Hans-Jürg Monstein1, Katarina Ellnebo-Svedlund. 1. Molecular Biology Laboratory-LMO, University Hospital, Department of Biomedicine and Surgery, Faculty of Health Sciences, Linköping University, S-58 185 Linköping, Sweden.
Abstract
BACKGROUND: In recent years, numerous PCR amplification typing methods have been developed for the identification of H. pylori specific virulence genes. To reduce the number of PCR amplifications needed we have previously developed virulence gene based multiplex PCR and RT-PCR assays. AIM: The aim of the present study was to characterise Helicobacter pylori strains by means of virulence-gene based multiplex PCR and reverse-transcription PCR. SUBJECTS: Helicobacter pylori clinical reference strains HP-HJM 1-25 originally obtained from routine cultures of clinical gastric biopsy specimens from dyspeptic patients were used in the present study. METHODS: Helicobacter pylori multiplex PCR and RT-PCR assays were carried out using primer pairs targeting the cagA, vacA, ureA, ureI, hspA (hsp60), and sodB genes and their cDNA. RESULTS: Unexpected multiplex PCR and RT-PCR patterns were observed by ethidium bromide staining of agarose gels and Southern blot hybridisation analysis. DNA sequence analysis revealed a complex pattern of point mutations, deletions and insertions in the sodB, cagA and vacA genes, respectively. Mutations occurring in the PCR and hybridisation primer binding sites might explain some of the discrepancies previously observed in the expression of these genes. Furthermore, the present data show that coexpression of cagA and vacA transcripts of different sizes may take place in the same strain. CONCLUSIONS: The multiplex PCR and RT-PCR assay described allows rapid characterisation of H. pylori virulence genes at the DNA and RNA (cDNA) levels. However, extensive DNA sequence analysis seems necessary if one wants to reveal details of mutations occurring in the cagA and vacA genes.
BACKGROUND: In recent years, numerous PCR amplification typing methods have been developed for the identification of H. pylori specific virulence genes. To reduce the number of PCR amplifications needed we have previously developed virulence gene based multiplex PCR and RT-PCR assays. AIM: The aim of the present study was to characterise Helicobacter pylori strains by means of virulence-gene based multiplex PCR and reverse-transcription PCR. SUBJECTS:Helicobacter pylori clinical reference strains HP-HJM 1-25 originally obtained from routine cultures of clinical gastric biopsy specimens from dyspeptic patients were used in the present study. METHODS:Helicobacter pylori multiplex PCR and RT-PCR assays were carried out using primer pairs targeting the cagA, vacA, ureA, ureI, hspA (hsp60), and sodB genes and their cDNA. RESULTS: Unexpected multiplex PCR and RT-PCR patterns were observed by ethidium bromide staining of agarose gels and Southern blot hybridisation analysis. DNA sequence analysis revealed a complex pattern of point mutations, deletions and insertions in the sodB, cagA and vacA genes, respectively. Mutations occurring in the PCR and hybridisation primer binding sites might explain some of the discrepancies previously observed in the expression of these genes. Furthermore, the present data show that coexpression of cagA and vacA transcripts of different sizes may take place in the same strain. CONCLUSIONS: The multiplex PCR and RT-PCR assay described allows rapid characterisation of H. pylori virulence genes at the DNA and RNA (cDNA) levels. However, extensive DNA sequence analysis seems necessary if one wants to reveal details of mutations occurring in the cagA and vacA genes.
Authors: Santanu Chattopadhyay; Rajashree Patra; T Ramamurthy; Abhijit Chowdhury; Amal Santra; G K Dhali; S K Bhattacharya; Douglas E Berg; G Balakrish Nair; Asish K Mukhopadhyay Journal: J Clin Microbiol Date: 2004-06 Impact factor: 5.948