磁分散固相萃取-酶联免疫吸附法检测金黄色葡萄球菌肠毒素A

目的 以金黄色葡萄球菌肠毒素A (staphylococcal enterotoxin A, SEA)为代表,设计并合成一种适配体修饰功能化磁性材料,与酶联免疫吸附(enzyme linked immunosorbent assay,ELISA)检测技术相结合,进一步提高检测方法灵敏度。方法 样品经SEA适配体修饰磁性微球富集后,结合SEA的ELISA试剂盒检测样品中SEA含量,建立SEA检测的磁分散固相萃取-酶联免疫吸附新方法。结果 与仅采用ELISA试剂盒测定SEA的方法相比,本研究方法的灵敏度可提高10倍,检测方法的线性范围为0.01～0.81μg/L,同时检测方法具有良好的特异性。将建立的检测方法用于牛奶中SEA的检测,回收率在84.45%～114.45%之间。结论 本方法操作简单、特异性强、灵敏度高,同时具有广泛的适用性,可通过特定适配体修饰磁性微球实现不同目标物的检测,同时适配体修饰功能化磁性材料也可与其他检测技术联合使用,提高检测的灵敏度。     

Gold nanoparticle-based colorimetric detection of staphylococcal enterotoxin B using ssDNA aptamers

Staphylococcal enterotoxin (SE) B is one of the most common serotype of SEs to cause staphylococcal food poisoning. To ensure food safety, rapid and low-cost methods have been constantly developed and applied to detect SEB worldwide. In the present investigation, a panel of aptamers of single-stranded DNA molecules against SEB was obtained by optimizing the procedure of systematic evolution of ligands by exponential enrichment, and five of them were selected for further analyzing the characteristics of sequences and second structures. Afterward, an aptamer-based colorimetric method of SEB detection was carried out by employing unmodified gold nanoparticle probes (AuNPs). Results showed that the main second structures of these aptamers were stem loop and hairpin forms. In addition, applying one of these aptamers (No. 15-1), SEB could be detected at the concentration of 10 ng/mL by AuNPs-based colorimetric method. Moreover, the aptamer also possessed a good selectivity toward SEB and SEC₁. Our work demonstrated that aptamers had their potential applications as a bioprobe for the detection of SEs in food products.     

Gold nanoparticle-based enhanced lateral flow immunoassay for detection of Cronobacter sakazakii in powdered infant formula

Cronobacter sakazakii is an opportunistic foodborne pathogen that can infect newborns through powdered infant formula (PIF). In this study, we developed a novel enhanced lateral flow immunoassay (LFA) with enhanced sensitivity for detection of C. sakazakii in PIF by the naked eye. The proposed strategy for signal enhancement of the traditional LFA used concentrated gold nanoparticles (AuNP) as the enhancer to conjugate with capture antibodies, which could increase the immobilized capture antibodies concentration at the detection zone to improve capture efficiency. Besides, the detection signal was further amplified by accumulated AuNP as the C. sakazakii labeled with AuNP probes was captured by antibodies conjugated with enhancer at the test line. We also studied the effect of different concentrations of capture antibodies and concentrated AuNP on detection performance, and found that 2.2 mg/mL of capture antibodies and 0.06 nM concentrated AuNP were the optimal combination that could avoid a false-positive signal and maximally amplify the detection signal of the enhanced LFA. Using this strategy, the detection sensitivity of the enhanced LFA was 10³ cfu/mL and improved 100-fold compared with traditional LFA. The strip was highly specific to C. sakazakii, and the time for detection of C. sakazakii in PIF was shortened by 3 h. In summary, the enhanced LFA developed by the addition of concentrated AuNP as the enhancer can be used as a sensitive, rapid, visual qualitative and point-of-care test method for detecting target analytes.     

