纳米金辅助不对称PCR扩增幽门螺杆菌23S rRNA基因的优化和建立  

Optimization and establishment of Au nanoparticles-assisted asymmetric PCR for amplification 23S rRNA of Helicobacter pylori

作　　者：宣世海[1] 周洁[1] 周玉贵[1] 王琴[1] 崔亚林[1] 徐康[1] 李明兰[1] 王惠民[2] 

机构地区：[1]南通大学附属东台人民医院检验科,江苏省东台市224200 [2]南通大学附属医院检验医学中心,226001

出　　处：《实用医学杂志》2010年第1期122-124,共3页The Journal of Practical Medicine

基　　金：江苏省医学重点学科建设基金资助项目(编号:200723);江苏省卫生厅"科教兴卫"基金资助项目

摘　　要：目的:建立纳米金辅助不对称PCR方法扩增幽门螺杆菌23S rRNA基因,为建立基因芯片法检测幽门螺杆菌耐药基因提供试验平台。方法:将幽门螺杆菌23S rRNA基因转化至感受态大肠杆菌JM109,小量抽提阳性构建质粒DNA,以质粒DNA为模板,设计引物对23S rRNA基因进行不对称PCR扩增。对PCR条件中引物浓度及浓度比例、纳米金浓度等进行优化。结果:限制性引物与非限制性引物浓度比例为1∶60,限制性引物与非限制性引物浓度分别为0.04pmol/L、2.4pmol/L时可获得理想的单链和双链DNA,纳米金浓度为0.8nmol/L时非特异性产物最少,PCR的扩增效率最高。结论:通过对PCR扩增条件的优化,确立了扩增幽门螺杆菌23S rRNA基因纳米金辅助不对称PCR方法的最佳条件,为建立基因芯片法检测幽门螺杆菌耐药基因奠定基础。Objective To establish the method of nanoparticles-assisted asymmetric PCR for amplification 23S rRNA of Helicobacter pylori. Methods 23S rRNA gene was transformed into competent Escherichia coli JM109. A small amount of plasmid DNA was extraced Plasmid DNA as template, the Helicobacter pylori 23S rRNA gene was asymmetry amplified by using self-designed primer.The primers concentrations, concentration ratios, Au nanoparticle concentrations were optimized. Results The ratio of primer vs non-restrictive was 1 ： 60. When the final restrictive primer concentration and non-restrictive primer concentration was 0.04 nmol/L and 2.4 pmol/L respectively, the expected products of single and double-stranded DNA could be obtained. The non- specific products was least and the PCR efficiency was highest when Au nanoparticle concentration was 0.8 nmol/L. Conclusion The optimized conditions of Au nanoparticles -assisted asymmetric PCR has been studied, and that has laid foundation for Helicobacter pylori resistance gene detection by gene chip.

关 键 词：螺杆菌 幽门 基因 芯片 克隆 纳米金 优化 

分 类 号：R450[医药卫生—治疗学] R573[医药卫生—临床医学]