Separation and detection of multiple pathogens in a food matrix by magnetic SERS nanoprobes

A rapid and sensitive method was developed here for separation and detection of multiple pathogens in food matrix by magnetic surface-enhanced Raman scattering (SERS) nanoprobes. Silica-coated magnetic probes (MNPs@SiO₂) of ∼100 nm in diameter were first prepared via the reverse microemulsion method using cetyltrimethylammonium bromide as a surfactant and tetraethyl orthosilicate as the silica precursor. The as-prepared MNPs@SiO₂ were functionalized with specific pathogen antibodies to first capture threat agents directly from a food matrix followed by detection using an optical approach enabled by SERS. In this scheme, pathogens were first immuno-magnetically captured with MNPs@SiO₂, and pathogen-specific SERS probes (gold nanoparticles integrated with a Raman reporter) were functionalized with corresponding antibodies to allow the formation of a sandwich assay to complete the sensor module for the detection of multiple pathogens in selected food matrices, just changing the kinds of Raman reporters on SERS probes. Here, up to two key pathogens, Salmonella enterica serovar Typhimurium and Staphylococcus aureus, were selected as a model to illustrate the probability of this scheme for multiple pathogens detection. The lowest cell concentration detected in spinach solution was 10³ CFU/mL. A blind test conducted in peanut butter validated the limit of detection as 10³ CFU/mL with high specificity, demonstrating the potential of this approach in complex matrices.     

Microarray of DNA–protein complexes on poly-3-hydroxybutyrate surface for pathogen detection

A novel strategy was developed for the specific immobilization of DNA probes on poly-3-hydroxybutyrate (PHB) surface by using the substrate-binding domain (SBD) of PHB depolymerase as an active binding motif. To demonstrate whether this method can be used for the detection of clinical pathogens, the pathogen-specific biotin-labeled DNA probes were immobilized via core streptavidin (cSA) fused to the SBD. The pathogen-specific 15-mer oligonucleotide probes were designed for four model pathogens, while the target DNAs were prepared by PCR using universal primers. The complex of pathogen-specific probes and cSA-SBD fusion protein was immobilized on the PHB-coated slide by microspotting. This DNA–protein complex microarray was able to successfully diagnose Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Furthermore, the specific pathogens could be diagnosed in the presence of other microorganisms. Thus, the DNA–protein complex microarray platform technology employing PHB and the SBD reported here can be widely used for the detection of DNA–DNA and DNA–biomolecule interactions without synthetic or chemical modification of biomolecules or solid surface.     

Development of a LIBS assay for the detection of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium from food

Laser-induced breakdown spectroscopy (LIBS) is used for the identification of the presence of hazardous bacteria in food. In this study, our main focus was centered on the identification of S. enterica serovar Typhimurium, a Gram-negative foodborne pathogen, in various liquids such as milk, chicken broth, and brain heart infusion due to the infection being most prevalent in raw meat and dairy products. A Nd:YAG laser of operating wavelength 266 nm was used to obtain the spectra from the artificially inoculated liquid samples. A series of experiments were performed to determine the effectiveness of LIBS to discriminate the bacteria from the background liquids. These results are compared with competing modern molecular methods of detection which include polymerase chain reaction (PCR) and quantitative real-time PCR. In addition to analyzing S. enterica serovar Typhimurium, another common Gram-negative, Escherichia coli, as well as Gram-positive pathogen, Staphlycoccus auerus, were used to determine the specificity of the LIBS technique.     

Label‐Free and Ultra‐Low Level Detection of Salmonella enterica Serovar Typhimurium Using Electrochemical Impedance Spectroscopy

An immunosensor for rapid and low level detection of the bacterial pathogen Salmonella enterica Serovar Typhimurium was designed and developed based upon label‐free electrochemical impedance spectroscopy and correlated to viable cell counts. The immunosensor was fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode by immobilizing monoclonal antibody (MAb) specific against Salmonella typhimurium cell surface lipopolysaccharide (LPS) onto the surface of the electrode. Use of mass‐fabricated and electroplated PCB electrodes allowed for disposable, highly sensitive, and rapid detection of Salmonella in an aqueous environment. Results demonstrate that in purified solution, Salmonella can be detected as low as 10 CFU in a 100 μL volume and label‐free and rapid manner in fewer than 90 s. The cost effective approach described here can be used for detection of pathogens with relevance for healthcare, food, and environmental applications.     

