RESUMO
Using the amino acid sequences and analysis of selected known structures of Bt Cry toxins, Cry1Ab, Cry1Ac, Cry1Ah, Cry1B, Cry1C and Cry1F we specifically designed immunogens. After antibodies selection, broad-spectrum polyclonal antibodies (pAbs) and monoclonal antibody (namely 1A0-mAb) were obtained from rabbit and mouse, respectively. The produced pAbs displayed broad spectrum activity by recognizing Cry1 toxin, Cry2Aa, Cry2Ab and Cry3Aa with half maximal inhibitory concentration (IC50) values of 0.12-9.86 µg/mL. Similarly, 1A0-mAb showed broad spectrum activity, recognizing all of the above Cry protein (IC50 values of 4.66-20.46 µg/mL) with the exception of Cry2Aa. Using optimizations studies, 1A10-mAb was used as a capture antibody and pAbs as detection antibody. Double antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) were established for Cry1 toxin, Cry2Ab and Cry3Aa with the limit of detection (LOD) values of 2.36-36.37 ng/mL, respectively. The present DAS-ELISAs had good accuracy and precisions for the determination of Cry toxin spiked tap water, corn, rice, soybeans and soil samples. In conclusion, the present study has successfully obtained broad-spectrum pAbs and mAb. Furthermore, the generated pAbs- and mAb-based DAS-ELISAs protocol can potentially be used for the broad-spectrum monitoring of eight common subtypes of Bt Cry toxins residues in food and environmental samples.
Assuntos
Anticorpos Monoclonais , Toxinas de Bacillus thuringiensis , Endotoxinas , Ensaio de Imunoadsorção Enzimática , Proteínas Hemolisinas , Animais , Ensaio de Imunoadsorção Enzimática/métodos , Coelhos , Camundongos , Endotoxinas/análise , Endotoxinas/imunologia , Proteínas Hemolisinas/imunologia , Proteínas Hemolisinas/análise , Proteínas Hemolisinas/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/análise , Bacillus thuringiensis/química , Camundongos Endogâmicos BALB CRESUMO
α-hemolysin (Hla), a toxin secreted by Staphylococcus aureus (S. aureus), has been proved to be involved in the occurrence and aggravation of food poisoning. Hence, it is quite essential to establish its rapid detection methods to guarantee food safety. Sandwich ELISA based on nanobody is well known to be viable for toxins, but there is absence of nanobody against Hla, let alone a pair for it. Therefore, in this paper, we screened specific nanobodies by bio-panning and obtained the optimal nanobody pair for sandwich ELISA firstly. Then, RANbody, a novel nanobody owning both recognition and catalytic capability, is generated in a single step and at low cost through molecular recombination technology. Subsequently, sandwich ELISA was developed to detect Hla based on the nanobody and RANbody, that not only eliminated the use of secondary antibodies and animal-derived antibody, but also reduced detection time and cost, compared with traditional sandwich ELISA. Lastly, the performance has been evaluated, especially for specificity which showed no response to other hemolysins and a low limit of detection of 10 ng/mL. Besides, the proposed sandwich ELISA exhibits favorable feasibility and was successfully employed for the detection of Hla in milk and pork samples.
Assuntos
Ensaio de Imunoadsorção Enzimática , Proteínas Hemolisinas , Leite , Proteínas Hemolisinas/imunologia , Proteínas Hemolisinas/análise , Proteínas Hemolisinas/química , Ensaio de Imunoadsorção Enzimática/métodos , Animais , Leite/química , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Contaminação de Alimentos/análise , Toxinas Bacterianas/análise , Toxinas Bacterianas/imunologia , Suínos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/imunologia , Limite de Detecção , Análise de Alimentos/métodosRESUMO
Although Vibrio parahaemolyticus (V. parahaemolyticus) is a pathogen frequently found in seafood, there is a possibility of its presence in other foods, such as dairy products. The main virulence factors of V. parahaemolyticus are thermostable direct hemolysins (TDHs) which are lethal toxins, so it is necessary to establish qualitative and quantitative methods for determining TDHs. HPLC-ESI-TOF was employed to establish a method for identifying TDHs. The identification and quantification ions of TDHs were confirmed by HPLC-ESI-TOF. The method was developed for detecting TDHs in milk powder using HPLC-ESI-TOF in this paper, and limits of detection (were between 0.20 and 0.40 mg/kg, limits of quantitation were between 0.5 and 1.0 mg/kg and recoveries of all TDHs were between from 78% to 94% with relative standard deviation lower than 10%. This research will provide a reference for developing methods of HPLC-MS/MS to detect TDHs in food samples, which can provide a tool for the government to monitor TDHs contamination in foods.
