基于表面增强拉曼光谱法快速检测细菌抗生素敏感性的研究  

Study on the rapid detection of the antibiotic susceptibility of bacteria based on surface-enhanced Raman spectroscopy(SERS)

作　　者：付世杰 李磊娇 聂鑫 王习文 李志萍 曲晗 王宇田 李颂 田栢会 郝良玉 王玥[5] 邵琳 卜思含 韩德明[1] FU Shi-jie;LI Ld-jiao;NIE Xin;WANG Xi-wen;LI Zhi-ping;QU Han;WANG Yu-tian;LI Song;TIAN Bai-hui;HAO Liang-yu;WANG Yue;SHAO Lin;BU Si-han;HAN De-ming(School of Life Science and Technology,Changchun University of Science and Technology,Changchun ,China 130022;Unit 65316of the PLA;Institute of Military Veterinary Medicine,AMMS;Jilin Agricultural University;Jilin Medical CAollege)

机构地区：[1]长春理工大学生命科学技术学院 [2]长春理工大学化学与环境工程学院 [3]65316部队 [4]军事科学院军事医学研究院军事兽医研究所 [5]吉林农业大学 [6]吉林医药学院

出　　处：《中国病原生物学杂志》2018年第12期1362-1367,1371,共7页Journal of Pathogen Biology

基　　金：吉林省教育厅"十三五"科学技术项目(No.JJKH20170604KJ);长春理工大学科技创新基金项目(No.XJJLG-2015-07)

摘　　要：目的基于核酸适配体表面增强拉曼光谱技术(SERS),建立一种快速检测细菌抗生素敏感性试验方法,评价不同抗生素对细菌拉曼光谱强度的影响。方法将大肠埃希菌O157:H7和金黄色葡萄球菌分别与不同抗生素作用,再将大肠埃希菌O157:H7和金黄色葡萄球分别与其特异性适配体共孵育,通过表面增强银纳米粒子的方法在金黄色葡萄球菌-适配体和大肠埃希菌-适配体表面还原银纳米粒子,通过SERS技术检测目标细菌。结果分别用大肠埃希菌O157:H7和金黄色葡萄球菌进行SERS试验,用不同浓度萘啶酸、硫酸粘菌素、土霉素、利福平作用于大肠埃希菌。结果表明在抗生素作用1h发现萘啶酸浓度达到1μg/ml、硫酸粘菌素浓度达到1μg/ml、土霉素浓度达到1μg/ml、利福平浓度为2μg/ml时,细菌拉曼光谱显著降低,并可快速确定此时抗生素浓度为大肠埃希菌最低抑制浓度(MIC)值;用不同浓度环丙沙星、氯霉素、红霉素、万古霉素作用于金黄色葡萄球菌,结果在抗生素作用1h发现环丙沙星浓度为1μg/ml、氯霉素浓度达到1μg/ml、红霉素浓度达到1μg/ml、万古霉素浓度达到1μg/ml,细菌拉曼光谱显著降低,并可快速确定此时抗生素浓度为金黄色葡萄球菌最低抑制浓度(MIC)值;当反应时间达到2h,可发现抗生素低于MIC时,细菌的拉曼强度与对照组相似,而MIC未发生变化,证明试验结果正确。结论基于核酸适配体SERS方法可快速评价抗生素对细菌的影响,并在短时间内确定MIC值,因此可用于检测细菌对抗生素敏感性的快速检测。Objectives To create a test method for the rapid detection of the antibiotic susceptibility of bacteria based on aptamer-mediated surface-enhanced Raman spectroscopy(SERS)in order to evaluate the effects of different antibiotics on the Raman intensity of bacteria. Methods Escherichia coli O157:H7 and Staphylococcus aureus were respectively treated with different antibiotics and incubated with specific aptamers.Silver nanoparticles were synthesized in situ on the surface of aptamers.The target bacteria were detected using SERS technology. Results E.coli O157:H7and S.aureus were respectively tested using SERS technology.E.coli O157:H7was treated with different concentrations of nalidixic acid,colistin sulfate,oxytetracycline,and rifampicin.Results indicated that the concentration of nalidixic acid reached 1μg/ml,the concentration of colistin sulfate reached 1μg/ml,the concentration of oxytetracycline reached 1μg/ml,and the concentration of rifampicin reached 2μg/ml at 1hour,the Raman spectra of bacteria decreased significantly,and the minimum inhibitory concentration(MIC)of E.coli O157:H7was quickly determined at that time.S.aureus was treated with different concentrations of ciprofloxacin,chloramphenicol,erythromycin,and vancomycin.Results indicatedthat the concentration of ciprofloxacin reached 1μg/ml,the concentration of chloramphenicol reached 1μg/ml,the concentration of erythromycin reached 1μg/ml,and the concentration of vancomycin reached 1μg/ml at 1 hour,the Raman spectra of bacteria decreased significantly,and the MIC of S.aureus was quickly determined at that time.When the reaction time reached 2 hours and the MIC of the antibiotic was lower,the Raman intensity of bacteria was similar to that of the control group,but the MIC did not change,which further indicated that the experimental results were correct. Conclusion The SERS method based on aptamer mediation can quickly evaluate the effect of antibiotics on bacteria and determine the MIC value in a short period of time,so it can be used to rapidl

关 键 词：核酸适配体 抗生素敏感性实验 表面增强拉曼光谱 大肠埃希菌O157:H7 金黄色葡萄球菌 

分 类 号：R378[医药卫生—病原生物学]