金黄色葡萄球菌肠毒素B检测方法的研究进展

金黄色葡萄球菌分泌的肠毒素B(staphylococcal enterotoxin B,SEB)作为与食品中毒相关的重要毒素之一而被广泛报道。同时由于其具有热稳定性、易制备、高毒及易传播等特点引起科研人员的高度重视,因此,建立SEB高效的检测方法非常重要。本文对SEB的检测方法进行了综述,主要讨论了生物学检测、免疫凝集实验、琼脂糖扩散法、酶联免疫检测技术、放射性免疫检测技术、免疫荧光检测技术、胶体金试纸条检测技术、基因探针、仪器分析、生物传感检测等检测方法的原理及相关国内外研究进展的应用和局限,这对提前预防金黄色葡萄球菌肠毒素B引起的食物中毒具有重要意义,同时也为今后开发新型检测方法提供理论参考和技术支持。     

A novel chemiluminescence immunoassay of staphylococcal enterotoxin B using HRP-functionalised mesoporous silica nanoparticle as label

A novel chemiluminescence immunoassay was developed, in which two monoclonal antibodies recognised different epitopes of staphylococcal enterotoxin B (SEB) using HRP-functionalised mesoporous silica nanoparticle (MSN) as label. The enzyme-functionalised MSN was fabricated by co-immobilizing HRP and anti-SEB onto the surface of MSN simultaneously using glutaraldehyde as the linkage.     

荧光微球免疫层析法定量检测牛乳中酪蛋白

目的研究用荧光微球免疫层析法定量检测牛乳中的酪蛋白。方法本文以酪蛋白为抗原,免疫新西兰大白兔制备抗酪蛋白的多克隆抗体,将纯化后的多克隆抗体通过EDC介导法与荧光微球进行偶联,将滤纸、样品垫、结合垫、NC膜和吸水纸组装成试纸条,用此荧光微球免疫层析法定量检测牛乳中的酪蛋白。结果试纸条在25 min内就能判定结果,最低检测限为100 ng/mL,该方法与BSA、OVA均无交叉反应,具有很好的特异性。检测酪蛋白浓度为100.0、500.0、1000.0 ng/mL的样品,试纸条的批内回收率分别为(89.03±5.2)%、(93.47±6.9)%和(91.2±7.8)%,批间回收率分别为(87.69±6.2)%、(92.73±8.3)%、(89.82±8.5)%。结论初步建立了一种快速、方便、高灵敏度的荧光微球免疫层析方法用以检测牛乳制品中的过敏原——酪蛋白。     

竞争抑制酶联免疫吸附法快速检测金黄色葡萄球菌肠毒素P

目的 建立一种稳定性强、灵敏度高的快速检测金黄色葡萄球菌新型肠毒素P(staphylococcal enterotoxin P,SEP)的单克隆抗体竞争抑制酶联免疫吸附法(Enzyme-linked Immunosorbent assay,ELISA)。方法 根据ELISA的检测程序,利用交叉方阵滴定法确定SEP抗原的最佳包被浓度及单克隆抗体的最佳倍比稀释浓度,再对辣根过氧化物酶标记的羊抗鼠IgG(HRP-IgG)最佳稀释浓度及最佳反应时间进行筛选,然后通过测定450nm处不同SEP抗原包被条件(包被液类型、包被环境)、封闭液类型及浓度、封闭时间、竞争反应温度、反应方式(预混反应、直接反应)下的OD值对检测条件进行优化,最后用灵敏度、批内变异、批间变异和加标样品回收率对方法进行评价。结果 SEP抗原最佳包被浓度为2.5 μg/mL,鼠单抗血清稀释度1:6000,酶标抗稀释度1:3000;反应1 h,最佳包被条件为磷酸盐缓冲液4 ℃过夜,10%脱脂乳封闭2 h,37 ℃直接竞争反应。此方法的线性回归方程为：y = 0.4166x - 0.7415(R2 = 0.9908),灵敏度为0.954 μg/mL,批内及批间变异系数均低于3 %,对人工污染的脱脂牛奶、LB液体培养基中肠毒素蛋白SEP的回收率高于98%。结论 本实验建立了一种能够快速检测葡萄球菌肠毒素SEP的ELISA方法。     

一种快速检测呕吐毒素的胶体金试纸条

利用胶体金免疫层析法,研制了一种能够快速检测黄豆中呕吐毒素含量的试纸条。通过试验,胶体金试纸条对呕吐毒素的检测限是1μg/g,对玉米赤霉烯酮毒素、黄曲霉毒素B1、赭曲毒素A等无交叉反应,假阳性率和假阴性率均为0,检测时间为10min。试纸条能够准确、可靠、简便、快速地检测黄豆中残留的呕吐毒素,适合大量样品的现场检测。     

