食源性致病菌适配体的筛选及分析应用研究
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摘要
寡核苷酸适配体（又称适配体，aptamer）是由SELEX（systematic evolution of ligandsby exponential enrichment，指数富集的配体系统进化）技术筛选得到的能够与相应配体特异性紧密结合的一小段DNA或RNA分子。适配体具有靶标广泛、亲和力高、特异性好等优点，在基础研究、蛋白组学研究、临床诊断与治疗等方面应用广泛。但在食品安全检测领域的研究还比较少，本研究以食源性致病菌为研究对象，通过体外SELEX技术筛选得到其特异性结合适配体，并将其作为识别元件，结合磁分离富集技术、上转换荧光纳米标记技术、流式细胞术、免标记传感器等构建了一系列新颖、灵敏、快速的食源性致病菌检测新方法，为丰富和提高食品安全检测技术提供了新思路和新方法。
首先，利用whole-bacteria SELEX技术，以食源性致病菌副溶血性弧菌为靶标，经历9轮的筛选富集，将产物进行克隆测序，并结合流式细胞术考察所得序列的亲和力和特异性，根据解离常数值分析比较得出亲和力高和特异性好的序列，得到了副溶血性弧菌的特异性适配体。基于相同原理和方法，筛选得到了鼠伤寒沙门氏菌的特异性适配体。同时，采取碱基逐个去除的方法分析了这两种适配体的保守序列。
其次，以适配体为识别元件，以适配体功能化磁性纳米颗粒为捕获探针，适配体功能化的双色上转换纳米颗粒为显示探针，结合上转换激光诱导荧光技术，构建了一种可同时高灵敏检测金黄色葡萄球菌和沙门氏菌的新方法，该方法对两种目标菌均具有良好的线性响应，其中鼠伤寒沙门氏菌的线性检测范围为101~105cfu/mL，（线性方程y=169.66x-35.2，R~2=0.9964），检测限为5cfu/mL，金黄色葡萄球菌线性检测范围为101~105cfu/mL（线性方程y=118.5x+15.6，R~2=0.9936），检测限为8cfu/mL。该方法已成功应用于实际水样中两种目标菌的同时检测。
第三，基于适配体的识别能力和量子点的荧光特性，制备了适配体功能化荧光量子点复合物，同时结合流式细胞术，构建了一种基于适配体识别和量子点标记技术的副溶血性弧菌和沙门氏菌同时检测新方法。该方法检测结果与平板计数法的检测结果无显著差异（其中副溶血性弧菌P<0.0001，R~2=0.9993；沙门氏菌P<0.0001，R~2=0.9995），可成功应用于实际虾肉样品的检测。这是首次将适配体识别技术与荧光量子点标记技术结合应用于流式细胞术检测细菌。
第四，基于AccuBlue荧光染料在游离或是与单链DNA共存时，只显示微弱的荧光，与双链DNA共存时，其荧光信号显著增强并且不具有膜穿透性这一特性，分别以副溶血性弧菌和沙门氏菌为模式分析物，采用signal on和signal off两种检测模式，构建了一种新型食源性致病菌免标记荧光适配体传感器。所构建传感器分析性能优异，其中副溶血性弧菌的线性检测范围50~106cfu/mL，（线性方程y=197.23x+15.688，R~2=0.9956），检出限为35cfu/mL；沙门氏菌的线性检测范围50~106cfu/mL，(线性方程y=183.72x+30.79，R~2=0.9957），检测限为25cfu/mL。所建立方法已成功应用于实际虾肉样品和鸡肉样品的检测。
最后，通过测定适配体与目标菌表面分子的结合力，研究了所筛选适配体与目标菌可能的结合机制。结果显示，细菌外膜蛋白和脂多糖是最有可能与适配体结合的表面分子，通过分离提取，将得到的外膜蛋白和脂多糖与其相应的适配体结合，利用荧光偏振技术分析二者与适配体的亲和力，检测结果显示两种菌适配体与其对应外膜蛋白的结合力均大于与脂多糖的结合力，从而可推断通过whole-bacteria SELEX技术筛选得到的副溶血性弧菌和鼠伤寒沙门氏菌适配体主要是与靶标的外膜蛋白发生了特异性结合。研究结果对于阐明适配体-目标菌结合机制具有积极意义。
总之，本论文筛选得到了食源性致病菌新型分子识别元件-适配体，利用其与靶标的高亲和力、高特异性和易于标记等特性，结合上转换荧光纳米材料、荧光量子点、流式细胞术等技术，构建了一系列新颖灵敏快速的分析方法，并将其成功应用于检测食品中一种或多种食源性致病菌，为基于新型分子识别原理的食源性致病菌检测新技术和新产品的研发提供了良好的技术支持。
Aptamers are short, single-stranded RNA or DNA molecules that can bind with highaffinity and specificity to a target molecules. They are generated in an in vitro systematiciterative process, which is known as SELEX (systematic evolution of ligands by exponentialenrichment). Due to its high affinity and specificity, aptamers are widely used in basicresearch, proteomics research, diagnostic and therapeutic. However, there is few research onfood safety. In this study, high-affinity ssDNA aptamers binding to foodborn pathogenicbacteria were screened. As a recognition element, it was used to combine magneticnanoparticles separation and concentration, upconversion nanoparticals, flow cytometry andlabel-free aptasensor to establish a series of novel, sensitive and rapid detection methods forfoodborn pathogenic bacteria, which provide a new means for enriching and improvingtechnology of detection in food safety.
