胶体金免疫层析技术快速定量检测金黄色葡萄球菌肠毒素B

目的建立胶体金免疫层析技术快速定量检测金黄色葡萄球菌肠毒素B的方法。方法利用胶体金标记和双抗体夹心免疫层析技术,建立金黄色葡萄球菌肠毒素B的快速检测方法,评价其特异性和敏感性,并拟合检测曲线进行定量检测;在牛奶样品中添加金黄色葡萄球菌肠毒素B作为模拟污染样品进行检测。结果该法可在5～10min内完成定性和半定量检测,检出限为8ng/ml,线性范围8～1000ng/ml。结论建立的检测金黄色葡萄球菌肠毒素B的胶体金免疫层析方法,能快速、灵敏、特异、准确地检测样品中的金黄色葡萄球菌肠毒素B,并可实现定量,适用于现场快速检测。     

金黄色葡萄球菌B型肠毒素间接竞争ELISA 检测方法的建立

以金黄色葡萄球菌B型肠毒素免疫Balb/c小鼠,待血清效价达到要求后,腹腔注射骨髓瘤细胞,制备抗金黄色葡萄球菌B型肠毒素抗体,并建立快速检测金黄色葡萄球菌B型肠毒素的间接竞争ELISA(ciELISA)方法。经方阵滴定确定B型肠毒素的最佳包被浓度为0.31μg/ml,抗B型肠毒素抗体的稀释度为1:1.2×104,羊抗鼠IgG-HRP的稀释度为1:103。本实验建立的B型肠毒素的间接竞争ELISA检测方法的最低检测量为1ng/ml,线性检测范围为1～103ng/ml,相关系数R2=0.9951,人工污染样品的平均回收率为94.5%。     

乳源性金黄色葡萄球菌肠毒素A的间接ELISA检测方法建立

从乳品中筛选出金黄色葡萄球菌,并对其所产的肠毒素进行分离纯化。制备出金黄色葡萄球菌肠毒素的特异性抗血清,建立金黄色葡萄球菌肠毒素A特异性血清间接ELISA方法。结果表明,最佳抗原包被浓度为10μg/mL,抗体最佳稀释倍数为1︰1 600,抗血清效价达到1︰3 200。通过建立此检测方法可以为乳源金黄色葡萄球菌肠毒素的检验提供依据。     

基于TaqMan探针荧光定量PCR技术快速检测乳中金黄色葡萄球菌肠毒素A

目的:建立检测金黄色葡萄球菌肠毒素A的Taq Man探针实时荧光定量PCR方法。方法:对金黄色葡萄球菌肠毒素A的基因序列分析、对比,设计特异性引物和Taq Man探针,建立对金黄色葡萄球菌肠毒素A的快速检测方法,并验证该方法的特异性、稳定性和灵敏性。结果:Taq Man探针荧光PCR检测金黄色葡萄球菌肠毒素A具有极强特异性,标准曲线相关系数为0.998,最低可检测出71个拷贝数的细菌DNA,检测乳中金黄色葡萄球菌肠毒素A菌灵敏度为1.3×102CFU/m L,不同浓度质粒重复性扩增试验Ct值的变异系数均3%,显示了良好重复性。结论:Taq Man探针荧光定量PCR方法可以在6h内快速、准确地检出金黄色葡萄球菌肠毒素A,为金黄色葡萄球菌快速检测和食物中毒快速诊断提供技术支撑,推动了荧光PCR技术在食品安全检测方面的实践应用。     

TaqMan探针荧光定量PCR检测金黄色葡萄球菌肠毒素D

建立了检测乳中产肠毒素D的金黄色葡萄球菌的荧光定量PCR方法,以SED基因为肠毒素D的检测靶序列,设计荧光定量PCR引物和Taq Man探针,将构建的重组质粒作为阳性对照,建立了对产肠毒素D金黄色葡萄球菌快速检测的Taq Man探针荧光定量PCR方法,并评价该方法的特异性、灵敏性和重复性。结果显示,Taq Man探针实时荧光定量PCR检测金黄色葡萄球菌肠毒素D的方法具有极强的特异性,标准曲线的相关系数为0.999,最低可检测到40copies/m L的阳性质粒,检测乳中产肠毒素D金黄色葡萄球菌的灵敏度为1.0×102CFU/m L。该检测方法具有较好的特异性和灵敏性,在产肠毒素D金黄色葡萄球菌快速筛查方面具有良好的应用前景。     

