用于Escherichia coli O157∶H7直接快速检测的倏逝波荧光核酸适配体传感器研究

doi:10.13560/j.cnki.biotech.bull.1985.2019-1132

摘要/Abstract

摘要： 通过融合倏逝波荧光光纤传感器和特异性核酸适配体的优势,提出了一种基于倏逝波荧光原理及其与病原菌尺寸效应的Escherichia coli O157∶H7（E.coli O157∶H7）直接快速检测方法。基本原理是当一定浓度荧光标记E.coli O157∶H7核酸适配体加入样品检测池时,倏逝波激发荧光分子发出荧光,利用倏逝波全光纤生物传感器即可实现荧光信号的定量检测;当荧光标记的核酸适配体与E.coli O157∶H7混合后加入样品检测池,因倏逝波渗入深度仅为100 nm,导致特异性结合E.coli O157∶H7的核酸适配体标记荧光分子不能被激发,从而使得检测荧光信号降低;利用荧光信号强度与E.coli O157∶H7浓度的比例关系即可实现其定量检测。结果表明：该方法检测E.coli O157∶H7的检测限可达610 CFU/mL,线性检测区间为1.1×10³-1.4×10⁷ CFU/mL。实际水样加标回收率在40%-180%之间,相对标准偏差在10%之内,水样基质对E.coli O157∶H7的检测没有明显影响。本研究建立基于倏逝波荧光原理及其与病原菌尺寸效应的生物传感分析方法具有普适性,仅需使用不同荧光标记的生物识别分子即可实现其他病原菌的直接快速检测。

关键词: E.coli O157:H7, 核酸适配体, 倏逝波荧光, 生物传感器

Abstract: By combining the advantages of evanescent wave fluorescence optical fiber biosensor and specific nucleic acid aptamer,a fast and direct method to detect Escherichia coli O157∶H7（E. coli O157∶H7）based on evanescent wave fluorescence principle and its size effect with pathogen was proposed. The basic principle is that when a certain concentration of fluorescence labeled E. coli O157∶H7 aptamer is added to the sample detection cell,the evanescent wave triggers the fluorescence molecules to emit fluorescence,and the evanescent wave all fiber biosensor is applied for the quantitative detection of fluorescence signal. After the fluorescence labeled aptamer is mixed with E. coli O157∶H7,the mixture is added to the sample detection cell. Because the penetration depth of evanescent wave is only 100 nm,the fluorescence molecules labeled aptamer specifically bind with cannot be triggered,which results in the decrease of detected fluorescence signal. The quantitative detection of E. coli O157∶H7 can be achieved by using the proportional relationship between the fluorescence intensity and its concentrations. The results showed that the detection limit of E. coli O157∶H7 was 610 CFU/ml,and the linear detection range was from 1.1×10³ to 1.4 × 10⁷ CFU/mL. The recovery of E. coli O157∶H7 spiked in real samples was 40%-180% and the relative standard deviation was within 10%. The matrix of water sample had no significant effect on the detection of E. coli O157∶H7. Based on the principle of evanescent wave fluorescence and its size effect on pathogens,this universal biosensing method can be applied for the direct and rapid detection of other pathogenic bacteria only using different fluorescence labeled bio-recognition molecules.

Key words: Escherichia coli O157:H7, nucleic acid aptamer, evanescent wave fluorescence, biosensor

方顺燕, 宋丹, 刘艳萍, 徐文娟, 刘佳瑶, 韩向峙, 龙峰. 用于Escherichia coli O157∶H7直接快速检测的倏逝波荧光核酸适配体传感器研究[J]. 生物技术通报, 2020, 36(7): 228-234.

FANG Shun-yan, SONG Dan, LIU Yan-ping, XU Wen-juan, LIU Jia-yao, HAN Xiang-zhi, LONG Feng. Study on Evanescent Wave Fluorescence Aptasensor for Direct and Rapid Detection of Escherichia coli O157∶H7[J]. Biotechnology Bulletin, 2020, 36(7): 228-234.

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