A novel fluorescence method based on loop-mediated isothermal amplification and universal molecular beacon in Mycobacterium tuberculosis detection

This article illustrated a novel method combining loop-mediated isothermal amplification (LAMP) with molec-ular beacon (MB) to specifically detect Mycobacterium tuberculosis (MTB) IS6110 gene. In this method, we designed a double-stranded DNA tail (S and Sc) at the 5' ends of the loop primers (LF and LB). When the target was present, the Sc would be continually generated by strand displacement reactions to hybridize with MB, then fluorescence signals were detected. Negligible background fluorescence would be detected in the absence of target due to the unparalleled advantage of MB. Under the optimized conditions (6 mM Mg2+, 0.48 U/mu L Bst 2.0 polymerase, 0.2 mu M MB and 2:1 annealing ratio of S-LF/LB to Sc), the detection limit of this assay was 4.3 x 104 copies mu L-1. The whole detection time was reduced to less than 30 min with a low cost of $2.37 per sample. We also successfully applied this method in real samples detection, which meant that this specific, fast and cost-effective method has the potential in clinical diagnosis of Tuberculosis (TB).