An antibody microarray,in multiwell plate format,for multiplex screening of foodborne pathogenic bacteria and biomolecules

Intoxication and infection caused by foodborne pathogens are important problems worldwide, and screening tests for multiple pathogens are needed because foods may be contaminated with multiple pathogens and/or toxic metabolites. We developed a 96-well microplate, multiplex antibody microarray method to simultaneously capture and detect Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium (S. typhimurium), as well as a biomolecule (chicken immunoglobulin G or IgG employed as a proteinaceous toxin analog) in a single sample. Microarrayed spots of capture antibodies against the targeted analytes were printed within individual wells of streptavidin-coated polystyrene 96-multiwell microtiter plates and a sandwich assay with fluorescein- or Cy3-labeled reporter antibodies was used for detection. (Printing was achieved with a conventional microarray printing robot that was operated with custom-developed microplate arraying software.) Detection of the IgG was realized from ca. 5 to 25 ng/mL, and detection of E. coli O157:H7 and S. typhimurium was realized from ca. 10⁶ to 10⁹ and ca. 10⁷ to 10⁹ cells/mL, respectively. Multiplex detection of the two bacteria and the IgG in buffer and in culture-enriched ground beef filtrate was established with a total assay (including detection) time of ca. 2.5 h. Detection of S. typhimurium was largely unaffected by high concentrations of the other bacteria and IgG as well as the ground beef filtrate, whereas a small decrease in response was observed for E. coli O157:H7. The multiwell plate, multiplex antibody microarray platform developed here demonstrates a powerful approach for high-throughput screening of large numbers of food samples for multiple pathogens and toxins.     

Development of a novel approach for the production of dried genomic DNA for use as standards for qualitative PCR testing of food-borne pathogens

As part of a multi-centre European project, FOOD-PCR, the feasibility of a novel approach for production of dried bacterial DNA that could be used as certified reference materials (CRM) was assessed. Selected strains of Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157, Campylobacter jejuni and Yersinia enterocolitica were used to produce genomic DNA (gDNA). These preparations gave support to method development for qualitative polymerase chain reaction (PCR) detection methods for food-borne pathogens. Purified gDNA was transformed into stable and dry gDNA by using polypropylene vials as carrier and applying a vacuum-drying technique. The gDNA preparations were shown to be sufficiently stable under ambient transport conditions without cooling and proved to have long-term stability at 5°C of at least 22 months. The dried DNA was easily reconstituted by addition of distilled water then gentle shaking. These studies have shown that production of stable and dry bacterial gDNA material is feasible and could help satisfy the increasing need for certified reference DNA positive control samples in the field of PCR testing for detection and verification of food-borne microbial pathogens.

     

Polymerase Chain Reaction-based Detection of Total and Specific Vibrio Species

The polymerase chain reaction (PCR) technique is widely used for efficient detection of food-borne pathogens because of speed and specificity. However, PCR methods have focused mostly on species-specific detection. In the present work, we describe a PCR-based method for the simultaneous detection of all Vibrio species because lots of them are notorious food-borne human pathogens. We then combined this total detection method with specific detection of Vibrio cholerae pathogen. Using a degenerate primer set based on the sequence of the potassium uptake gene, trkA, we were able to successfully detect all Vibrio species. Specific detection of V. cholerae was also possible using primer sets based on putative flagellin sequence. Importantly, simultaneous total and species-specific Vibrio detection was possible using all two primer sets in a multiplexed PCR strategy. Thus, the PCR method we have developed is applicable to both simultaneous and two-step detection of total and specific Vibrio species.     

