Simple and accurate PCR-based system for typing vacuolating cytotoxin alleles of Helicobacter pylori

Abstract

Alleles of the vacuolating cytotoxin gene (vacA) of Helicobacter 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 possessed cagA (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.

FIG. 1

Schematic of vacA midregions for the m1 and m2 allelic types, showing the original midregion allelic type-specific typing system (1) and the new PCR strategies. The diagram is drawn to scale except for the sizes of the arrows representing the PCR primers. The boxed area is 910 bp for strain 60190 (coordinates 1395 to 2304 numbered from the adenine of the start codon [8]) and 1,000 bp for strain Tx30a (coordinates 1371 to 2370 [1]). X represents an insertion of 75 bp, and Y represents an insertion of 15 bp; both are present in m2 alleles but not in m1 alleles. The arrows labeled VA3-F and VA3-R and those labeled VA4-F and VA4-R are the allelic type-specific primers used in our original PCR-based system (1). The solid lines labeled pCTB4 and VA6 are the allelic type-specific probes used for hybridization. VAG-F and VAG-R are the primers used in new strategy 1, and they anneal to regions of vacA conserved between m1 and m2 alleles; the types are then differentiated on the basis of PCR product size. Primers VA7-F and VA7-R and primers VA4-F and VA4-R are allelic type-specific primers used together in new strategy 2. Product size depends on which specific primers anneal, and the expected annealing primers are shown below the m1 and m2 regions.

FIG. 2

One percent agarose gels with ethidium bromide showing examples of PCR-based vacA midregion typing by new strategy 1 (A) and new strategy 2 (B). Details about the PCR primers and expected product sizes are given in the text and in Fig. 1. The PCR products shown were amplified from the following nine strains (in the lanes from left to right), respectively: J238 (from the United States, m1 control), J226 (from the United States, m2 control), Ch2 (from China, m1/m2 hybrid), Ch3 (from China, m1), Ch4 (from China, m2), Ch5 (from China, m1), Ch7 (from China, m1), Ch8 (from China, m1), and 92-24 (from the United States m1 control).