Assuntos
Proteínas Hemolisinas , Limite de Detecção , Leite , Espectrometria de Massas por Ionização por Electrospray , Cromatografia Líquida de Alta Pressão/métodos , Leite/química , Leite/microbiologia , Animais , Espectrometria de Massas por Ionização por Electrospray/métodos , Proteínas Hemolisinas/análise , Proteínas Hemolisinas/química , Toxinas Bacterianas/análise , Toxinas Bacterianas/química , Reprodutibilidade dos Testes , Modelos Lineares , Espectrometria de Massas em Tandem/métodos , Pós/química , Contaminação de Alimentos/análise , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/isolamento & purificaçãoRESUMO
Thirteen previously undescribed iridoids (1-13), together with five known iridoids (14-18) were isolated from the roots and rhizomes of Valeriana jatamansi Jones. Their structures with absolute configurations were elucidated by analysis of MS, NMR, optical rotation and their experimental and calculated electronic circular dichroism spectra. All of the isolated compounds were tested for their protective effects against α-hemolysin-induced cell death in A549 cells. Compounds 14, 16 and 17 showed moderate protective effects, and compounds 15 and 18 showed weak protective effects.
Assuntos
Nardostachys , Valeriana , Rizoma , Valeriana/química , Proteínas Hemolisinas/análise , Estrutura Molecular , Iridoides/farmacologia , Iridoides/química , Raízes de Plantas/químicaRESUMO
Vibrio parahaemolyticus is a common foodborne pathogen in seafood, which often causes seafood borne bacterial gastroenteritis or food poisoning. Thermostable direct hemolysin (TDH) is considered to be one of the main virulence factors involved in this pathogen. The most clinical V. parahaemolyticus isolates produce TDH. Therefore, high sensitivity and specificity detection of TDH are of great significance for food safety and early diagnosis of diseases caused by V. parahaemolyticus. In this study, we developed a rapid, sensitive immunochromatographic test paper assay for the quantitative detection of TDH in seafood samples using time-resolved fluorescence techniques. First, we completed the preparation of fluorescent detection antibodies by coupling lanthanide fluorescent nanospheres with homemade high-affinity polyclonal antibodies based on the principle of the double-antibody sandwich. The lanthanide fluorescent nanospheres used in this study are characterized by a large stokes shift and a long fluorescence lifetime, which effectively reduces background noise and improves detection sensitivity. In addition, the method can be completed within 15 min for the detection of TDH, has a detection limit below 50 ng/mL and good linearity in the range of 50-5000 ng/mL. Moreover, it has good specificity and no cross-reactivity with Vibrio vulnificus hemolysin (VVH), Clostridium perfringens α toxin (CPA) or C. perfringens ε toxin (ETX). Finally, the sensitivity of this method was unchanged when the three simulated samples of Patinopecten yessoensis, Ruditapes philippinarum, and Scapharca broughtonii tested, indicating that the method is not affected by samples in a complex matrix. In conclusion, this study establishes a practical new method for on-site rapid detection of TDH, which is easy to operate, fast response, easy to carry and can be implemented under the field conditions without expensive equipment and professional person.
