The Development Of Nested PCR Assay For Genus Helicobacter And Clinic Application

With 34 Helicobacter species now formally named according to LPSN(List of Prokaryotic Names with Standing in Nomenclature)until 2013,it is clear that Helicobacter spp.can infect human and various animal hosts,as well as colonize different anatomical regions of the gastrointestinal system.About half of human in the world have H.pylori infection,which causes gastric and duodenal inflammation,peptic ulcer and malignant tumor.Besides,some NHPHs(Non-Helicobacter pylori helicobacters),such as Helicobacter heilmanni,H.hepaticus and H.bilis can also infect human.However,the prevalence of NHPHs in human is low and the pathogenicity is poor;furthermore,the association between some NHPHs and human is ambiguous.At present,the detection assay of H.pylori and H.heilmanni are enough,but the assay of H.hepaticus and H.bilis don't meet clinical practice and experiment.And the increasing helicobacter specieses,a newly designed assay is going to detect helicobacters.Nested PCR has been used frequently in recent years due to its higher specificity.The most sensitive and widely used method for detecting helicobacter infections is PCR targeting at a genus-specific and conserved region of 16 S r RNA.Feces samples is apt to be obtained.In this study,based on Ribosomal Database Project,we have tried to establish and optimize nested PCR assay that would simultaneously detect and differentiate the formally named helicobacters from feces samples.Part 1 Nested PCR Assay for Genus Helicobacter1 A Hemi-nested PCR Diagnostic Assay for Genus Helicobacter Based on Nucleotide Sequence of Its 16 S r RNA GeneIn the present report,the objective is to establish a genus-specific PCR assay to detect helicobacters using 16 S r RNA gene as the target.The hemi-nested primers were designed based on sequences of 16 S r RNA gene of 34 types of Helicobacter spp..The inclusivity,sensitivity,and specificity of the PCR assay using these primers were examined via feces simulated samples,mice infection model and clinic patients samples.The detection sensitivity of H.pylori,H.hepaticus and H.bilis strains for feces simulated samples was all 102 CFU/ml.H.hepaticus and H.bilis were successfully detected in the liver,caecum and feces of experimentally infected mice.H.pylori was successfully detected in the feces samples from 3 patients infected with H.pylori while not in the feces samples from 3 healthy human.Due to the PCR assay's excellent inclusivity,high sensitivity and specificity it may be used to detect the presence of Helicobacters.2 Development and Clinical Application of A Nested-PCR Assay on Helicobacter Hepaticus DetectionAIM: The objective is to establish a nested-PCR assay for the detection of H.hepaticus with high sensitivity and specificity and analyze its application.METHODS: The nested primers were designed based on sequences of 16 S r RNA gene of nine subtypes of H.hepaticus.After optimizing reaction condition,the sensitivity and specificity of the assay were examined via related bacteria,feces simulated samples,mice infection model and clinic patients samples.RESULTS: The detection limit of H.hepaticus strain for feces simulated samples was 102 CFU/ml.No specific PCR product was detected with DNA from Helicobacter prlori,Helicobacter bilis,Campylobacter jejuni,Escherichia coli,Enterococcus faecalis,Pseudomonas aeruginosa and Staphylococcus aureus.H.hepaticus were successfully detected in the liver,caecum and feces of experimentally infected mice.Moreover,H.hepaticus was successfully detected in the bile,cholecyst mucous membrane and feces samples from two of ten patients with cholelithiasis.Medical intervention,samples type and management,PCR methods may all influence the detection.CONCLUSION: Due to the PCR assay's high sensitivity and specificity it may be used to detect the infection of H.hepaticus.H.hepaticus may be associated with the pathogenesis of cholelilthiasis.Medical intervention,samples type and management,PCR methods may all influence the detection.3 Development of A Nested PCR Assay for Detection of Helicobacter BilisIn this study,the objective is to establish a nested-PCR assay for the detection of H.bilis with high sensitivity and specificity.The nested primers were designed based on sequences of 16 S r RNA gene of seventeen subtypes of H.bilis.After optimizing reaction condition,the sensitivity and specificity of the assay were examined via the detection of feces simulated samples,mice infection model samples and clinic patients samples.The detection sensitivity of H.bilis strain for feces simulated samples was 102 CFU/ml.H.bilis was successfully detected in the liver,caecum and feces of experimentally infected mice.Moreover,H.bilis was successfully detected in the bile,cholecyst mucous membrane and feces samples from two of ten patients with cholelithiasis.Due to the PCR assay's high sensitivity and specificity,the method may be used to detect the infection of H.bilis.Part 2 Clinical Application of Helicobacter Specific PCR AssaysAIM: The aim is to evaluate the efficacy of helicobacter specific PCR assays.METHODS: We used Hp SA method and the hemi-nested PCR assay(G34 assay),respectively,to examined 100 children with upper gastrointestinal discomfort.The data were analyzed by IBM-SPSS 22.0?RESULTS: Twenty-six children with upper gastrointestinal discomfort were positive using Hp SA method.While 28 patients had helicobacter infection using G34 assay for Genus Helicobacter specific 16 S r DNA(30.0%),including 26(26.0%)H.pylori and 2(2.0%)H.bilis.Then the two H.bilis infection patients were detected positively by H.bilis-specific nested PCR assay.CONCLUSION: G34 assay may be used to detect the presence of Helicobacters in children.