Stability of randomly amplified polymorphic DNA fingerprinting in genotyping clinical isolates of Helicobacter pylori.

AIM: H pylori genomes are highly diversified. This project was designed to genotype H pylori isolates by the polymerase chain reaction (PCR)-based randomly amplified polymorphic DNA (RAPD) fingerprinting technique and to verify its stability by Southern blotting and DNA sequencing. METHODS: Clinical isolates of H pylori were cultured from gastric antra and cardia of 73 individuals, and genomic DNA was prepared for each isolate. RAPD was carried out under optimized conditions. 23S rDNA was regarded as an internal control, and a 361 bp rDNA fragment (RDF) was used as a probe to screen the RAPD products by Southern blotting. Ten RDFs from different clinical isolates and the flanking regions (both upstream and downstream) of four RDFs were amplified and sequenced. RESULTS: H pylori isolates from different individuals had different RAPD profiles, but the profiles for isolates cultured from different gastric sites of a given individual were identical in all but one case. Isolates from 27 individuals were RDF positive by Southern blotting. Sequences of the RDFs and their flanking regions were almost the same between the RDF positive and negative isolates as determined by Southern blotting. There was no binding site for random PCR primer inside the sequences. CONCLUSION: RAPD is very useful in genotyping H pylori grossly on a large scale. However, it seems unstable in amplification of low yield fragments, especially those that do not appear as visible bands on the agarose gel stained with EB, since the primer is partially matched to the template.

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

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 1 036 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.

Serum IgG response to differentiated antigens of Helicobacter pylori.

AIM:To detect antibodies against Helicobacter pylori spiral and coccoid antigens in human sera.METHODS:Blood samples were collected from 278 patients with gastric diseases. A 3-day-old culture of H. pylori on chocolate blood agar was used to providespiral form. Synchronous coccoids were cultured in (BHY) (brain heart infusion supplemented with 10% horse serum and 0.4% yeast extract) medium in a chemostat.Antigens from spiral and coccoid form were prepared using acid glycine extraction.Enzyme-linked immunosorbent assay (ELISA) was performed to detect serum IgG anti-bodies against spiral and coccoid forms of H. pylori.RESULTS:Seroprevalence of H. pylori infection was higher in patients with gastric ulcer (79%) and gastric cancer (83%) than those with non-ulcer dyspepsia (NUD)(44%) and other diseases (45%) (P <0.05). IgG antibodies against spiral and coccoid antigens were detected in 50.7% (141/278) and 49.6% (138/278), respectively.CONCLUSION:The spiral and coccoid forms of H.pylori coexist in patients infected with the bacterium.