适配体识别-化学发光技术检测福氏志贺菌

福氏志贺菌(Shigella flexneri)是一种危害人类健康的食源性致病菌。该实验首先制备鲁米诺功能化花状纳米金和氨基化磁性纳米Fe₃O₄,然后将福氏志贺菌适配体的互补序列(FSZ-2HH)连接在鲁米诺功能化花状纳米金的表面构建信号探针,并将福氏志贺菌适配体(FSZ-1)通过亲和素的桥接作用修饰到氨基化磁性纳米Fe₃O₄表面构建捕获探针。当目标物存在时,目标物和信号探针会竞争性结合捕获探针,进而通过测定化学发光来达到检测目的。实验结果表明：在浓度2.6×10～2.6×10⁵ CFU/mL,福氏志贺菌的浓度与化学发光强度呈良好的线性关系,检出限为12 CFU/mL,特异性良好。该实验建立的方法为食品中福氏志贺菌的快速高灵敏检测提供了良好思路。     

全细胞SELEX法筛选单增李斯特菌的ssDNA适配体

以单增李斯特菌活细胞为靶标,采用指数富集配基的系统进化技术(Systematic Evolution of Ligands by Exponential Enrichment,SELEX)从含40个随机碱基序列的单链DNA(single-stranded DNA,ss DNA)文库中筛选与之特异性结合的适配体。酶联配体吸附法(ELASA)测定筛选过程中次级文库与单增李斯特菌结合力的变化。对筛选得到的适配体进行序列测定,RNA structure软件预测二级结构,ELASA测定适配体与靶标的结合能力和特异性。结果显示,每轮筛选后次级文库与靶标的亲和力增强,特异性适配体逐步得到富集。测序获得23条适配体,其序列同源性不高,但预测的二级结构有些相似,根据二级结构将其分为3个群,分析结果表明二级结构与适配体的亲和力有关。挑选亲和力较强的21号(Lma-21)和35号(Lma-35)适配体能特异性地识别单增李斯特菌。本研究为开发食源性病原菌单增李斯特菌的新型检测试剂提供了基础材料。     

Capture-SELEX技术筛选β-苯乙胺核酸适配体

利用指数富集配体的系统进化技术(SELEX)中基于固定ssDNA文库的捕获SELEX(Capture-SELEX)技术,筛选能特异性识别β-苯乙胺的核酸适配体.通过在Capture-SELEX技术中结合熔解曲线监测ssDNA文库的富集,并经过氧化石墨烯(GO)荧光法验证候选适配体的亲和力和特异性,证实获得一条特异性识别...     

原儿茶醛对福氏志贺菌的抑制作用

为寻求安全、有效的福氏志贺菌控制方法,探究原儿茶醛对福氏志贺菌的抑制作用。测定原儿茶醛对福氏志贺菌的最小抑菌浓度、最小杀菌浓度以及其对菌体细胞形态、胞内ATP浓度、膜电位、菌体蛋白质合成和破坏及生长曲线的影响,结果表明：原儿茶醛对福氏志贺菌的最小抑菌浓度为0.5 mg/mL,最小杀菌浓度为4.0 mg/mL。场发射扫描电镜观测到原儿茶醛使福氏志贺菌形态受损,菌体皱缩干瘪;原儿茶醛影响福氏志贺菌细胞膜的通透性,表现为质量浓度为2×MIC和4×MIC的原儿茶醛使细菌胞内ATP浓度显著降低,引起细菌膜电位超极化,破坏菌体蛋白质并抑制细胞蛋白质合成;原儿茶醛延长了福氏志贺菌的生长迟滞期,减小其最大生长速率。结论：原儿茶醛对福氏志贺菌具有良好的抑制作用,其有作为天然、安全的抑菌物质应用于食品工业中的潜力。     

农药核酸适配体筛选及应用的研究进展

核酸适配体是通过指数富集的配体系统进化(systematic evolution of ligands by exponential enrichment,SELEX)技术在体外筛选到的单链寡核苷酸,与靶标具有高亲和力和特异性,且稳定性好,易于合成和修饰,被认为是生物传感器的理想识别元件之一,在农药残留快速检测应用中表...     