利用简并引物检测金黄色葡萄球菌肠毒素A、B基因

通过设计简并引物建立一种PCR技术同步快速检测金黄色葡萄球菌肠毒素A和B基因的方法。根据金黄色葡萄球菌肠毒素A、B基因编码序列,设计一对特异性简并引物SEAB来扩增靶基因片段,长度分别为105bp和135bp,通过对金黄色葡萄球菌肠毒素A、B菌株和4株对照菌株进行PCR检测,评价该引物的特异性;对金黄色葡萄球菌肠毒素B的DNA系列10倍稀释,对其灵敏性进行PCR检测。结果显示,金黄色葡萄球菌肠毒素A和B菌株的DNA检测结果呈阳性,4株对照菌株的检测结果呈阴性,通过基因测序证实了PCR产物的特异性,SEB的DNA最低检测浓度为3.58ng,整个检测过程不超过20h。建立了一个特异、快速灵敏的在同一条件下,金黄色葡萄球菌肠毒素A、B基因的PCR检测方法。     

多重实时荧光定量PCR快速检测婴幼儿奶粉中沙门氏菌、克罗诺杆菌和金黄色葡萄球菌

目的建立多重实时荧光定量PCR(multiplex quantitative real-time PCR,multiplex qPCR)快速检测奶粉中金黄色葡萄球菌、沙门氏菌和克罗诺杆菌3种常见致病菌的方法。方法筛选目标菌株的特异性引物与探针,优化反应体系,建立稳定的多重q PCR反应体系。通过阳性菌株加标的方式验证体系的特异性,并确定人工污染奶粉的检出限。结果各对引物探针对目标菌株均能扩增,多重实时荧光PCR未发现交叉反应,对17株非目标菌进行检测均未检出,人工污染奶粉中克罗诺杆菌和沙门氏菌的检出限均为10~3 CFU/mL,金黄色葡萄球菌的检出限为10~4 CFU/mL。结论本研究方法可实现婴幼儿奶粉样品中3种致病菌qPCR高效率检测。     

基于qPCR检测金黄色葡萄球菌3种肠毒素基因的方法研究

目的建立同时检测3种可引起食物中毒金黄色葡萄球菌肠毒素sea、seb、sec基因的方法。方法对GenBank中公布的sea、seb、sec基因序列进行分析,针对3种肠毒素基因分别设计3对特异性引物和探针,通过条件优化建立检测肠毒素sea、seb、sec基因的三重qPCR方法。结果该方法可以特异性检测到sea、seb、sec基因,而与其他病原菌或肠毒素基因无交叉反应;对肠毒素sea、seb、sec基因的最低检测量分别为10.2、1.63、9.4 copies/μL;组内和组间的变异系数均小于5%。结论该方法快速灵敏、准确可靠,为金黄色葡萄球菌肠毒素的快速检测及多重qPCR试剂盒的开发奠定基础。     

蘑菇罐头中肠毒素的检测

金黄色葡萄球菌肠毒素(简称肠毒素)的检测,以前多以猫、猴等动物实验为主。由于动物的来源有限,个体差异较大而不能定量等原因,使得动物实验的局限性很大。血清学试验方法具有灵敏度高、特异性强、简便快速等优点,在毒素检测方面发展很快。为了检测出     

原辅料中金黄色葡萄球菌带入量对萨拉米香肠栅栏因子、安全性的影响

将不同浓度的金黄色葡萄球菌添加到萨拉米香肠中,研究其在萨拉米香肠中的生长与致死规律,以及其对萨拉米香肠栅栏因子p H、水分活度的影响。根据金黄色葡萄球菌最低产肠毒素的浓度,以及干燥结束时萨拉米香肠中金黄色葡萄球菌的残留量来判断产品的安全性。结果表明,金黄色葡萄球菌与发酵剂存在竞争关系,其存在影响发酵剂的产酸速率,而对萨拉米香肠的水分活度影响不大。金黄色葡萄球菌添加量为10 cfu/g时,发酵阶段金黄色葡萄球菌最大值约为103.88 cfu/g,未达到产肠毒素的水平,干燥结束时最终产品检测不到金黄色葡萄球菌的存在,产品较为安全;金黄色葡萄球菌添加量为103、105、107 cfu/g时,发酵阶段菌落数最大浓度分别为105.60、106.23、108.57 cfu/g,达到了产肠毒素的水平,干燥阶段金黄色葡萄球菌致死缓慢,到结束时产品中均能检测到金黄色葡萄球菌的存在,产品存在安全隐患。     

