Chen Shaohua, Li Youming, Yu Chaohui
First Affiliated Hospital, Medical College, Zhejiang University, Zhejiang Province, China.
J Gastroenterol Hepatol. 2008 Jan;23(1):126-31. doi: 10.1111/j.1440-1746.2007.04900.x.
Resistance to antibiotics in Helicobacter pylori is increasing and becoming a serious problem in eradication treatment. Resistance of H. pylori to clarithromycin has been found to be associated with 2142 A-to-G, 2143 A-to-G and 2182 C-to-T point mutations in the 23S rRNA gene. Thus, the purpose of the present study was to develop a new method to analyze single nucleotide polymorphism (SNPs) of 23S rRNA gene using oligonucleotide microarray and to determine the prevalence of each mutation in H. pylori-positive patients.
Gastric tissue biopsy specimens were obtained from patients undergoing upper gastrointestinal endoscopy. After DNA extraction, asymmetric PCR was employed to prepare single-stranded target DNA labeled with a fluorescent dye. The PCR products that amplified a portion of 23S rRNA from H. pylori isolates were hybridized on DNA microarray, specific for SNP genotyping and mutation detection. The optimal signal intensity and efficiency of hybridization were observed for capture probes in the detection of DNA sequence variation. The relevant mutation was confirmed by DNA sequencing analysis.
Fifty-four gastric biopsy specimens yielded H. pylori-positive results and were studied to detect mutations in the 23S rRNA gene. There were no samples with A-to-G transition at position 2142. The 2143 A-to-G and 2182 C-to-T mutations were present in 11.11% and 12.96% of H. pylori strains examined, respectively. The relevant mutation was confirmed by analysis of DNA sequencing to be the same as that described at position 2142, 2143 and 2182 using oligonucleotide microarray.
Oligonucleotide microarray of the PCR product permits a rapid and accurate screening of SNPs of 23S rRNA gene from H. pylori. It is now possible to apply this hybridization technology in clinical diagnosis and treatment.
幽门螺杆菌对抗生素的耐药性不断增加，已成为根除治疗中的一个严重问题。幽门螺杆菌对克拉霉素的耐药性与23S rRNA基因中的2142 A→G、2143 A→G和2182 C→T点突变有关。因此，本研究的目的是开发一种利用寡核苷酸微阵列分析23S rRNA基因单核苷酸多态性（SNP）的新方法，并确定幽门螺杆菌阳性患者中每种突变的发生率。
从接受上消化道内镜检查的患者中获取胃组织活检标本。DNA提取后，采用不对称PCR制备用荧光染料标记的单链靶DNA。从幽门螺杆菌分离株中扩增23S rRNA一部分的PCR产物与用于SNP基因分型和突变检测的DNA微阵列杂交。在检测DNA序列变异时观察捕获探针的最佳信号强度和杂交效率。通过DNA测序分析确认相关突变。
54份胃活检标本幽门螺杆菌检测呈阳性，用于检测23S rRNA基因中的突变。在2142位没有A→G转换的样本。在检测的幽门螺杆菌菌株中，2143 A→G和2182 C→T突变分别占11.11%和12.96%。通过DNA测序分析确认相关突变与使用寡核苷酸微阵列在2142、2143和2182位描述的突变相同。
PCR产物的寡核苷酸微阵列可快速、准确地筛选幽门螺杆菌23S rRNA基因的SNP。现在有可能将这种杂交技术应用于临床诊断和治疗。
