可视化抗体阵列快速联合检测大肠杆菌O157:H7和鼠伤寒沙门氏菌

【目的】建立一种同时快速检测大肠杆菌O157:H7(E.coli O157:H7)和鼠伤寒沙门氏菌(Salmonella typhimurium)的可视化抗体阵列技术。【方法】将免疫学技术与蛋白芯片技术相结合,基于双抗体夹心法检测原理利用蛋白质芯片技术的高通量,结合可视化结果判定技术,用一份样品,同步检测大肠杆菌O157:H7和鼠伤寒沙门氏菌两种病原。【结果】检测结果肉眼可见,检测周期短至90 min,纯菌液检测灵敏度达105 CFU/mL,模拟带菌检测灵敏度为106 CFU/mL,与常规的ELISA灵敏度等同且具有良好的特异性和重复性。【结论】该可视化抗体阵列检测结果肉眼可见,检测通量高,无需大型设备,操作简便,检测成本低廉,同时为快速检测致病菌提供一种新途径。     

噬菌体裂解酶Lysin1902原核表达及其与ε-聚赖氨酸联用效果评价北大核心

【目的】研究裂解酶Lysin1902与ε-聚赖氨酸(ε-PL)对大肠杆菌O157:H7的协同抗菌作用。【方法】通过Mega构建进化树、使用在线工具等分析大肠杆菌噬菌体裂解酶Lysin1902氨基酸序列组成和结构等;原核表达并纯化Lysin1902;通过平板裂解实验检测Lysin1902对大肠杆菌O157:H7灭活菌株的裂解活性;用96孔板法检测Lysin1902或ε-PL的活菌裂解能力;棋盘法验证Lysin1902和ε-PL联用效果。【结果】体外成功表达并纯化了Lysin1902。Lysin1902对大肠杆菌O157:H7灭活菌株具有裂解活性,但不能有效裂解活的大肠杆菌。噬菌体裂解酶与ε-PL联用结果表明,加入Lysin1902后,ε-PL能够完全控制大肠杆菌O157:H7增殖的使用浓度由0.7mg/mL降低到0.1 mg/mL。【结论】体外原核表达并纯化Lysin1902,其单独使用对活的大肠杆菌O157:H7无裂解活性,但与ε-PL联用可显著提高ε-PL对大肠杆菌O157:H7的裂解能力。     

检测大肠杆菌(Escherichia coli) O157:H7免疫磁珠的制备、保存与应用

【背景】大肠杆菌(Escherichia coli) O157:H7是导致肠出血性大肠杆菌食源性疾病暴发的主要血清型,免疫磁珠(Immunomagnetic beads,IMBS)在E. coli O157的检测中发挥着重要作用,而免疫磁珠的稳定性、特异性、广谱性等性能指标关系着在实际应用中的使用效果。【目的】制备高效、稳定且具有广谱性的免疫磁珠,联合分子检测技术如环介导恒温扩增 (Loop-mediated isothermal amplification,LAMP)技术、PCR等,提高目标菌的检出率。【方法】采用新型的磁珠活化剂MIX&GO制备E. coli O157免疫磁珠,并进行广谱性以及特异性检测;针对6种试剂牛血清白蛋白(Bovine serum albumin,BSA)、酪蛋白(Casein)、海藻糖(Trehalose)、聚乙烯吡咯烷酮(Polyvinyl pyrrolidone,PVP)、抗坏血酸(Vitamin C)和防腐剂ProClin 300,利用正交试验L18(37)优化免疫磁珠保存液组分;采用IMBS-LAMP、IMBS-PCR、IMBS-生化、菌液-LAMP、菌液-PCR、显色平板-生化鉴定6种方式对20份生猪肉样品进行检测。【结果】利用MIX&GO活化剂制备的免疫磁珠捕获率最高达到81.5%±1.3%;免疫磁珠保存液最优配方为：牛血清白蛋白15.0 g/L,酪蛋白10.0 g/L,海藻糖10.0 g/L,PVP 2.0 g/L,抗坏血酸5.0 g/L,ProClin 300 2.5 g/L,保存6个月后免疫磁珠捕获率为75.5%;在20份生猪肉样品的检测中,自制磁珠和商品化磁珠与LAMP联用均检出9例阳性样品;IMBS-LAMP在6种检测方式中具有最高的检测灵敏度,但检出的样品会因磁珠抗体的差异而有所不同。【结论】与商品化磁珠相比,实验制备的免疫磁珠具有良好的特异性和广谱性,免疫磁珠-LAMP联用提高了目标菌的检出率,是一种高灵敏度、具有应用前景的检测方法。     