Assuntos
Bivalves , Vibrio parahaemolyticus , Animais , Humanos , Proteínas Hemolisinas/análise , Vibrio parahaemolyticus/genética , Reação em Cadeia da Polimerase/métodos , Bivalves/microbiologia , Proteínas de Bactérias/genéticaRESUMO
Perturbation of cell membranes by amyloid ß (Ab) peptide oligomers is one possible mechanism of cytotoxicity in Alzheimer's disease, but the structure of such Ab-membrane complexes is unknown. Here we examine the stability of several putative structures by implicit membrane and all-atom molecular dynamics simulations. The structures include (a) a variety of models proposed by other researchers in the past, (b) a heptameric ß barrel determined by grafting the Ab sequence onto α-hemolysin, (c) a similar structure with modified strand orientation and turn location based on an experimental ß-hairpin structure, (d) oligomers inserting C-terminal ß hairpins into one leaflet of the bilayer, (e) oligomers forming parallel C-terminal ß barrels, and (f) a helical hexamer made of C-terminal fragments. The α-hemolysin-grafted structure and its alternately oriented variant are stable in the membrane and form an aqueous pore. In contrast, the C-terminal parallel barrels are not stable, presumably due to excessive hydrophobicity of their inner surface. The helical hexamer also failed to stabilize an aqueous pore for the same reason. The C-terminal hairpin-inserting structures remain stably inserted but, again, do not form an aqueous pore. Our results suggest that only ß-barrels inserting a combination of C-terminal and other residues can form stable aqueous pores.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Peptídeos beta-Amiloides/metabolismo , Proteínas Hemolisinas/análise , Membrana Celular/metabolismo , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Amiloide/análiseRESUMO
Cadmium (Cd) pollution poses a serious threat to agricultural production and paddy field fauna. Crystalline proteins (e.g., Cry1Ab and Cry1Ac) are secreted by Bacillus thuringiensis, which can manage pests via a complicated toxic mechanism and have been widely used for pest control due to the commercialization of transgenic crops (e.g., cotton and rice) that expresses Bt insecticidal proteins. Nonetheless, studies on the effects of combined stress of Cd and Cry1Ab protein on field indicator species are limited. In the present study, we showed that spiders, Pirata subpiraticus, fed with Cd-containing flies+Cry1Ab had dramatically higher Cd accumulation than that in the spiders fed with Cd-containing flies (p < 0.05). In addition, the enrichment of Cd led to the activation of the protective mechanism by elevating the concentrations of glutathione peroxidase, glutathione S-transferase, and metallothionein in the spiders (p < 0.05). An in-depth transcriptome analysis revealed that the activities of ion metal binding proteins, transporters, and channels might play essential roles in the Cd accumulation process. More importantly, the higher Cd concentration in the combined Cd+Cry1Ab exposure prolonged developmental duration of P. subpiraticus, due to the down-regulated cuticle proteins (CPs) encoding genes involved in the molting process, which was regulated by a series of putative transcriptional factors such as ZBTB and zf-C2H2. Collectively, this integrated analysis illustrates that the combined Cd+Cry1Ab exposure increases the adverse effects of Cd stress on the growth, antioxidase, and CPs encoding genes of P. subpiraticus, thus providing a research basis and prospect for the rationality of transgenic Cry1Ab crops in the cultivation of heavy metal contaminated soil.
Assuntos
Bacillus thuringiensis , Aranhas , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Cádmio/metabolismo , Cádmio/toxicidade , Produtos Agrícolas/metabolismo , Endotoxinas/análise , Endotoxinas/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas/análise , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Plantas Geneticamente Modificadas/metabolismo , Aranhas/metabolismo , TranscriptomaRESUMO
Protein detection is a universal tool critical to many applications in medicine, agriculture, and biotechnology. We developed a novel protein detection method combining light transmission spectroscopy and particle-size analysis of gold nanospheres monovalently functionalized with polyclonal antibodies and applied it to an emerging challenge for such technologiesâthe monitoring of environmental proteins (eProteins) present in natural aquatic systems. These are an underreported source of pollution and include the pseudopersistent Cry toxins that enter aquatic ecosystems from surrounding genetically engineered crops. The assay is capable of detecting proteins in complex matrices, such as water samples collected in the field, making it a competitive assay for eProtein detection. It is sensitive, reaching 1.25 ng mL-1, and we demonstrate its application to the detection of Cry1Ab from subsurface tile-drain and streamwater samples from agricultural waterways. The assay can also be quickly adapted for other protein detection applications in the future.
