The Application Of Real-time Fluorescent PCR In The Detection Of Helicobacter Pylori In Gastric Mucosa

【Objective】The purpose of this project is to investigate the consistency of toluidine blue tissue section staining and real-time quantitative PCR(qPCR)in regard to the positive rate and infectious degree of Helicobacter pylori(HP).Moreover,this study aims to determine HP’s genotype as well as its correlation with gastric mucosa biopsy morphology(including the degree of atrophy,degree of intestinalization,presence or absence of lymphoid tissue hyperplasia,and degree of glandular epithelial neoplasia),as well as the positive degree of HP infection.The mutation of clarithromycin resistance sites,including A2142 G and A2143 G,are analyzed for further research,and the association between mutation incidence of resistant genes and age,gender and the HPpositive degree of patientsare also investigated.【Methods】1.From January 2018 to June 2018 at the 900 th Hospital of the Chinese People’s Liberation Army’s Joint Service and Support Corps,a total of 230 cases of gastric mucosa paraffin sectionsfrom patients who received gastroscopy due to digestive tract discomfort were included in this study.Among them,180 cases of active gastritis and50 cases of non-active gastritis were diagnosed pathologically.All patients were examined for HP infection using toluidine blue tissue staining and real-time fluorescent PCR.These twotechniques were adopted in order to compare and analyze the consistency of toluidine blue tissue staining and real-time fluorescent PCR in the diagnosis of HP infection,as well as the degree of infection.2.The Cag A genotyping of HP positive samples was detected by qPCR,where patients were divided into groups of type I and type II strains.Accordingly,the correlation between the type I and type II strains and the degree of HP positivity in gastric mucosa biopsy and pathological morphology were examined by analyzing the atrophy degree,intestinal degree,lymphoid hyperplasia,and adenoepithelioma changes.3.Clarithromycin resistant mutations at A2142 G and A2143 G were detected via qPCR in patients having a positive HP,and the relationship between the incidence of mutations in the drug-resistant genes at A2142 G and A2143 G and the age,gender,and the degree of HP positivity was determined.【Results】1.The consistency of toluidine blue tissue staining and qPCRin detecting HP infection is significant(Kappa=0.923),and similar results were obtained in recognizing the degree of HP infection(Kappa=0.706).2.The expression levels of Cag A in the type I strains of the gastric mucosa paraffin specimens were found to be significantly correlated with the degree of atrophy and intestinal morphology of gastric biopsy tissues(P<0.001).Additionally,Cag A levels increased with the development of atrophy and intestinal metaplasia(P<0.05),while having a certain correlation with lymphoid tissues hyperplasia(P=0.014).Cag A expression was also found to be elevated ccording to the degree of lymphoid hyperplasia,however,it was not statistically significant(P =0.083).Moreover,no significant correlation was present between Cag A expression and epithelial neoplasia(P =0.059),but Cag A expression increased with the development of epithelial neoplasia(P<0.001).Furthermore,a significant correlation was identified between Cag A levels and the degree of HP positivity(P<0.001),where expression was markedly increased with the increase in HP positivity(P<0.001).3.No significant correlation between A2142 G mutation with age and gender of patients was observed in the detection of A2142 G and A2143 G mutations at clarithromycin resistance sites(P>0.05).However,the mutation of A2142 G was positively correlated with the HP positive level(P<0.01),and A2142 G mutation levels increased with the HP positive level,but was not statistically significant(P=0.08).Interestingly,A2143 G levels increased in women(P=0.03),though this mutation possessed no correlation with age and patient gender p>0.05).Moreover,the A2143 G mutation site was significantly correlated with the HP infection level(P=0001),and expression levels increased with increasing HP infection levels(P=0.002).【Conclusion】1.Compared to toluidine blue tissue staining method,real-time fluorescent PCR is simple,convenient,and does not rely on human skill.Moreover,the obtained test results were found to not be significantly different.2.Real-time fluorescent PCR analysis of the expression of Cag A in gastric mucosa samples may identify type I HP strains,and the Cag A gene was found to be correlated with gastric mucosal atrophy,intestinalization,HP infection,and lymphocyte proliferation.3.A2142 G and A2143 G mutations at clarithromycin resistance sites were significantly correlated with the degree of HP infection,and expression levels were also positively correlated with the degree of HP infection.4.Real-time fluorescent PCR method has very potential in HP infection and histopathological evaluation of gastric mucosa.