Chemiluminescent enzyme-linked immunosorbent assay on a strip to detect Escherichia coli O157:H7

A fast and sensitive chemiluminescent enzyme-linked immunosorbent assay method to measure pathogenic bacteria, Escherichia coli O157:H7, on immuno-chromatographic membrane was studied. Non-specific binding of proteins on membrane strip was controlled to attain the best performance of immunosensor by optimising the composition of a running buffer. The specificity of the proposed immunostrip was confirmed by conducting experiments for four different micro-organisms. A chemiluminescent signal could be successfully generated from a proposed immunostrip sensing system, and a significant change in the chemiluminescent light intensity with the concentration of target microbes was obtained. E. coli O157:H7 could be quantitatively measured in the range of 1.1?×?10^3?–1.1?×?10⁷ CFU (colony forming units) mL^?1 within 16?min by using the developed chemiluminescent immunostrip.     

大肠杆菌O157:H7微滴数字PCR定量方法的建立

以大肠杆菌O157：H7(E. coli O157：H7)rfbE基因为靶基因,建立了可对其准确定量的微滴数字PCR( ddPCR)方法。对ddPCR反应中的探针浓度进行了优化,考察了方法的线性范围、精密度、定量限和检出限。最终确定ddPCR 反应中的最佳探针浓度为300 nmol/L。 E. coli O157：H7基因组 DNA 浓度范围为4~1.25×105拷贝/20μL ddPCR反应液时,ddPCR方法线性相关系数( R2)为0.999。当DNA浓度为760~88400拷贝/20μL 时,方法的精密度最好( RSD<5%)。本方法的定量限为4拷贝/20μL,检出限为3拷贝/20μL。特异性验证结果表明,建立的ddPCR方法特异性良好,对13份猪肉、牛肉和鸡肉样品的检测结果与定量PCR方法检出结果一致。     

亲水作用色谱/质谱联用方法用于大肠杆菌代谢组分析

建立了两性离子亲水作用色谱/质谱联用方法用于大肠杆菌胞内极性代谢物的分离分析。选取52个代表性极性物质对方法进行考察,发现此方法有较好的线性范围,且大部分物质最低检测限均在ng/mL数量级。平行制备6份样品进行分析,结果显示85%以上代谢物峰面积的RSD值小于30%。6个内标物质在低、中、高3个浓度下的日内精密度（RSD）均小于20%,大部分物质的相对回收率都在可接受的范围内（70%~130%）。把此方法用于yfcC基因改造的3株大肠杆菌代谢组分析,发现一些小肽、氨基酸、核苷、有机酸、磷脂等物质在基因改造后发生明显变化。此研究结果表明,建立的两性离子亲水作用色谱/质谱方法检测到的物质化学性质分布广,跨越了极性磷脂到小肽的各个范围,且具有良好的重复性、稳定性和适用性。     

Rapid detection of Escherichia coli O157:H7 spiked into food matrices

Food poisoning causes untold discomfort to many people each year. One of the primary culprits in food poisoning is Escherichia coli O157:H7. While most cases cause intestinal discomfort, up to 7% of the incidences lead to a severe complication called hemolytic uremic syndrome which may be fatal. The traditional method for detection of E. coli O157:H7 in cases of food poisoning is to culture the food matrices and/or human stool. Additional performance-based antibody methods are also being used. The NRL array biosensor was developed to detect multiple antigens in multiple samples with little sample pretreatment in under 30 min. An assay for the specific detection of E. coli O157:H7 was developed, optimized and tested with a variety of spiked food matrices in this study. With no sample pre-enrichment, 5 × 10³ cells mL⁻¹ were detected in buffer in less than 30 min. Slight losses of sensitivity (1-5 × 10⁻⁴ cell mL⁻¹⁾ but not specificity occur in the presence of high levels of extraneous bacteria and in various food matrices (ground beef, turkey sausage, carcass wash, and apple juice). No significant difference was observed in the detection of E. coli O157:H7 in typical culture media (Luria Broth and Tryptic Soy Broth).     

