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.     

免疫磁捕获-实时荧光PCR快速检测鸡肉中沙门氏菌

对免疫磁捕获-实时荧光PCR检测鸡肉中沙门氏菌进行了初步研究。主要包括免疫磁珠制备条件的选择及优化,通过实验最终确定了以6 mg/mL EDC和5 mg/mL NHS活化直径为700 nm的羧基聚苯乙烯磁性微球,与浓度为100μg/mL的沙门氏菌抗体在37℃振荡孵育1.5 h,制备得到抗沙门氏菌免疫磁珠,与市售沙门氏菌免疫磁珠试剂盒(Dynal产品)捕获效率相当。免疫磁捕获-实时荧光PCR检测方法检测限为104 CFU/mL左右,能在16 h内检测出鸡肉中104 CFU/mL的沙门氏菌,可用于食品中沙门氏菌的快速检测。     

免疫磁珠结合显色平板快速检测婴儿配方奶粉中阪崎克罗诺杆菌

该研究利用羧基化磁珠偶联兔多克隆抗体,制备特异性免疫磁珠,并优化制备条件,然后与显色培养基联用,建立免疫磁珠-显色平板检测方法,用于富集分离婴儿奶粉中的阪崎克罗诺杆菌。结果表明,磁珠与抗体的最佳偶联温度37℃、偶联时间2.0 h,1 mg磁珠的最佳抗体添加量为200μg,饱和偶联量为182.77μg。制得的免疫磁珠,捕获目标菌的浓度范围为10²～10⁷ CFU/mL,捕获率为68.4%～82.4%,与非目标菌反应率低于10%。检测方法应用于模拟污染样品,在目标菌浓度10²～10⁵ CFU/mL时,捕获率为66.2%～71.5%。相比传统方法,该方法的检测周期缩短了2～3 d。     

免疫富集联合基质辅助激光解吸电离飞行时间质谱法测定牛奶、鸡蛋中的沙门氏菌

目的建立免疫富集联合基质辅助激光解吸电离飞行时间质谱法(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF MS)检测牛奶、鸡蛋中沙门氏菌的方法。方法沙门氏菌经免疫磁珠特异性富集后采用MALDI-TOF MS鉴定,并优化富集过程中的抗体、免疫磁珠添加量以及捕获时间,研究其特异性,并用于实际样品中沙门氏菌的检测。结果富集过程最佳反应条件为:每1 mg磁珠中抗体添加量为250μg,1 mL菌样中免疫磁珠添加量为600μL,捕获时间为30 min,免疫磁珠捕获沙门氏菌具有特异性;用于检测牛奶和鸡蛋中的沙门氏菌时,将浓度为3 CFU/mL的沙门氏菌特异性富集后选择性增菌9h即可被MALDI-TOF MS准确鉴定。结论该方法具有选择性好,特异性高,耗时短,结果准确的优点,为食品中沙门氏菌的检测提供相关参考。     

免疫磁珠-环介导等温扩增快速检测牛肉中的鼠伤寒沙门氏菌与金黄色葡萄球菌

建立免疫磁珠分离（immunomagnetic separation,IMS）联合环介导等温扩增（loop-mediated isothermal amplification,LAMP）技术检测牛肉中鼠伤寒沙门氏菌与金黄色葡萄球菌的方法。用生物素标记的鼠伤寒沙门氏菌抗体和生物素标记的金黄色葡萄球菌菌体蛋白抗体对链霉亲和素磁珠进行功能化,从牛肉中捕获和分离目标致病菌。结果表明：经优化后,每毫克磁珠与10 μL鼠伤寒沙门氏菌抗体偶联,400 μL鼠伤寒沙门氏菌免疫磁珠在45 min内对104 CFU/mL鼠伤寒沙门氏菌的捕获率为52.16%;每毫克磁珠与6 μL金黄色葡萄球菌抗体偶联,300 μL金黄色葡萄球菌免疫磁珠在30 min内对104 CFU/mL金黄色葡萄球菌的捕获率为56.80%;建立的IMS-LAMP方法特异性高,对牛肉中鼠伤寒沙门氏菌的检测灵敏度为1.2×103 CFU/mL,对金黄色葡萄球菌的检测灵敏度为4.4×104 CFU/mL;富集5 h后,鼠伤寒沙门氏菌的检测限可至1.2 CFU/mL,富集7 h后,金黄色葡萄球菌的检测限可降至4.4 CFU/mL。建立的IMS-LAMP方法用时短,灵敏度高,操作简单,可以有效检测牛肉中的鼠伤寒沙门氏菌和金黄色葡萄球菌。     

