Modified allele-specific primer-polymerase chain reaction method for analysis of susceptibility of Helicobacter pylori strains to clarithromycin.
OBJECTIVE
Most clarithromycin-resistant strains of Helicobacter pylori have a mutation from adenine (A) to guanine (G) at position 2142 or 2143 of the 23S rRNA gene. Our aim in this study was to develop a polymerase chain reaction (PCR)-based assay that could determine these mutations in a single reaction tube.


METHODS
We designed the forward primer FP2143G and the reverse primer RP2142G, which specifically anneal with the 2143G- and 2142G-mutated sequences, respectively, of the 23S rRNA gene of H. pylori. We also designed the forward primer FP-1 and reverse primer RP-1 upstream and downstream from the positions 2142 and 2143, respectively, to distinguish the wild-type A2142G and A2143G mutations from each other by amplicon sizes. DNA was extracted from 292 gastric tissue samples positive for rapid urease test, and the DNA underwent the PCR reaction. The results were compared with minimum inhibitory concentrations (MIC) for clarithromycin.


RESULTS
Helicobacter pylori strains with A2142G, A2143G and wild type could be distinguished by amplicon sizes by a single PCR reaction. The genotyping results were correlated well with the MIC values for clarithromycin. The median MIC for clarithromycin of the wild-type strains was <0.015 microg/mL. Those of strains with 2142G or 2143G were > or =1.0 microg/mL.


CONCLUSIONS
Our new PCR-based assay for 23S rRNA mutations of H. pylori is a useful method for detecting clarithromycin-resistant strains of H. pylori easily.
Furuta
Takahisa
T
Center for Clinical Research, Hamamatsu University School of Medicine, Hamamatsu, Japan. furuta@hama-med.ac.jp
Soya
Yoshihiro
Y
Sugimoto
Mitsushige
M
Shirai
Naohito
N
Nakamura
Akiko
A
Kodaira
Chise
C
Nishino
Masafumi
M
Okuda
Masumi
M
Okimoto
Tadayoshi
T
Murakami
Kazunari
K
Fujioka
Toshio
T
Hishida
Akira
A
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000900:Anti-Bacterial Agents / Q000494:pharmacology / Q000627:therapeutic use*; D017291:Clarithromycin / Q000494:pharmacology / Q000627:therapeutic use*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005260:Female; D005756:Gastritis / Q000188:drug therapy* / Q000382:microbiology; D005838:Genotype; D016481:Helicobacter Infections / Q000188:drug therapy* / Q000382:microbiology; D016480:Helicobacter pylori / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests; D008875:Middle Aged; D009154:Mutation; D010437:Peptic Ulcer / Q000188:drug therapy* / Q000382:microbiology; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D012338:RNA, Ribosomal, 23S
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D017291:Clarithromycin