Objective To establish a duplex fluorescent PCR for detecting the point mutation of moxifloxacin resistant gene gyrA of Clostridioides difficile.
Methods The specific primers of gyrA and different TaqMan-MGB probes were designed, which targets moxifloxacin sensitive strains, to optimize the duplex fluorescent PCR to detect ACT and ATT mutation sites simultaneously. This duplex fluorescent PCR system was further evaluated. The sensitivity, specificity and repeatability of the method were evaluated.
Results The detection limits of this assay for ACT and ATT mutations in gene gyrA were 4 copies/μL and 5 copies/μL, respectively. The results were negative for other common intestinal bacteria. The intra and inter group coefficients of variation were all less than 5% in repeatability test. And the result of this method was highly consistent with the sequencing results of gene gyrA (k = 0.98).
Conclusion This duplex fluorescent PCR is sensitive, specific and reproducible for the detection of the point mutation of moxifloxacin resistant gene gyrA of C. difficile, which is effective to identify the moxifloxacin resistant or sensitive C. difficile, and aid the recognizing of the hyper virulent BI/NAP1/RT027.