Rapid and ultrasensitive E. coli O157:H7 quantitation by combination of ligandmagnetic nanoparticles enrichment with fluorescent nanoparticles based two-color flow cytometry

A novel, fast and sensitive determination strategy for E. coli O157:H7 has been developed by combination of ligandmagnetic nanoparticles (LMNPs) enrichment with a fluorescent silica nanoparticles (FSiNPs) based two-color flow cytometry assay (LMNPs@FSiNPs-FCM). E. coli O157:H7 was first captured and enriched through the lectin concanavalin A (Con A) favored strong adhesion of E. coli O157:H7 to the mannose-conjugated magnetic nanoparticles. The enriched E. coli O157:H7 was further specially labeled with goat anti-E. coli O157:H7 antibody modified RuBpy-doped FSiNPs, and then stained with a nucleic acid dye SYBR Green I (SYBR-I). After dual-labeling with FSiNPs and SYBR-I, the enriched E. coli O157:H7 was determined using multiparameter FCM analysis. With this method, the detection sensitivity was greatly improved due to the LMNPs enrichment and the signal amplification of the FSiNPs labelling method. Furthermore, the false positives caused by aggregates of FSiNPs conjugates and nonspecific binding of FSiNPs to background debris could be significantly decreased. This assay allowed the detection of E. coli O157:H7 in PB buffer at levels as low as 7 cells mL(-1). The total assay time including E. coli O157:H7 sample enrichment and detection was less than 4 h. An artificially contaminated bottled mineral water sample with a concentration of 6 cells mL(-1) can be detected by this method. It is believed that the proposed method will find wide applications in biomedical fields demanding higher sensitive bacterial identification.     

基于二氧化硅荧光纳米颗粒与核酸染料SYBRGreenⅠ的双色显微成像技术用于E.coliO157:H7的检测

将免疫荧光纳米标记技术与激光共聚焦显微成像方法相结合,发展了一种基于二氧化硅荧光纳米颗粒和核酸染料SYBR Green Ⅰ的双色显微成像技术用于大肠杆菌O157:H7的检测.采用联吡啶钌(RuBpy)二氧化硅荧光纳米颗粒对羊抗大肠杆菌O157:H7抗体进行修饰,基于抗体-抗原相互作用实现了其对目标大肠杆菌O157:H7...     

A sensitive lateral flow biosensor for Escherichia coli O157:H7 detection based on aptamer mediated strand displacement amplification

Foodborne diseases caused by pathogens are one of the major problems in food safety. Convenient and sensitive point-of-care rapid diagnostic tests for food-borne pathogens have been a long-felt need of clinicians. Commonly used methods for pathogen detection rely on conventional culture-based tests, antibody-based assays and polymerase chain reaction (PCR)-based techniques. These methods are costly, laborious and time-consuming. Herein, we present a simple and sensitive aptamer based biosensor for rapid detection of Escherichia coli O157:H7 (E. coli O157:H7). In this assay, two different aptamers specific for the outmembrane of E. coli O157:H7 were used. One of the aptamers was used for magnetic bead enrichment, and the other was used as a signal reporter for this pathogen, which was amplified by isothermal strand displacement amplification (SDA) and further detected by a lateral flow biosensor. Only the captured aptamers on cell membrane were amplified, limitations of conventional DNA amplification based method such as false-positive can be largely reduced. The generated signals (red bands on the test zone of a lateral flow strip) can be unambiguously read out by the naked eye. As low as 10 colony forming units (CFU) of E. coli O157:H7 were detected in this study. Without DNA extraction, the reduced handling and simpler equipment requirement render this assay a simple and rapid alternative to conventional methods.     

