| Helicobacter pylori can cause gastritis,gastric ulcer,gastric cancer and other digestive tract-related diseases,and the World Health Organization lists it as a class I carcinogen.Eradication of H.pylori significantly reduces gastric inflammation,promotes ulcer healing,and may prevent gastric cancer.Due to the overuse and even abuse of antibiotics,the prevalence of antibiotic-resistant strains continues to increase,resulting in a decline in the efficacy of H.pylori eradication treatments.Therefore,in medical institutions,in addition to general H.pylori diagnosis,antibiotic resistance assessment is also required to guide clinicians to provide effective treatment options.However,traditional in vitro drug susceptibility testing,E-test and other drug resistance detection methods are complicated to operate,require skilled technicians,and take a long time.In recent years,the newly released Maastricht V consensus mentioned for the first time"the use of molecular diagnostic methods to detect the resistance of H.pylori and clarithromycin and/or fluoroquinolones on biopsy specimens".Nucleic acid extraction from H.pylori samples,establishment of nucleic acid detection methods for H.pylori clarithromycin and levofloxacin resistance,and clinical sample detection of H.pylori infection.Quantitative PCR system for one-step diagnosis of H.pylori infection and detection of levofloxacin and clarithromycin resistance mutations.The first part is the research on the optimal sampling site and nucleic acid extraction method for the detection of H.pylori.A total of 86 samples of saliva,dental plaque and gastric mucosa from three different parts of patients undergoing gastroscopy were selected for nucleic acid extraction from the pretreated samples by thermal lysis,spin column method and magnetic bead extraction.H.pylori 16S r DNA and human internal reference gene RPP30 were detected by establishing double q PCR amplification.The experimental results showed that H.pylori was not detected in saliva and dental plaque,and the consistency between gastric mucosa samples and clinicopathological biopsy was 100%.Nucleic acid extraction was performed on known H.pylori-positive gastric mucosa using three extraction methods.By comparing the nucleic acid concentration and amplified Ct value,the nucleic acid concentration(80-100 ng/μL)extracted by magnetic bead method and the average Ct value of subsequent amplification Both are better than thermal cracking method and spin column extraction method.The second part is the establishment of a single-plex q PCR detection system for levofloxacin and clarithromycin resistance sites of H.pylori.The levofloxacin-resistant H.pylori gyr A gene of C261A/G,G271A/T and A272G mutants and the clarithromycin resistance of A2142G/C and A2143G mutants were synthesized by artificially synthesizing plasmids containing wild-type and drug-resistant mutant gene sequences.Different Taq Man-MGB probes were synthesized from H.pylori 23S r DNA,and the primers and probes were screened to verify the feasibility and specificity of single-plex q PCR amplification.The results showed that the system could accurately distinguish recombinant plasmids with different mutation markers.In addition,the method has good specificity,and the detection of H.pylori containing levofloxacin and clarithromycin resistance mutation sites can be distinguished from other 8 pathogenic bacteria that also cause gastrointestinal diseases.The third part is the establishment of the multiplex q PCR system and the detection of clinical samples.The integrated primers and probes were added into the two reaction tubes of A and B to establish a multiplex q PCR detection system of levofloxacin and clarithromycin-resistant H.pylori Taq Man-MGB probes,and verified.The specificity,feasibility and sensitivity of the system were investigated.The system can detect whether H.pylori infection can reach 10~1 copies/μL,and the detection of drug resistance mutation sites can reach 10~2 copies/μL,with high sensitivity and strong specificity.The detection system was used to detect gastric mucosa samples from 697 patients with severe stomach pain,and the results were in good agreement with biopsy and Sanger sequencing results,with Kappa values exceeding 0.90.The system’s test results can also be used to count the prevalence of drug resistance patterns in patients by factors such as age,gender,and geographic location.This simple and fast system will allow clinicians to personalize treatment based on a patient’s H.pylori strain and avoid the overuse of antibiotics. |
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