Firstly, a whole-bacteria Systemic Evolution of Ligands by Exponential Enrichment(SELEX) method was applied to identify DNA aptamers demonstrating specific binding toVibrio parahaemolyticus. After nine rounds of selection, a highly enriched oligonucleotidepool was sequenced. The binding affinity and specificity assay were analysed by flowcytometry. The sequence, which presented high affinity and specificity with the target, wasthe aptamer of Vibrio parahaemolyticus. Based on the same principle and method, ahigh-affinity ssDNAaptamer binding to Salmonella typhimurium was obtained. Moreover, theconsensus sequence of the two aptamers were also analysed by removing base one by one.
Secondly, a sensitive luminescent bioassay for the simultaneous detection of Salmonellatyphimurium and Staphylococcus aureus was developed using aptamer-conjugated magneticnanoparticles (MNPs) for both recognition and concentration elements and usingupconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The correlationbetween the concentration of Salmonella typhimurium and luminescent signal was found to belinear within the range of101~105cfu/mL (y=169.66X-35.2, R~2=0.9964), with the limits ofdetection5cfu/mL, and101~105cfu/mL (y=118.5X+15.6, R~2=0.9936) for Staphylococcusaureu, with the limits of detection8cfu/mL. The bioassay was successfully applied to detectSalmonella typhimurium and Staphylococcus aureus in real water samples.
Thirdly, a dual functional platform was demonstrated for the simultaneous detection oftwo pathogenic bacteria using quantum dots as fluorescence markers coupled with aptamersas the molecular recognition element by flow cytometry. The results were compared with theexperimental results from plate-counting methods (for Vibrio parahaemolyticus P<0.0001,R~2=0.9993; for Salmonella typhimurium P<0.0001, R~2=0.9995). The bioassay wassuccessfully applied to detect Vibrio parahaemolyticus and Salmonella typhimurium in realshrimp samples. This is the first time for the application of aptamer-conjugated QDs in flowcytometry for bacteria determination.
Fourthly, a fluorescence detection of foodbore pathogenic bacteria using a label-freeaptasensor was researched in this section. The fluorescence of AccuBlue reagent itself is veryweak, but it becomes highly fluorescent upon binding to dsDNA, while no significant fluorescence change can be observed with its binding to ssDNA and impermeable to cellmembranes. This unique phenomenon of AccuBlue has laid the foundation for thedevelopment of the label-free aptasensor herein. Two strategy of “signal on” and “signal off”was proposed to detect Vibrio parahaemolyticus and Salmonella typhimurium, respectively.The correlation between the concentration of Vibrio parahaemolyticus and luminescent signalwas found to be linear within the range of50~106cfu/mL (y=197.23x+15.688, R~2=0.9956),and the limits of detection was35cfu/mL. The correlation between the concentration ofSalmonella typhimurium and luminescent signal was found to be linear within the range of50~106cfu/mL (y=183.72x+30.79, R~2=0.9957), and the limits of detection was25cfu/mL.The proposed methods had the ability to effectively detect the real shimp sample and chickensample.
Fifthly, the binding mechanism of aptamer and target molecules was preliminary studiedby detecting the affinity between aptamers and surface molecules of the bacteria. The bacteriaouter membrane proteins (OMPs) and lipopolysaccharide (LPS) was analysed to be the mostlikely surface molecules to aptamer. The OMPs and LPS were isolated and incubated withtheir corresponding aptamers, and the binding affinity was analysed using fluorescencepolarization. The results indicated that the affinity of Vibrio parahaemolyticus, or Salmonellatyphimurium aptamer and OMPs is higher than that of LPS. That is to say aptamers, generatedby whole-bacteria SELEX, bind to OMPs with specificity. The results had a positive value forclarifying the binding mechanism of aptamer and target molecules.