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。     

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.     

Disposable Immunosensor for Escherichia Coli O157:H7 Based on a Multi-Walled Carbon Nanotube-Sodium Alginate Nanocomposite Film Modified Screen-Printed Carbon Electrode

A disposable immunosensor for the detection of Escherichia coli O157:H7 based on a multiwalled carbon nanotube–sodium alginate nanocomposite film was constructed. The nanocomposite was placed on a screen-printed carbon electrode, and horseradish peroxidase-labeled antibodies were immobilized to E. coli O157:H7 on the modified electrode to construct the immunosensor. The modification procedure was characterized by atomic force microscopy and cyclic voltammetry. Under optimal conditions, the proposed immunosensor exhibited good electrochemical sensitivity to E. coli O157:H7 in a concentration range of 10³–10¹⁰ cfu/mL, with a relatively low detection limit of 2.94 × 10² cfu/mL (S/N = 3). This immunosensor exhibited satisfactory specificity, reproducibility, stability, and accuracy, making it a potential alternative tool for early assessment of E. coli O157:H7.     

一种实用的基于化学发光和磁性纳米颗粒的E.coli O157:H7免疫鉴定方法

化学发光磁酶免疫已经被应用于检测病原体,但是由于针对相应病原体的抗体筛选和修饰等的步骤耗时费力,不适于对多种病原体进行筛查.制备了兔抗大肠杆菌(E.coli)O157:H7的免疫磁性纳米颗粒,富集病原菌后与鼠抗E.coli O157:H7的单克隆抗体形成双抗夹心,采用碱性磷酸酶标记的马抗鼠IgG与单抗结合,加入碱性磷酸酶的化学发光底物试剂3-(2'-螺旋金刚烷)-4-甲氧基-4-(3'-羟基)苯-1,2-二氧杂环丁烷磷酸检测化学发光.实验研究了底物缓冲液、碱性磷酸酶浓度对化学发光强度的影响,比较了NaBH4和甘氨酸对免疫磁珠剩余活性醛基的封闭效果以及本方法检测E.coli O157:H7的特异性和敏感性.结果表明,碱性磷酸酶与底物在c缓冲液中反应的化学发光强度最高,碱性磷酸酶浓度决定了化学发光的强度和持续时间,NaBH4对活性醛基的封闭效果优于甘氨酸,以D群宋内氏志贺氏菌、B群福氏志贺氏菌、鼠伤寒沙门氏菌、金黄色葡萄球菌和霍乱弧菌及E.coli Top10f'为对照的比较实验显示,该检测方法具有良好的特异性,以1mL的菌液为检测体积时对E.coli O157:H7的检测灵敏度为103cell/mL,整个方法的检测时间约为3h.该方法适用于对多样本进行筛查.     

Characterization of complex,heterogeneous lipid A samples using HPLC–MS/MS technique II. Structural elucidation of non‐phosphorylated lipid A by negative‐ion mode tandem mass spectrometry

Non‐phosphorylated lipid A species confer reduced inflammatory potential for the bacteria. Knowledge on their chemical structure and presence in bacterial pathogens may contribute to the understanding of bacterial resistance and activation of the host innate immune system. In this study, we report the fragmentation pathways of negatively charged, non‐phosphorylated lipid A species under low‐energy collision‐induced dissociation conditions of an electrospray ionization quadrupole time‐of‐flight instrument. Charge‐promoted consecutive and competitive eliminations of the acyl chains and cross‐ring cleavages of the sugar residues were observed. The A‐type fragment ion series and the complementary X‐type fragment(s) with corresponding deprotonated carboxamide(s) were diagnostic for the distribution of the primary and secondary acyl residues on the non‐reducing and the reducing ends, respectively, of the non‐phosphorylated lipid A backbone. Reversed‐phase liquid chromatography in combination with negative‐ion electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry could provide sufficient information on the primary and secondary acyl residues of a non‐phosphorylated lipid A. As a standard, the hexa‐acylated ion at m/z 1636 with the Escherichia coli‐type acyl distribution (from E. coli O111) was used. The method was tested and refined with the analysis of other non‐phosphorylated hexa‐ and several hepta‐, penta‐, and tetra‐acylated lipid A species detected in crude lipid A fractions from E. coli O111 and Proteus morganii O34 bacteria. Copyright © 2016 John Wiley & Sons, Ltd.     