Assuntos
Ouro , Nanopartículas Metálicas , Proteínas de Bactérias/genética , Ecossistema , Ouro/química , Proteínas Hemolisinas/análise , Nanopartículas Metálicas/química , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/metabolismo , Análise EspectralRESUMO
Cry protein residue and accumulation in soil are two important components of the environmental safety assessment for the plantation of transgenic Bt crops. Several Bt rice lines with good commercial prospects have been developed in China, but it is unclear whether Cry proteins will accumulate in soils after multiple years of Bt rice cultivation. We planted the transgenic Bt rice lines cry1Ab/1Ac Minghui 63 (Huahui No. 1) and cry2A Minghui 63 for 9 years in the same field. The Cry proteins in the rhizosphere soil were estimated with enzyme linked immunosorbent assay (ELISA) at tillering stage and on the 60th day after harvest in each year. The Cry protein residues during the seedling, flowering and ripening stages were estimated in the first year (2012) and the last year (2020) of the experiment. In 2012, the concentration of Cry1Ab/1Ac in the rhizosphere soil of Huahui No. 1 was 1.25, 1.77, 1.97, 1.71 and 0.30 ng·g-1 at the seedling, tillering, flowering, ripening stages and on the 60th day after harvest, respectively. In 2020, the corresponding values were 1.30, 1.69, 2.03, 1.77, and 0.43 ng·g-1. In 2012, the concentration of Cry2A in rhizosphere soil of line cry2A Minghui 63 was 0.91, 1.52, 1.53, 1.37, and 0.12 ng·g-1 at the seedling, tillering, flowering, ripening stages and on the 60th day after harvest, respectively. The corresponding values in 2020 were 0.95, 1.43, 1.61, 1.40, and 0.15 ng·g-1. Results of multi-way ANOVA showed that the effect of year was not significant, but the effects of rice variety and growth stage were significant. Our results indicated that Cry proteins could be detected in rhizosphere soil during the growth stages of Bt rice, but would be degraded by 60 d after harvest, and that the concentrations of Cry proteins in the soil would not accumulate across multiple planting years.
Assuntos
Toxinas de Bacillus thuringiensis/análise , Endotoxinas/análise , Proteínas Hemolisinas/análise , Oryza , Solo/química , Oryza/genética , Plantas Geneticamente Modificadas , RizosferaRESUMO
Adequate water supply is one of the public health issues among the population living in low-income settings. Vibriosis remain a significant health challenge drawing the attention of both healthcare planners and researchers in South West districts of Uganda. Intending to clamp down the disease cases in the safest water deprive locality, we investigated the virulent toxins as contaminants and epidemiologic potentials of Vibrio species recovered from surface waters in greater Bushenyi districts, Uganda. Surface water sources within 46 villages located in the study districts were obtained between June and October 2018. Standard microbiological and molecular methods were used to analyse samples. Our results showed that 981 presumptive isolates retrieved cell counts of 10-100 CFU/g, with, with (640) 65% confirmed as Vibrio genus using polymerase chain reaction, which is distributed as follows; V. vulnificus 46/640 (7.2%), V. fluvialis 30/594 (5.1), V. parahaemolyticus 21/564 (3.7), V. cholera 5/543 (0.9), V. alginolyticus 62/538 (11.5) and V. mimicus 20/476 (4.2). The virulence toxins observed were heat-stable enterotoxin (stn) 46 (82.10%), V. vulnificus virulence gene (vcgCPI) 40 (87.00%), extracellular haemolysin gene {vfh 21 (70.00)} and Heme utilization protein gene {hupO 5 (16.70)}. The cluster analysis depicts hupO (4.46% n = 112); vfh (18.75%, n = 112); vcgCPI and stn (35.71%, & 41.07%, n = 112). The principal component analysis revealed the toxins (hupO, vfh) were correlated with the isolate recovered from Bohole water (BW) source, while (vcgCPI, stn) toxins are correlated with natural raw water (NRW) and open springs (OS) water sources isolates. Such observation indicates that surface waters sources are highly contaminated with an odds ratio of 1.00, 95% CI (70.48-90.5), attributed risk of (aR = 64.29) and relative risk of (RR = 73.91). In addition, it also implies that the surface waters sources have > 1 risk of contamination with vfh and > six times of contamination with hupO (aR = 40, - 66). This is a call of utmost importance to the population, which depends on these water sources to undertake appropriate sanitation, personal hygienic practices and potential measures that ensure water quality.