In conclusion, aptamers targeted to foodborn pathogenic bacteria were selected as anovel recognition element. Benefit from its high affinity and specificity to targets and simplemodification, a series of novel, sensitive and rapid detection methods were established for oneor two bacteria determination combining with UCNPs, QDs, flow cytometry. That provides atechnical support for developing detection methods and relative products based on aptamersfor pathogenic bacteria.
首先，利用whole-bacteria SELEX技术，以食源性致病菌副溶血性弧菌为靶标，经历9轮的筛选富集，将产物进行克隆测序，并结合流式细胞术考察所得序列的亲和力和特异性，根据解离常数值分析比较得出亲和力高和特异性好的序列，得到了副溶血性弧菌的特异性适配体。基于相同原理和方法，筛选得到了鼠伤寒沙门氏菌的特异性适配体。同时，采取碱基逐个去除的方法分析了这两种适配体的保守序列。
其次，以适配体为识别元件，以适配体功能化磁性纳米颗粒为捕获探针，适配体功能化的双色上转换纳米颗粒为显示探针，结合上转换激光诱导荧光技术，构建了一种可同时高灵敏检测金黄色葡萄球菌和沙门氏菌的新方法，该方法对两种目标菌均具有良好的线性响应，其中鼠伤寒沙门氏菌的线性检测范围为101~105cfu/mL，（线性方程y=169.66x-35.2，R~2=0.9964），检测限为5cfu/mL，金黄色葡萄球菌线性检测范围为101~105cfu/mL（线性方程y=118.5x+15.6，R~2=0.9936），检测限为8cfu/mL。该方法已成功应用于实际水样中两种目标菌的同时检测。
第三，基于适配体的识别能力和量子点的荧光特性，制备了适配体功能化荧光量子点复合物，同时结合流式细胞术，构建了一种基于适配体识别和量子点标记技术的副溶血性弧菌和沙门氏菌同时检测新方法。该方法检测结果与平板计数法的检测结果无显著差异（其中副溶血性弧菌P<0.0001，R~2=0.9993；沙门氏菌P<0.0001，R~2=0.9995），可成功应用于实际虾肉样品的检测。这是首次将适配体识别技术与荧光量子点标记技术结合应用于流式细胞术检测细菌。
第四，基于AccuBlue荧光染料在游离或是与单链DNA共存时，只显示微弱的荧光，与双链DNA共存时，其荧光信号显著增强并且不具有膜穿透性这一特性，分别以副溶血性弧菌和沙门氏菌为模式分析物，采用signal on和signal off两种检测模式，构建了一种新型食源性致病菌免标记荧光适配体传感器。所构建传感器分析性能优异，其中副溶血性弧菌的线性检测范围50~106cfu/mL，（线性方程y=197.23x+15.688，R~2=0.9956），检出限为35cfu/mL；沙门氏菌的线性检测范围50~106cfu/mL，(线性方程y=183.72x+30.79，R~2=0.9957），检测限为25cfu/mL。所建立方法已成功应用于实际虾肉样品和鸡肉样品的检测。
最后，通过测定适配体与目标菌表面分子的结合力，研究了所筛选适配体与目标菌可能的结合机制。结果显示，细菌外膜蛋白和脂多糖是最有可能与适配体结合的表面分子，通过分离提取，将得到的外膜蛋白和脂多糖与其相应的适配体结合，利用荧光偏振技术分析二者与适配体的亲和力，检测结果显示两种菌适配体与其对应外膜蛋白的结合力均大于与脂多糖的结合力，从而可推断通过whole-bacteria SELEX技术筛选得到的副溶血性弧菌和鼠伤寒沙门氏菌适配体主要是与靶标的外膜蛋白发生了特异性结合。研究结果对于阐明适配体-目标菌结合机制具有积极意义。
总之，本论文筛选得到了食源性致病菌新型分子识别元件-适配体，利用其与靶标的高亲和力、高特异性和易于标记等特性，结合上转换荧光纳米材料、荧光量子点、流式细胞术等技术，构建了一系列新颖灵敏快速的分析方法，并将其成功应用于检测食品中一种或多种食源性致病菌，为基于新型分子识别原理的食源性致病菌检测新技术和新产品的研发提供了良好的技术支持。
Aptamers are short, single-stranded RNA or DNA molecules that can bind with highaffinity and specificity to a target molecules. They are generated in an in vitro systematiciterative process, which is known as SELEX (systematic evolution of ligands by exponentialenrichment). Due to its high affinity and specificity, aptamers are widely used in basicresearch, proteomics research, diagnostic and therapeutic. However, there is few research onfood safety. In this study, high-affinity ssDNA aptamers binding to foodborn pathogenicbacteria were screened. As a recognition element, it was used to combine magneticnanoparticles separation and concentration, upconversion nanoparticals, flow cytometry andlabel-free aptasensor to establish a series of novel, sensitive and rapid detection methods forfoodborn pathogenic bacteria, which provide a new means for enriching and improvingtechnology of detection in food safety.
