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2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2条探针的比例和不同的检测方法对杂交反应条件和产物检测进行优化。结果金纳米探针呈稳定的酒红色,与互补的靶序列DNA进行液相杂交后颜色呈现明显的蓝色变化。当2条探针不对称加入时颜色变化更明显。采用反向色谱反应盘检测反应产物也可获得满意的效果。产物离心后检测灵敏度可达2.36 fmol/L。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的快速检测提供一种新的方法。
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 were hybridized with its paired mecA gene sequence in liquidphrase. The optical reaction condition was explored by using different concentration of two nanopartieles probes and different methods to detection of hybridization products. Results DNA modified gold nanoparticles probes were exhibited the color of win red. The visible blue color changes of the reaction solution were observed when the DNA-modified colloid gold nanoparticles probes hybridized with its paired target sequence. The more obvious results were obtained by the means of asymmetry adding of two probes to reaction solution. The detection of reaction products by TLC could also acquire satisfactory results. The sensitivity of the method was 2.36 fmol/L if the reaction products were centrifuged and sedimentations were detected by TLC. Conclusion 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.
出处
《重庆医学》
CAS
CSCD
北大核心
2010年第7期782-783,786,I0001,共4页
Chongqing medicine
基金
重庆市卫生局科研基金资助项目(07-02-231)