Assuntos
Toxinas Bacterianas/análise , Toxinas Bacterianas/genética , Nascentes Naturais/microbiologia , Vibrioses/prevenção & controle , Vibrio/genética , Vibrio/patogenicidade , Fatores de Virulência/análise , Fatores de Virulência/genética , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Genes Bacterianos , Proteínas Hemolisinas/análise , Proteínas Hemolisinas/genética , Reação em Cadeia da Polimerase/métodos , Uganda/epidemiologia , Vibrio/classificação , Vibrioses/epidemiologia , Vibrioses/microbiologia , Virulência/genéticaRESUMO
Many strains of Staphylococcus aureus produce a variety of cytolysins that target many different cell types to both fight the immune system and acquire nutrients. This includes hemolysins which destroy erythrocytes and are well studied virulence factors. Traditionally, hemolysin activity is measured on blood agar plates due to the simplicity of the assay. While this is telling, it cannot encapsulate the full story because S. aureus is known to behave differently in broth and on agar. Furthermore, plate-based assays are primarily semiquantitative and often a more accurate determination of hemolytic potential is needed to discern differences between strains. Here, we describe a method to quantify hemolysin activity from broth or similarly grown cells.
Assuntos
Eritrócitos/fisiologia , Proteínas Hemolisinas/análise , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Proteínas Hemolisinas/metabolismo , Hemólise , Humanos , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/análise , Fatores de Virulência/metabolismoRESUMO
A colloidal gold immunochromatographic strip (ICS) for simultaneous detection of multiple transgenic proteins, including CP4 EPSPS, BT-Cry1Ab and BT-Cry1Ac, was developed in this study. The sensitivity of the strip to the target protein was 5 ng/mL for CP4 EPSPS, 100 ng/mL for BT-Cry1Ab and Cry1Ac, respectively. Parallel analysis for maize, soybean, sugar beet and cotton showed the strip could detect 1% of transgenic content in crops containing BT-Cry1Ab and Cry1Ac, and, at least, 0.1% of content in crops containing CP4 EPSPS. The detection results for seed samples indicated the multicomponent analysis ICS had good accuracy. The analysis could be completed within 10 min and had the advantages of being high-throughput, easy to operate and visual detection. This is the first report of semi-quantitative ICS for detecting three transgenic proteins simultaneously. The developed approach may provide insights into the development of ICS for analyzing simultaneously multiple components in genetically modified crops.
Assuntos
Proteínas de Bactérias/análise , Produtos Agrícolas/genética , Endotoxinas/análise , Proteínas Hemolisinas/análise , Imunoensaio/instrumentação , Plantas Geneticamente Modificadas , Animais , Toxinas de Bacillus thuringiensis , Coloide de Ouro/química , Fitas Reagentes , Fatores de TempoRESUMO
The purpose of this study was to culture and characterise bacteria from an intact abscess on the skin of a dead Bryde's whale (Balaenoptera edeni) which stranded in the northern Beibu Gulf, China. To grow bacteria, samples from the abscess were added to blood agar. After incubation, yellowish mucous colonies were visualized. The bacterium was firstly recognised as Shewanella algae by the VITEK® 2 System. However, by using 16S rRNA gene sequencing the bacterium was finally identified as S. indica. To characterise the bacterium, antibiotic susceptibility and virulence factors, such as hemolysis and biofilm formation were investigated. The bacterium is capable of ß-hemolysis and biofilm formation and it is also sensitive to several different classes of antibiotics, such as ß-lactams, quinolones, and aminoglycosides. To date there have been no reports of this bacterium causing infections in humans or animals. However, in this study we described the first case of S. indica isolated from an intact abscess on the back of a Bryde's whale.