氨基化磁珠非靶向富集食源性致病菌的条件筛选及优化

该研究基于氨基化磁珠静电富集细菌技术,建立了一种可同时富集鼠伤寒沙门氏菌,金黄色葡萄球菌,单增李斯特氏菌的方法。在每2 mL单菌液中（103 CFU/mL）分别加入5~200 μg的3种粒径的氨基化磁珠（1 μm、300 nm、100 nm）,当孵育5~90 min时检测各细菌的富集效率。将单菌液体系的富集正交试验结果应用至混和菌体系的单因素试验和正交试验,将最终结果应用至大体积实验体系以及实际样本（市售牛奶、水果沙拉）。结果表明,当氨基化磁珠添加量为50 μg、孵育时间为30 min时对3种病原菌可以达到60%以上的捕获率,所有反应均在pH值7.4的PBS中进行。磁珠粒径选择300 nm（p<0.05）。在实际样本牛奶、水果沙拉中,当三种混合菌的最低终浓度为2×102 CFU/g（mL）时,捕获率高于55%,结果可达到荧光定量PCR的检测低限。氨基化磁珠与免疫磁珠比较,其具有稳定保存、低成本,高效率等优势,其非靶向富集的能力为下游食源性致病菌的快速检测提供有效前处理富集手段。     

免疫磁珠法富集沙门氏菌的优化及应用

采用活泼酯法将磁珠和抗体偶联,制备沙门氏菌免疫磁珠,系统地优化了偶联条件和免疫磁珠捕获工艺.结果表明,最佳偶联条件:每毫克磁珠抗体偶联量为50μg,免疫磁珠的添加量为0.4mg,菌液浓度为102～106 CFU/mL时,捕获效率为65％～82％;免疫磁珠捕获工艺:目的菌最佳孵育时间为45min,最佳磁分离时间为3min,磁场强度为0.8T,磁珠粒径为180nm;对鼠伤寒沙门氏菌、猪霍乱沙门氏菌、甲型副伤寒沙门氏菌、鸭沙门氏菌和肠炎沙门氏菌的捕获效率分别为61.4％、45.8％、48.2％、78.5％、64.1％,对鸡蛋清、蛋黄、牛奶和纯培养基中沙门氏菌捕获效率分别为44.3％、43.2％、65.2％和72.5％.免疫磁分离富集技术具有分离速度快、操作简单、捕获效率高、可重复性好等特点,可在食品样品预处理中广泛应用.     

基于免疫磁珠快速分离金黄色葡萄球菌的研究

通过实验优化建立基于免疫磁分离技术对金黄色葡萄球菌的快速分离方法,为后续快速检测提供基础.利用金黄色葡萄球菌多抗制备的免疫磁珠对其捕获.结合平板菌落计数,考察包被磁珠时多抗加入量、免疫磁珠加入量及捕获时间等因素对捕获效率的影响.在单因素实验基础上进行正交实验,并对该方法的灵敏度、特异性及其在食品中的应用效果初步探索.结果表明,最佳捕获条件为抗体加入量30μL,免疫磁珠加入量80μL,捕获时间45min,在此条件下捕获效率均超过30％,该方法捕获限可达到101CFU/mL,特异性良好,并初步用于羊肉卷洗水中金黄色葡萄球菌的快速分离,捕获时间小于1h.免疫磁分离作为一种快速的分离筛选方法,可用于食源性金黄色葡萄球菌的快速分离.     

利用人免疫球蛋白构建免疫磁珠富集金葡菌条件优化

利用人免疫球蛋白制备富集金黄色葡萄球菌的免疫磁珠及优化条件;利用化学键法制备免疫磁珠,并用矩阵法和单因素法优化免疫磁珠制备条件,通过磁珠对抗体饱和吸附量及加入量相关性评价磁珠与抗体固定化程度,根据免疫磁珠的特异性、灵敏性及饱和捕捉量来考察IMBS.aureus对金葡菌的富集能力;结果显示采用MB-M型磁珠,加入质量浓度均为5 mg/L的活化剂EDC和NHS,于24℃偶联人IgG,24 h得到IMBS.aureus,其饱和吸附量为393.27μg,磁珠与人IgG加入量具有相关性,IMBS.aureus特异性好、灵敏度高(26 CFU/10 mL),0.2 mL IMBS.aureus饱和捕捉菌液浓度为106CFU/mL;通过优化,磁珠装载人IgG分子的能力提高了5倍,检测限提高了4倍,富集金葡菌的效率大大增强,成本降低,为人免疫球蛋白磁珠富集和分离金黄色葡萄球菌实际应用创造了条件。     

