A Mismatch-Tolerant Reverse Transcription Loop-Mediated Isothermal Amplification Method and Its Application on Simultaneous Detection of All Four Serotype of Dengue Viruses

Abstract

A Mismatch-Tolerant LAMP a visual determination method by adding colorimetric dyes. Because of its simplicity, all LAMP-based diagnostic assays may be easily updated to the newly improved version. The novel mismatch-tolerant LAMP method represents a simple, sensitive and promising approach for molecular diagnosis of highly variable viruses, and it is especially suited for application in resource-limited settings.

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

“…and then selected the primers having high amplification efficiency using the conventional RT-LAMP method. The RdRp primers showed higher amplification efficiency, and were selected to establish the SARS-CoV-2 detection assay using the mismatch-tolerant RT-LAMP method Zhou et al 2019). The RdRp primer information is shown in Fig.…”

“…Recently, we developed a mismatchtolerant LAMP method by adding 0.15 U of high-fidelity DNA polymerase . The main difference between the mismatch-tolerant LAMP and the conventional ones is the inclusion of an additional amount of highfidelity DNA polymerase in the former, which largely improves the sensitivity and reaction speed Zhou et al 2019). Using this mismatch-tolerant technique, we developed a one-step single-tube RT-LAMP assay for detection of SARS-CoV-2.…”

A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

“…One promising POCT method, LAMP, has been used for the detection of various pathogens because of its high sensitivity, rapid reaction speed, relatively simple operation, and visual determination capability [14,15,22,23]. We recently upgraded the LAMP method to a mismatch-tolerant version [14,15]. Compared to the conventional LAMP method, the novel version contains an additional 0.15 U of high-fidelity DNA polymerase, which confers upon it a higher applicability to highly variable viruses, and a 10-15 min faster reaction speed.…”

“…The assay can be performed at 63 • C for 50 min in a real-time PCR instrument, for real-time monitoring using fluorescent dye, or for 40 min in a regular PCR machine or heating block (e.g., dry incubator or water bath), for visual detection using pH-sensitive indicator dyes. In the real-time monitoring system, SYTO9 is used as a fluorescent dye which has a minimal inhibitory effect on LAMP amplification [10,14]. In the visual detection system, cresol red is used as a pH indicator dye because of a clear color contrast between negative (red) and positive (yellow) reactions after 40 min of incubation at 63 • C; a color change from red to orange or yellow is defined as SARS-CoV-2 positive [24].…”

“…LAMP is a simple, fast (within 50 min) and sensitive isothermal amplification technique, that is less dependent on sophisticated equipment, and has been widely used for the development of POCT techniques for the detection of viruses and other pathogens in field and/or resource-limited settings [13]. Recently, we developed a mismatch-tolerant version of the LAMP method that has higher sensitivity and a faster reaction speed than the conventional ones [14,15]. In this study, we applied the mismatch-tolerant technique to develop novel real-time fluorescent and visual RT-LAMP assays for the rapid and sensitive detection of SARS-CoV-2 RNA, and evaluated the novel assays using clinical samples.…”