Development of a novel approach for the production of dried genomic DNA for use as standards for qualitative PCR testing of food-borne pathogens

As part of a multi-centre European project, FOOD-PCR, the feasibility of a novel approach for production of dried bacterial DNA that could be used as certified reference materials (CRM) was assessed. Selected strains of Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157, Campylobacter jejuni and Yersinia enterocolitica were used to produce genomic DNA (gDNA). These preparations gave support to method development for qualitative polymerase chain reaction (PCR) detection methods for food-borne pathogens. Purified gDNA was transformed into stable and dry gDNA by using polypropylene vials as carrier and applying a vacuum-drying technique. The gDNA preparations were shown to be sufficiently stable under ambient transport conditions without cooling and proved to have long-term stability at 5°C of at least 22 months. The dried DNA was easily reconstituted by addition of distilled water then gentle shaking. These studies have shown that production of stable and dry bacterial gDNA material is feasible and could help satisfy the increasing need for certified reference DNA positive control samples in the field of PCR testing for detection and verification of food-borne microbial pathogens.     

Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop-mediated isothermal amplification coupled to a new bioluminescent assay

Testing for bioluminescent pyrophosphate is a convenient method of DNA detection without complex equipments, but it is insufficiently sensitive and offers no particular time advantage over other rapid detection methods. The shortcomings of the traditional bioluminescent pyrophosphate method have been addressed by using 2-deoxyadenosine-5-(α-thio)-triphosphate (dATPαS) instead of dATP for LAMP, thus reducing the high background signal and generating a constant background value. In this study, LAMP coupled to a novel bioluminescent pyrophosphate assay was developed to detect E. coli O157:H7. The new method has a limit of detection of <10 copies/μL or 5 CFU/mL; its sensitivity is higher than that of the conventional LAMP assay. Moreover, a food-borne pathogen can be detected when a single DNA template is included in the LAMP assay, making it 100 times more sensitive than the traditional LAMP method. Three hundred food samples were tested with this assay and the accuracy of detection was verified with a culture method and MALDI Biotyper. The assay only took 90–120 min and detected <10 copies of the pathogen. This method had the advantages of rapidity, sensitivity, and simplicity, so it is very competitive for the rapid and highly sensitive detection of food-borne pathogens.     

Simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes with an array-based immunosorbent assay using universal protein G-liposomal nanovesicles

A novel universal reagent for immunoassays, protein G-liposomal nanovesicles has been developed and successfully used in an immunomagnetic bead sandwich assay for the detection of Escherichia coli O157:H7 [C.-S. Chen, A.J. Baeumner, R.A. Durst, Talanta 67 (2005) 205]. To demonstrate the universal capability of protein G-liposomal nanovesicles, this reagent was used to develop an array-based immunosorbent assay for the simultaneous detection of E. coli O157:H7, Salmonella, and Listeria monocytogenes. Both direct and competitive immunoassay formats were used to demonstrate the feasibility of detecting multiple analytes in a single test by using universal protein G-liposomal nanovesicles. Both pure and mixed cultures were examined in the direct immunoassay format. Results indicate that the limits of detection (LODs) of the direct assay for E. coli O157:H7, Salmonella enterica serovar Typhimurium and L. monocytogenes in pure cultures were approximately 100, 500 and 1.5 × 10⁴ CFU/ml, respectively. In mixed cultures, the LODs were approximately 3.1 × 10³, 7.8 × 10⁴, and 7.9 × 10⁵ CFU/ml. In the competitive assay format, the LODs for E. coli O157:H7, S. enterica serovar Typhimurium, and L. monocytogenes were approximately 1.5 × 10⁴, 5 × 10⁴, and 1.2 × 10⁵ CFU/ml for the pure cultures. These results showed that protein G-liposomal nanovesicles can be successfully used in a simultaneous immunoassay for several food-borne pathogens, thereby demonstrating that they are effective universal reagents for use in immunoassays.     