Multitoxin biosensor-mass spectrometry analysis: a new approach for rapid, real-time, sensitive analysis of staphylococcal toxins in food

Biomolecular interaction analysis mass spectrometry (BIA-MS) was applied to detection of bacterial toxins in food samples. This two-step approach utilizes surface plasmon resonance (SPR) to detect the binding of the toxin(s) to antibodies immobilized on a surface of a sensor chip. SPR detection is then followed by identification of the bound toxin(s) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Staphylococcal enterotoxin B (SEB) was readily detected in milk and mushroom samples at levels of 1 ng/ml. In addition, non-specific binding of food components to the immobilized antibody and to the sensor chip surface was detected. To evaluate the applicability of BIA-MS in the analysis of materials containing multiple toxic components, sample containing both SEB and toxic-shock syndrome toxin-1 was analyzed. Both toxins were successfully and simultaneously detected through the utilization of multiaffinity sensor chip surfaces.     

COMPARISON OF REVERSE PASSIVE LATEX AGGLUTINATION TEST AND IMMUNOBLOTTING FOR DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN A AND B

Staphylococcal foodborne diseases resulting from consumption of food contaminated with staphylococcal enterotoxins (SEs) produced by certain strains of Staphylococcus aureus are the second most common foodborne illnesses in the world. Analytical methods are essential for routine monitoring purposes and safeguard public health. Different methods for SE detection have been proposed although their use in a complex matrix is often limited by the presence of substances that interfere with tests. In this article reverse passive latex agglutination (RPLA) and immunoblotting methods based on specific antibodies and currently available for SE detection have been compared. Culture filtrates from enterotoxin S. aureus strains isolated from cheese samples were identified by SET‐RPLA. Then the culture filtrates identified as staphylococcal enterotoxin A and staphylococcal enterotoxin B by RPLA test were analyzed with immunoblotting. The results obtained suggest that either SET‐RPLA or immunoblotting may be applied to culture filtrates for the detection of SEs with good correspondence of results. Although SET‐RPLA represents a simple method for routine monitoring purposes, a positive result by a rapid method (RPLA) is only regarded as presumptive and must be confirmed by standard methods ( Feng 1996 ), such as immunoblotting method.     

Rapid immunofluorescence assay for staphylococcal enterotoxin A using magnetic nanoparticles

A competitive immunoassay for staphylococcal enterotoxin A (SEA) detection in milk was developed, using immobilised antibody onto magnetic nanoparticles (MNPs). MNPs were prepared and then modified to introduce amino groups on them. The morphology and size of the obtained both unmodified and modified MNPs were characterized using TEM analyses. Monoclonal anti-SEA antibody was immobilised onto the modified MNPs (MNP-Ab). Staphylococcal enterotoxin A was conjugated with fluorescent dye ATTO620NHS. The characteristics of fluorescence conjugate were examined. The amount of MNP-Ab and concentration of the fluorescent conjugate used for competitive immunoassay were optimized: 0.25 mg and 53 μg mL⁻¹, respectively. The detection limit of developed immunoassay was determined – 0.23 ng mL⁻¹ SEA in spiked milk samples. The immunoassay takes only 30 min, the magnetic separation is fast (<10 s) and the volume of the sample for analysis is very small (200 μL).     

