Identification of H. pylori strain specific DNA sequences between two clinical isolates from NUD and gastric ulcer by SSH

摘要

AIM: The genomes of Helicobacter pylori (H. pylori) from different individuals are different. This project was to identify the strain specific DNA sequences between two clinical H. pylori isolates by suppression subtractive hybridization (SSH).METHODS: Two clinical H. pylori isolates, one from gastric ulcer (GU, tester) and the other from non-ulcer dyspepsia (NUD, driver), were cultured and the genomic DNA was prepared and submitted to Alu I digestion. Then two different adaptors were ligated respectively to the 5’-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNA. The un-hybridized tester DNA sequences were amplified by two sequential PCR and cloned into pGEM-T-Easy Vector. The tester strain specific inserts were screened and disease related DNA sequences were identified by dot blotting.RESULTS: Among the 240 colonies randomly chosen, 50 contained the tester strain specific DNA sequences. Twenty three inserts were sequenced and the sizes ranged from 261 bp to 1036 bp. Fifteen inserts belonged to the H.pylori plasmid pHPO100 that is about 3.5 kb and codes a replication protein A. Other inserts had patches of homologous to the genes of H.pylori in GenBank. Various patterns of dot blots were given and no GU strain unique DNA sequences were found when 4 inserts were used as probes to screen the genomic DNA from 27 clinical isolates, 8 from GU, 12 from duodenum ulcer (DU), 4 from GU-DU, 2 from NUD and 1 from gastric cancer (GC). But a 670 bp DNA fragment (GU198) that was a bit homologous to the 3’-end of the gene of thymidylate kinase was positive in 7 GU strains (7/8), 3 GU-DU strains (3/4) and 3 DU strains (3/12). A 384 bp fragment (GU79) of the replication gene A (repA) was positive only in 4 H.pylori isolates, 2 from GU and 2 from GU-DU.CONCLUSION: Differences exist in the genes of different H.pylori isolates. SSH is very effective to screen H.pylori strain specific DNA sequences between two clinical isolates, and some of these sequences may have clinical significance.