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

• Published in: Biochip journal Vol. 13; no. 4; pp. 341 - 351
• Main Authors: Kim, Jin Hwa, Kang, Minhee, Park, Eunkyoung, Chung, Doo Ryeon, Kim, Jiyeon, Hwang, Eung Soo
• Format: Journal Article
• Language: English
• Published: Seoul The Korean Society for Applied Biological Chemistry 01.12.2019; Springer Nature B.V; The Korean BioChip Society (KBCS); 한국바이오칩학회
• Subjects: Assaying; Biomedical Engineering and Bioengineering; Biotechnology; Chemistry; Colorimetry; Coronaviruses; Developing countries; Diagnosis; Epidemics; Fluorescence; Gene amplification; Gene sequencing; Genes; Genomes; Infections; LDCs; Multiplexing; Nucleocapsids; Original; Original Article; Polymerase chain reaction; Real time; Respiratory diseases; Serology; Severe acute respiratory syndrome; Target detection; Viral diseases; 생물공학; Loop-mediated isothermal amplification; Point-of-care test; SARS-CoV; Colorimetric detection
• ISSN: 1976-0280; 2092-7843
• DOI: 10.1007/s13206-019-3404-3

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 10 4 -10 5 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 10 5 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.