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REP-PCR fragments as biomarkers for differentiating gastroduodenal disease-specific Helicobacter pylori strains
D H Kwon1, F A El-Zaatari, J S Woo
1Department of Medicine, Veterans Affairs Medical Center, Baylor College of Medicine, Houston, Texas 77030, USA.
Abstract:
We previously identified four potential putative gastroduodenal disease fragments by using the interspersed repetitive extragenic palindromic DNA sequence based PCR (REP-PCR) technique. We investigated these fragments with regard to their disease specificity. The putative disease-specific REP-PCR fragments were cloned, mapped by restriction enzymes, cross-hybridized, and confirmed by Southern hybridization. The four fragments were also used as probes against REP-PCR amplicons from H. pylori isolates obtained from gastritis (N = 20), duodenal ulcer (N = 30), and gastric cancer patients (N = 30). Three of these fragments (1.4- and 0.76-kb for gastritis; 1.35 kb for duodenal ulcer) were amplified without any discrimination between any disease-specific H. pylori isolates. However, amplification following hybridization with the fourth 0.81-kb fragment was observed only from gastritis (60%) and duodenal ulcer (52%) but with none (0%) of gastric cancer patients. Nucleotide sequence analysis of the 0.81-kb fragment revealed that it was an open reading frame of the hypothetical protein HP0373 matched to the position of 380,966 to 383,068 nucleotides of the H. pylori complete genome sequence. Hence, the REP-PCR sequence was not a extragenic palindromic DNA sequence. The hypothetical protein was also present in all the tested isolates. The REP-PCR fingerprinting technique is useful to differentiate disease-specific H. pylori strains based on the interspersed repetitive extragenic palindromic DNA sequences; however, it may not be useful to identify disease-specific virulence determinant(s) without being confirmed by DNA sequence analysis and functional studies.
Insights
Repetitive extragenic palindromic DNA sequence PCR (REP-PCR) can differentiate Helicobacter pylori strains. However, sequence analysis is crucial to confirm disease-specific virulence determinants, as one fragment was misidentified.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Helicobacter pylori (H. pylori) infection is a major cause of gastroduodenal diseases.
- Identifying specific H. pylori strains associated with different diseases is crucial for effective treatment.
- Repetitive extragenic palindromic DNA sequence based PCR (REP-PCR) is a molecular typing technique.
Purpose of the Study:
- To investigate the disease specificity of four potential H. pylori REP-PCR fragments.
- To determine if these fragments can differentiate H. pylori isolates from gastritis, duodenal ulcer, and gastric cancer patients.
- To confirm the identity and function of disease-specific fragments.
Main Methods:
- REP-PCR was used to identify potential disease-specific DNA fragments.
- Fragments were cloned, mapped, cross-hybridized, and confirmed by Southern hybridization.
- Fragments were used as probes against H. pylori isolates from different patient groups.
Main Results:
- Three fragments showed no discrimination between H. pylori isolates from different diseases.
- A fourth 0.81-kb fragment hybridized only with isolates from gastritis (60%) and duodenal ulcer (52%), but not gastric cancer (0%).
- Sequence analysis revealed the 0.81-kb fragment was an open reading frame for hypothetical protein HP0373, not an extragenic palindromic sequence.
Conclusions:
- REP-PCR fingerprinting can differentiate H. pylori strains based on interspersed repetitive extragenic palindromic DNA sequences.
- However, it may not identify disease-specific virulence determinants without DNA sequence analysis and functional studies.
- The 0.81-kb fragment, identified as HP0373, was present in all tested isolates, indicating its sequence was misidentified as REP-PCR based.