Establishment of a triplex real-time quantitative fluorescence PCR method for detecting drug resistance genes mcr-1, bla, and vanA

This study establishes a triplex real-time quantitative fluorescence polymerase chain reaction (PCR) method for simultaneous and rapid MCR-1, vanA, and blaNDM-1 detection. Different primer-probe combinations for mcr-1 , vanA , and bla NDM-1 gene sequences were designed and manually screened to obtain the optimal combination. The optimal triplex quantitative fluorescence PCR system and amplification conditions were obtained by optimizing the reaction conditions, such as the annealing temperature and primer-probe concentration. The sensitivity, reproducibility, and specificity were verified using the established real-time quantitative fluorescence PCR method, and actual samples were used for detection. The results show that this method has strong specificity, high sensitivity, and good reproducibility. The standard curves of the three target genes showed good linear relationships with linear correlation coefficients (R 2 ) of 0.990, 0.996, and 0.993. The detection limit of plasmid standards for three target genes was 10 2 copies/μL. The relative standard deviations within and between groups were less than 3.0 %. This study's real-time quantitative fluorescence PCR method accurately detected three genes: mcr-1 , vanA , and bla NDM-1 .