寡核苷酸微阵列技术用于检测幽门螺杆菌克拉霉素耐药相关23S rRNA基因多态性

Screening of single nucleotide polymorphism of a 23S rRNA gene from Helicobacter pylori associated with clarithromycin resistance using oligonucleotide microarray

目的幽门螺杆菌(Hp)克拉霉素耐药与23S rRNA基因A2142G、A2143G和C2182T点突变相关。本研究旨在探索建立一种快速、简便的检测方法,获取Hp克拉霉素耐药相关23S rRNA基因多态性的流行病学资料,为临床合理用药提供依据。方法收集胃黏膜标本,用于病理检查、提取DNA。根据核苷酸位点设计相应探针,探针3′端以氨基修饰,氨基与探针序列之间以间隔臂相连,将探针用芯片点样仪点至玻片上。下游引物5′末端用cy3荧光标记,进行不对称PCR扩增。其产物与芯片杂交,杂交后洗涤、扫描芯片,观察信号强度。非荧光标记引物扩增PCR产物克隆至T载体,测序验证芯片结果。结果(1)寡核苷酸微阵列技术与测序检测Hp 23S rRNA基因多态性结果完全一致。(2)经病理和PCR证实为Hp阳性的54例标本,杂交结果显示A2142位点均为野生型(54/54)；A2143G突变率为11．11％(6/54),尚未发现A2143C和A2143T的突变；C2182T突变率为12．96％(7/ 54),尚未发现C2182A和C2182G的突变,其余均为野生型。结论(1)利用寡核苷酸微阵列技术检测Hp克拉霉素耐药的23S rRNA基因多态性,快速、简便而准确,可以高通量并直接检测胃黏膜而不需进行细菌培养,为根除Hp选择用药提供科学依据,推动个体化治疗方案的实施。(2)本研究没有发现Hp 23S rRNA基因2142点突变,A2143G和C2182T突变率分别为11．11％和12．96％。

Objective To develop a new method to analyze single nucleofide polymorphism （SNP） of 23S rRNA gene using oligonucleotide microarray and to determine the prevalence of each mutation in 54 Helicobacter pylori （ H. pylori ） positive patients. Methods Gastric tissue biopsy specimens were obtained from patients followed by DNA extraction and a PCR assay that amplified a portion of 23S rRNA from H. priori isolates. An oligonucleotide microarray system which included oligonucleotide synthesis, preparation of the oligonucleotide microarray, hybridization and signal detection for the analysis of the known SNP of 23S rRNA gene was fabricated. The relevant mutation was confirmed by transformation and culturing of colonies followed by DNA sequencing analysis. Results Fifty-four gastric biopsy specimens yielded H. pylori-positive results and were studied to detect mutations in the 23S rRNA gene. No A-to-G transition at position 2142 was found. Both A2143G and C2182T mutations were respectively present in 11.11% and 12.96% of H.pylori strains examined. The relevant mutation in transformants was confirmed by DNA sequencing to be the same as that described at position 2142, 2143 and 2182 using oligonucleotide microarray. Conclusion Oligonucleotide microarray of the PCR product offers a rapid and accurate screening of SNP of 23S rRNA gene from H. priori.