Typing of Helicobacter pylori vacA gene and detection of cagA gene by PCR and reverse hybridization

Abstract

The present report describes an analysis of two virulence genes of Helicobacter pylori. Parts of the cagA gene, as well as parts from the signal (s) and middle (m) regions of the mosaic vacA gene, were amplified with biotin-labelled PCR primers and the products were subsequently analyzed by a single-step reverse hybridization line probe assay (LiPA). This assay comprises a strip containing multiple specific probes for the vacA s region (sla, slb, and s2 alleles), the vacA m region (ml and m2 alleles), and the cagA gene. A total of 103 H. pylori-positive materials, including cultured isolates, gastric biopsy specimens, and surgical specimens from patients living in Portugal (n = 55) and The Netherlands (n = 48) were tested by the PCR-LiPA. cagA was detected in 84 and 73% of the Portuguese and Dutch patients, respectively. vacA typing results, as determined by reverse hybridization, were completely concordant with those of sequence analysis. Most Portuguese patients (72%) contained type slb, whereas most Dutch patients (61%) contained type sla (P < 0.001). The method is also very effective at detecting the presence of multiple genotypes in a single biopsy specimen. The prevalence of multiple strains in Portuguese patient samples was significantly higher (29%) than that in Dutch patient samples (8%) (P = 0.001). There was a significant association between the presence of ulcers or gastric carcinoma and the presence of vacA type sl (sla or slb; P = 0.008) and cagA (P = 0.003) genes.

FIG. 1

Mosaic structure of the vacA gene comprising the variable s and m regions. The locations of the various primer target sites are indicated by arrows, and the expected amplimer sizes are shown. Primer sets A through F were described by Atherton et al. (3). Primer sets G and H were described in the present study and permit general amplification of the vacA s and m regions with single primer sets.

FIG. 2

Nucleotide sequence alignment of vacA s1 and s2. The alignment shows nucleotides 36 to 108 and 36 to 135 of the s1 (GenBank accession no. U05676) and s2 (GenBank accession no. U29401) allelic types, respectively. Dots indicate the presence of a nucleotide identical to the nucleotide in the top sequence. A dash indicates a gap. U29401 and NL4600 represent s2 variants; U07145, PO30, and PO32 represent s1a variants; and NL4601 and PO12 represent s1b variants. The positions of the allele-specific probes are indicated by shading.

FIG. 3

Nucleotide sequence alignment of vacA m1 and m2. The alignment shows nucleotides 1495 to 1607 and 1471 to 1658 of m1 (GenBank accession no. U5676) and m2 (GenBank accession no. U29401) allelic types, respectively. Dots indicate the presence of a nucleotide identical to the nucleotide in the top sequence. A dash indicates a gap. U29401 and NL4600 represent m2 variants, and U07145, PO30, PO32, NL4601, and PO12 represent m2 variants. The positions of the universal primers as well as those of the allele-specific probes are indicated by shading.

FIG. 4

Outline and examples of the LiPA for the identification of the different vacA alleles and the cagA gene. Different representative examples are shown, including a sample containing multiple strains.