Simple and Accurate PCR-Based System for Typing Vacuolating Cytotoxin Alleles of Helicobacter pylori

Abstract

Alleles of the vacuolating cytotoxin gene (vacA) ofHelicobacter pylori vary between strains, particularly in the region encoding the signal sequence (which may be type s1 or s2) and the midregion (which may be type m1 or m2). Using a PCR-based typing system developed in the United States, we showed that 36 strains from Asia and South America were all vacA signal sequence type s1; 3 were midregion type m1 and 11 were m2, but 22 could not be typed for the vacA midregion. All strains possessedcagA (cytotoxin-associated gene A), another virulence marker. vacA nucleotide sequence analysis showed that midregion typing failure was due to base substitutions at the primer annealing sites. Using the new sequence data, we developed two new PCR-based vacA midregion typing systems, both of which correctly typed 41 U.S. strains previously typed by the old system and successfully typed all 36 of the non-U.S. strains. All previously untypeable strains were vacA m1, other than one m1/m2 hybrid. In summary, we describe and validate a simple PCR-based system for typing vacuolating cytotoxin (vacA) alleles of H. pylori and show that this system correctly identifies the signal and midregion types of vacA in 77 strains from Asia and North and South America.

Conservation and microdiversity of the phospholipase A (pldA) gene of Helicobacter pylori infecting dyspeptics from different countries

“…PCR conditions for the two assays for cagA (a marker for the right-hand end of the cag PAI and for the cagI region) were as described for the F1/B1 primers and the D008/R008 primers [26,27]. Genotyping based on vacA (signal and mid-regions) were also performed as previously described [14].…”

“…Various pathogenicity mechanisms that could be contributing to the role of H. pylori in peptic ulcer disease have been proposed [11^13]. Most widely investigated are vacuolating cytotoxin (vacA) activity [14], lipopolysaccharides (LPS) [15], and factors involved in cytokine (IL-8) induction mediated by genes located on the cag pathogenicity island (PAI) [16]. Degradation of the gastric mucosal barrier by phospholipase (PL) activity is another potential pathogenicity mechanism of H. pylori that may have an important role in establishing and maintaining long-term infection [17].…”

High relative content of lysophospholipids ofHelicobacter pylorimediates increased risk for ulcer disease

“…Various pathogenicity mechanisms that could be contributing to the role of H. pylori in peptic ulcer disease have been proposed. Most widely investigated are urease activity [2], vacuolating cytotoxin (vacA) activity [3], lipopolysaccharides (LPS) [4], and factors involved in cytokine induction mediated by genes located on the cag pathogenicity island (PAI) [5]. Strains that are cagA positive have been found to be more infectious, achieve higher bacterial density on the gastric mucosa and cause more inflammation than cagA negative strains [6].…”

Molecular Typing of Helicobacter pylori by Virulence‐Gene Based Multiplex PCR and RT‐PCR Analysis

“…M olecular typing methods such as ribotyping [1] and randomly amplified polymorphic DNA (RAPD) analysis [2] have demonstrated considerable genomic diversity among Helicobacter pylori isolates. During the past few years, numerous PCR assays have been developed for the identification of specific virulence genes in H. pylori including ureA [3][4][5][6], ureC [7,8], cagA [3,4,9], vacA [10][11][12][13][14][15][16] and the 16S rRNA genes [17,18]. Recently, we have described a method for the rapid simultaneous molecular identification and subtyping of H. pylori by pyrosequencing of the 16S-rDNA variable V1 and V3 region [19].…”