A novel extraction method for peanut allergenic proteins in chocolate and their detection by a liposome-based lateral flow assay

In this study, conditions for extracting the major peanut allergen (Ara h1) from chocolate were optimized, and the extracted samples were analyzed by a lateral flow assay (LFA) using liposomal nanovesicles. The optimal conditions using peanut-spiked chocolate were found to be extraction with a mixture of phosphate buffered saline and hexane for 30 min at 35 °C. After centrifugation, the buffer portion was treated with insoluble poly(vinylpolypyrrolidone) to remove phenolic compounds, and then analyzed by the LFA. The entire analysis, including sample preparation and LFA, could be easily completed within 2 h, and the detection limit was 158 g of peanuts/g of chocolate.     

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).     

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.     

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。     

胶体金免疫层析法快速检测T-2毒素的研究

应用胶体金免疫层析技术,建立了一种快速检测谷物和饲料中T-2毒素的方法.采用柠檬酸三钠还原法制备胶体金颗粒,标记抗T-2毒素单克隆抗体并喷涂于玻璃纤维上,T-2毒素偶联抗原和羊抗鼠二抗分别喷涂于硝酸纤维素膜上,作为检测线和质控线,依次将样品垫、胶体金垫、硝酸纤维素膜和吸水垫组装成试纸条并装入检测卡中.测试结果表明,T-2毒素快速检测试纸条的检测限为0.Smg/L,检测时间为10min,假阳性率和假阴性率均为0.该法使用简单方便,非常适合现场快速检测谷物和饲料中的T-2毒素.     

Gold nanoparticle-based method for detection of calcium carbide in artificially ripened mangoes (Magnifera indica)

A new approach was developed for a simple and easy colorimetric detection assay to detect the use of calcium carbide in artificial ripening of fruits. Residues of arsenic on the fruit surface were used as an indicator for this. Use of calcium carbide in artificial ripening has been banned in many countries including India. In the present study, we have used a gold nanoparticle (AuNP)-based colorimetric detection method for determination of artificial ripening of fruits. ICP-MS analysis showed the presence of higher amounts of arsenic on fruits ripened using calcium carbide. Lauryl sulphate (LS)-capped AuNP aggregates in the presence of arsenic, replacing the LS, resulting in a colour change from red to purple. Hence, the developed method can be used for easy and rapid detection of use of calcium carbide in artificial ripening of fruits.     

辣椒制品中罗丹明B胶体金免疫层析法的建立

目的建立灵敏的胶体金免疫层析法快速检测辣椒制品中的罗丹明B (rhodamine B,RB)。方法用柠檬酸三钠还原法制备胶体金,并通过物理吸附标记罗丹明B单克隆抗体。在优化条件下,建立了基于竞争性抑制的罗丹明B的可视化胶体金免疫层析法。结果使用建立的方法检测辣椒制品中罗丹明B,定性检测限为5μg/kg,满足了国家安全限量要求;对实际样本的检测结果与液质联用检测结果一致。结论本研究建立的方法可以实现对辣椒制品中非法使用罗丹明B的灵敏、快速、高效的检测。     

胶体金免疫层析技术快速检测蜂蜜中5-羟甲基糠醛

建立快速检测蜂蜜中有毒物质5-羟甲基糠醛(HMF)的胶体金免疫层析试纸条。文中采用免疫竞争法,将制备的胶体金颗粒与抗HMF单克隆抗体结合成抗HMF-金标复合物标记在胶体金结合垫上,并将HMF-载体蛋白包被在硝酸纤维素膜上作为检验线(T线),其与待测蜂蜜中HMF竞争结合胶体金标记的抗HMF单克隆抗体,并能以颜色直观显示检测的定性结果。结果显示:若蜂蜜液中HMF的含量<40 mg/kg,则过量的胶体金标记物与T线上HMF-载体蛋白特异性结合形成紫红色条带,即阴性;若蜂蜜液HMF含量≥40 mg/kg,不能产生紫红色条带,即阳性。该方法是对分光光度法及HPLC法测定蜂蜜中HMF含量的补充,并且该法灵敏度高,操作简便快速等优点。     

Nanozyme-based lateral flow assay for the sensitive detection of Escherichia coli O157:H7 in milk