检测腹蛇毒的压电免疫传感器的研究

目的将石英晶振的高灵敏度与免疫反应的特异性相结合,研制检测蝮蛇毒的压电免疫传感器方法用巯基丙酸在镀银电极石英晶体自组装巯基丙酸单分子膜偶联抗蝮蛇毒球蛋白构建传感器探头结果组成的检测器对蝮蛇毒响应良好。结论采用免疫传感技术将是实现快速、仪器化检测蛇伤的可能途径,本文结果为蛇毒免疫传感器的进一步深入研究打下了基础。     

大肠杆菌O157显色培养基检测效果的评价

【目的】评价显色培养基对大肠杆菌O157:H7(Escherichia coli O157:H7)的检测效果。【方法】本实验室研制的大肠杆菌O157显色培养基(HKM),与国外梅理埃、科玛嘉及国内厂家的同类产品及传统培养基CT-SMAC作比较,对相关菌株以及污染样品和实际样品进行对比测试。【结果】实验室研制的HKM大肠杆菌O157显色培养基与科玛嘉同类产品在特异性、灵敏度及检测效果方面均无明显差异,均优于梅里埃、国内厂家产品及CT-SMAC。【结论】HKM大肠杆菌O157显色培养基具有高检出率及高特异性的特点,具有较好的应用价值和前景。     

大肠杆菌O157:H7核酸探针检测方法的建立

目的：应用核酸探针方法快速检测大肠杆菌O157：H7。方法：通过使用吖啶酯标记的特异DNA探针方法检测大肠杆菌O157：H7,对此种方法的特异性、敏感性、准确性进行研究,比较该方法与传统国标法的检测结果。结果：核酸探针方法检测大肠杆菌O157：H7特异性以及敏感性强,检出大肠杆菌O157：H7菌液浓度最低限约为106cfu/ml,检测大肠杆菌O157：H7的结果与国标法相一致;对O157：H7鉴定时间仅需30min,简便快捷。结论：核酸探针方法可用于大肠杆菌O157：H7的快速检测。     

一种酶免疫传感器的制备和性能测试

这种酶免疫传感器是采用丝素蛋白溶液将待测抗原(兔IgG)固定在基础电极表面,选用抗体(山羊抗兔IgG-HRP)与其识别结合。利用H2O2将抗原抗体结合的电位响应信号放大而检测抗原的浓度。该传感器测定抗原的最低检测浓度1.0×10-10 mol/L,线性范围1.0×10-8~1.0×10-10 mol/L,响应时间为10s。通过电泳的方法加速抗原抗体的识别结合,反应时间由原来的90min缩短到30min,这在国内外鲜有报道。这种以固定化抗原结合酶标抗体量的多少作为检测抗原标准的新型酶免疫传感器,在临床检测、生物医学研究等领域将会有广阔的应用前景。     

畜禽肉沙门氏菌和大肠杆菌O157多重PCR检测研究

沙门氏菌和大肠杆菌O157都是目前世界公认引起食源性疾病的重要致病菌.本研究针对致病茵传统检测方法耗时长、过程繁琐的缺点,建立了同时检测畜禽肉及其制品中沙门氏菌和大肠杆菌O157的多重PCR分子检测方法.结果表明:分别针对沙门氏茵侵袭基因invA、大肠杆菌O157抗原基因rfbE建立的多重PCR方法可简便、快速、灵敏地实现对沙门氏菌和大肠杆菌O157的同时检测,整个过程在9h～10h内完成,人工污染猪肉检测限分别达到2.4×102cfu/mL(沙门氏菌)和2.2×102 cfu/mL(大肠杆菌O157);为食源性致病菌的检测提供了理想手段,有良好的应用前景.     

