TaqMan-MGB荧光探针法检测北京地区幽门螺杆菌gyrA基因第87位密码子和第91位密码子耐药突变

Employment of TaqMan-MGB fluorescent probe real-time PCR for detection of the quinolones resistant mutations of Helicobacter pylori in Beijing area

目的采用TaqM an-MGB荧光探针法检测北京地区幽门螺杆菌喹诺酮类耐药位点gyr A基因第87位密码子和第91位密码子突变情况,比较与药敏试验和一代测序法的一致性。方法收集北京大学第一医院消化内科门诊尿素酶试验阳性的患者252例,所有患者均使用MGB荧光探针法和一代测序法对喹诺酮类耐药位点gyr A基因第87位密码子和第91位密码子进行检测。其中158例患者同时进行药敏试验检测,同时分析耐药位点突变发生率与耐药表型的关系。结果 158例传统培养联合药敏法检测的标本中成功培养出85例,占53.8%,其中耐药型菌株40例,敏感型菌株42例。而MGB荧光探针法成功检出155例,占98.1%,其中野生型93例,突变型62例。在药敏试验检测出的85例阳性标本中,两种方法检出的符合率为94.1%。252例标本中,一代测序法成功检测出234例,占92.9%,其中野生型160例,突变型74例。在74例突变型标本中,含gyr A基因第87位密码子突变的标本占60.8%,含gyr A基因第91位密码子突变的标本占39.2%。MGB荧光探针法成功检测出250例,占99.2%,其中野生型161例,突变型89例。在89例突变组标本中,含gyr A基因第87位密码子突变的标本占64.0%,含gyr A基因第91位密码子突变的标本占36.0%。一代测序法和MGB荧光探针法在区分是否含有突变上的符合率为95.3%。结论 Taq Man-MGB荧光探针法可快速、敏感地检测患者胃黏膜标本中幽门螺杆菌喹诺酮类耐药位点gyr A基因第87位密码子和第91位密码子的突变情况,与药敏试验和一代测序法有较高的符合性。

Objective To use the method based on TaqMan-MGB fluorescent probe real-time PCR for detection of the prevalence of primary quinolones-resistance and the distribution involved Helicobacterpylori mutations in Beijing area. Methods A total of 252 Helicobacter pylori-positive patients undergoing gastroscope in the First Hospital of Beijing University was enrolled. Helicobacter pylori infection was identified by histology and rapid urease test. The quinolones resistant status of gastric mucosa tissues from 158 patients was detected by traditional culture with E-test, TaqMan-MGB fluorescent probe real-time PCR and Sanger sequencing. The detection for the rest of 94 patients was performed by only TaqMan-MGB fluorescent probe real-time PCR and Sanger sequencing. Results Among the 158 cases using both traditional culture with E-test and TaqMan-MGB fluorescent probe real-time PCR, 53.8% and 98.1% of Helicobacterpylori positive cases were detected by traditional assay and TaqMan-MGB method, respectively. Total of 94.1% consistency were showed in both assays in 85 positive case with traditional culture. In the 252 cases, 99.2% and 92.9% Helicobacter pylori positive cases was detected using TaqMan-MGB fluorescent probe real-time PCR and Sanger sequencing, respectively. Coincidence rate of 95.3% was found in both assays to distinguish mutations. For the 234 positive cases tested by Sanger sequencing, the mutations were detected in 74 cases （including 60.8% mutations with gyrA 87 codon or 39.2% mutations with gyrA 91 codon）, while wild type was detected in 160 cases. For the 250 positive cases tested by TaqMan-MGB fluorescent probe real-time PCR, mutations were detected in 89 cases （including 64.0% mutations with gyrA 87 eodon or 36.0% mutations with gyrA 91 codon）, while wild type （without gyrA 87 eodon mutation or gyrA 91 codon mutation） was detectable in 161 cases. Conclusion The method based on TaqMan-MGB fluorescent probe real-time PCR could be applied for rapid and sensitive clinical detection of quinolones resistance to Helicobacterpylori.