Assuntos
Balaenoptera/microbiologia , Filogenia , Shewanella/classificação , Animais , Proteínas de Bactérias/análise , Técnicas de Tipagem Bacteriana , Biofilmes/crescimento & desenvolvimento , China , DNA Bacteriano/genética , Proteínas Hemolisinas/análise , Testes de Sensibilidade Microbiana , RNA Ribossômico 16S/genética , Shewanella/isolamento & purificaçãoRESUMO
The insecticidal crystal proteins of Cry2A family from Bacillus thuringiensis (Bt) are important candidate proteins expressed in gene pyramiding Bt crops. A transgenic rice line (T2A-1) harboring a synthetic Cry2A* (Cry2Aa) gene showed effective resistance to some lepidopteran rice pests. As a generalist predator in rice ecosystems, the rove beetle (Paederus fuscipes) can prey on many rice insect pests such as planthoppers. Considering the possible exposure of Cry2Aa to P. fuscipes through tritrophic food chain, it is necessary to assess the potential risks of T2A-1 rice to this predator. In this study, a tritrophic experiment was conducted to assess the prey-mediated effects of Cry2Aa on P. fuscipes through the T2A-1 rice-Nilaparvata lugens-P. fuscipes food chain. After preying on N. lugens nymphs reared on T2A-1, no accumulated Cry2Aa could be detected in P. fuscipes adults, despite Cry2Aa being detected in N. lugens. In addition, no harmful effects were detected on the life table parameters of P. fuscipes in this tritrophic chain. Additionally, direct exposure to a high dose of purified Cry2Aa protein, representing the worst case scenario, showed no significant adverse effects on the development of P. fuscipes. These results showed that transgenic Cry2Aa rice had no harmful effects on P. fuscipes.
Assuntos
Proteínas de Bactérias/genética , Besouros , Endotoxinas/genética , Proteínas Hemolisinas/genética , Oryza/genética , Plantas Geneticamente Modificadas/efeitos adversos , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/análise , Besouros/química , Produtos Agrícolas/genética , Endotoxinas/análise , Cadeia Alimentar , Hemípteros/química , Hemípteros/crescimento & desenvolvimento , Proteínas Hemolisinas/análise , Ninfa/químicaRESUMO
In this work, comparative studies on DNA-PNA and polyarginine-conjugated DNA-PNA duplexes unzipping inside the α-hemolysin nanopore (α-HL) are presented. We identified significant differences in the blockade currents, as the applied voltage across the nanopore facilitated the duplex capture inside the nanopore's vestibule against the constriction region, subsequent cDNA strand insertion inside the nanopore's ß-barrel past the constriction site, its complete unzip from the duplex, and translocation. We observed that inside the voltage-biased nanopore, polyarginine-conjugated DNA-PNA duplexes dehybridize faster than their DNA-PNA counterparts and proposed a model to describe the duplex unzipping. This study identifies key particularities of DNA-PNA duplex unzipping as it takes place inside the nanopore and being preceded by entrapment in the vestibule domain of the α-HL. Our results are a crucial step toward understanding the nucleic acids duplexes unzipping kinetics variability, in confined, variable geometries.
