| Objective:1The presences of12virulence-associated genes and8drugresistance associated genes from3GBS-patients were examined by PCR.Results were compared with the C.jejuni strain NCTC11168to find thepathogenic mechanism of Campylobacter jejuni associated withGuillain-Barré Syndrome.2The traditional identify method for Campylobacter jejuni is the platingmethod. After morphological and biochemical identification, some suspectedbacterial colonies are chosed to do molecular identification, the procedure atleast takes3days. Common specific biochemical experiment is hippuric acidexperiment, due to the difference of the time there will be false negativeresults which will reduce the separation rate. As to the plating methods, weneed experienced laboratory assistant to pick up suspicious single colony,even so, through the unnatural culture some of the virulence and resistancewill miss, finally it must be influencing the molecular research. Our researchis focusing on PCR to directly detect and distinguish Campylobacter speciesin human feces without enrichment.Methods:1In this previous study we tested for the presence of12virulence-associated genes. FlaA was the flagellin gene; cadF and racR wereselected as pathogenic genes responsible for the expression of adherence; cdtA,cdtB, cdtC, wlaN and virB11plasmid genes were selected as pathogenic generesponsible for toxin regulatory; dnaJ, ceuE, CiaB and pldA while for theexpression heat shock protein and transport related protein. The eight ofresistance genes included in this study, gyrA and parC which wereciprofloxacin resistance gene, tet (o) associated to tetracycline resistance,mutation(s), CJ1/DP1, CJ18/CJ19and CJ20/SJ21these were the macrolide resistant gene. In this study, Polymerase Chain Reaction (Polymerase ChainReaction, referred to as PCR) was applied to analyse the relationship betweenpathogenic genes and sources of C. jejuni.2After plating methods to transfer of culture NC11168strain to the P2generation, we did the biochemical and molecular identification. Fresh fecesof16patients who were not with diarrhea or Guillain-Barre syndrome wererandomly collected, then placed in Brinell broth. After shaking to the particles,the feces samples were put into membrane filtration and mixedphosphate-buffered Saline. According to the turbidity of bacteria, sampleswere divided into five groups, respectively for103/ml,104/ml,105/ml,106/ml and control group. Multiple PCR and ordinary PCR were applied to detectfeces.Results:1FlaA, cadF, racR, pldA and cdtB were detected in all fourstrains, while the cdtA and dnaJ genes were detected in11168, lulei andlichang strains. VirB11was detected in no strain, while the CiaB and cdtConly in lulei strain. The wlaN gene existed in lulei, lichang and qiaoyuntaostrains. The ceuE gene was detected in lulei, qiaoyuntao and11168. Drugresistance gene CJ1/DP1and mutation were not found in four strains. TheCJ18/CJ19can be detdcted in lichang, lulei and11168strains. The CJ20/CJ21gene was detected in qiaoyuntao,11168and lulei strains, whileciprofloxacin resistance gene gyrA and parC existed in all four strains.Tetracycline resistance plasmid gene tet (o) was only detected in lichang. Fortobramycin-gentamicin resistance gene int1was detected in qiaoyuntaostrains.2Due to multiple PCR results of the five group, there were two sampleswith a16s gene fragment in group2, one sample with a hipo gene fragment ingroup3, one sample with16s and hipo gene fragment in group4. Single-copy16S gene PCR results showed that there was one sample amplifying objectivebanding in the experimental group1,2samples in group2,3all samples ingroup3, and2samples in group4. Ordinary PCR samples to map geneamplification did not get the purpose gene amplification. Single-copy FlaA for amplification only in the experimental group2with2samples and1samplesin group4amplied the purpose gene.Conclusions:1Campylobacter jejuni has not only multiple regulationand monitoring gene, but also multiple virulence and resistance genes. Thevirulence genes of GBS-associated Campylobacter jejuni more thanNCTC11168strain, and different GBS-associated Campylobacter jejunistrains have difference virulence genes. Adherence, invasion and cytotoxinproduction appear to be different virulence factors. The strongly lulei straincontains more among12virulence genes. More kinds of drug resistance genesin the GBS-associated Campylobacter jejuni strains to NCTC11168, this maybe as to medical source environmental selection. Could the different strainshave different pathogenic mechanisms? There is not enough evidence to prove.However, the difference truly exsits between infection and resistance throughthe research for virulence and resistance genes.2The experiment through the boiling method for DNA extraction,identify the Campylobacter jejuni, experimental results show that the multiplePCR does not apply to the Campylobacter jejuni of human feces, when theconcentration between106and103, the accuracy rate lower than30%, in theprocess identification Campylobacter jejuni of circulation system, thecomposition and temperature requirement of multiple PCR is relatively strict.In the experiment,16s, map and flaA gene were selected for amplification.The positive rate of16S gene amplification was about81.15%, but map geneamplification didn’t get a positive result. In flaA genes,12experimental groupsamples amplification out four positive results, positive rate is about33.33%.In the three results, single-copy16S gene were highly specific for C. jejuni,and the other two genes result is not ideal, but because of16S specificityconfined in campylobacter, unable to Campylobacter jejuni for identityrecognition, and so for the Campylobacter jejuni with the purpose of gene selection is also there are a lot of place. |
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