and Monitoring of Loop Mediated Amplification (LAMP) Reaction Using Self-quenching and De-quenching Fluorogenic Probes. Sci. Rep. DOI 10.1038/s41598-018-23930-1 (2018). [DOI] [PMC free article] [PubMed]
  • 35.Deng H.-W., Zhou Y., Recker R.R., Johnson M.L., Li J. Fragment Size Difference between Multiplex and Singleplex PCR Products and Their Practical Implications. BioTechniques. 2000;29(2):298–308. doi: 10.2144/00292st05. [DOI] [PubMed] [Google Scholar]
  • 36.Lim Yvonne, Ng Yan, Tam James, Liu Ding. Human Coronaviruses: A Review of Virus–Host Interactions. Diseases. 2016;4(4):26. doi: 10.3390/diseases4030026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Marra M. A. The Genome Sequence of the SARS-Associated Coronavirus. Science. 2003;300(5624):1399–1404. doi: 10.1126/science.1085953. [DOI] [PubMed] [Google Scholar]
  • 38.Almazán Fernando, Sola Isabel, Zuñiga Sonia, Marquez-Jurado Silvia, Morales Lucia, Becares Martina, Enjuanes Luis. Coronavirus reverse genetic systems: Infectious clones and replicons. Virus Research. 2014;189:262–270. doi: 10.1016/j.virusres.2014.05.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Zhang Xin, Zhang He, Pu Jinji, Qi Yanxiang, Yu Qunfang, Xie Yixian, Peng Jun. Development of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 In Soil. PLoS ONE. 2013;8(12):e82841. doi: 10.1371/journal.pone.0082841. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

13206_2019_3404_MOESM1_ESM.pdf (1.7MB, pdf)

Supplementary Table 1. The target sequences for amplification.

Supplementary Figure 1S. Color change in LAMP Assay. Primer set II (six primers) for N genes was used for amplification. A, Before amplification; B, After amplification. An orange color indicates a positive and a neutral pink color indicates a negative reaction. C, Gel electrophoresis of LAMP products (POS: positive; NEG: negative; C: control).

Supplementary Figure 2S. LAMP primer set optimization with two LAMP primer set. A, four primers (F3, B3, FIP, BIP); and B, six primers (added Loop F and Loop B primers to A primer set). Detection of LAMP products by (a) naked eye with color change and (b) agarose gel electrophoresis (Top: negative control (non-template control, NTC); Bottom: positive control with 50 ng of genomic DNA). An orange color indicates a positive and a neutral pink color indicates a negative reaction. (b) Agarose electrophoresis results of LAMP assay (temperature gradient from 50°C (lane 2) to 72°C (lane 12), 1kb DNA ladder; lane 1). Optimal temperature was determined to be 65°C for both targets.

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