Binding Characteristics Study of DNA based Aptamers for E. coli O157:H7

E. coli O157:H7 is a pathogenic bacterium producing verotoxins that could lead to serious complications such as hemolytic uremia syndrome. Fast detection of such pathogens is important. For rapid detection, aptamers are quickly gaining traction as alternative biorecognition molecules besides conventional antibodies. Several DNA aptamers have been selected for E. coli O157:H7. Nonetheless, there has not been a comparative study of the binding characteristics of these aptamers. In this work, we present a comprehensive analysis of binding characteristics including binding affinity (K_d) and binding capacity (B_max) of DNA-based aptamers for E. coli O157:H7 using qPCR. Our results show that aptamer E18R has the highest binding capacity to E. coli 157:H7 and the highest specificity over non-pathogenic E. coli strains K12 and DH5α. Our study also finds that the common biotin-tag modification at 5′ end typically changes the binding capacity significantly. For most of the selected aptamers, the binding capacity after a biotin-tag modification decreases. There exists a discrepancy in the binding capability between the selected aptamer and the aptamer used for detection. Our study also shows that a lower concentration of Mg²⁺ ions in the binding buffer leads to a decrease in the binding capacity of E17F and E18R, while it does not affect the binding capacity of S1 and EcoR1.     

化学发光磁酶免疫法检测O157:H7大肠埃希菌

通过自行制备的免疫磁珠结合新型发光底物(AMPPD), 改进了化学发光磁酶免疫检测法, 对人工猪肉样品中O157:H7大肠埃希菌进行了检测, 并与人工计数法进行了比较.     

Lipid A components from Pseudomonas aeruginosa PAO1 (serotype O5) and mutant strains investigated by electrospray ionization ion-trap mass spectrometry

The lipid A components of the Pseudomonas aeruginosa strains PAO1 (wild-type) and derived mutants PAO1 algC::tet and PAO1 PDO100 were isolated after mild acetic acid hydrolysis of LPS. Their structural heterogeneities were characterized using electrospray ionization (ESI) ion-trap mass spectrometry (MS) with direct infusion in the negative ion mode without prior derivatization. The ESI-mass spectra revealed monophosphorylated molecules corresponding to known tetra-, penta- and hexaacylated structures of P. aeruginosa lipid A. The MS/MS fragmentation patterns allowed the location of fatty acyl chains on the disaccharide backbone of lipid A. In addition, a hexaacylated lipid A containing a hexadecanoyl chain was detected for the first time in strain P. aeruginosa PAO1. With multiple stages of fragmentation (MS(n)), the position of this hexadecanoyl chain O-linked to the decanoyl chain at the C-3(') position of the glucosamine backbone was determined. This sensitive method is suitable to reveal lipid A heterogeneity, i.e. the nature, number and distribution of acyl chains, without prior lipopolysaccharide purification. The lipid A from mutant strains were also characterized and significant differences were shown in the abundance of monophosphorylated lipid A components between the wild-type and the mutant strains.     