基于环介导恒温扩增技术的大肠杆菌O157:H7快速检测试剂盒的评价

【目的】评价基于环介导恒温扩增技术(LAMP)的大肠杆菌O157:H7(Escherichia coli O157:H7)快速检测试剂盒的实效性。【方法】测定快速检测试剂盒的特异性、灵敏度、重复性、保质期以及运输稳定性,并与传统方法对比检测实际样品。【结果】大肠杆菌O157:H7标准菌株样品均检测为阳性,非大肠杆菌O157:H7标准菌株样品均检测为阴性,未发现有交叉反应;试剂盒最低检验限为29 CFU;该试剂盒的特异性、灵敏度及准确度与传统方法相比具有较高的一致性;试剂盒对高菌量目标菌和阴性菌样品的检测重复率均为100%,对低菌量目标菌样品的批间检测重复率为94%。试剂盒可在4°C保存9个月以上,并且可进行变温储存72 h以上。【结论】该试剂盒特异性好,灵敏度高,重复性好,储存方便,检测结果稳定、可靠,适用于对食品中大肠杆菌O157:H7的检测需求。     

原核表达MurA蛋白并制备其多克隆抗体用于免疫磁珠快速检测单增李斯特菌

【目的】制备MurA多抗,结合免疫磁珠与选择平板进行单增李斯特菌的快速检测,建立单增李斯特菌的免疫磁珠快速检测方法。【方法】构建MurA的原核表达载体,转化大肠杆菌进行优化表达。镍柱纯化表达产物,质谱鉴定重组蛋白,再免疫小鼠,制备其多克隆抗体。用所获多抗制备免疫磁珠,建立单增李斯特菌免疫磁珠-选择性培养基检测方法,并对人工污染牛奶样品进行检测。【结果】在大肠杆菌中高效表达了分子量约为72 kD的可溶性融合蛋白,质谱鉴定其为MurA蛋白;免疫小鼠获得的抗血清效价达1:10 000,与伤寒沙门氏菌、副溶血弧菌、大肠杆菌及属内其它病原菌均无交叉;所建立的免疫磁珠-选择性培养基检测法可检出浓度为103 CFU/mL及以上的单增李斯特菌,仅与英诺克李斯特菌存在一定交叉反应;牛奶样品单次仅需9 h增菌就能被检出,较常规增菌时间缩短39 h;检测限为0.4 CFU/mL。【结论】表达并纯化得到高纯度的单增李斯特菌MurA蛋白,制备的鼠源多克隆抗体亲和力高,特异性好;建立了快速检测单增李斯特菌的免疫磁珠联合选择性培养基法,在灵敏度不变的情况下,实现24 h内成功对牛奶样品的检测,较国标法减少42 h以上。     

QCM immunosensor detection of Escherichia coli O157:H7 based on beacon immunomagnetic nanoparticles and catalytic growth of colloidal gold

In this paper, we describe a novel method for detecting Escherichia coli (E. coli) O157:H7 by using a quartz crystal microbalance (QCM) immunosensor based on beacon immunomagnetic nanoparticles (BIMPs), streptavidin-gold, and growth solution. E. coli O157-BIMPs were magnetic nanoparticles loaded with polyclonal anti-E. coli O157:H7 antibody (target antibody, T-Ab) and biotin-IgG (beacon antibody, B-Ab) at an optimized ratio of 1:60 (T-Ab:B-Ab). E. coli O157:H7 was captured and separated by E. coli O157-BIMPs in a sample, and the streptavidin-gold was subsequently conjugated to E. coli O157-BIMPs by using a biotin-avidin system. Finally, the gold particles on E. coli O157-BIMPs were enlarged in growth solution, and the compounds containing E. coli O157:H7, E. coli O157-BIMPs, and enlarged gold particles were collected using a magnetic plate. The QCM immunosensor was fabricated with protein A from Staphylococcus aureus and monoclonal anti-E. coli O157:H7 antibody. The compounds decreased the immunosensor's resonant frequency. E. coli O157-BIMPs and enlarged gold particles were used as "mass enhancers" to amplify the frequency change. The frequency shift was correlated to the bacterial concentration. The detection limit was 23 CFU/ml in phosphate-buffered saline and 53 CFU/ml in milk. This method could successfully detect E. coli O157:H7 with high specificity and stability. The entire procedure for the detection of E. coli O157:H7 took only 4 h.     

