Detection Of Antibiotic Resistant Mutations In Helicobacter Pylori In Fecal Samples Based On Next Generation Sequencing Technology

Helicobacter pylori（H.pylori）infection is associated with a range of gastrointestinal diseases and has been classed as group I carcinogen for gastric cancer,which poses a great threat to public health.The large-scale and irrational use of antibiotics over the years has led to increasing resistant rates of H.pylori to antibiotics.Therefore,a more accurate and cost-effective detection of antibiotic resistance is required to achieve the goal of achieving the first successful eradication of H.pylori.H.pylori mainly colonizes the epithelial cells of the host gastric mucosa,and is also present in both oral cavity and feces due to esophageal reflux and natural excretion,respectively.given that few number of H.pylori in saliva and inconvenient sampling of gastric mucosa,as a non-invasive detection method,the use of fecal samples for Helicobacter pylori gene detection is receiving high attention in clinical practice.Gene testing of H.pylori using fecal samples is difficult because of few number of H.pylori as well as DNA fragmentation,and the presence of a large number of other species of bacteria and PCR inhibitors.PBP1 has a high mutation rate in H.pylori.Although individual mutations or combinations of mutations may lead to amoxicillin resistance,there is no definitive conclusion on how to guide clinical treatment.In addition,the resistant rate of rdx A mutations of H.pylori is very high in China,and Rifamycin is not used for H.pylori eradication treatment in China.In view of this,23S r RNA,gyr A,16S r RNA,por D and oor D were selected in this study for the detection of antibiotic resistant mutations of H.pylori in fecal samples.In this study,a two-step nested PCR combined with Sanger sequencing was used to detect antibiotic resistant mutations of H.pylori in fecal samples and analyzed its diagnostic performance.Based on this,the feasibility of next-generation sequencing（NGS）in detecting H.pylori antibiotic resistant mutations in fecal samples was further explored by using multiplex PCR amplicon targeted sequencing.The main results were as follows:1.The Magen kit was considered as more suitable for DNA extraction from fecal sample by comparing the extraction effect through three commercially available fecal DNA extraction kits.2.Among 135 fecal samples,63 samples were H.pylori positive by nested PCR combined with Sanger sequencing,with a positive rate of 46.67%.The frequencies of antibiotic resistance mutations of 23S r RNA,gyr A,and 16S r RNA were 65.08%,38.10%,and 9.52%,respectively.The frequency of por D and oor D co-mutation（furazolidone resistance）rate was 37.93%.The overall frequency of resistance mutations in this study was high,which may be due to the small sample size and sample selection bias.Further lager scale studies are required to validate these results.3.To evaluate the diagnostic performance of nested PCR combined with Sanger sequencing for H.pylori,the ¹³C breath test and fecal antigen test of H.pylori were used as standard.Kappa and receiver operating characteristic（ROC）curves were analyzed.The Kappa values for 23S r RNA,gyr A,16S r RNA,por D and oor D were higher than 0.830,and the area under curve（AUC）values were higher than 0.910,suggesting that nested PCR combined with Sanger sequencing had good diagnostic performance.4.Five genes（23S r RNA,gyr A,16S r RNA,por D and oor D）were successfully detected by multiplex PCR amplicon targeted sequencing in 63 H.pylori-positive fecal samples.The detection sensitivity of NGS was higher than that of nested PCR combined with Sanger sequencing.The frequencies of antibiotic resistance mutations23S r RNA,gyr A and 16S r RNA were 74.60%,47.62%and 9.52%.The frequency of por D and oor D co-mutations was 34.92%.5.To investigate the feasibility of multiplex PCR amplicon targeted sequencing for the detection of antibiotic resistant mutations of H.pylori in feces samples,the results of resistant mutations were compared with those of nested PCR combined with Sanger sequencing.Amplicon targeted sequencing can sensitively detect antibiotic resistance mutations at low allele frequencies compared with Sanger sequencing,which was the main reason for the inconsistent results between the two assayes.The concordance of results between the two assays increased with the increase of allele frequency.The Kappa values for 23S r RNA,gyr A,16S r RNA,por D and oor D were higher than 0.747,and the area under curve（AUC）values were higher than 0.875,suggesting that NGS had good diagnostic performance.NGS assay can be used as a novel non-invasive test for patients with H.pylori infection,providing reliable technical assurance for clinical H.pylori eradication treatment.The thesis has 36 figures,41 tables,and 147 references.