基于甘露糖功能化的水凝胶用于E.coli O157: H7的检测

利用伴刀豆球蛋白A(Con A)的多价结合能力, 结合水凝胶技术与核酸染色技术发展了一种基于甘露糖功能化的水凝胶检测大肠杆菌(E.coli)O157: H7的方法. 以过硫酸铵(APS)为催化剂, 四甲基乙二胺(TEMED)为加速剂, 用丙烯酰胺(AAm)、N,N-二甲基双丙烯酰胺和N-丙烯酰氧琥珀酰亚胺(NAS)合成水凝胶, 通过氨基化甘露糖与NAS发生交联反应, 制备了甘露糖功能化的水凝胶. 当甘露糖功能化的水凝胶加入与Con A共孵育后的菌悬液中时, 由于Con A既能与甘露糖特异性结合, 又能与E.coli O157: H7表面的O-抗原发生免疫反应而紧密连接, 使目标菌被捕获到水凝胶表面, 采用核酸染料SYBR Green Ⅰ对捕获细菌进行染色, 实现了对E.coli O157: H7的核酸标记, 最后通过活体荧光成像系统对水凝胶进行荧光成像, 从而实现对待测样品的检测. 研究结果表明, 该方法可应用于缓冲液体系和混合细菌样品中E.coli O157: H7的特异性检测, 且整个检测步骤包括样品预处理可在2 h内完成. 该方法成本低、易操作, 且具有较好的灵敏度, 可检出3.7×10¹ Cells/mL的目标细菌样品.     

Microfluidic based impedance biosensor for pathogens detection in food products

A MEMS‐based impedance biosensor was designed, fabricated, and tested to effectively detect the presence of bacterial cells including E. coli O157:H7 and Salmonella typhimurium in raw chicken products using detection region made of multiple interdigitated electrode arrays. A positive dielectrophoresis based focusing electrode was used in order to focus and concentrate the bacterial cells at the centerline of the fluidic microchannel and direct them toward the detection microchannel. The biosensor was fabricated using surface micromachining technology on a glass substrate. The results demonstrate that the device can detect Salmonella with concentrations as low as 10 cells/mL in less than 1 h. The device sensitivity was improved by the addition of the focusing electrodes, which increased the signal response by a factor between 6 and 18 times higher than without the use of the focusing electrodes. The biosensor is selective and can detect other types of pathogen by changing the type of the antibody immobilized on the detection electrodes. The device was able to differentiate live from dead bacteria.     

An anti E. coli O157:H7 antibody-immobilized microcantilever for the detection of Escherichia coli (E. coli).

A silicon microcantilever sensor was developed for the detection of Escherichia coli O157:H7. The microcantilever was modified by anti-E. coli O157:H7 antibodies on the silicon surface of the cantilever. When the aquaria E. coli O157:H7 positive sample is injected into the fluid cell where the microcantilever is held, the microcantilever bends upon the recognition of the E. coli O157:H7 antigen by the antibodies on the surface of the microcantilever. A negative control sample that does not contain E. coli O157:H7 antigen did not cause any bending of the microcantilever. The detection limit of the sensor was 1 x 10(6) cfu/mL when the assay time was < 2 h.     

纳米金标记抗体增强SPR检测大肠杆菌O157∶H7

将金纳米粒子(AuNPs)标记的大肠杆菌O157∶H7(E.coli O157∶H7)的多克隆抗体(PAb)作为二抗,采用氨基偶联法将PAb固定在传感器表面作为一抗,通过三明治方法用双通道表面等离子体子共振(SPR)传感器对E.coli O157∶H7进行检测,并与SPR直接法检测进行了比较.结果表明,直接法的检出限为103cfu/mL,线性范围为103～109cfu/mL;AuNPs增强三明治法的检出限为10 cfu/mL,线性范围为10～1010cfu/mL,灵敏度比直接法提高了100倍,且具有更宽的检测范围.本方法不仅检测时间短,而且具有良好的选择性和重现性.     

Nano-ZnO微叉指电极生物传感器检测大肠杆菌

设计了一种基于纳米ZnO材料检测大肠杆菌(E.coli O157:H7)的微叉指阻抗生物传感器,利用电化学方法在氧化铟锡(ITO)叉指电极表面沉积上纳米ZnO,然后将链霉亲和素固定在纳米ZnO表面,利用生物素亲和素的高亲和性原理将大肠杆菌抗体绑定在传感器表面,完成传感器的构建。实验表明,传感器检测E.coli O157:H7线性范围为40~4×10^6cfu/mL,检出限为40 cfu/mL,传感器的特异性、重现性、实用性较好。     