Amperometric immunosensor for the detection of Escherichia coli O157:H7 in food specimens

A novel, label-free amperometric immunosensor has been developed for the rapid detection of heat-killed Escherichia coli O157:H7 (E. coli O157:H7). This immunosensor was prepared as follows. First, the long-chain, amine-terminated alkanethiol 11-amino-1-undecanethiol hydrochloride (AUT) was self-assembled onto a gold electrode surface to form an ordered, oriented, compact, and stable monolayer possessing -NH(2) functional groups that could immobilize massive gold nanoparticles (GNPs). Next, chitosan-multiwalled carbon nanotubes-SiO(2)/thionine (CHIT-MWNTs-SiO(2)@THI) nanocomposites and GNPs multilayer films were prepared via layer-by-layer (LBL) assembly. The surface area enhancement from the LBL assembly of the multilayer films improves the stability of the immobilized CHIT-MWNTs-SiO(2)@THI. More important, the sensitivity and stability of the immunosensor can be enhanced proportionally to the quantity of the THI mediator immobilized on the electrode surface. Finally, the E. coli O157:H7 antibody (anti-E. coli O157:H7) was covalently bound to the GNP monolayer and its bioactivity was measured by enzyme-linked immunosorbent assay (ELISA). Transmission electron microscopy (TEM) was employed to characterize the morphology of the MWNTs, CHIT-MWNTs, and CHIT-MWNTs-SiO(2)@THI. Under optimal conditions, the calibration curve for heat-killed E. coli O157:H7 has a working range of 4.12×10(2)-4.12×10(5) colony-forming units (CFU)/ml, and the total assay time was less than 45 min.     

A mixed self-assembled monolayer-based surface plasmon immunosensor for detection of E. coli O157:H7

The sensitivity and specificity of a polyethylene glycol terminated alkanethiol mixed self-assembled monolayers (SAM) on surface plasmon resonance (SPR) immunosensor to detect Escherichia coli O157:H7 is demonstrated. Purified monoclonal (Mabs) or polyclonal antibodies (PAbs) against E. coli O157:H7 were immobilized on an activated sensor chip and direct and sandwich assays were carried to detect E. coli O157:H7. Effect of Protein G based detection and effect of concentrations of primary and secondary antibodies in sandwich assay were investigated. The sensor surface was observed under an optical microscope at various stages of the detection process. The sensor could detect as low as 10(3)CFU/ml of E. coli O157:H7 in a sandwich assay, with high specificity against Salmonella Enteritidis. The detection limit using direct assay and Protein G were 10(6)CFU/ml and 10(4)CFU/ml, respectively. Results indicate that an alkanethiol SAM based SPR biosensor has the potential for rapid and specific detection of E. coli O157:H7, using a sandwich assay.     

Fate of Escherichia coli O157:H7 during the manufacture of Mozzarella cheese

AIMS: The fate of Escherichia coli O157:H7 was investigated during the manufacture of Mozzarella cheese. METHODS AND RESULTS: The Mozzarella cheese was made from unpasteurized milk which was inoculated to contain ca 10(5) cfu ml(-1)E. coli O157:H7. Two different heating temperatures (70 and 80 degrees C), commonly used during curd stretching, were investigated to determine their effects on the viability of E. coli O157:H7 in Mozzarella cheese. Stretching at 80 degrees C for 5 min resulted in the loss of culturability of E. coli O157:H7 strains, whereas stretching at 70 degrees C reduced the number of culturable E. coli O157:H7 by a factor of 10. CONCLUSIONS: The results show that stretching curd at 80 degrees C for 5 min is effective in controlling E. coli O157:H7 during the production of Mozzarella cheese. Brining and storage at 4 degrees C for 12 h was less effective than the stretching. Significance and Impact of the Study: Mozzarella cheese should be free of E. coli O157:H7 only if temperatures higher than or equal to 80 degrees C are used during milk processing.     

A self-assembled monolayer-based piezoelectric immunosensor for rapid detection of Escherichia coli O157:H7

A piezoelectric immunosensor was developed for rapid detection of Escherichia coli O157:H7. It was based on the immobilization of affinity-purified antibodies onto a monolayer of 16-mercaptohexadecanoic acid (MHDA), a long-chain carboxylic acid-terminating alkanethiol, self-assembled on an AT-cut quartz crystal's Au electrode surface with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. The binding of target bacteria onto the immobilized antibodies decreased the sensor's resonant frequency, and the frequency shift was correlated to the bacterial concentration. The stepwise assembly of the immunosensor was characterized by means of both quartz crystal microbalance (QCM) and cyclic voltammetry techniques. Three analytical procedures, namely immersion, dip-and-dry and flow-through methods, were investigated. The immunosensor could detect the target bacteria in a range of 10(3)-10(8)CFU/ml within 30-50 min, and the sensor-to-sensor reproducibility obtained at 10(3) and 10(5) colony-forming units (CFU)/ml was 18 and 11% R.S.D., respectively. The proposed sensor was comparable to Protein A-based piezoelectric immunosensor in terms of the amount of immobilized antibodies and detection sensitivity.     