Two-dimensional separation system of coupling capillary liquid chromatography to capillary electrophoresis for analysis of Escherichia coli metabolites

A two-dimensional (2-D) separation system of coupling chromatography to electrophoresis was developed for profiling Escherichia coli metabolites. Capillary liquid chromatography (LC) with a monolithic silica-octadecyl silica column (500 x 0.2 mm ID) was used as the first dimension, from which the effluent fractions were further analyzed by capillary electrophoresis (CE) acting as the second dimension. Field-enhanced stacking was selectively employed as a concentration strategy to interface the two dimensions, which proved to be beneficial for the detection of metabolites. An artificial sample containing 118 standards, some of which lack chromophores or have weak UV absorbance, was used to optimize the 2-D separation system. Under the optimum conditions, 63 components in the artificial sample having absorbance at 254 nm could be well resolved and detected. The utility of the system was demonstrated by comprehensive analysis of E. coli metabolites. Comparing with the previous 2-D separation system we published in Anal. Chem. 2004, 76, 1419-1428, using a longer monolithic column in the first dimension improved the separation efficiency and offered the possibility of increasing the injection volume without compromising the separation efficiency. In the second dimension, field-enhanced stacking was used to improve the concentration sensitivity of the metabolites, and more metabolites in E. coli cell extract were detected and identified using the developed 2-D separation system. In addition, preliminary investigation for future CE-mass spectrometry coupling was also made in the study by using volatile buffers in the capillary LC and CE techniques.     

Study on the photodegradation and microbiological degradation of pirimicarb insecticide by using liquid chromatography coupled with ion-trap mass spectrometry

The intermediates of photodegradation and microbial degradation of pirimicarb insecticide were investigated by liquid chromatography coupled with ion-trap mass spectrometry (LC–IT-MS). Different intermediates were detected in the photodegradation and microbial degradation of pirimicarb. In the photodegradation of pirimicarb in aqueous solution 2-dimethylamino-5,6-dimethyl-4-hydroxypyrimidine (MW = 167), 2-methylamino-5,6-dimethylpyrimidin-4-yl-dimethylcarbamate (MW = 224) and 2-formylamino-5,6-dimethylpyrimidin-4-yl-dimethylcarbamate (MW = 252) were the main products. It was found that 2-dimethylamino-5,6-dimethyl-4-hydroxypyrimidine (MW = 167) was the major product in the microbial degradation of pirimicarb in soil.     

Investigation of Symphytum cordatum alkaloids by liquid-liquid partitioning, thin-layer chromatography and liquid chromatography-ion-trap mass spectrometry

From the alkalised crude extract of Symphytum cordatum (L.) W.K. roots, pyrrolizidine alkaloids (PAs) were extracted as free tertiary bases and polar N-oxides in a merely one-step liquid-liquid partitioning (LLP) in separation funnel and subsequently pre-fractionated by preparative multiple-development (MD) thin-layer chromatography (TLC) on silica gel plates. In this way three alkaloid fractions of different polarities and retention on silica gel plates were obtained as: the most polar N-oxides of the highest retention, the tertiary bases of medium retention, and diesterified N-oxides of the lowest retention. The former fraction was reduced into free bases by sodium hydrosulfite and purified by LLP on Extrelut-NT3 cartridge. It was further analysed together with the two other fractions by high-performance liquid chromatography (HPLC)-ion-trap mass spectrometry with atmospheric pressure chemical ionization (APCI) interface on XTerra C₁₈ column using a gradient elution. Based on MSⁿ spectra, 18 various alkaloids have been tentatively determined for the first time in this plant as the following types of structure: echimidine-N-oxide (three diasteroisomers), 7-sarracinyl-9-viridiflorylretronecine (two diasteroisomers), echimidine (two diasteroisomers), lycopsamine (two diasteroisomers), dihydroechinatine-N-oxide, dihydroheliospathuline-N-oxide, lycopsamine-N-oxide (three diasteroisomers), 7-acetyllycopsamine-N-oxide, symphytine-N-oxide (two diasteroisomers) and 2″,3″-epoxyechiumine-N-oxide.     