Firstly, a whole-bacteria Systemic Evolution of Ligands by Exponential Enrichment(SELEX) method was applied to identify DNA aptamers demonstrating specific binding toVibrio parahaemolyticus. After nine rounds of selection, a highly enriched oligonucleotidepool was sequenced. The binding affinity and specificity assay were analysed by flowcytometry. The sequence, which presented high affinity and specificity with the target, wasthe aptamer of Vibrio parahaemolyticus. Based on the same principle and method, ahigh-affinity ssDNAaptamer binding to Salmonella typhimurium was obtained. Moreover, theconsensus sequence of the two aptamers were also analysed by removing base one by one.
Secondly, a sensitive luminescent bioassay for the simultaneous detection of Salmonellatyphimurium and Staphylococcus aureus was developed using aptamer-conjugated magneticnanoparticles (MNPs) for both recognition and concentration elements and usingupconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The correlationbetween the concentration of Salmonella typhimurium and luminescent signal was found to belinear within the range of101~105cfu/mL (y=169.66X-35.2, R~2=0.9964), with the limits ofdetection5cfu/mL, and101~105cfu/mL (y=118.5X+15.6, R~2=0.9936) for Staphylococcusaureu, with the limits of detection8cfu/mL. The bioassay was successfully applied to detectSalmonella typhimurium and Staphylococcus aureus in real water samples.
Thirdly, a dual functional platform was demonstrated for the simultaneous detection oftwo pathogenic bacteria using quantum dots as fluorescence markers coupled with aptamersas the molecular recognition element by flow cytometry. The results were compared with theexperimental results from plate-counting methods (for Vibrio parahaemolyticus P<0.0001,R~2=0.9993; for Salmonella typhimurium P<0.0001, R~2=0.9995). The bioassay wassuccessfully applied to detect Vibrio parahaemolyticus and Salmonella typhimurium in realshrimp samples. This is the first time for the application of aptamer-conjugated QDs in flowcytometry for bacteria determination.
Fourthly, a fluorescence detection of foodbore pathogenic bacteria using a label-freeaptasensor was researched in this section. The fluorescence of AccuBlue reagent itself is veryweak, but it becomes highly fluorescent upon binding to dsDNA, while no significant fluorescence change can be observed with its binding to ssDNA and impermeable to cellmembranes. This unique phenomenon of AccuBlue has laid the foundation for thedevelopment of the label-free aptasensor herein. Two strategy of “signal on” and “signal off”was proposed to detect Vibrio parahaemolyticus and Salmonella typhimurium, respectively.The correlation between the concentration of Vibrio parahaemolyticus and luminescent signalwas found to be linear within the range of50~106cfu/mL (y=197.23x+15.688, R~2=0.9956),and the limits of detection was35cfu/mL. The correlation between the concentration ofSalmonella typhimurium and luminescent signal was found to be linear within the range of50~106cfu/mL (y=183.72x+30.79, R~2=0.9957), and the limits of detection was25cfu/mL.The proposed methods had the ability to effectively detect the real shimp sample and chickensample.
Fifthly, the binding mechanism of aptamer and target molecules was preliminary studiedby detecting the affinity between aptamers and surface molecules of the bacteria. The bacteriaouter membrane proteins (OMPs) and lipopolysaccharide (LPS) was analysed to be the mostlikely surface molecules to aptamer. The OMPs and LPS were isolated and incubated withtheir corresponding aptamers, and the binding affinity was analysed using fluorescencepolarization. The results indicated that the affinity of Vibrio parahaemolyticus, or Salmonellatyphimurium aptamer and OMPs is higher than that of LPS. That is to say aptamers, generatedby whole-bacteria SELEX, bind to OMPs with specificity. The results had a positive value forclarifying the binding mechanism of aptamer and target molecules.
In conclusion, aptamers targeted to foodborn pathogenic bacteria were selected as anovel recognition element. Benefit from its high affinity and specificity to targets and simplemodification, a series of novel, sensitive and rapid detection methods were established for oneor two bacteria determination combining with UCNPs, QDs, flow cytometry. That provides atechnical support for developing detection methods and relative products based on aptamersfor pathogenic bacteria.
引文
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