液相固化纳米胶体金探针杂交快速检测MRSA的.doc

固化DNA纳米胶体金探针杂交目视化比色检测耐甲氧西林金黄色葡萄球菌mecA基因的实验研究 夏云1， 黎颖1，何云燕2（1重庆医科大学附属第一医院检验科，重庆400016；2重庆市中山医院检验科，重庆400014） 摘要：目的 建立以纳米金颗粒为载体的DNA探针杂交方法，快速、特异地检测耐甲氧西林金黄色葡萄球菌（MRSA）特异性mecA基因。方法 采用直径为60nm的胶体金纳米颗粒与巯基修饰的两条mecA基因寡核苷酸探针共价结合，制备成固化的DNA 金纳米探针。将金纳米探针与其互补的mecA基因靶序列在液相中杂交，通过调节两条探针的比例和不同的检察方法对杂交反应条件和产物检测进行优化。结果：金纳米探针与互补的靶序列进行液相杂交后颜色呈现明显的变化。两条探针不对称加入、采用反向色谱反应盘检测反应产物等可获得满意的效果。结论：固化纳米胶体金探针杂交技术具有快速简便的特点，有望为MRSA的快速检测提供一个新的方法。 关键词： 金纳米颗粒；DNA探针；MRSA；比色法 中图法分类号：R378 文献标识码：A Rapid colorimetric detction of methicillin resistant Staphylococcus aureus mecA gene by DNA-modified colloid gold nanoparticles peobes XIA Yun1, LI Ying1, HE Yun-yan2 （1 Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016; 2 Department of Laboratory Medicine, Chongqing Zhongshan Hospital, Chongqing 400013, China）Abstract: OBJECTIVE To stablish the DNA-modified colloid gold nanoparticles probes hybridization method for rapid and specified detection of methicillin resistant Staphylococcus aureus(MRSA) mecA gene. Methods: DNA modified nanoparticles probes were prepared by using two 5’S modified mecA gene oligonucleotide probes bounding with 60nm diameter colloid gold nanoparticles through covalent binding of Au-S. The prepared nanoparticles probes hybridized with it paired mecA gene sequence. The optical reaction condition were explored by using different concentration of two nanoparticles probes and different methods to detection of hybridization products. Results: The visible color changes of the reaction solution were observed when the DNA-modified colloid gold nanoparticles probes hybridized with its paired target sequence. The satisfactory results were obtained by the means of asymmetry adding of two probes and detection of products by TLC. Conclusions: The technique of the DNA-modified colloid gold nanoparticles probes hybridization has character of rapid and convenient performing and is potent to be a new method for rapid detection of (MRSA).