检测艰难梭菌耐莫西沙星<i>gyrA</i>基因点突变的双重荧光PCR方法的建立和评价

贾筱溪; 张文竹; 王媛媛; 李文革; 卢金星; 吴媛; 马超锋

doi:10.3784/jbjc.202102260090

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

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

摘要

摘要:
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。
结果该方法对莫西沙星耐药株和敏感株gyrA基因突变位点的检测下限分别可达4 copies/μL和5 copies/μL，对艰难梭菌以外其他常见肠道菌群的检测均为阴性。重复性检测结果显示，批间和批内变异系数均<5%，该方法的检测结果与测序结果一致性高（k=0.98）。
结论针对耐莫西沙星艰难梭菌gyrA基因点突变建立的双重荧光定量PCR方法，灵敏且具有很高的特异性和重复性，可有效鉴别艰难梭菌莫西沙星敏感株和耐药株，并可辅助识别流行株BI/NAP1/RT027的暴发。

Abstract:
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.

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