用于分析幽门螺杆菌菌株对克拉霉素敏感性的改良等位基因特异性引物聚合酶链反应方法

Modified allele-specific primer-polymerase chain reaction method for analysis of susceptibility of Helicobacter pylori strains to clarithromycin.

作者信息

Furuta Takahisa, Soya Yoshihiro, Sugimoto Mitsushige, Shirai Naohito, Nakamura Akiko, Kodaira Chise, Nishino Masafumi, Okuda Masumi, Okimoto Tadayoshi, Murakami Kazunari, Fujioka Toshio, Hishida Akira

机构信息

Center for Clinical Research, Hamamatsu University School of Medicine, Hamamatsu, Japan.

出版信息

J Gastroenterol Hepatol. 2007 Nov;22(11):1810-5. doi: 10.1111/j.1440-1746.2007.04919.x.

DOI:10.1111/j.1440-1746.2007.04919.x

PMID:17914955

Abstract

BACKGROUND AND AIM

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.

CONCLUSION

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.

摘要

背景与目的

大多数对克拉霉素耐药的幽门螺杆菌菌株在23S rRNA基因的2142或2143位存在从腺嘌呤（A）到鸟嘌呤（G）的突变。本研究的目的是开发一种基于聚合酶链反应（PCR）的检测方法，能够在单个反应管中确定这些突变。

方法

我们设计了正向引物FP2143G和反向引物RP2142G，它们分别与幽门螺杆菌23S rRNA基因的2143G和2142G突变序列特异性退火。我们还分别在2142和2143位的上游和下游设计了正向引物FP - 1和反向引物RP - 1，通过扩增片段大小区分野生型A2142G和A2143G突变。从292份快速尿素酶试验阳性的胃组织样本中提取DNA，并进行PCR反应。将结果与克拉霉素的最低抑菌浓度（MIC）进行比较。