Enhancement of deoxyribonucleic acid microarray performance using post-hybridization signal amplification

Microarray performance depends upon the ability to screen samples against a vast array of probes with the appropriate sensitivity and selectivity. While these factors are significantly influenced by probe design, they are also subject to the particular detection methodology and reagents employed. Herein we describe the incorporation of super avidin-biotin system (SABS) and secondary enzymatic enhancement (SEE) as post-hybridization signal amplification techniques to improve the sensitivity of oligonucleotide microarrays. To these ends, we tested these methods on electrochemically interrogated arrays using both purified influenza A PCR products and randomly amplified genomic Francisella tularensis DNA as targets. While SABS treatment did not improve sensitivity for CombiMatrix ElectraSense(?) arrays using purified influenza A cDNA, chip sensitivity was improved 10-fold for randomly amplified targets. SEE improved performance to a greater degree and was able to lower the detection limits 10-fold for influenza A and 100-fold for F. tularensis DNA. These results indicate the promising capability of post-hybridization amplification techniques for enhancing microarray performance.     

Glutaraldehyde-modified electrode for nonlabeling voltammetric detection of <Emphasis Type="Italic">p16</Emphasis><Superscript><Emphasis Type="Italic">INK4A</Emphasis></Superscript> gene

A nonlabeling electrochemical detection method for analyzing the polymerase-chain-reaction-amplified sequence-specific p16 ^INK4A gene, in which the basis for the covalent immobilization of deoxyribonucleic acid (DNA) probe is described, has been developed. The self-assembly process was based on the covalent coupling of glutaraldehyde (GA) as an arm molecule onto an amino-functional surface. The p16 ^INK4A gene was used as the model target for the methylation detection of early cancer diagnosis. An amino-modified DNA probe was successfully assembled on the GA-coupling surface through the formation of Schiff base under potential control. The hybridization of amino-modified DNA probes with the target was investigated by means of electrochemical measurements, including cyclic voltammetry and square wave voltammetry. Furthermore, the functions of GA coupling for sequence-specific detection were compared with those obtained based on mercaptopropionic acid. Hybridization experiments indicated that the covalent coupling of GA was suitable for the immobilization of DNA probe and was sensitive to the electrochemical detection of single-base mismatches of label-free DNA targets in hybridization. Moreover, reported probe-modified surfaces exhibited excellent stability, and the hybridization reactions were found to be completely reversible and highly specific for recognition in subsequent hybridization processes. The strategy provided the potential for taking full advantage of existing modified electrode technologies and was verified in microarray technology, which could be applied as a useful and powerful tool in electrochemical biosensor and microarray technology.     

Proteome-Based Serotyping of the Food-Borne Pathogens Salmonella Enterica by Label-Free Mass Spectrometry

Food-borne diseases caused by Salmonella enterica of 2500 serovars represent a serious public health problem worldwide. A quick identification for the pathogen serovars is critical for controlling food pollution and disease spreading. Here, we applied a mass spectrum-based proteomic profiling for identifying five epidemiologically important Salmonella enterica subsp. enterica serovars (Enteritidis, Typhimurium, London, Rissen and Derby) in China. By label-free analysis, the 53 most variable serovar-related peptides, which were almost all enzymes related to nucleoside phosphate and energy metabolism, were screened as potential peptide biomarkers, and based on which a C5.0 predicted model for Salmonella enterica serotyping with four predictor peptides was generated with the accuracy of 94.12%. In comparison to the classic gene patterns by PFGE analysis, the high-throughput proteomic fingerprints were also effective to determine the genotypic similarity among Salmonella enteric isolates according to each strain of proteome profiling, which is indicative of the potential breakout of food contamination. Generally, the proteomic dissection on Salmonella enteric serovars provides a novel insight and real-time monitoring of food-borne pathogens.     