Nickel species analysis of human colonic tissue using liquid chromatography,gel electrophoresis and mass spectrometry

Studies to specify metal-binding species, such as metalloproteins that are present in trace amounts in colonic cell cytosol, using chromatographic separation methods in combination with inductively coupled plasma mass spectrometry (ICP-MS) as element-specific detection require an optimised sample preparation regarding the solubilisation of the proteins. Focus should be taken to avoid metal contamination, enzymatic digestion by different proteases and oxidation. In this article different sample preparation methods are studied to find a suitable method for the isolation and characterisation of Ni species previously found in cytosols from normal and malignant tissues of the human colon. The total Ni concentrations of the cytosols were determined as well as the total protein content. Thus, a Ni-containing protein could be isolated from cytosols of malignant human colonic tissues using size-exclusion chromatography with ICP-MS for element-specific detection. Ni-containing species in the molecular mass range from 10,000 to 20,000 Da were found and pre-concentrated. The determination of the molecular mass of the species was performed through online coupling of reversed-phase chromatography with electrospray ionisation quadrupole time-of-flight MS. Using identical chromatographic conditions and ICP-MS the detected protein was shown to contain Ni.     

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.     

Combining microwave-assisted extraction and liquid chromatography–ion-trap mass spectrometry for the analysis of hexabromocyclododecane diastereoisomers in marine sediments

An efficient microwave-assisted extraction (MAE) procedure coupled with high performance liquid chromatography–electrospray-ion-trap mass spectrometry (HPLC–ESI-ITMS) has been evaluated to determine hexabromocyclododecane diastereoisomers (α-, β- and γ-HBCD) in marine sediments. The composition of the LC mobile phase (consisting of water, methanol and acetonitrile) and the parameters of electrospray ionization (ESI) were evaluated to obtain chromatographic baseline separation and high sensitivity for the detection of these diastereoisomers. The effects of various operating parameters on the quantitative extraction of the HBCDs through MAE were systematically investigated. The three diastereoisomers were then quantitated by HPLC–ITMS employing ESI operated in the negative ionization mode. The HBCDs were extracted from the sediments through MAE using 40 mL of acetone/n-hexane (1/3, v/v) at 90 °C for 12 min. The limits of quantitation (LOQ) ranged from 25 to 40 pg/g (dry weight) in 5 g of the sediment samples. The recoveries of the HBCDs in spiked sediment samples ranged from 68 to 91% (relative standard derivation: 2–11%). The extraction efficiency of the MAE technique was also compared with Soxhlet extraction and pressurized liquid extraction.     

Screening antiallergic components from Carthamus tinctorius using rat basophilic leukemia 2H3 cell membrane chromatography combined with high‐performance liquid chromatography and tandem mass spectrometry

Carthamus tinctorius, used in traditional Chinese medicine, has many pharmacological effects, such as anticoagulant effects, antioxidant effects, antiaging effects, regulation of gene expression, and antitumor effects. However, there is no report on the antiallergic effects of the components in C. tinctorius. In the present study, we investigated the antiallergic components of C. tinctorius and its mechanism of action. A rat basophilic leukemia 2H3/cell membrane chromatography coupled online with high‐performance liquid chromatography and tandem mass spectrometry method was developed to screen antiallergic components from C. tinctorius. The screening results showed that Hydroxysafflor yellow A, from C. tinctorius, was the targeted component that retained on the rat basophilic leukemia 2H3/cell membrane chromatography column. We measured the amount of β‐hexosaminidase and histamine released in mast cells and the key markers of degranulation. The release assays showed that Hydroxysafflor yellow A could attenuate the immunoglobulin E induced release of allergic cytokines without affecting cell viability from 1.0 to 50.0 μM. In conclusion, the established rat basophilic leukemia 2H3 cell membrane chromatography coupled with online high‐performance liquid chromatography and tandem mass spectrometry method successfully screened and identified Hydroxysafflor yellow A from C. tinctorius as a potential antiallergic component. Pharmacological analysis elucidated that Hydroxysafflor yellow A is an effective natural component for inhibiting immunoglobulin E–antigen‐mediated degranulation.