铜绿假单胞菌特异性核酸适配体的筛选及其在菌体检测方面的应用  

Selection and Application of Specific Nucleic Acid Aptamers for the Detection of Pseudomonas aeruginosa

作　　者：李旭 成洋洋 杨凯 刘本康 李成[1] LI Xu;CHENG Yangyang;YANG Kai;LIU Benkang;LI Cheng(School of Biological Engineering,Dalian Polytechnic University,Dalian 116034,China;School of Physics and Materials Engineering,Dalian Minzu University,Dalian 116600,China)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034 [2]大连民族大学物理与材料工程学院,辽宁大连116600

出　　处：《食品科学》2025年第9期322-328,共7页Food Science

基　　金：辽宁省自然科学基金项目(2021-MS-302);辽宁省教育厅科研基金项目(LJKZ0520);辽宁省教育厅科研基金项目(LJKZZ20220060)。

摘　　要：为实现对致病菌铜绿假单胞菌(Pseudomonas aeruginosa)的快速微量检测,本研究首先利用全细胞指数富集的配体系统进化技术,筛选靶向铜绿假单胞菌的特异性单链DNA(single-stranded DNA,ssDNA)适配体。经15轮富集后共获得30个ssDNA序列,比较吉布斯自由能ΔG和解离常数Kd后,确定适配体Apt_(13)的特异性和亲和力最佳,可用于铜绿假单胞菌的检测。异硫氰酸荧光素(fluorescein isothiocyanate,FITC)标记菌体形成的FITC-P.aeruginosa复合物具有荧光,适配体Apt_(13)的加入可猝灭其荧光,而反筛菌大肠杆菌、金黄色葡萄球菌、枯草芽孢杆菌和溶壁微球菌不影响Apt_(13)对FITC-P.aeruginosa荧光的猝灭作用。基于此现象构建了用于铜绿假单胞菌检测的荧光系统。通过适配体Apt_(13)对待测样本中荧光的猝灭情况判断是否存在铜绿假单胞菌。在确定Apt_(13)最佳使用浓度及其与P.aeruginosa孵育的最优时间基础上,Apt_(13)猝灭的荧光强度与铜绿假单胞菌在10^(1)~10^(8)CFU/mL范围内呈线性关系,检出限为2 CFU/mL,检测可在2.0 h内完成,并可对水样中铜绿假单胞菌实现有效检测。For rapid and sensitive detection of pathogenic bacterium Pseudomonas aeruginosa,the whole-cell systematic evolution of ligands by exponential enrichment(SELEX)technique was used to select single-stranded DNA(ssDNA)aptamers specific for P.aeruginosa.After 15 rounds of enrichment,30 ssDNA sequences were obtained.By comparing the Gibbs free energy(ΔG)and dissociation constant(Kd),aptamer Apt_(13) was identified to have the best specificity and affinity for P.aeruginosa.P.aeruginosa labeled with fluorescein isothiocyanate(FITC)exhibited fluorescence,which could be quenched by the addition of Apt_(13).Counter-screening with other bacteria,including Escherichia coli,Staphylococcus aureus,Bacillus subtilis,and Micrococcus luteus,did not affect the fluorescence quenching effect of Apt_(13) on FITC-labelled P.aeruginosa.Based on this phenomenon,a fluorescence detection system for P.aeruginosa was developed.Whether or not P.aeruginosa is present in samples could be judged from the fluorescence quenching effect of Apt_(13).Under optimized concentration of Apt_(13) and incubation time of P.aeruginosa,the fluorescence quenching intensity exhibited a linear relationship with the concentration of P.aeruginosa ranging from 10^(1) to 10^(8) CFU/mL.The limit of detection(LOD)was 2 CFU/mL,and the entire detection process took less than 2.0 h.In conclusion,the developed method allowed effective detection of P.aeruginosa in water samples.

关 键 词：铜绿假单胞菌 全细胞指数富集的配体系统进化技术 适配体 荧光传感器 

分 类 号：TS207.4[轻工技术与工程—食品科学]