国产沙门氏菌免疫磁珠的制备及其应用

目的优化免疫磁珠制备条件,获得捕获性能高、成本低的国产沙门氏菌免疫磁珠,为国产沙门氏菌免疫磁珠的产业化制备奠定基础。方法采用亚微米级羧基磁珠及沙门氏菌多克隆抗体,利用碳二亚胺缩合法制备2 mg级免疫磁珠。以目标菌捕获率、磁珠表面抗体偶联率为评价指标,对4种粒径(100、300、500、1000 nm)的磁珠原材料进行筛选;以目标菌捕获率、磁珠表面抗体偶联率、免疫磁珠表面FITC荧光强度为评价指标,对磁珠与抗体不同的投料比(100:1、100:1.5、100:2、100:2.5、100:3)进行优化;同时,对国产沙门氏菌免疫磁珠与进口免疫磁珠(Dynalbeads~?沙门氏菌免疫磁珠、Bac Trace~?沙门氏菌免疫磁珠)的性能进行比较分析,考察了捕获率、免疫磁珠与目标菌的结合情况、特异性捕获能力等,并在实际样品中进行了应用检测。结果以粒径为300 nm的磁珠为原材料制备的免疫磁珠对浓度为10~2 CFU/mL的目标菌的捕获率为99%、抗体偶联率为34.64%,明显高于其他粒径的免疫磁珠;当投料比为100:2(磁珠:抗体)时,制备的免疫磁珠对浓度为10~2 CFU/m L的目标菌的捕获率为100%,偶联率为52.34%,优于其他投料比;针对不同浓度的鼠伤寒沙门氏菌(10~0~10~6 CFU/mL),国产免疫磁珠的捕获率高于进口免疫磁珠(国产:83%~100%;Dynalbeads~?:57%~74%;Bac Trace~?:9%~40%);在25 mL牛奶中添加(11.9±3.7)CFU鼠伤寒沙门氏标准菌株,国产沙门氏菌免疫磁珠的检出率高于进口免疫磁珠(国产:100%;Dynalbeads~?:70%;Bac Trace~?:40%)。结论优化了制备条件后所制备的国产沙门氏菌免疫磁珠的捕获能力强、价格低、能够实现大体积实际样品中痕量目标菌的100%检出,具有广阔的应用前景,本研究为沙门氏菌免疫磁珠的国产化应用提供了科学数据。     

免疫磁性糊精微球的制备及快速分离检测单增李斯特菌

研究免疫磁性糊精微球的制备及在快速分离检测单增李斯特菌中的应用。将磁性糊精微球由环氧氯丙烷活化后,与抗体交联制得免疫磁性糊精微球,通过单因素和正交试验对合成条件进行优化。再利用合成好的免疫磁性糊精微球捕获单増李斯特菌,在外加磁场的作用下,富集免疫磁性糊精微球,提取细菌DNA,经过PCR扩增后,进行琼脂糖凝胶电泳。结果表明:活化反应的最佳条件,环氧氯丙烷用量0.4 mL/g,NaOH浓度2.0 mol/L,最佳反应时间5 h;制备免疫磁性糊精微球的最优合成条件:抗体质量浓度1.0 mg/mL,反应温度28℃,反应时间2.5 h;捕获单增李斯特菌时,免疫磁性糊精微球的最佳用量100μL、最佳捕获时间1 h。通过研究得出免疫磁性糊精微球可以快速捕获单增李斯特菌,并能扩增出特异性产物,检出限达到每mL菌液中10个单增李斯特菌。     

荧光免疫层析法结合免疫磁珠分离技术快速检测单增李斯特菌

为建立单增李斯特菌简单快速、灵敏度高和特异性强的检测方法,本研究以抗单增李斯特菌单克隆抗体偶联磁珠制备免疫磁珠;以羧基荧光微球标记的抗单增李斯特菌多克隆抗体及鼠IgG为标记抗体,抗单增李斯特菌多克隆抗体和羊抗鼠二抗分别作为检测线和质控线制备荧光免疫层析试纸条。将免疫磁珠分离与荧光免疫层析法相结合应用于单增李斯特菌的现场快速检测中。结果表明:荧光免疫层析试纸条对纯培养单增李斯特菌的检测限为4×105CFU/mL,联合检测方法10倍、100倍浓缩时,检测限分别为4×104CFU/mL和1×104CFU/mL。联合检测体系特异性较好,与实验室保存的10株细菌无交叉反应。人工污染样本检测限为1×104CFU/mL,同纯培养物相比检测灵敏度并没有降低。本方法的建立对于食品中单增李斯特菌的现场快速检测具有重要意义。     

