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GENOTYPING OF VIRULENCE FACTORS IN H. PYLORI USING A MICROARRAY WITH FOCUS ON THE CAG PATHOGENICITY ISLAND
Author(s) -
Sillèn Anna,
Eriksson Lena,
Engstrand Lars
Publication year - 2000
Publication title -
journal of gastroenterology and hepatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.214
H-Index - 130
eISSN - 1440-1746
pISSN - 0815-9319
DOI - 10.1046/j.1440-1746.2000.00a46.x
Subject(s) - caga , pathogenicity island , virulence , helicobacter pylori , genotyping , gene , biology , microarray , genotype , genetics , microbiology and biotechnology , dna microarray , gene expression
Objective The aim of this work was to identify genetic differences regarding virulence factors in Helicobacter pylori, including the cag pathogenicity island (PAI). Sixty strains isolated from patients belonging to case‐control study was analyzed H. pylori infection is associated with chronic gastritis, duodenal ulcer and gastric cancer. Vacuolating cytotoxin (VacA) producing strains of H. pylori are mostly isolated from patients with the more severe diseases. The CagA (cytotoxin associated gene A) protein is associated with cytotoxin production, and the corresponding gene cagA is found in ca. 60% of H. pylori strains. The CagA gene is located in the cag pathogenicity island, which is 38 kb in size and contains 28 open reading frames. The function of the CagA protein is still unknown. Hitherto, no study has been made to investigate if it actually is the cagA gene which determines the pathogenicity or if it can be one (or several) of the neighbouring genes. Materials & Methods The cases have adenocarcinoma and the controls have different non‐cancer GI diseases. We have set up a microarray system, which enables us to screen for genotypic differences for a large number of strains. The microarray consist of 45 PCR products spotted onto poly‐L‐lysine coated slides. These PCR products were amplified from all 28 predicted genes in the H. pylori cag PAI and from 17 other virulence genes. As template in the PCR reactions, DNA from the sequenced strain 26695 was used. The PCR primers were designed based on the public whole genome sequence from 26695 strains. The arrays were made by a GMS 417 Arrayer. Genomic DNA was prepared using bacteria from a whole agar plate. The DNA was digested by AluI and fluorescently labelled with Cy3‐dCTP by random priming. Hybridisation was performed o.n. and the arrays were then scanned for signals using the GMS 418 Array Scanner. Results Analysis of the clinical isolates showed a great variety in the genetic make‐up concerning the cag PAI. Strains that are cagA negative can still contain a large fragment of the PAI and vice versa. Partial deletions of the PAI was especially observed among the control strains. Furthermore, the array is sensitive enough to differentiate between the vacA gene subtypes (s1/s2 and m1/m2) which earlier have been made by PCR. Conclusion We have shown that it is very important to distinguish between a cagA negative strain and a cag PAI negative one. Statistics from these 155 strains will hopefully pinpoint which gene/‐s that is the real virulence determinant in the cag PAI ‐ if it is cagA or some other gene.