Ultrasensitive detection of drug resistance genes in Mycobacterium tuberculosis based on molecular crowding conditions coupled with padlock probe amplification

In this work, we developed a novel approach driven by molecular crowding conditions combined with padlock probe amplification (MCC-PPA) to detect the rpoB 531 (TCG > TTG) mutation in Mycobacterium tuberculosis associated with rifampicin resistance. Polyethylene glycol (PEG) was employed to create molecular crowding conditions in the ligation of the padlock probe, enhancing padlock probe cyclization and increasing the amplification efficiency. This proposed assay displayed a broader linear range (10 aM-1 nM) and a lower detection limit (4.52 aM) within 1.5 hours. The sensitivity of this assay was enhanced 400-fold over the PEG-free padlock probe amplification assay. When evaluated on 105 clinical specimens, including 21 with the rpoB 531 mutation and 84 without this mutation, the assay demonstrated 100% sensitivity and 100% specificity relative to DNA sequencing results. When compared against fluorescence PCR melting curve analysis, the clinical sensitivity for detecting Mycobacterium tuberculosis rifampicin resistance gene rpoB 531 TCG > TTG mutation was also 100%. These findings highlight our proposed assay's strong applicability for the detection of antimicrobial resistance mutations in Mycobacterium tuberculosis.