Polymerase chain reaction detection of Listeria monocytogenes on frankfurters using oligonucleotide primers targeting the genes encoding internalin AB

A polymerase chain reaction (PCR) assay targeting the genes encoding internalin AB (inlAB) was developed for detecting Listeria monocytogenes in pure cell cultures and on artificially contaminated frankfurters. Four sets of oligonucleotide primers were evaluated. The set targeting a 902-bp region of the inlAB gene was the most specific. This PCR product was detected in 51 L. monocytogenes strains belonging to four different serogroups (1/2a, 1/2b, 1/2c, and 4b). In contrast, the PCR product was not detected in other Listeria spp. (Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, or Listeria grayi) or in gram-positive, non-Listeria bacteria, indicating that the primer set was highly specific for L monocytogenes. The detection limit of the PCR assay was 10(5) CFU per ml of pure cell culture. However, the assay could detect as few as 10(1) CFU of L. monocytogenes in 25 g of frankfurter with 16 h of enrichment in modified Listeria enrichment broth at 30 degrees C. The total assay time including enrichment was approximately 24 h. These results suggest that the PCR assay can be used to rapidly detect L. monocytogenes on frankfurters and possibly other types of ready-to-eat meat products.     

Simultaneous detection by PCR of Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain

A multiplex PCR procedure was established to detect Escherichia coli, Listeria monocytogenes and Salmonella typhimurium in artificially inoculated wheat grain. The PCR protocol with an enrichment step successfully detected all three organisms inoculated together in non-autoclaved wheat grain. After a one day enrichment, E. coli, L. monocytogenes and S. typhimurium were detected at levels of 56, 1800 and <54 CFU/mL, respectively, in the initial sample. For L. monocytogenes, an improved detection limit of <62 CFU/mL was achieved using singleplex PCR. For autoclaved wheat grain inoculated with the three bacterial strains individually, a detection limit of 3 CFU/mL was achieved after an enrichment step. The ability to test for the three bacteria simultaneously will save time and increase the ability to assure grain quality.     

Enhanced rapidity for qualitative detection of Listeria monocytogenes using an enzyme-linked immunosorbent assay and immunochromatography strip test combined with immunomagnetic bead separation

An enzyme-linked immunosorbent assay (ELISA), immunochromatography (ICG) strip test, and immunomagnetic bead separation (IMBS) system based on a monoclonal antibody were individually developed for the detection and isolation of Listeria monocytogenes in meat samples. The three methods showed a strong reaction with Listeria species and a weak reaction with Staphylococcus aureus. To increase the rapidity of L. monocytogenes detection, combinations of the ELISA and ICG strip test with the IMBS system (ELISA-IMBS and ICG-IMBS) were investigated. In comparative analyses of artificially inoculated meat and samples of processed meat, the ELISA and ICG strip test required 24 h of enrichment time to detect the inoculated meat samples with > or =1 X 10(2) CFU/10 g, whereas the ELISA-IMBS and ICG-IMBS required only 14 h of enrichment. Analyses of naturally contaminated meat samples (30 pork samples, 20 beef samples, 26 chicken samples, 20 fish samples, and 20 processed meat samples) performed by ELISA-IMBS, ICG-IMBS, and API kit produced similar results. The ELISA-IMBS and ICG-IMBS provide a more rapid assay than the individual ELISA and the ICG strip test and are appropriate for rapid and qualitative detection of L. monocytogenes (or Listeria species) in meat samples. With the ICG-IMBS, L. monocytogenes could be detected in meat samples within 15 h and the method has potential as a rapid, cost-effective on-site screening tool for the detection of L. monocytogenes in food samples and agricultural products at a minimum detection level of approximately 100 CFU/10 g.     

