Locked nucleic acid molecular beacon for multiplex detection of loop mediated isothermal amplification

Abstract

Loop mediated isothermal amplification (LAMP) holds incredible promise for point – of – care molecular diagnostics because of its high sensitivity and isothermal amplification behaviour. The issues related to the spurious non-specific amplification caused by the template independent amplification of primers itself causes false positive detection. This can be exacerbated by the common indirect methods used for detection of LAMP. Developing robust and specific detection methods for LAMP is a challenge due to the complex nature of the LAMP amplicons. To see wider adaptation of LAMP, we employ locked nucleic acid bases in molecular beacon to provide the structural stability to the hairpin probes that enable specific and multiplex detection of LAMP. Locked nucleic acid (LNA) modification provides ultra – high thermal stability to the molecular beacons resulting in negligible background fluorescence in the closed state. In this study, various combinations of LNA modifications in the stem and loop region were used and characterized for their thermal stability and influence on hybridization efficiency. The sequence specificity and ultra – high thermal stability of the LNA bases was exploited to develop a multiplex LAMP assay for detection of clinically important antibiotic resistance in S.aureus in 30 min. Multiplex approaches hold a significant advancement in LAMP and would find widespread applications in molecular diagnostics.