适配体及其研究进展

综述了适配体的筛选方法以及近几年来在分析检测、食品安全、临床医疗等领域的应用。适配体是一类能与靶分子高亲和力、高特异性结合的寡核苷酸单链。它可以通过指数级富集的配体进化技术(SELEX)从体外筛选而得。适配体类似于抗体而优于抗体,愈渐成为各种研究项目、检测方面等的重要课题之一。     

氧化石墨烯指数富集法筛选食品中的组胺适配体

组胺(HA)是影响人类健康和食品质量的重要生化物质.对其进行快速、准确检测和鉴定至关重要.本试验利用氧化石墨烯对单链寡核苷酸(ssDNA)的强吸附作用,分离与靶标结合和未结合的ssDNA,对组胺适配体进行11轮的体外指数富集筛选,最终得到15条高亲和力和特异性的适配体.通过后续的适配体二级结构、亲和性和特异性分析,HA...     

真菌毒素适配体筛选技术研究进展

真菌毒素遍布于粮食生产、储藏、运输和加工等各个环节,其污染已经成为全球亟待解决的问题。对真菌毒素进行监测并配合使用各种有效的降毒手段,有助于真菌毒素污染问题的解决。目前,真菌毒素检测、监控和靶向降解所用识别元件多为抗体,但抗体存在价格昂贵、批次间差异性大、热稳定性差等缺点,限制了其应用,而适配体合成简单、热稳定性强、免疫原性小、几乎无批次间差异等优点,有望取代抗体,但目前有关适配体商业化应用仍旧很少。本文概括总结了以真菌毒素为靶标的适配体筛选技术及其所筛适配体情况,并分析了适配体商业化应用仍旧很少的原因,以及各种筛选技术所面临的问题和挑战,以期为真菌毒素适配体的筛选提供参考,促进粮食中真菌毒素污染问题的解决。     

基于核酸适配体结合纳米金模拟酶用于单增李斯特菌的快速检测

利用纳米金模拟酶在一定条件下能够催化过氧化氢氧化3,3’,5,5’-四甲基联苯胺发生颜色反应,同时结合特异性高、亲和力强的核酸适配体建立了单增李斯特菌快速检测方法。通过对实验条件进行优化,建立了单增李斯特菌浓度与特定波长下吸光度值之间的关系式。线性回归方程为y=0.032 1x+0.024 8,R²=0.996 3,检出限约为5 CFU/mL,实际样品测定的回收率为93.5%～105.4%,相对标准偏差小于10%。该文方法特异性强、操作简单、成本低,能快速对食品中单增李斯特菌进行定量检测。     

基于计算机模拟技术优化筛选核酸适配体的初探

目的:通过分子模拟技术优化筛选5’单磷酸腺苷(AMP)的高特异性适配体,利用胶体金可视化试验验证筛选结果。方法:研究文献中已知的AMP原始适配体(S0)的结合位点,对7个关键碱基进行多轮迭代单点突变,获得新的适配体,进一步比较结合自由能,优化获得特异性高的适配体序列。结果:历经5轮筛选,优化筛选出4个特异性高的新序列(S1-S4),使用胶体金的可视化检测对最佳新序列(S4)和原始序列(S0)作比较,结果发现试验结果与模拟结果一致性良好。结论:计算机模拟技术优化筛选适配体具有省时、易制备、分子质量小、储存运输简单等多种优势,为快速获得特异性适配体提供了一个新方法、新思路。     

应用SELEX技术筛选沙门氏菌抗原的适配子

目的:应用指数富集配体系统进化(SELEX)技术筛选沙门氏菌抗原的高亲和性适配子。方法:首先合成一个全长78个核苷酸中间含35个随机序列的随机单链寡核苷酸序列(ssDNA)文库,再以环氧乙烷丙烯酸珠子作为筛选介质,利用生物素-抗地高辛碱性磷酸酶显色系统检测DNA适配子与沙门氏菌抗原的亲和力,以获得沙门氏菌抗原的高亲和性适配子。结果:随着筛选轮数的增加,DNA适配子与沙门氏菌抗原结合后显色,吸光度逐渐增加,初步获得了沙门氏菌抗原的高亲和性适配子。结论:实验所用的筛选流程是适当的,可以考虑推广用于类似靶目标的筛选。     