Multiplex identification of sepsis‐causing Gram‐negative pathogens from the plasma of infected blood

Early and accurate detection of bacterial pathogens in the blood is the most crucial step for sepsis management. Gram‐negative bacteria are the most common organisms causing severe sepsis and responsible for high morbidity and mortality. We aimed to develop a method for rapid multiplex identification of clinically important Gram‐negative pathogens and also validated whether our system can identify Gram‐negative pathogens with the cell‐free plasm DNA from infected blood. We designed five MLPA probe sets targeting the genes specific to major Gram‐negative pathogens (uidA and lacY for E. coli, ompA for A. baumannii, phoE for K. pneumoniae, and ecfX for P. aeruginosa) and one set targeting the CTX‐M group 1 to identify the ESBL producing Gram‐negative pathogens. All six target‐specific peaks were clearly separated without any non‐specific peaks in a multiplex reaction condition. The minimum detection limit was 100 fg of pathogen DNA. When we tested 28 Gram‐negative clinical isolates, all of them were successfully identified without any non‐specific peaks. To evaluate the clinical applicability, we tested seven blood samples from febrile patients. Three blood culture positive cases showed E. coli specific peaks, while no peak was detected in the other four culture negative samples. This technology can be useful for detection of major sepsis‐causing, drug‐resistant Gram‐negative pathogens and also the major ESBL producing Gram‐negatives from the blood of sepsis patients in a clinical setting. This system can help early initiation of effective antimicrobial treatment against Gram‐negative pathogens for sepsis patients, which is very crucial for better treatment outcomes.     

Detection of Mutations in RNA Polymerase Beta Subunit Gene Encoding Resistance to Rifampin in Mycobacterium tuberculosis by DNA Microarray

Abstract

Rapid and efficient diagnosis is essential in the management of drug‐resistant tuberculosis. A DNA microarray technique based on differential hybridization method was described in the present study for detecting mutations in the RNA polymerase beta subunit (rpoB) gene of Mycobacterium tuberculosis (M. tuberculosis) cultures and in clinical specimens. The mutations in rpoB confer resistance to rifampin, an important first‐line antituberculosis drug. The differential hybridization approach was mainly based on the effect of a single base mismatch on the melting temperature of the hybridized DNA; therefore, any point mutation of rpoB gene resulting in the rifampin resistance can be detected efficiently. The development of the DNA microarray involves the design of dozens of oligonucleotide probes for identifying rifampin‐resistant and ‐sensitive strains. The method comprises isolating genomic DNA from the samples containing M. tuberculosis cells, amplifying rpoB gene coding sequence to produce fluorescently labelled product, and hybridization with the oligonucleotide arrays. The results demonstrated the capability of DNA microarray to provide important clinically relevant information about the rpoB gene of mycobacterial organisms. The DNA microarray offers a reliable diagnostic test for rapidly detecting multidrug resistance caused by gene mutations of mycobacteria.     

Detection of Listeria Monocytogenes by Electrochemical Impedance Spectroscopy

Listeria monocytogenes is detected by electrochemical impedance spectroscopy using a mouse monoclonal antibody immobilized onto an Au electrode. This yields sensitivities of 0.825 kΩ cm²/(CFU/mL) and 1.129 kΩ cm²/(CFU/mL) and detection limits of 5 CFU/mL and 4 CFU/mL for ideal solutions and filtered tomato extract, respectively. Control experiments with an Au electrode onto which a mouse monoclonal antibody to GAPDH is immobilized demonstrate that non‐specific adsorption is insignificant for the system and methodology studied here. Control experiments with Salmonella enterica demonstrate no cross‐reactivity to this food pathogen. Potential technological hurdles to development of a multiplexed impedance biosensor for food pathogens are also discussed.     

Development and evaluation of a novel nucleic acid sequence-based amplification method using one specific primer and one degenerate primer for simultaneous detection of Salmonella Enteritidis and Salmonella Typhimurium