Lateral flow assay (LFA) has been applied in many fields due to its relative ease of use and cost-effectiveness. However, it has low sensitivity and its applications are limited. Probe materials play a significant role in improving the detection efficiency and sensitivity of LFA. In this study, by using concave palladium-platinum (Pd-Pt) nanoparticles as a nanozyme probe, we developed a sensitive LFA based on the sandwich format for qualitative and quantitative detection of Escherichia coli O157:H7. The sensitivity of the LFA was improved by applying the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate onto the test line where the nanozyme was accumulated in the presence of analytes. The nanozyme showed high catalytic performance toward TMB and greatly enhanced the signal intensity of the test line. The sensitivity of the nanozyme-based LFA was 9.0 × 10² cfu/mL in milk, which was 111-fold higher than that of traditional colloidal gold-based LFA. The proposed method has remarkable potential in the detection of various pathogens in real samples.     

Immunomagnetic flow cytometric detection of staphylococcal enterotoxin B in raw and dry milk

A rapid and sensitive method for detection of staphylococcal enterotoxin B (SEB) in raw and dry milk samples with the use of antibody-based immunomagnetic separation (IMS) in conjunction with flow cytometry (FCM) was developed. Sheep anti-SEB immunoglobulin G (IgG) was immobilized on Dynabeads M-280. The SEB initially binds to the capturing antibody, which is bound on the magnetic beads. The rabbit anti-SEB IgG binds to the captured toxin and is further labeled with a Cy5-labeled goat anti-rabbit IgG antibody. The percentage of the beads that were fluorescent was measured by FCM. FCM was carried out for 1 min, and the data obtained were expressed as histograms for particle size (forward light scatter) and histograms for fluorescence intensity. A peak corresponding to the magnetic beads was clearly distinguished from a peak derived from contaminating particles in the sample solution. In the absence of SEB, about 10% of the beads emitted fluorescence. The percentage of fluorescent beads and the fluorescence intensity increased with increasing SEB concentrations. For this IMS-FCM assay, the lower limits of detection for SEB were estimated to be 0.01 and 0.25 ng/ml for buffer and milk samples, respectively.     

胶体金免疫层析法快速检测黄曲霉毒素B1的研究

本文应用胶体金免疫层析技术,建立了一种快速检测食品中黄曲霉毒素B1的方法。采用柠檬酸三钠还原法制备胶体金颗粒,标记抗黄曲霉毒素B1单克隆抗体并喷于玻璃纤维上,黄曲霉毒素B1偶联抗原和二抗鼠抗驴分别结合于硝酸纤维膜上,依次将样本垫、胶金垫、硝酸纤维膜和吸水纸组装切割成胶体金试纸条并装入检测卡中。测试结果表明黄曲霉毒素B1快速检测试纸条的灵敏度为5ng/ml,检测时间为10min,批内和批间重复性为100%,假阳性率和假阴性率均为0。使用简单方便,非常适合现场快速检测黄曲霉毒素B1。     

Advantages of immunoglobulin Y for the detection of Staphylococcal enterotoxin A in a double‐antibody sandwich enzyme‐linked immunosorbent assay

To determine the amounts of staphylococcal enterotoxin A (SEA), a novel and sensitive enzyme‐linked immunosorbent assay (ELISA) was developed. Protein A, which is produced by Staphylococcus aureus, interferes with the reaction between SEA and anti‐SEA immunoglobulin G (IgG), resulting in a false‐positive reaction. Chicken IgY was introduced as a capture antibody in the sandwich ELISA system, as IgY binds less efficiently to protein A. When the anti‐SEA IgG antibody was used as the capture and detection antibodies (IgG‐IgG ELISA), the background levels of protein A increased, thus resulting in a false‐positive reaction. A 0.01 ng mL^?1 concentration of protein A significantly increased the absorbance value of the blank wells. When the anti‐SEA IgY antibody was used as the capture antibody, 1000 ng mL^?1 of protein A did not affect the absorbance value. The ELISA system using anti‐SEA IgY as a capture antibody and anti‐SEA IgG as a detection antibody (IgY‐IgG ELISA) showed a detection limit of <0.25 ng mL^?1 and a creditability of R² = 0.98. These findings demonstrate the advantage of chicken IgY for the detection of SEA by means of double‐antibody sandwich ELISA.