Assuntos
DNA/química , Proteínas Hemolisinas/análise , Nanoporos , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , CinéticaRESUMO
In Brazil and in other countries of the world, studies have been conducted to identify Listeria monocytogenes in cattle meat that is preferably consumed undercooked and, when marketed without meeting strict phytosanitary requirements, may cause outbreaks of listeriosis. In the such, foodborne outbreaks, the methods used for the detection of the pathogen and the efficiency associated with them are crucial for the proper assessment. In this study, we used the techniques biochemical and molecular for identification of the L. monocytogenes isolated from 30 samples of the fresh beef, marketed in ten butchers' shop of the free-fair from a municipality from the Bahia, Brazil. The results obtained from biochemical tests (catalase, motility, ß-hemolysis and carbohydrate fermentation), as well as PCR analysis for the hly gene (hemolysin production is an important factor in the pathogenesis of listeriosis) revealed that 50% of butchers shops presented bovine meat contaminated with bacteria of the Listeria sp. and confirmed that 54.16% of the analyzed meat samples were positive for L. monocytogenes. This study highlights the importance of microbiological surveillance in free-fair to minimize the exposure of consumers to this foodborne pathogen.
Assuntos
DNA Bacteriano/análise , Proteínas Hemolisinas/genética , Listeria monocytogenes/isolamento & purificação , Produtos da Carne/microbiologia , Animais , Brasil , Bovinos , Microbiologia de Alimentos , Proteínas Hemolisinas/análise , Listeria monocytogenes/genéticaRESUMO
The wide cultivation of genetically modified (GM) insect-resistant crops has raised concerns on the risks to the eco-environment resulting from a release of Cry proteins. Therefore, it is vital to develop a method for the quantification of GM crops. Herein, A highly sensitive immunosensing platform has been developed for both colorimetric and chemiluminescent (CL) detection of Cry 1Ab using dual-functionalized gold nanoparticles (AuNPs) as signal amplification nanoprobes for the first time. In this work, anti-Cry 1Ab monoclonal antibody and horseradish peroxidase (HRP) are simultaneously functionalized on the surface of AuNPs with an exceptionally simple synthesis method. Combined with immunomagnetic separation, this immunosensing platform based on colorimetric method could detect Cry 1Ab in one step in a linear range from 1.0 to 40 ng mL-1 within 1.5 h, with a limit of detection of 0.50 ng mL-1. The sensitivity of fabricated nanoprobes was 15.3 times higher than that using commercial HRP-conjugated antibody. Meanwhile, the fabricated nanoprobes coupled with CL detection was successfully applied for Cry 1Ab detection with a minimum detection concentration of 0.050 ng mL-1 within a linear range of 0.10-20 ng mL-1. The proposed approach was validated with genuine GM crops, and the results showed a good correlation coefficient of 0.9906 compared to those of a commercial ELISA kit. Compared with ELISA, the developed immunosensing platform significantly simplified the assay procedure and shortened the analytical time, thus providing a new platform for the detection of genetically modified crops with high sensitivity, rapidity and simplicity.
Assuntos
Colorimetria/métodos , Produtos Agrícolas/química , Imunoensaio/métodos , Medições Luminescentes/métodos , Plantas Geneticamente Modificadas/química , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/análise , Produtos Agrícolas/genética , Endotoxinas/análise , Ouro/química , Proteínas Hemolisinas/análise , Limite de Detecção , Nanopartículas Metálicas/química , Plantas Geneticamente Modificadas/genéticaRESUMO
Fluorescent polymer dots (PDs) with maximum excitation/emission wavelengths of 410/515 nm were prepared in water solution from 1,4-benzoquinone and ethylenediamine. The green fluorescence of these PDs is screened off by the red-colored oxidation product (PPDox, maximum absorption at 510 nm) formed by horseradish peroxidase (HRP)-catalyzed oxidation of p-phenylenediamine (PPD). It causes the reduction of the fluorescence intensity of the PDs due to spectral overlap and an inner filter effect (IFE). If glucose is enzymatically oxidized under the formation of H2O2, the formed H2O2 can be quantified by the above IFE. The assay for HRP activity and glucose have detection limits of 0.2 U·L-1 and 0.1 µM, respectively. The nanoprobe was further extended to an immunosorbent assay (ELISA) for the determination of insecticidal Cry1Ab/Ac protein with a detection limit of 0.25 ng·mL-1. The ELISA was applied to rice leaf analysis. Graphic abstract Schematic representation of fluorometrict enzyme-linked immunosorbent assay for Cry1Ab/Ac protein detection based on horseradish peroxidase (HRP)-triggered fluorescence quenching of polymer dots (PDs). Quenching is caused by an inner filter effect (IFE) caused by PPDox, the oxidation product of p-phenylenediamine (PPD).
