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Abstract

Helicobacter pylori, Gram-negative microaerobic bacterium is associated with human gastritis, gastric ulcer and gastric cancer. Conventional culture methods for identification of H. pylori are complex, labor intensive, and time-consuming. The objective of the present investigation was to establish a multiplex PCR for the identification of H. pylori. Reference strains including H. pylori LMG 8775 and NCTC 11638 were used for the validation in this study. H. pylori were cultured on Brain Heart Infusion blood agar at 37癈 for 48 h at microaerobic atmosphere. Bacterial DNA was extracted and purified by QIAamp DNA Mini Kit. Multiplex PCR detected three genes of 16S rRNA, cagA encoding for virulence factor cytotoxin-associated gene A, and ureC for housekeeping urease gene C. The best combination of primers and the annealing temperature of multiplex PCR were examined. The result indicated that multiplex PCR with annealing temperature at 57癈 was able to effectively amplify specific products. In total, 55 gastric biopsies from dyspeptic patients were comparatively studied using rapid urease test (CLO test) and multiplex PCR. The results revealed that CLO test detected H. pylori in 7 gastric biopsies (12.73%). Whereas, 6 specimens evaluated by the multiplex PCR were identified as H. pylori (10.91%). In addition, cagA gene was detected in all positive samples. This system is useful for the detection of the presence of cagA gene that is responsible for toxin activity in H. pylori.

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