金黄色葡萄球菌肠毒素A诱导的S180肉瘤细胞凋亡分析

分析了金黄色葡萄球菌肠毒素A(SEA)诱导的S180肉瘤细胞凋亡。将S180肉瘤细胞接种于小鼠腋下,建立小鼠肿瘤模型,随机分为2组,即SEA实验组和生理盐水对照组,隔日进行腹腔注射,九天后解剖,将肿瘤组织做成石蜡切片。用末端转移酶介导的dUTP切口末端标记法(TUNEL)检测S180肉瘤细胞凋亡,并用免疫组化方法观察CD8+T细胞和颗粒酶B的分布,最后用ELISA法检测血清、脾脏中TNF-α的含量。SEA组的凋亡指数明显大于对照组,SEA组肿瘤组织周围的CD8+T细胞、颗粒酶B明显多于对照组,且血清、脾脏中TNF-α的含量也高于对照组。SEA诱导S180细胞凋亡与颗粒酶B和TNF-α的分泌有关。     

熟食肉制品中金黄色葡萄球菌风险评估基础研究

通过对上海市场所监测的396份散装熟食肉制品样品进行金黄色葡萄球菌及其肠毒素分析检测,样品中金黄色葡萄球菌阳性数70分,检出率为17.7%,金黄色葡萄球菌阳性样品中,金黄色葡萄球菌肠毒素阳性数60份,检出率85.7%。按样品种类和样品来源进行分析,金黄色葡萄球菌阳性数比例最高的样品是熏烧烤制品,为23.9%;农贸市场样品金黄色葡萄球菌阳性数比例最高,为22.1%。熟食肉制品中存在金黄色葡萄球菌及其肠毒素污染而且污染程度较高,因此有必要建立熟食肉制品中金黄色葡萄球菌及肠毒素的剂量-反应关系和风险评估模型,加强对熟食肉制品中金黄色葡萄球菌及肠毒素的检验和对食品企业生产安全监管。     

应用通用引物PCR法检测葡萄球菌A、D和E型肠毒素基因

本实验采用20bp通用引物P3和P4的PCR方法对产A、D和E肠毒素的葡萄球菌进行了检测。结果表明,产SEE菌株能产生666bp的特异扩增片段,产SEA菌株产生666bp和约400bp两条扩增带,产SED菌株产生400bp片段;SEE菌株的扩增产物经EcoRV酶切能产生251和415bp两个片段。扩增敏感性实验表明,该方法可检出10~6个细菌。     

Staphylococcal enterotoxins

Staphylococcus aureus is a major human pathogen that produces a wide array of toxins, thus causing various types of disease symptoms. Staphylococcal enterotoxins (SEs), a family of nine major serological types of heat stable enterotoxins, are a leading cause of gastroenteritis resulting from consumption of contaminated food. In addition, SEs are powerful superantigens that stimulate non-specific T-cell proliferation. SEs share close phylogenetic relationships, with similar structures and activities. Here we review the structure and function of each known enterotoxin.     

Quantum dot bead-based competitive immunochromatographic assay for enterotoxin aureus A detection in pasteurized milk

Staphylococcal enterotoxin A (SEA) is an important biotoxin, produced by Staphylococcus aureus under appropriate conditions, and often contaminates milk and dairy products. Herein, an anti-SEA monoclonal antibody (anti-SEA mAb) was prepared by injecting the SEA protein in BALB/c mice, and a novel immunochromatographic assay (ICA) was developed for the rapid and sensitive determination of SEA in pasteurized milk by using highly luminescent quantum dot beads (QB) as signal amplification probe. Given the 1020-fold enhancement in the photoluminescence intensity of QB to the original quantum dot, the proposed QB-ICA exhibits high sensitivity for SEA determination in real milk samples with a limit of detection of 1.89 ng/mL, and shows good dynamic linearity for SEA quantitative detection from 2 to 150 ng/mL within 15 min of test time. The proposed QB-ICA also shows good selectivity to SEA detection with a negligible cross-reaction to common analogs, including staphylococcal enterotoxins B, C, D, and E. In addition, the accuracy and precision of QB-ICA were assessed by analyzing SEA-fortified milk samples. The average recoveries of intra- and interassays range from 85.5 to 128.1%, and the coefficients of variation range from 4.6 to 14.2%, indicating an acceptable accuracy for the quantitative detection of SEA in real milk samples. In summary, this work provides a powerful and rapid analysis tool for the sensitive monitoring of SEA contamination in pasteurized milk samples.