Detection of Escherichia coli O157:H7 bacteria by a combination of immunofluorescent staining and capillary electrophoresis

As the number of incidents of bacterial infections continues to rise around the globe, simpler, faster, and more sensitive diagnostic techniques are required to improve the safety of the food supply and to screen for potential bacterial infections in humans. We present here direct and indirect approaches for the detection of bacteria, which are based upon a combination of immunofluorescent staining and capillary electrophoresis. In the direct approach, Escherichia coli O157:H7 bacteria stained with fluorescein-tagged specific antibodies are detected by CE, while in the indirect approach fluorescein-tagged specific antibodies to E. coli are first captured by E. coli O157:H7 bacteria and then released and detected by CE. We have identified suitable bacteria staining and CE protocols, which involved a 10 mM Tris-borate-EDTA (TBE) buffer, 0.25 micro g antibody/1 million bacteria, and capillaries dynamically coated with poly-N-hydroxyethylacrylamide (polyDuramide). We have also successfully detected the presence of E. coli O157:H7 in contaminated meat. The total time required for analysis was 6-8 h, which is less than that realized in most commercial assays presently available.     

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC methods 996.09, Vip for EHEC and 996.10, assurance Eia EHEC with the reference culture method for the detection of Escherichia coli O157:H7 in beef

The Visual Immunoprecipitate Assay (VIP) method for the detection of enterohemorrhagic Escherichia coli O157:H7 (VIP for EHEC) and Assurance Enzyme Immunoassay (EIA) method for the detection of EHEC (EHEC EIA) are AOAC INTERNATIONAL Official Methods 996.09 and 996.10, respectively. A minor modification to the enrichment medium used in both methods has been developed. This modification, the BioControl modified EHEC medium (BioControl mEHEC) provides a more cost-effective procedure with performance equivalent to that of the cultural method for detection of E. coli O157:H7 in beef.     

Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels

In this study, we explored the use of semiconductor quantum dots (QDs) as fluorescence labels in immunoassays for simultaneous detection of two species of foodborne pathogenic bacteria, Escherichia coli O157:H7 and Salmonella Typhimurium. QDs with different sizes can be excited with a single wavelength of light, resulting in different emission peaks that can be measured simultaneously. Highly fluorescent semiconductor quantum dots with different emission wavelengths (525 nm and 705 nm) were conjugated to anti-E. coli O157 and anti-Salmonella antibodies, respectively. Target bacteria were separated from samples by using specific antibody coated magnetic beads. The bead-cell complexes reacted with QD-antibody conjugates to form bead-cell-QD complexes. Fluorescent microscopic images of QD labeled E. coli and Salmonella cells demonstrated that QD-antibody conjugates could evenly and completely attach to the surface of bacterial cells, indicating that the conjugated QD molecules still retain their effective fluorescence, while the conjugated antibody molecules remain active and are able to recognize their specific target bacteria in a complex mixture. The intensities of fluorescence emission peaks at 525 nm and 705 nm of the final complexes were measured for quantitative detection of E. coli O157:H7 and S. Typhimurium simultaneously. The fluorescence intensity (FI) as a function of cell number (N) was found for Salmonella and E. coli, respectively. The regression models can be expressed as: FI = 60.6 log N- 250.9 with R(2) = 0.97 for S. Typhimurium, and FI = 77.8 log N- 245.2 with R(2) = 0.91 for E. coli O157:H7 in the range of cell numbers from 10(4) to 10(7) cfu ml(-1). The detection limit of this method was 10(4) cfu ml(-1). The detection could be completed within 2 hours. The principle of this method could be extended to detect multiple species of bacteria (3-4 species) simultaneously, depending on the availability of each type of QD-antibody conjugates with a unique emission peak and the antibody coated magnetic beads specific to each species of bacteria.     

