| Helicobacter pylori (H. pylori) can induce chronic gastritis gastric ulcer and other gastroenterology diseases. It is the only one I cancerogenic in bacteria which considered by WHO. Proton inhibitor combined with amoxicillin and clarithromycin or metronidazlo was recommended as the first line therapeutic regimen by the Maastricht III and Chinese consensus. And it was also used in Children in China. While the eradication rate of this regimen is declining with the increasing resistance of clarithromycin. The gene type of CYP2C19 of P450 in human gene also influences the eradication therapy, which decided the speed of metabolism of PPI. Thus, it is necessary to make clear the susceptibility of clarithromycin and gene type of CYP2C19 before the prescription was decided which can ensure the success of therapy. Traditional method of H. pylori can't give the information of clarithromycin susceptibility and CYP2C19. We try to set up a real time PCR method which can detect the infection of H. pylori, access the susceptibility of calrithromycin and make the gene type of CYP2C19 clear in one test from a gastric biopsy sample.In this study, we have detected the primary antibiotic resistance of H. pylori isolated from children in Beijing, and analyzed the population of mutations in 23 S rDNA, gyrA and gyrB. The primary resistance to 9 antibiotics of 73 H. pylori strains isolated from 120 gastric biopsies of children recruited at Beijing Children's Hospital was assessed, and the mutations in 23S rRNA gene of 65 macrolide-resistant strains and in gyrA and gyrB of 12 quinolone-resistant strains were investigated. The resistance rate to clarithromycin, azithromycin, metronidazole, levofloxacin, moxifloxacin, and rifampicin was 84.9%,87.7%,61.6%, 13.7%,15.1%, and 6.8%, respectively. No resistance to amoxicillin, gentamicin, and tetracycline was observed. Dual, triple and quadruple antibacterial resistant percentage was 46.6%(34/73),15.1%(11/73) and 2.7%(2/73). The gene mutation rate of A2142C, A2142G and A2143G in 23S rRNA gene was 1.5%(1/65),6.2%(4/65), and 84.6%(55/65). The detection rate of mutations of Asn87, Asp91 and Met 191 in GyrA was 41.7%(5/12),25%(3/12) and 25%(3/12). The high prevalence of primary antibiotic resistance was out of expectation in H. pylori strains isolated from the children in Beijing. Antibiotic susceptibility should be made clear before the antibiotic was used in the anti-H. pylori therapy in this population. The A2143G was the most populated mutation in macrolide-resistant strains, and Asn87 and Asp91 of GyrA were the most common mutation points in quinolone resistance strains.A multi real time PCR has been set up after the population of mutations associated with the clarithromycin has been made clear. And the specific gene of H. pylori and the allele of CYP2C19 have also been used. A probe aim at the ribonuclease P (RNP) of human epithelium was composed and used as the internal reference. HEX was marked in this probe. The probe for H. pylori was composed aim to a conserve gene of cagH in the cytotoxin associate gene pathogenicity island (cagPAI) which was selected from the sequence of cagPAI in 74 H. pylori isolated from difference area in China. FAM was marked in this probe. Three probes were composed aim to the wild, A2142G and A2143G in 23S rDNA of H. pylori. FAM was marked in the wild probe and HEX was marked in mutate probes. Two couples of probes were composed aimed to the alleles of G681A in the fifth extron and G681A in the forth extron of CYP2C19. FAM was marked in the wild probe and HEX was marked in mutate probes. The specificity of each probe was tested and the reaction condition of each probe was optimized. Then, the probes were combined in four reactions as follow:cagH and RNP, three probes of 23S rRNA, a couple probe of the fifth extron in CYP2C19, a couple probe of the forth extron in CYP2C19. The specificity of combination was testified.Thus, we have set up a multiple real time PCR which using four reactions in one test to give the information of H. pylori infection, carithromycin susceptibility and gene type of CYP2C19.At last, we have used the multi real time PCR to detect 1791 gastric samples isolated from 13 A rating-three level hospitals in different areas in China. All the samples were collected from the 13C urea breath test (13C-UBT) positive persons or the rapid urease tests (RUT) of the samples were positive. The results were compared with the results of culture and E-test by kappa coincidence test. The positive of culture, PCR and real time PCR was 62.37%,72.47% and 84.81% respectively.The resistance rate of clarithromycin tested by E-test and real time PCR was 29.50% and 31.67% respectively, the coincidence rate was 88.88%, and the Kappa value was 0.681. It was conspicuous that the detect rate of multi real time PCR was higher than other method, and the results of the susceptibility test to clarithromycin between multi real time PCR and E-test was highly coincidence.Concern to above all, we have set up a multi real time PCR in foundation of the population of mutations of H. pylori associated with clarithromycin resistance, which can detect H. pylori, access the susceptibility of clarithromycin and analyze the gene type of CYP2C19 from the gastric biopsy in one test. This method can get result rapidly and exactly, and had high coincidence with E-test to the susceptibility of clarithromycin. The results of our study showed a good prospect for multi real time PCR being used in the diagnosis of H. pylroi and evaluation of susceptibility to clarithromycin. |
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