免疫磁珠捕获结合荧光PCR 技术检测食品中黄曲霉菌的研究

目的：研究利用免疫磁珠对食品中黄曲霉菌进行捕获的技术,为检测食品中污染的黄曲霉菌提供新方法。方法：应用山羊抗黄曲霉菌血清的免疫磁珠捕获食品中的黄曲霉菌,不需要进行增菌培养,应用磁捕获- 荧光聚合酶链式反应(IMC-FPCR)技术快速检测鉴定食品中的黄曲霉菌。结果：IMC-FPCR 方法检测黄曲霉菌的最低检测值为2CFU/mL。应用IMC-FPCR 对从市场上购买的12 种食品进行检测,发现有两份样品含有产毒素相关基因(即为可能的致癌菌株),检出率为16.7%,其中花生中检出1 株黄曲霉菌,面线中检出1 株黄曲霉菌。结论：建立了黄曲霉菌的磁捕获- 荧光聚合酶链式反应(IMC-FPCR)方法,该方法可用于食品中黄曲霉菌的快速检测及鉴定,实物检测结果提示市场上食品有被真菌污染。     

Rapid detection of Listeria monocytogenes by nanoparticle-based immunomagnetic separation and real-time PCR

The objective of this study was to develop a method combining nanoparticle-based immunomagnetic separation (IMS) with real-time PCR for a rapid and quantitative detection of Listeria monocytogenes. Carboxyl modified magnetic nanoparticles were covalently bound with rabbit anti-L. monocytogenes via the amine groups. Several factors, such as the amount of immunomagnetic nanoparticles (IMNPs), reaction and collection times, and washing step, were optimized, and the nanoparticle-based IMS in combination with real-time PCR was further evaluated for detecting L. monocytogenes from artificially contaminated milk. The cell numbers calculated from the means of threshold cycles (CT) of PCR amplification curves were compared to those from plate counts in order to determine the correspondence degree of quantitative data. The capture efficiency (CE) by plating from IMNP-based IMS was 1.4 to 26 times higher than those of Dynabeads-based IMS depending on the initial cell concentrations inoculated into milk samples. When combined with real-time PCR, L. monocytogenes DNA was detected in milk samples with L. monocytogenes >or=10(2) CFU/0.5 ml. In the range of 10(3) to 10(7)L. monocytogenes CFU/0.5 ml, cell numbers calculated from CT values were 1.5 to 7 times higher than those derived from plate counts. Our results demonstrated that both the use of nanoparticles and the choice of anti-L. monocytogenes in our IMNP-based IMS in combination with real-time PCR has improved the sensitivity of L. monocytogenes detection from both nutrient broth and milk samples.     

基于磁性金属有机框架分离的纳米金比色法检测单核细胞增生李斯特菌

以磁性金属有机框架复合材料作为捕获探针,免疫功能化纳米金作为信号探针,建立一种纳米金比色法快速检测单核细胞增生李斯特菌的方法。结果表明：在最适条件下,单核细胞增生李斯特菌的可视化检出限为1.2×10³ CFU/m L,紫外光谱检测结果在1.2×10¹～1.2×10⁸ CFU/mL范围内有良好的线性关系(Y=0.519-0.043X,R²=0.978),检出限低至0.45 CFU/mL,各浓度梯度的批内变异系数在0.31%～1.30%之间。将单核细胞增生李斯特菌接种到鸡胸肉上进行检测,其加标回收率在91.1%～108.7%之间,变异系数不高于7.4%,且结果与平板计数法一致。本方法具有准确、灵敏、快速等特点,在食源性致病菌检测方面具有较好的应用前景。     

Development of a loop-mediated isothermal amplification assay for detecting Listeria monocytogenes prfA in milk

A rapid and sensitive loop-mediated isothermal amplification (LAMP) assay was developed for detecting Listeria monocytogenes prfA in milk. The inclusivity of 23 L. monocytogenes and the exclusivity of 16 non-L. monocytogenes strains were both 100% in the assay. The limit of detection (LoD) of the LAMP assay in Listeria enrichment broth (LEB) was 2.22 CFU/mL after 12 h and 24 h of incubation. The LoDs of the LAMP assay in LEB with artificially contaminated milk (LEB-M) incubated for 12 h (2.22×10¹ CFU/mL) and 24 h (2.22 CFU/mL) were lower than those of the PCR and real-time PCR assays. Comparison of the LoDs in LEB with those in LEB-M showed that the LAMP assay was less influenced by the milk compounds than the real-time PCR assay. Our results indicate that the LAMP assay can be utilized as a potential screening tool for L. monocytogenes in milk.