Comparison of PCR-restriction fragment length polymorphism analysis and PCR-direct sequencing methods for differentiating Helicobacter pylori ureB gene variants

Abstract

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the Helicobacter pylori genes is widely used to differentiate strains. However, with this typing method only a single base change at a specific restriction site can be detected. In addition, it is unclear whether the nucleotide base change recognized by RFLP is related to a substitution of encoded amino acid. To examine the validity of the PCR-RFLP method, 933-bp PCR products were obtained from 41 different clinical H. pylori isolates and were digested with Sau3A restriction endonuclease. Furthermore, the nucleotides of the same region in the ureB gene were directly sequenced and compared. PCR-RFLP confirmed that there was genetic diversity within the ureB gene with three distinct types, one being well conserved and the other two being variations. However, the direct sequencing method revealed that there was no difference at the nucleotide level among these RFLP types. Base substitutions recognized by Sau3A occurred in the third-base position and did not change the encoded amino acid. In addition, many nucleotide mutations, which could not be recognized by Sau3A, were frequently found. These results suggest that the PCR-RFLP method provides for an easy typing scheme of isolates, but does not reveal the true extent of genetic diversity. It is proposed that careful observation is required for the interpretation of results when clinical isolates are differentiated.

FIG. 1

Restriction digest types of the 933-bp PCR product from the H. pylori ureB gene of six representative clinical isolates. Amplified DNA was digested with Sau3A and separated by electrophoresis on a 5% gel. Lane M is the molecular mass standard of 100 bp. Lanes show type A (A), type B (B), and type C (C) by PCR-RFLP analysis. The B and C types were considered variants.

FIG. 2

The region of the H. pylori ureB gene PCR was amplified and sequenced. The PCR-amplified region corresponding to nucleotides 96 to 1029 of strain 85P is indicated as a hatched bar. The sequenced region corresponding to nucleotides 226 to 886 is indicated as a black bar. White arrowheads show the recognition sites for Sau3A. Closed circles indicate the nucleotide mutation site found by Sau3A. The B and C PCR-RFLP types were considered to be variants.

FIG. 3

Partial nucleotide sequences of the 933-bp ureB gene PCR product obtained from five H. pylori strains representing two different PCR-RFLP types and one reference strain (85P), as previously reported. PCR-RFLP of KP48a and KP48b strains showed them to be type A. KP72b, KP96a, and KP96b were considered to be variant strains showing type B by PCR-RFLP. Numbers on the left indicate the base positions corresponding to nucleotides 316 to 765 of strain 85P. Bases included in the Sau3A restriction site (GATC) are double underlined. Asterisks indicate complete identity of the nucleotides, and dots indicate base mutations. The positions of the encoded amino acid substitution are underlined.