The Establishment And Application Of Rapid Multiple Genetic Quantitative Analysis System To Identify Helicobacter Pylori And Its Virulence By 16S RRNA And Other Associated Genes

Objectives1. To build a 25-genetic analysis system for H.pylori identification and virulence detection.2. To evaluate the sensitivity and specificity of the multiple genetic analysis system3. To construct the standard quantitative curve by H.pylori ureC gene.Methods1. The establishment of the multiple genetic quantitative analysis system for H.pylori identification and virulence detection.25 kinds of identification and virulence genes sequences were obtained from NCBI. Pairs of primers were designed by the primer designing tool of NCBI-primer, primer 3, primer premier 5.0. The specificity of these primers were evaluated over and over again by H.pylori standard strains NCTC11637 and Sydney SS1. Meanwhile, the reacting conditions were changed (mainly for annealing temperature) over and over again, in order to explore the best combination of 25 pairs of primers and the optimum reaction conditions of the multiple genetic analysis system.2. The evaluation of sensitivity and specificity of the multiple genetic analysis system.The 10 times concentration dilutions were established by constructing plasmid by the single copy ureC gene. The single copy number was 10, 1× 102, 1×103, 1×104, 1×105,1×106,1×107 and 1×108, respectively. The sensitivity of the multiple genetic analysis system was evaluated by targeted amplify signals generated by GeXP system for each copy dilution.The E. coli ATCC25922, Pseudomonas aeruginosa strain ATCC27853, Hepatitis B virus (HBV) quality control strain and water was used as the negative template to evaluate the specificity of the technique by using the 25pair of primers according to whether there were multiple PCR amplification signals.3. The construction of the quantitative analysis system by drawing standard quantitative curve with the H.pylori ureC gene.The 10 times concentration dilutions were established by constructing plasmid by the single copy ureC gene. The single copy number was 10, 1×102, 1×103,1×104, 1×105,1×106,1×107 and 1×108. The under curve area was obtained automaticly by the multiple genetic analysis system. The function relationship model between the peak area and copies was obtained by Eurve Expert statistics software.Results1. The establishment of the multiple genetic quantitative analysis system for H.pylori identification and virulence detection.4 important identifying genes (16 SrRNA gene, ureC gene,26 KDa gene and ureA gene) primers and 21 of virulence-related genes were effectively expressed in the ONE reacting system after optimizing the primers and reacting conditions over and over again. Optimum conditions were as follows:Initial denaturation 94 ℃,1 min; denaturation 94℃,30s; extention 60℃,30s; 35 cycles; extention 70℃, lmin. Optimum concentration of primers:the Forward primer of ureC gene was 200nmol/μL, the reverse primer of ureC gene was 1μmol/μL; the others primers was 200nmol/μL. Optimum concentration of template was 10nmol～20nmol.2. The sensitivity and specificity of the multiple genetic analysis system was evaluated.According to the results, the system could at least amplify 10 copies of ureC genes. It was indicated that the sensitivity was very high; The multiple genetic analysis system did not appear any amplification signal by using standard strains of E. coli ATCC25922, Pseudomonas aeruginosa strain ATCC27853, Hepatitis B virus (HBV) quality control stain and water as the negative control system,. It was suggested that the system was highly specific.3. The quantitative analysis system was constructed by drawing standard quantitative curve with the H.pylori ureC gene.1×108, 1×107, 1×106, 1×107, 1×104, 1×103, 1×102,10 copy dilutions were chosen to establish the standard quantitative curve. The corresponding multiple genetic analysis system peak urea of the eight copy points was 105032,62315,56476,47014, 33849,23843,11106,7023, respectively. The function relationship model between the peak area and copies was obtained by Eurve Expert statistics software. The correlation coefficient r= 0.99817222> 0.99, indicated that the standard quantitative curve would perform well when used for H.pylori loads quantification.Conclusions1. Multiple gene loci of H.pylori could be detected by using multiple genetic analysis system. H.pylori infection could be confirmed and the pathogenicity could be predicted by analyzing the expression of virulence-related genes. The clinical significance would be great.2. The new platform was very suitable for H.pylori testing. It not only permited rapid, high sensitivity and high specificity detection, but could also perform absolute quantification analysis simultaneously.