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

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. © The Korean BioChip Society and Springer 2019.

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.