酶显色法基因芯片检测幽门螺杆菌克拉霉素耐药基因  

Detection of Gene from Helicobacter pylori Associated with Clarithromycin Resistance by Using Coloration Gene Chips

作　　者：宣世海[1] 周玉贵[1] 邵柏[1] 崔亚林[1] 周洁[1] 王琴[1] 徐康[1] 王惠民[2] 褚少朋[2] 景奉香[3] 金庆辉[3] 

机构地区：[1]南通大学附属东台医院检验科,江苏东台224200 [2]南通大学附属医院检验医学中心,江苏南通226001 [3]中国科学院上海微系统与信息技术研究所生物芯片实验室,上海200050

出　　处：《中华医院感染学杂志》2009年第24期3325-3329,共5页Chinese Journal of Nosocomiology

基　　金：江苏省医学重点建设学科资助项目(XK200723)

摘　　要：目的建立酶显色法基因芯片快速、准确检测幽门螺杆菌(Hp)克拉霉素耐药基因。方法设计一对引物,以地高辛标记其中一条引物,扩增Hp 23S rRNA部分基因,得到285 bp DNA片段,根据23S rRNA上的5个位点设计探针和制作芯片,扩增产物与芯片杂交,酶显色后扫描判读结果。结果63例Hp阳性标本中,芯片结果显示突变率:A2143G为3.17%、T2182C为11.11%、A2143G+T2182C为14.29%;酶显色法芯片和DNA直接测序法检测Hp 23S rRNA基因多态性结果符合率为96.83%;质粒浓度为1.53×102拷贝/μl时芯片法仍可检测到野生和突变信号。结论酶显色法芯片检测实现了高通量的同时具有较高敏感性和特异性,能同时检测23S rRNA的多种突变型,无需特殊仪器,可以用于指导临床合理用药和进行流行病学调查,有一定的临床应用价值。OBJECTIVE To establish a rapid and accurate assay for detection of clarithromycin resistance genes in Helicobacter pylori by coloration gene chips.METHODS Collected gastric tissue biopsy specimens from patients followed by DNA extraction,designed a pair of primers and characterized one of the primers by digoxin,a PCR assay was used to amplifing a portion of 23S rRNA from H.pylori,and the gene fragments about 285 bp were amplified.Probes were designed and the gene chip was composed of single nucleotide polymorphism of 5 sites in 23S rRNA associated with clarithromycin resistance.RESULTS Sixty-three H.pylori positive gastric tissue biopsy specimens were studied to detect mutations in the 23S rRNA gene.The rates of A2143G,T2182C and A2143G＋T2182C mutations were respectively present in 3.17%,11.11% and 14.29%.The results of the coloration gene chip were concordant with DNA sequencing in 96.83%.The gene chip may detect the mutation in every site even when the concentration of plasmid was 1.53×102 copies/μl.CONCLUSIONS Coloration gene chip for detecting resistance gene in H.pylori is highly sensitive,specific and throughput,and is technically feasible for clinical application.

关 键 词：基因芯片 幽门螺杆菌 克拉霉素 耐药 

分 类 号：R378[医药卫生—病原生物学]