Liposome-based microcapillary immunosensor for detection of Escherichia coli O157:H7

Our group has previously reported a sandwich-based strip immunoassay for rapid detection of Escherichia coli O157:H7 [Anal. Chem. 75 (2003) 4330]. In the present study, a microcapillary flow injection liposome immunoanalysis (mFILIA) system was developed for the detection of heat-killed E. coli O157:H7. A fused-silica microcapillary with anti-E. coli O157:H7 antibodies chemically immobilized on the internal surface via protein A served as an immunoreactor/immunoseparator for the mFILIA system. Liposomes tagged with anti-E. coli O157:H7 and encapsulating a fluorescent dye were used as the detectable label. In the presence of E. coli O157:H7, sandwich complexes were formed between the immobilized antibodies in the column, the sample of E. coli O157:H7 and the antibody-tagged sulforhodamine-dye-loaded liposomes. Signals generated by lysing the bound liposomes with 30 mM n-octyl-beta-D-glucopyranoside were measured by a fluorometer. The detected signal was directly proportional to the amount of E. coli O157:H7 in the test sample. The mFILIA system successfully detected as low as 360 cells/mL (equivalent to 53 heat-killed bacteria in the 150 microL of the sample solution injected). MeOH (30%) was used for the regeneration of antibody binding sites in the capillary after each measurement, which allowed the immunoreactor/immunoseparator to be used for at least 50 repeated assays. The calibration curve for heat-killed E. coli O157:H7 has a working range of 6 x 10(3)-6 x 10(7)cells, and the total assay time was less than 45 min. A coefficient of variation for triplicate measurements was < or =8.9%, which indicates an acceptable level of reproducibility for this newly developed method.     

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.     

Antimicrobial activity of a 14-residue peptide against Escherichia coli O157:H7

An amphiphilic, cationic peptide composed of eight leucines and six lysines was synthesized by solid phase peptide synthesis (SPPS). The synthetic peptide was bactericidal within 10 min at concentrations as low as 3 microg ml - 1 against mid-exponential Escherichia coli O157:H7 suspended in buffer. Concentrations of 25 microg ml - 1 caused up to 7 log10 cfu ml - 1 reductions. When tested against E. coli O157:H7 grown in TSB, the peptide was bactericidal and bacteriostatic at concentrations of 50 and 25 microg ml - 1, respectively. An inhibitory effect was also observed against stationary phase cells. The synthetic peptide caused the release of u.v.-absorbing materials from the E. coli O157:H7 as well as an increase in its O.D.600 nm. Intracellular K+ and ATP depletion were also observed. These results suggest that the peptide increased the cell membrane permeability but it did not lyse the cells.     

Rapid electrochemical detection of polyaniline-labeled Escherichia coli O157:H7

There is a high demand for rapid, sensitive, and field-ready detection methods for Escherichia coli O157:H7, a highly infectious and potentially fatal food and water borne pathogen. In this study, E. coli O157:H7 cells are isolated via immunomagnetic separation (IMS) and labeled with biofunctionalized electroactive polyaniline (immuno-PANI). Labeled cell complexes are deposited onto a disposable screen-printed carbon electrode (SPCE) sensor and pulled to the electrode surface by an external magnetic field, to amplify the electrochemical signal generated by the polyaniline. Cyclic voltammetry is used to detect polyaniline and signal magnitude indicates the presence or absence of E. coli O157:H7. As few as 7CFU of E. coli O157:H7 (corresponding to an original concentration of 70 CFU/ml) were successfully detected on the SPCE sensor. The assay requires 70 min from sampling to detection, giving it a major advantage over standard culture methods in applications requiring high-throughput screening of samples and rapid results. The method can be performed with portable, handheld instrumentation and no biological modification of the sensor surface is required. Potential applications include field-based pathogen detection for food and water safety, environmental monitoring, healthcare, and biodefense.