Modified allele-specific primer-polymerase chain reaction method for analysis of susceptibility of Helicobacter pylori strains to clarithromycin
Modified allele-specific primer-polymerase chain reaction method for analysis of susceptibility of Helicobacter pylori strains to clarithromycin
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DOI：
10.1111/j.1440-1746.2007.04919.x
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发表时间：
2007-11-01
影响因子：
4.1
通讯作者：
Hishida, Akira
中科院分区：
文献类型：
--
作者：
Furuta, Takahisa;Soya, Yoshihiro;Hishida, Akira
背景与目的：大多数耐克拉霉素幽门螺杆菌菌株在23S rRNA基因2142或2143位点发生腺嘌呤(a)突变为鸟嘌呤(G)突变。我们在这项研究中的目的是开发一种基于聚合酶链反应（PCR）的检测方法，可以在单个反应管中确定这些突变。方法：设计了正向引物FP2143G和反向引物RP2142G，分别对H. pylori 23S rRNA基因的2143G和2142g突变序列进行特异性退火。我们还在2142和2143位点的上游和下游分别设计了正向引物FP-1和反向引物RP-1，通过扩增子大小区分野生型A2142G和A2143G突变。从292份快速脲酶试验阳性的胃组织样本中提取DNA，并进行PCR反应。结果与克拉霉素的最低抑菌浓度（MIC）进行比较。结果：通过单次PCR反应可区分出A2142G、A2143G和野生型幽门螺杆菌。基因分型结果与克拉霉素的MIC值有很好的相关性。野生型菌株对克拉霉素的中位MIC < 0.015 μ g/mL。2142G和2143G菌株的浓度为>= 1.0 μ g/mL。结论：基于pcr的幽门螺杆菌23S rRNA突变检测方法是一种简便易行的检测耐克拉霉素幽门螺杆菌的方法。
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 mu g/mL. Those of strains with 2142G or 2143G were >= 1.0 mu g/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.