靶向mecA基因纳米胶体金探针快速检测耐甲氧西林金黄色葡萄球菌方法的建立  

Rapid detection of methicillin-resistant Staphylococcus aureus by mecA gene targeted colloid gold nanoparticles probes

作　　者：夏云[1] 何云燕[2] 黎颖[1] 张莉萍[1] 

机构地区：[1]重庆医科大学附属第一医院检验科,400016 [2]重庆市中山医院检验科

出　　处：《中华检验医学杂志》2009年第7期781-784,共4页Chinese Journal of Laboratory Medicine

基　　金：重庆市卫生局资助项目（07-02-231）

摘　　要：目的用以纳米胶体金颗粒为载体的DNA探针杂交技术，建立快速、简便和特异的检测耐甲氧西林金黄色葡萄球菌（MRSA）的方法。方法以直径60nm的胶体金纳米颗粒与巯基修饰的2条mecA基因特异性寡核苷酸探针共价结合，制备成纳米胶体金探针。直接提取金黄色葡萄球菌的基因组DNA，经超声破碎后与纳米胶体金探针进行液相杂交。杂交产物经高速离心后再用反向色谱反应盘检测有无胶体金颗粒沉淀产生。用该法和PCR扩增试验同时检测临床分离株的mecA基因，以PCR法为金标准，评价纳米胶体金探针液相杂交法的准确性。结果在95株临床试验菌株中PCR法共检出71株MRSA，24株MSSA。在71株MRSA中，纳米胶体金探针杂交试验有69株为阳性，敏感度为97．2％，最低检测限为4．72fmol／L。而在24株甲氧西林敏感的金黄色葡萄球菌（MSSA）中，纳米胶体金探针杂交试验无一株阳性，特异度为100％。95株临床试验菌株中纳米胶体金探针杂交试验正确检出93株，准确度97．9％。结论纳米胶体金探针杂交试验与PCR检测MRSA具有很好的一致性。应用胶体金探针杂交技术检测MRSA具有快速、简便、准确的特点，有望成为MRSA的快速诊断的新方法。Objective To establish hybridization method by using the DNA-modified colloid gold nanoparticles probes for rapid and specified detection of methieillin resistant Staphylococcus aureus （MRSA）. Methods DNA-modified nanopartieles probes were prepared by using two mereapto-modified meeA genespecific oligonucleotide probes bounding with 60nm-diameter colloid gold nanopartieles through covalent binding. Genomic DNA of Staphylococcus aureus strains were extracted and then fragmented by uhrasonie waves. The fragmentized DNA was hybridized with the DNA-modified colloid gold nanopartieles probes. The reaction products were centrifuged and then detected by reversed-phase thinlayer chromatography plate to observe any colloid gold nanopartieles precipitation. The results of mecA gene detected by the colloid gold nanoparticles probes hybridization were compared with the results of PCR and the accuracy of the hybridization method was evaluated. Results Of total 95 tested strains, 71 strains were confirmed as MRSA and 24 strains were confirmed as methicillin susceptible Staphylococcus aureus （MSSA） by PCR. Of 71 MRSA strains, 69 strains were positive by colloid gold nanopartieles probes hybridization, the sensitivity of this method was 97.2%. All of the 24 MSSA strains were negative by using this technique. The specificity of this method was 100%. Of total 95 test strains,93 strains were detected correctly. The accuracy was 97.9%. Conclusions Colloid gold nanoparticles probes hybridization test is well consistent with the gold standard method of PCR in detection of MRSA. Detection of MRSA by using the technique of the DNA-modified colloid gold nanoparticles probes hybridization is rapid, simple and accurate. It is potent to be a new method for rapid diagnosis of MRSA infection.

关 键 词：甲氧西林抗药性 葡萄球菌 金黄色 DNA探针 纳米粒子 胶体金 

分 类 号：R686[医药卫生—骨科学]