检测艰难梭菌耐莫西沙星gyrA基因点突变的双重荧光PCR方法的建立和评价

目的 建立一种检测艰难梭菌耐莫西沙星gyrA基因点突变的双重荧光PCR方法.方法 设计针对艰难梭菌gyrA基因的特异性引物,并针对莫西沙星耐药株和敏感株的gyrA基因突变位点设计不同的TaqMan-MGB探针,优化可同时检测ATT、ACT突变点的双重荧光定量PCR方法,验证该方法的灵敏性、特异性和重复性,并进行应用评价...

Establishment and evaluation of a duplex fluorescent PCR for detection of point mutation of moxifloxacin resistant gyrA gene in Clostridioides difficile

  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.