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Info. Vol.13 - No.4 (2019.12.20)
Title A Simple and Multiplex Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of SARS-CoV
Authors Jin Hwa Kim1,†, Minhee Kang1,2,†,*, Eunkyoung Park1,2, Doo Ryeon Chung3,4,5,*, Jiyeon Kim6,7 & Eung Soo Hwang6,7
Institutions 1Biomedical Engineering Research Center, Smart Healthcare Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
2Department of Medical Device Management and Research, SAIHST, Sungkyunkwan University, Seoul, Republic of Korea
3Center for Infection Prevention and Control, Samsung Medical Center, Seoul, Republic of Korea
4Asia Pacific Foundation for Infectious Diseases (APFID), Seoul, Republic of Korea
5Division of Infectious Diseases, Department of Internal Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea
6Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, South Korea
7Institute of Endemic Diseases, Seoul National University Medical Research Center, Seoul, South Korea
These authors contrilbuted equally.
*Correspondence and requests for materials should be addressed to M.H. Kang (minhee.kang@samsung.com) and D.R. Chung ( minikang@skku.edu)
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.
Keyword SARS-CoV, Loop-mediated isothermal amplification, Colorimetric detection, Point-of-care test
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