Antimicrobial effect of spices and herbs on Shigella sonnei and Shigella flexneri

Of 17 spices and herbs tested at 1% (wt/vol) in Mueller-Hinton (MH) agar, only cloves, thyme, oregano, allspice, basil, rosemary, and marjoram showed antimicrobial effects on Shigella. The MICs of thyme, oregano, basil, and rosemary (as determined by the agar dilution method) ranged from 0.5 to 1% (wt/vol) depending on the Shigella strain used. With the use of various combinations of temperatures (12, 22, and 37 degrees C), pHs (5.0, 5.5, and 6.0), and NaCl concentrations (1, 2, 3, and 4%, wt/vol) and the inclusion or exclusion of thyme or basil at 1% (wt/vol) in an MH agar model system, it was established that basil or thyme can contribute to combination processing as a growth-inhibitory factor for Shigella spp. In the presence of basil and thyme, Shigella flexneri did not develop CFU during the 7-day incubation period for, respectively, 14 and 16 of the 18 tested combinations, while growth was noted in the corresponding temperature-pH-NaCl concentration combinations without basil or thyme. A growth-inhibitory effect on Shigella sonnei was also noted. The results of an orientation study involving the addition of basil and thyme to spaghetti sauce prior to autoclaving and S. sonnei inoculation indicated that basil and thyme contributed to the reduction of S. sonnei after 16 days at 12 degrees C but not at 4 degrees C.     

量子点免疫标记法检测福氏志贺氏菌

研究基于抗原抗体反应捕获目标菌,结合生物素与亲和素间的特异性相互作用,联合免疫纳米磁珠磁性分离、免疫量子点荧光标记技术,运用荧光检测系统,建立了福氏志贺氏菌的定量检测模型。结果表明,福氏志贺氏菌菌浓度为102CFU/mL~105CFU/mL,相对荧光强度与菌浓度关系为FI=12.78lgN+15.941,呈良好的线性关系,决定系数R2=0.9761。进一步对模型的准确度和精确度进行验证,得到菌落浓度预测值与真实值差异小,检测相对标准偏差为1.8%,表明模型的准确度好,精密度高。该方法可简单、快速（2h）、高效地检测福氏志贺氏菌。     

Inhibition of Shigella sonnei and Shigella flexneri in Hummus Using Citric Acid and Garlic Extract

Hummus (chickpea dip) is a ready‐to‐eat product that may pose a significant risk to human if pathogens are present. Several organisms including Shigella spp. have been isolated from hummus. However, studies on the survival and inhibition of Shigella spp. in food are scarce. This study investigated the growth pattern of Sh. sonnei and Sh. flexneri in hummus at different temperatures (4, 10, and 24 °C). Additionally, the inhibitory activity of different concentrations of citric acid (CA) (0.5%, 1.0%, and 2.0%) and garlic extract (GE) (1.0%, 2.0%, and 3.0%) against Sh. sonnei and Sh. flexneri inoculated into hummus and stored at 4 and 10 °C was investigated. Both Shigella spp. survived well at 4 °C, while both grew to >7.0 log₁₀ after 4 d at 10 °C or 1 d at 24 °C. At 4 °C, CA at 0.5% and 1.0% resulted in a slight reduction in the count (approximately 1.0 log₁₀); a complete elimination of Sh. sonnei was attained by using 2.0% CA. However, approximately 3.0 log₁₀ reduction in Sh. sonnei was obtained at 10 °C. For Sh. flexneri, CA at 0.5% and 1.0% resulted in a bacteriostatic inhibition. GE at 1.0% and 2.0% resulted in approximately 1.0 to 2.0 log₁₀ reduction in Sh. sonnei count at 4 °C, while at 3.0% GE, approximately 4.0 and 3.0 log₁₀ reductions were obtained at 4 and 10 °C, respectively. In comparison, the 2.0% and 3.0% GE resulted in a bacteriostatic effect against Sh. flexneri at 4 and 10 °C.     