Assuntos
Proteínas de Bactérias/análise , Endotoxinas/análise , Glucose/análise , Proteínas Hemolisinas/análise , Peroxidase do Rábano Silvestre/análise , Polímeros/química , Pontos Quânticos/química , Anticorpos Imobilizados/imunologia , Armoracia/enzimologia , Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/imunologia , Endotoxinas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Glucose/química , Glucose Oxidase/química , Proteínas Hemolisinas/imunologia , Peróxido de Hidrogênio/química , Limite de Detecção , Oryza/química , Fenilenodiaminas/química , Plantas Geneticamente Modificadas/química , Espectrometria de Fluorescência/métodosRESUMO
Membrane biosensors that can rapidly sense pathogen interaction and disrupting agents are needed to identify and screen new drugs to combat antibiotic resistance. Bioelectronic devices have the capability to read out both ionic and electrical signals, but their compatibility with biological membranes is somewhat limited. Supported lipid bilayers (SLBs) have served as useful biomimetics for a myriad of research topics involving biological membranes. However, SLBs are traditionally made on inert, rigid, inorganic surfaces. Here, we demonstrate a versatile and facile method for generating SLBs on a conducting polymer device using a solvent-assisted lipid bilayer (SALB) technique. We use this bioelectronic device to form both mammalian and bacterial membrane mimetics to sense the membrane interactions with a bacterial toxin (α-hemolysin) and an antibiotic compound (polymyxin B), respectively. Our results show that we can form high quality bilayers of both types and sense these particular interactions with them, discriminating between pore formation, in the case of α-hemolysin, and disruption of the bilayer, in the case of polymyxin B. The SALB formation method is compatible with many membrane compositions that will not form via common vesicle fusion methods and works well in microfluidic devices. This, combined with the massive parallelization possible for the fabrication of electronic devices, can lead to miniaturized multiplexed devices for rapid data acquisition necessary to identify antibiotic targets that specifically disrupt bacterial, but not mammalian membranes, or identify bacterial toxins that strongly interact with mammalian membranes.
Assuntos
Biomimética/métodos , Técnicas Biossensoriais/métodos , Bicamadas Lipídicas/química , Biomimética/instrumentação , Técnicas Biossensoriais/instrumentação , Membrana Celular/química , Proteínas Hemolisinas/análise , Polímeros/química , Polimixina B/análiseRESUMO
The ability to produce enterohemolysin is regarded as a potential virulence factor for enterohemorrhagic Escherichia coli (EHEC) and is frequently associated with severe human diseases such as hemorrhagic colitis (HC) and the hemolytic uremic syndrome (HUS). The responsible toxin, which has also been termed EHEC-hemolysin (EHEC-Hly, syn. Ehx), belongs to the Repeats in Toxin (RTX)-family of pore-forming cytolysins and is characterized by the formation of incomplete turbid lysis zones on blood agar plates containing defibrinated sheep erythrocytes. Besides the expression of Shiga toxins (Stx) and the locus of enterocyte effacement (LEE), EHEC-Hly is a commonly used marker for the detection of potential pathogenic E. coli strains, although its exact role in pathogenesis is not completely understood. Based on the current knowledge of EHEC-Hly, this review describes the influence of various regulator proteins, explains the different mechanisms leading to damage of target cells, discusses the diagnostic role, and gives an insight of the prevalence and genetic evolution of the toxin.