Multiplex PCR assay for rapid detection and genotyping of Helicobacter pylori directly from biopsy specimens

Abstract

We developed and evaluated a simple, novel multiplex PCR assay for rapid detection of Helicobacter pylori infection and for the determination of vacA and cagA genotypes directly from gastric biopsy specimens. This assay did not require culturing of strains or extraction of DNA from biopsy samples. This multiplex PCR assay would be of particularly great value for laboratories in developing countries.

FIG. 1.

Amplification of vacA s1, vacA s2, vacA m1, and vacA m2 alleles and the cagA gene of H. pylori by multiplex PCR assay. (a) Lane M, 100-bp marker (New England Biolab); lanes 1 to 5, H. pylori strains isolated from India. Lanes 1 to 3, s1m1 cagA⁺ genotype; lane 2, s1m2 cagA⁺ genotype; lane 5, genotype of s2m2 cagA-negative strain. (b) Lane M, 100-bp marker (New England Biolab); lanes 1 to 5, H. pylori strains isolated from different part of the world. Lane 1, strain HUP 77 (s1m1 cagA⁺) from Spain; lane 2, strain TN2 (s1m1 cagA⁺) from Japan; lane 3, strain SS1(s2m2 cagA⁺) from Australia; lane 4, strain J99 (s1m1 cagA⁺) from the United States; lane 5, strain 26695 (s1m1 cagA⁺) from the United Kingdom. (c) Amplification of 550-bp fragment of cag-pathogenicity island empty site for cagA-negative strains discerned by multiplex PCR assay. Lanes 1 to 5, cagA-negative strains isolated from India; lane 6, HUP67, a cagA-negative strain isolated from Spain.

FIG. 2.

Genotype of H. pylori obtained by multiplex PCR format. (a) Lane M, 100-bp marker (New England Biolab); lanes 1 to 5, amplification of H. pylori virulence genes by multiplex PCR from biopsy specimens. As shown in lane 2, biopsy 23 gave a shorter amplicon for the vacA middle region. (b) Lane M, 100-bp marker (New England Biolab); lanes 1 to 5, multiplex PCR results obtained from DNA extracted from strains isolated from the respective patients. As shown in lane 2, strain PCR 23 gave a shorter amplicon similar to that obtained with biopsy 23.

FIG. 3.

Multiplex PCR assay for simulated mixed infection of strains 26695 (s1m1 cagA⁺) and I-80 (s2m2 cagA-negative strain). Lane M, 100-bp marker (New England Biolab); lanes 1 to 6, strains 26695 and I-80 at ratios of 1:1 to 1:32; lane 7, strain 26695; lane 8, strain I-80.

FIG. 4.

Analysis of vacA middle region of strain PCR 23. (a) Amplification of vacA middle region by vacA m1 allele-specific primers VAm-F3-Vam-R3. M, 100-bp marker (New England Biolab); lane 1, PCR 23; lane 2, known vacA m1 allele containing H. pylori DNA. (b) Amplification of vacA middle region by vacA m2 allele-specific primers VA4-F-VA4-R. M, 100-bp marker (New England Biolab); lane 1, PCR 23; lane 2, known vacA m2 allele containing H. pylori DNA. (c) vacA sequence alignment of strain PCR 23 with strain 26695 showing that a 120-bp deletion from strain PCR 23 resulted in a frameshift after the deletion. This gives rise to a premature termination of the protein synthesis. The first bases of the codons are shown in boldface characters in a larger font.