Bead-Based Optical Immunoassay Using Quantum-Dot Labeling and Immunocomplex Dissociation for Detection of Escherichia coli O157:H7

An immunoassay for Escherichia coli O157:H7 using quantum-dot (QD) labeling and subsequent release of the QD labels from immunocomplexes has been developed. The assay principle is that anti-E. coli O157:H7 conjugated immunomagnetic beads are used to capture E. coli O157:H7; this is followed by the binding of QD labeled antibodies to form sandwich immunocomplexes (Bead-Cell-QD); a dissociation buffer then elutes QDs from immunocomplexes by denaturing antibody or lysing cell; the fluorescence signal of the eluted QDs is measured to quantify E. coli O157:H7. This proposed approach avoids the interference of bead autofluorescence in signal transduction and, thus, enhances the detection resolution, while keeping the fast magnetic separation and sandwich binding of two selective antibodies for high specificity.     

An electrochemical method for simultaneous detection and identification of Escherichia coli, Staphylococcus aureus and Salmonella choleraesuis using a glucose oxidase-peroxidase composite biosensor

Quantification of bacterial pollution by amperometric detection at 0.0 V of glucose consumption at a graphite-Teflon-glucose oxidase-peroxidase-ferrocene composite biosensor under flow injection conditions is reported. Using Escherichia coli as the model bacterium, the composition of the growing medium was optimized. A constant glucose concentration of 4.0 x 10(-4) M was added to the culture medium. The relative response to glucose, expressed as the ratio between the amperometric signal and the signal at incubation time t = 0 multiplied by 100, as a function of E. coli concentration, showed a typical behaviour. Limits of detection of 6.5 x 10(2) or 6.5 cfu mL(-1) were achieved after 3 or 7 h of incubation, respectively, with no pre-concentration step. The detection of bacteria did not affect the lifetime of the biosensor. The feasibility of the detection of Staphylococcus aureus and Salmonella choleraesuis throughout the glucose consumption measurement at the composite biosensor is also demonstrated. The capability of bacterial identification by evaluation of bacterial growth in the culture medium containing the antibiotics polymyxin B, vancomycin, erythromycin, bacitracin, chloramphenicol, tetracycline and ciprofloxacin, was investigated. Each micro-organism tested exhibited a different antibiotic susceptibility profile, thus suggesting the possibility of bacteria differentiation. A rapid methodology for screening of bacteria is proposed.     

Escherichia coli O157:H7 detection limit of millimeter-sized PZT cantilever sensors is 700 cells/mL.

A composite self-excited millimeter-sized lead zirconate titanate (PZT) glass cantilever (2 mm x 1.8 mm; sensing area of 6 mm2) was fabricated for the detection of Escherichia coli (E. coli) O157:H7. The fundamental and second mode resonance in air was 10.95 +/- 0.05 kHz and 43.45 +/- 0.05 kHz, respectively. Affinity purified monoclonal antibody (anti-E. coli O157:H7) specific to the pathogen E. coli O157:H7 was immobilized at the cantilever glass tip, and then immersed in liquid containing the pathogen (70 to 7 x 10(7) cells/mL). The resonant frequency showed a reduction and reached a steady state shift of 0 +/- 5, 46 +/- 5, 260 +/- 5, and 1010 +/- 5 Hz corresponding to 0, 700, 7000, and 7 x 10(7) cells/mL. From the experiments conducted, the detection limit of the sensor was 700 cells/mL.     

Reveal E. coli 2.0 method for detection of Escherichia coli O157:H7 in raw beef

Reveal E. coli 2.0 is a new lateral-flow immunodiagnostic test for detection of E. coli O157:H7 and O157:NM in raw beef trim and ground beef. Compared with the original Reveal E. coli O157:H7 assay, the new test utilizes a unique antibody combination resulting in improved test specificity. The device architecture and test procedure have also been modified, and a single enrichment protocol was developed which allows the test to be performed at any point during an enrichment period of 12 to 20 h. Results of inclusivity and exclusivity testing showed that the test is specific for E. coli serotypes O157:H7 and O157:NM, with the exception of two strains of O157:H38 and one strain of O157:H43 which produced positive reactions. In internal and independent laboratory trials comparing the Reveal 2.0 method to the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of E. coli O157:H7 in 65 and 375 g raw beef trim and ground beef samples, there were no statistically significant differences in method performance with the exception of a single internal trial with 375 g ground beef samples in which the Reveal method produced significantly more positive results. There were no unconfirmed positive results by the Reveal assay, for specificity of 100%. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device incubation time or temperature. However, addition of the promoter reagent to the test sample prior to introducing the test device is essential to proper test performance.     

