| Purpose: To research and find a simple and convineous method of isolating , culturing , storing and reviving Helicobacter pylori (H.pylori). Observing the morphology of H. pylori's colony and the organism itself under the microscope after Gram stain on different culturing period ( 3days and 7days ). Using a new method of storing H. pylori to keep it and investigating the survival rate of the organism stored at different culturing period after 1 year later. To detect the minimal inhibitory concentration (MIC) of the H. pylori isolates to four different antibiotics as clarithromycin, metronidazole, amoxycilline and tetracycline by E-test method, then observe the resistance rate of H. pylori isolated from Shenyang patients with digestive disease to above four antibiotics. To amplify the DNA segment from 2047 to 2347 of 23S rRNA gene in H. pylori resistant to clarithromycin, then perform the sequence analysis of such segment to investigate the mechanism of H.pylori resistance.Methods: In the endoscopic examination of thepatients with digestive symptoms, we collected such patients with peptic ulcer N chronic gastritis and gastric carcinoma as our subjects. First, one piece of gastric antra mucosa biopsy specimen was obtained from the patients for the purpose of rapid urease enzyme test. Then two pieces of antra mucosa biopsy specimens were obtained from the same patient with H. pylori infected diagnosed by the positive rapid urease enzyme test . The biopsy specimens were then cut up in sterile plate and then cultured on the Columbia agar base with 10% (vol/vol) no-fiber fresh rabbit blood and 3mg/L Bacitracin. Put the agar base in anaerobic culturing jar, output the air in the jar and then input the microaerobic gas (5%02, 10%C02, 85%N2), seal the lid tightly. Culturing the organism at 37 and 90% humidity condition . 3 days later, observe the shape of H. pylori colony with naked eyes and bacterial morphology under microscopy. The organisms were identified as H. pylori by Gram stain morphology N colony morphology and positive urease. catalase and oxidase activities. Subculturing the typical bacterial to obtain the pure H. pylori and storing it at different culturing period (three days and seven days) in 3ml sterile no-fat milk at -80 deep low temperature. One year later, reviving 8 cases of H. pylori isolates stored at different culturing period (three days and seven days) randomly. In the subculturing , prepared the pure H. pylori suspension of 1 McFrand unit with sterile 0.9% natrium chloride, spread the suspension onto the Mueller-Hinton agar base with 10% fresh rabbit blood. Within fifteen minutes, the E-test strip must be put in the center of the base and cultured and appropriate conditions. 3 to 5days later, the Mueller-Hinton base were studied and the Minimal Inhibitory Concentrationof H. pylori to 4 kind of different antibiotics were read. The resistance of the bacteria was judged by the standard as follows: MIC of clarithromycin 8mg/L, Metronidazole 8mg/L, Amoxycillin 2mg/L, Tetracycline 8mg/L. Choose three cases of the resistant H. pylori isolates and one case of sensitive H. pylori isolates stored at deep low temperature of -80 .that is No. 13 (MIC 8mg/L), No. 17 (MIC 64mg/L), No. 22 (MIC>256 mg/L) and No. 33 (MIC 0. 125mg/L), and revive them. Wash the H.pylori with 0.01M PBS buffer into 1.5ml enphendoff tube , then centrifuge at 3000 rpm for 5 minutes. Collect the deposition (full of H. pylori) and extract the DNA from the bacteria with the phenol-chloroform extraction method. We designed primers using the 23S rRNA gene sequence repeated by Hiratsuka (Genebank accession number U27270). The primers were synthesized by Sangon and its sequence were as follows: forward primer, 5'- CTG CAT GAA TGG CGT AAC GAG - 3' (complementary to 23S rRNA gene sequence from 2047 to 2067), reverse primer, 5' - GAG CGA CCG CCC CGA TCA AAC - 3' (complementary to 23S rRNA gene sequence from 2327 to 2347), which will generate a 301 bp product. PCR amplifications reaction mixture (20ul) contained double distilled...
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