幽门螺杆菌耐药相关基因突变检测芯片的建立

Development of Oligonucleotide Microarray for Detection of Mutations Associated with Antibiotic Resistance of Helicobacter pylori

背景:幽门螺杆菌(H.pylori)感染与慢性胃炎、消化性溃疡、胃癌之间的相关性已得到确认。H.pyori对抗生素耐药是导致根除治疗失败的主要原因。目的:建立H.pylori耐药相关基因突变检测芯片,用于指导临床个体化治疗。方法:根据文献数据筛选出7个与根除治疗常用抗生素(克拉霉素、甲硝唑、阿莫西林、喹诺酮类、四环素、呋喃唑酮)耐药相关的基因及其单核苷酸多态性(SNP)位点,优化芯片探针设计和反应条件,采用多重PCR技术联合所建立的芯片,检测25例胃黏膜样本的H.pylori耐药相关基因突变情况。测序验证芯片检测结果。结果:25例H.pylori阳性胃黏膜样本的芯片检测结果均与测序结果相符。包括DNA抽提、多重PCR、芯片杂交、数据分析等在内的整个检测流程仅需6 h,且重复性和灵敏度十分理想。结论:H.pylori耐药相关基因突变检测芯片能快速、平行、高通量地检测H.pyori耐药相关突变位点,结果准确、可靠。

Helicobacter pylori （H. pylori） infection is linked to the development of chronic gastritis, peptic ulcer and even gastric cancer. Resistance of H. pylori to antibiotics is the major cause of eradication failure. Aims： To develop an oligonucleotide microarray for detection of mutations associated with antibiotic resistance of H. pylori that can be used to guide the individualized treatment in clinical practice. Methods： Seven genes associated with antibiotic resistance of H. pylori, including clarithromycin, metronidazole, amoxicillin, quinolones, tetracycline and furazolidone, and their single nucleotide polymorphism （SNP） locus were selected by literature review. Probe design and hybridization condition of the oligonueleotide microarray were optimized, and then the microarray was used in combination with multiplex PCR to detect mutations associated with antibiotic resistance in 25 gastric mucosa samples. Results of microarray were validated by direct sequencing. Results： The genotypes of H. pylori strains in 25 gastric mucosa samples tested by microarray were consistent with genotypes identified by sequencing. The assay, including DNA extraction, multiplex PCR, hybridization and image analysis, can be performed within 6 hours with high reproducibility and sensitivity. Conclusions： Microarray technology is a rapid and high throughput option for detection of mutations associated with antibiotic resistance of H. pylori. Precise and reliable results can be obtained by using this oligonucleotide microarray.