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目的用Taq Man-MGB荧光探针法检测北京地区幽门螺杆菌克拉霉素耐药位点A2142G、A2143G突变情况。方法收集北京大学人民医院消化科门诊尿素酶试验及病理活检均阳性的患者233例,所有患者均应用MGB荧光探针法和一代测序法对耐药位点A2142G/A2143G进行检测,其中79例患者同时进行传统培养联合药敏法检测。分析耐药位点突变发生率与患者年龄、性别和消化性溃疡的关系。结果 79例传统培养联合药敏法检测的标本中成功培养出42例,占53%(42/79),其中耐药型菌株22例,敏感型菌株20例,而MGB荧光探针法成功检出77例,占97%(77/79),其中野生型28例,突变型49例。在传统培养+药敏法培养出的42例阳性标本中,两种方法检出的符合率为86%(36/42)。233例标本中,一代测序法成功检测出214例,占92%(214/233),其中野生型138例,突变型76例。在76例突变型标本中,含A2142G突变(包括A2142G和A2142G+野生)的标本占5%(4/76),含A2143G突变(包括A2143G和A2143G+野生)的标本占95%(72/76)。MGB荧光探针法成功检测出231例,占99%(231/233),其中野生型141例,突变型90例。在90例突变型标本中,含A2142G突变的标本占14%(13/90),含A2143G突变的标本占86%(77/90)。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%(203/214)。根据MGB荧光探针法检测结果将患者分为突变组与野生组,2组在年龄和性别上差异无统计学意义(t年龄=-0.685,P=0.493;χ~2_(性别)=0.065,P=0.890),而在消化性溃疡发生率上差异存在统计学意义(χ~2=6.653,P=0.044)。结论TaqMan MGB荧光探针法可应用于临床快速、敏感地检测患者胃黏膜标本中幽门螺杆菌克拉霉素A2142G、A2143G耐药位点突变情况。
Objective To detect the mutations of A2142G and A2143G at the clarithromycin resistance site of Helicobacter pylori in Beijing using Taq Man-MGB fluorescent probe. Methods A total of 233 patients with positive urethral urease test and biopsy in Department of Gastroenterology of People’s Hospital of Peking University were collected. All patients were tested for resistance to A2142G / A2143G using MGB fluorescent probe and one-generation sequencing. 79 patients At the same time the traditional culture combined with drug sensitivity test. To analyze the relationship between the incidence of drug-resistant site mutation and patient’s age, gender and peptic ulcer. Results 42 cases (53% (42/79)) were successfully cultivated in 79 cases of traditional culture combined with drug susceptibility test, of which 22 cases were resistant to drug-resistant strains and 20 cases were sensitive strains. MGB fluorescent probe method was successfully applied Out of 77 cases, accounting for 97% (77/79), of which 28 cases of wild type, 49 cases of mutation. Among 42 positive samples cultured by traditional culture + drug sensitivity method, the coincidence rate of the two methods was 86% (36/42). Among 233 cases, 214 cases (92/214%) were successfully detected by one-generation sequencing, including 138 cases of wild type and 76 cases of mutated type. Of the 76 mutant specimens, 5% (4/76) contained A2142G mutation (including A2142G and A2142G + wild), and 95% (72/76) contained A2143G mutation (including A2143G and A2143G + wild). MGB fluorescence probe method successfully detected 231 cases, accounting for 99% (231/233), of which 141 cases of wild-type, 90 cases of mutation. Of the 90 mutant samples, 1414% (13/90) contained A2142G mutation and 86% (77/90) contained A2143G mutation. The 95% (203/214) coincidence rate between the first-generation sequencing and the MGB fluorescent probe method in distinguishing between mutations. The patients were divided into mutated group and wild group according to MGB fluorescence probe method. There was no significant difference in age and sex between the two groups (t = -0.685, P = 0.493; χ ~ 2_ (sex) = 0.065, P = 0.890), but there was a significant difference in the incidence of peptic ulcer (χ ~ 2 = 6.653, P = 0.044). Conclusion The TaqMan MGB fluorescence probe method can be used to detect the mutation of Klebsiella pneumonia A2142G and A2143G in gastric mucosa rapidly and sensitively.