基于嵌段式互补序列的无标记核酸适体荧光探针用于食品中金黄色葡萄球菌的快速检测  

Label-free aptamer and piecewise complementary sequences-based fluorescent probes for rapid detection of Staphylococcus aureus in food

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作  者:郑毅 陈果 严杭丽 王婧毓 赵凤霞 柏琴琴[1] 胡红梅[2] 梁好 牛湘衡 ZHENG Yi;CHEN Guo;YAN Hangli;WANG Jingyu;ZHAO Fengxia;BAI Qinqin;HU Hongmei;LIANG Hao;NIU Xiangheng(School of Public Health,University of South China,Hengyang 421001,China;Hengyang Center for Disease Control and Prevention,Hengyang 421001,China)

机构地区:[1]南华大学公共卫生学院,衡阳421001 [2]衡阳市疾病预防控制中心,衡阳421001

出  处:《分析试验室》2024年第11期1556-1562,共7页Chinese Journal of Analysis Laboratory

基  金:国家自然科学基金青年项目(81903369)项目资助。

摘  要:以金黄色葡萄球菌(S.aureus)为研究目标菌,开发了一种无标记核酸适体(aptamer)荧光探针,用于复杂食品基质中致病菌的快速检测。针对目标菌的aptamer,设计了全链的嵌段式互补序列,结合可特异性插入DNA双链结构并显著增强荧光发射的SYBR Green I(SG-I)染料,实现了探针的特异性识别和起始信号的双重放大。当目标菌存在时,由于探针中的互补序列为嵌段式,杂交作用力弱于S.aureus和aptamer的特异性结合。因此,目标菌能够与互补序列竞争aptamer,破坏DNA双链结构而释放SG-I,使得探针荧光显著降低。结果表明,在最佳实验条件下,荧光信号变化值(ΔF)与S.aureus浓度(c)的对数在1.00×10^(2)~1.00×10^(9) CFU/m L范围内呈良好的线性关系,回归方程为ΔF=221.0logc+687.1,线性相关系数R^(2)=0.9918,检出限可低至13 CFU/m L。该方法用于鸡肉、牛奶和鸡蛋样品测定,加标回收率在95.2%~104.7%之间,相对标准偏差在3.9%~7.6%。A label-free aptamer-based fluorescent probe was developed for the rapid and specific detection of Staphylococcus aureus (S.aureus) in complex food samples.The probe involves the use of four piecewise complementary sequences for full-strand based on a S.aureus-specific aptamer,and as a fluorescent dye,SYBR Green I (SG-I) can specifically insert into double-stranded structure of DNA to significantly enhance fluorescence emission and achieve double amplification of the initial signal.In the presence of S.aureus,the specific binding affinity between S.aureus and its aptamer surpassed the hybridization force between the aptamer and its four piecewise complementary sequences.Consequently,S.aureus competed with the complementary sequence to bind the aptamer,leading to the disruption of the double-stranded DNA structure,release of SG-I,and a substantial reduction in the fluorescence signal of the probe.Under the optimized experimental conditions,the logarithm of S.aureus concentration(c) and the change of fluorescence signal(ΔF) showed a good linear relationship in the range of 1.00×10^(2)-1.00×10^(9) CFU/m L,and the linear regression equation wasΔF=221.0logc+687.1,with the correlation coefficient R^(2)=0.9918.The limit of detection (LOD) for S.aureus was as low as 13 CFU/m L.This strategy was applied to detect S.aureus in chicken,milk,and egg samples,and the recoveries were 95.2%-104.7%,with the relative standard deviations of 3.9%-7.6%.

关 键 词:金黄色葡萄球菌 核酸适体 荧光探针 食品安全 

分 类 号:TS207.4[轻工技术与工程—食品科学]

 

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