Survival,growth, and inactivation of acid-stressed Shigella flexneri as affected by pH and temperature

A study was done to determine the survival, growth, and inactivation characteristics of unadapted, acid-adapted, and acid-shocked Shigella flexneri 2a cells as affected by pH and temperature. The pathogen was grown at 37 degrees C for 18 h in tryptic soy broth containing no glucose (TSBNG) (unadapted cells) and TSBNG supplemented with 1% glucose (TSBG) (acid-adapted cells). Cells grown in TSBNG were acid-shocked by adjusting 18-h cultures to pH 4.5+/-0.05 with lactic acid. All three cell types were separately inoculated into tryptic soy broth (6.6-7.0 log(10) cfu/ml) containing 0.25% glucose (TSB) acidified to pH 3.5-5.5 with lactic acid and incubated at 4, 12, 21, 30, and 48 degrees C for up to 144 h. Overall, inactivation of S. flexneri cells at low pH was enhanced with an increase in incubation temperature. All three types of cells survived for 144 h at 4 degrees C in TSB acidified to pH 3.5, compared to < 24 h at 30 degrees C and 2 h at 48 degrees C. The population of all three cell types increased significantly (alpha = 0.05) within 24 h when cells were incubated at 12, 21, or 30 degrees C in TSB at pH 5.0, 5.5, or 7.3. Prior exposure of the S. flexneri to an acidic environment (acid-adapted or acid-shocked cells) resulted in increased resistance to extreme acid and temperature conditions. Acid-adapted cells decreased by approximately 2.5 log(10) cfu/ml when incubated at 4 degrees C for 144 h, compared to a 6-log(10) reduction in control (unadapted) cells. When cells were exposed to low pH (3.5-4.5) and high temperature (48 degrees C), significantly higher (alpha = 0.05) populations were recovered on tryptic soy agar (TSA) than on TSA supplemented with 4% NaCl (TSAS), indicating that a portion of S. flexneri cells were injured. Results show that the ability of S. flexneri to survive and grow at a given pH is influenced by previous exposure to acidic environments and by incubation temperature.     

基于免疫纳米磁珠对福氏志贺氏菌的快速富集研究

利用纳米磁珠的超顺磁性,结合传统平板计数的方法,通过捕获效率,研究免疫纳米磁珠制备过程中抗体加入量以及免疫纳米磁珠捕获目标细菌时的加入量,并利用免疫纳米磁珠对纯菌液以及羊肉洗水中100、101、102、103CFU/mL的福氏志贺氏菌进行捕获。结果表明,志贺氏菌多克隆抗体(4～5mg/mL)结合1mL链酶亲和素纳米磁珠((180±20)nm,2mg/mL)的最佳量为70μL,免疫纳米磁珠捕获高浓度目标细菌(102～104CFU/mL)时的最佳加入量为60μL。对于纯菌液和羊肉洗水,其捕获限可分别达到100CFU/mL和101CFU/mL,并且捕获时间在1h之内。本研究优化了实验条件,为免疫磁珠快速富集目标菌提供了理论依据。      

基于免疫纳米磁珠对福氏志贺氏菌的快速富集研究

利用纳米磁珠的超顺磁性,结合传统平板计数的方法,通过捕获效率,研究免疫纳米磁珠制备过程中抗体加入量以及免疫纳米磁珠捕获目标细菌时的加入量,并利用免疫纳米磁珠对纯菌液以及羊肉洗水中100、101、102、103 CFU/mL的福氏志贺氏菌进行捕获.结果表明,志贺氏菌多克隆抗体(4～5mg/mL)结合1mL链酶亲和素纳米磁珠((180±20) nm,2mg/mL)的最佳量为70μL,免疫纳米磁珠捕获高浓度目标细菌(102 ～ 104 CFU/mL)时的最佳加入量为60μL.对于纯菌液和羊肉洗水,其捕获限可分别达到100CFU/mL和101CFU/mL,并且捕获时间在1h之内.本研究优化了实验条件,为免疫磁珠快速富集目标菌提供了理论依据.