Label-free capacitive immunosensor based on quartz crystal Au electrode for rapid and sensitive detection of Escherichia coli O157:H7

A label-free capacitive immunosensor based on quartz crystal Au electrode was developed for rapid and sensitive detection of Escherichia coli O157:H7. The immunosensor was fabricated by immobilizing affinity-purified anti-E. coli O157:H7 antibodies onto self-assembled monolayers (SAMs) of 3-mercaptopropionic acid (MPA) on the surface of a quartz crystal Au electrode. Bacteria suspended in solution became attached to the immobilized antibodies when the immunosensor was tested in liquid samples. The change in capacitance caused by the bacteria was directly measured by an electrochemical detector. An equivalent circuit was introduced to simulate the capacitive immunosensor. The immunosensor was evaluated for E. coli O157:H7 detection in pure culture and inoculated food samples. The experimental results indicated that the capacitance change was linearly correlated with the cell concentration of E. coli O157:H7. The immunosensor was able to discriminate between cellular concentrations of 10²–10⁵ cfu mL⁻¹ and has applications in detecting pathogens in food samples. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were also employed to characterize the stepwise assembly of the immunosensor.     

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 996.09 [Visual Immunoprecipitate (VIP) for E. coli O157:H7] to the reference culture methods

The Visual Immunoprecipitate (VIP) for the Detection of E. coli O157:H7 in Foods, AOAC Official Method 996.09, has been modified to use a simplified plastic housing for the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Three foods were analyzed. In total, valid results were obtained from 240 samples and controls. Results showed that the VIP for E. coli O157:H7 is equivalent to the reference culture methods for the detection of E. coli O157:H7.     

Sensitivity and specificity of the test kit BAX for screening/E. coli O157:H7 in ground beef: independent laboratory study

An independent laboratory study of the BAX for Screening/E. coli O157:H7 kit was conducted at the National Food Laboratory, Inc., Dublin, CA, to complete AOAC Performance Tested Method certification. The BAX system kit was compared with the BAM culture method and a modified BAM culture method for detection of E. coli O157:H7 in ground beef. The BAX system kit detected the target organism at levels approximately 10-fold lower than those that gave positive BAM results. This study validated product claims, and Performance Tested Method status was granted.     

DNA detection on lateral flow test strips: enhanced signal sensitivity using LNA-conjugated gold nanoparticles

A lateral flow test strip assay, enabling sensitive detection of DNA specific to the foodborne pathogen E. coli O157:H7, is described. The use of LNA-conjugated gold nanoparticle probes, along with signal amplification protocols, results in minimum detectable concentrations of ~0.4 nM.     

Bioassay-guided purification and identification of antimicrobial components in Chinese green tea extract

The Chinese green tea extract was found to strongly inhibit the growth of major food-borne pathogens, Escherichia coli O157:H7, Salmonella Typhimurium DT104, Listeria monocytogenes, Staphylococcus aureus, and a diarrhoea food-poisoning pathogen Bacillus cereus, by 44-100% with the highest activity found against S. aureus and lowest against E. coli O157:H7. A bioassay-guided fractionation technique was used for identifying the principal active component. A simple and efficient reversed-phase high-speed counter-current chromatography (HSCCC) method was developed for the separation and purification of four bioactive polyphenol compounds, epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epicatechin (EC), and caffeine (CN). The structures of these polyphenols were confirmed with mass spectrometry. Among the four compounds, ECG and EGCG were the most active, particularly EGCG against S. aureus. EGCG had the lowest MIC90 values against S. aureus (MSSA) (58 mg/L) and its methicilin-resistant S. aureus (MRSA) (37 mg/L). Scanning electron microscopy (SEM) studies showed that these two compounds altered bacterial cell morphology, which might have resulted from disturbed cell division. This study demonstrated a direct link between the antimicrobial activity of tea and its specific polyphenolic compositions. The activity of tea polyphenols, particularly EGCG on antibiotics-resistant strains of S. aureus, suggests that these compounds are potential natural alternatives for the control of bovine mastitis and food poisoning caused by S. aureus.