Salmonella Enteritidis and Salmonella Typhimurium are the most widespread causes of salmonellosis and gastrointestinal diseases worldwide. Thus, their simple and sensitive detection is significantly important in biosafety and point-of-care diagnostics. In that regard, although present nucleic acid-based attempts are mainly focused on the detection methods encompassing all Salmonella enterica members in a single reaction, serotypes other than S. Enteritidis and S. Typhimurium are clinically and epidemiologically rare to humans. Therefore, regarding high ribosomal RNA (rRNA) copy numbers in a cell, isothermal nucleic acid sequence-based amplification (NASBA) technique was employed for simple, sensitive and simultaneous detection of the bacteria. However, due to high sequence homology among 16S rRNA genes and consequently, very few specific regions, we developed a novel NASBA method called “single specific primer-NASBA or SSP-NASBA” in which the specificity of the antisense primer is sufficient to perform a specific NASBA reaction. Accordingly, we designed highly specific NASBA antisense and degenerate sense primers for a segment of 16S rRNA variable region by universal sequence alignment to simultaneously detect S. Enteritidis and S. Typhimurium. Meanwhile, the approach was successfully evaluated for various Salmonella as well as closely related non-Salmonella serovars. Specific and simultaneous detection of both bacteria was achieved with the designed primer set in a single reaction environment with a detection limit of less than 10 CFUs mL⁻¹. The developed NASBA assay should facilitate the overall process and provide a simple, fast, specific and sensitive approach for molecular diagnostics of pathogens under various circumstances, e.g. outbreaks.     

Development of ssDNA Aptamers for the Sensitive Detection of Salmonella typhimurium and Salmonella enteritidis

Salmonella enterica subsp. enterica ser. enteritidis and Salmonella enterica subsp. enterica ser. typhimurium are the most common and severe food-borne pathogens responsible for causing salmonellosis in humans and animals. The development of an early and ultra-sensitive detection system is the first critical step in controlling this disease. To accomplish this, we used the cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) technique to identify single-stranded DNA (ssDNA) aptamers to be used as detection probes that can specifically bind to S. enteritidis and S. typhimurium. A total of 12 target-specific ssDNA aptamers were obtained through ten rounds of Cell-SELEX under stringent selection conditions, and negative selection further enhanced the selectivity among these aptamers. Aptamer specificity was investigated using the gram-negative bacteria E. coli and P. aeruginosa and was found to be much higher towards S. enteritidis and S. typhimurium. Importantly, three candidate aptamers demonstrated higher binding affinities and the dissociation constants (Kd) were found to be in the range of nanomolar to submicromolar levels. Furthermore, individual aptamers were conjugated onto polyvalent directed aptamer polymer, which led to 100-fold increase in binding affinity compared to the individual aptamers alone. Taken together, this study reports the identification of higher affinity and specificity ssDNA aptamers (30mer), which may be useful as capture and detection probes in biosensor-based detection systems for salmonellosis.     

Fungal pathogenic nucleic acid detection achieved with a microfluidic microarray device

Detection of polymerase chain reaction (PCR) products obtained from cultured greenhouse fungal pathogens, Botrytis cinerea and Didymella bryoniae has been achieved using a previously developed microfluidic microarray assembly (MMA) device. The flexible probe construction and rapid DNA detection resulted from the use of centrifugal pumping in the steps of probe introduction and sample delivery, respectively. The line arrays of the oligonucleotide probes were “printed” on a CD-like glass chip using a polydimethylsiloxane (PDMS) polymer plate with radial microfluidic channels, and the sample hybridizations were conducted within the spiral channels on the second plate. The experimental conditions of probe immobilization and sample hybridization were optimized, and both complementary oligonucleotides and PCR products were tested. We were able to achieve adequate fluorescent signals with a sample load as small as 0.5 nM (1 μL) for oligonucleotide samples; for PCR products, we achieved detection at the level of 3 ng.     

蛋白质DNA混合微点阵和微流控芯片的整合

在活化的石英片上制作蛋白质和DNA微点阵, 并可逆地将其与含有通道的多聚二甲基硅氧烷弹性橡胶封接在一起, 使蛋白质和DNA微点阵组装在微通道列阵内; 实现在微通道列阵内同时检测和分析蛋白质与DNA的功能. 为了降低多聚二甲基硅氧烷弹性橡胶的疏水性, 增强其生物相容性, 实验通过多聚赖氨酸对多聚二甲基硅氧烷弹性橡胶的修饰, 提高了它的亲水性, 使溶液能够在微通道内顺畅地流通. 实验表明, 这种混合芯片能够提高检测速度和增加检测的信息量.