Establishment Of Molecular Detection Method For Clarithromycin Resistance Gene Of Helicobacter Pylori

Helicobacter pylori is a spiral,micro-aerobic gram-negative bacilli.The International Agency for Research on Cancer of the World Health Organization confirmed Helicobacter pylori infection as a class of carcinogen in October 2017.At present,clarithromycin is the most widely used and effective antibiotic for Helicobacter pylori eradication.With the in-depth study of bacterial drug resistance,it is found that the increase of Helicobacter pylori resistance to clarithromycin is the main reason for the failure of eradication therapy with this kind of antibiotics.Identifying the drug resistance of Helicobacter pylori in clinical samples can help doctors to make effective treatment plans so as to eradicate Helicobacter pylori infection.In this paper,the artificial synthetic plasmid was used to simulate the genome of wild-type Helicobacter pylori,and the site-directed mutation of clarithromycin resistance was carried out on the wild-type synthetic plasmid by single point mutation technique to simulate the genome of drug-resistant Helicobacter pylori.Using wild-type and drug-resistant mutant plasmids as templates,the gene detection method of clarithromycin resistance of Helicobacter pylori was established.They are probe method and dye method respectively,and their principles are based on mutation amplification hysteresis and real-time fluorescence PCR.The results showed that the specificity and reproducibility of the dye method were good(CV=2.25%),and the sensitivity was 10~1 copies(ΔCt>6),and the probe method used Taq Man-MGB probe and Blocker primers to improve the specificity and reproducibility(CV=1.21%),while the sensitivity reached 10~1 copies,and the specificity(ΔCt>7)was better than that of the dye method.Therefore,a method for detecting clarithromycin resistance mutant gene of Helicobacter pylori was successfully established.At the same time,203strains of Helicobacter pylori were successfully isolated and cultivated.Urease test strip,catalase test,ure A amplification of urease gene fragment and16s r RNA sequencing were used to identify Helicobacter pylori.The results were compared with real-time fluorescence PCR.The results showed that the specificity of real-time fluorescence PCR was better.Finally,using the established drug resistance mutation detection method and commercial E-test detection kit to identify clarithromycin resistance of Helicobacter pylori,E-test results showed that 18 clarithromycin resistant strains were found,which was consistent with the results of the detection method established in this paper.One column of E-test negative strains was positive by this method,and there was a drug resistant mutation site A2115G after sequencing.The results show that a rapid,sensitive and accurate molecular biological detection method has been successfully established.The established method for detecting clarithromycin resistant mutations of Helicobacter pylori can detect 19 clarithromycin resistant sites with good methodological evaluation.In the process of clinical application,real-time fluorescence PCR method can be used to identify Helicobacter pylori isolated from samples,and then clarithromycin resistance gene detection method can be used to accurately determine the drug resistance of Helicobacter pylori strains.The isolation,culture and detection period of 10-15 days in the traditional method was shortened to a few hours.