Detection Of Food Pathogens Research Articles

Pathogenic Escherichia coli biosensor detection on chicken food samples

Abstract Escherichia coli is an important pollution indicator and is the most important foodborne pathogens of public health concern. The chicken meat is one of the most important sources of good quality protein, and it is also susceptible to microbial contamination and often implicated in foodborne disease. In such context, the detection of E. coli K12 in frozen chicken meat was investigated by electrochemical impedance spectroscopy and surface plasmon resonance imaging techniques. The anti‐E. coli antibody was first immobilized onto gold surface by physisorption technique. The electrical and optical properties of the immobilized anti‐E. coli K12 antibody were studied. The binding of the E. coli K12 bacteria with the anti‐E. coli antibody layer was measured with a detection limit of 103 cfu/ml. This detection limit is better than those obtained with the enzyme‐linked immunosorbent assay (ELISA) technique. Moreover, the developed biosensor was used for E. coli K12 detection in inoculated frozen chicken meat.Practical applications Escherichia coli is an important pollution indicator and is the most important foodborne pathogens of public health concern. The present study deals with the development of bacteria biosensors based on electrochemical impedance spectroscopy and surface plasmon resonance imaging techniques for E. coli K12 detection in inoculated frozen chicken meat. A detection limit of 103 cfu/ml was obtained with the developed biosensors that was better than those obtained with classical methods such as ELISA (enzyme‐linked immunosorbent assay). This research work opens tremendous potential applications for the detection of pathogens in food at the early stage particularly with the development of miniaturized multiarrays platform based on impedance spectroscopy.

Single-Step Recombinase Polymerase Amplification Assay Based on a Paper Chip for Simultaneous Detection of Multiple Foodborne Pathogens.

A paper chip device-based recombinase polymerase amplification (RPA) method was developed for highly sensitive and selective single-step detection of foodborne pathogens. A paper chip was manufactured by simply stacking functional papers. RPA reagents and fluorescent probe were dried on the reaction zone of a patterned poly(ether sulfone) membrane. The RPA reaction was initiated by adding pathogen DNAs into an injection hole. Paper chip-based analysis of pathogens showed optimal performance at 37 °C for 20 min and the results were comparable to those obtained with solution-based RPA reactions. Based on the paper chip-based fluorescence signal, Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium were simultaneously detected with detection limits of 102 cfu/mL. The diagnostic utility of the device was demonstrated by the reliable detection of E. coli and S. aureus present in spiked milk. This ready-to-use device could be integrated with simple nucleic acid extraction for food pathogen detection in resource-limited settings.

Biosensors for pathogen surveillance

Biologists, chemists, and physicists are collaborating to develop highly sensitive and specific biosensors for pathogen detection in the food, healthcare, and environmental sectors. Those novel biosensors allow quick detection and are thus expected to solve the issues of the emergence of highly virulent or antibiotic-resistant pathogens. This article reviews different types of biosensors used for pathogen detection, classified based on the type of transducer used. Optical biosensors integrate labeled means, e.g., fluorophores, quantum dots, and carbon dots to overcome photobleaching. Surface plasmon resonance is also used for enhanced sensitivity. Mechanical biosensors with piezoelectric crystals and cantilevers are adapted for the detection of food pathogens without sample preparation or labels. Conventional methods using electrodes for the measurement of electrochemical changes with differential pulse voltammetry or impedance spectroscopy are fast and highly sensitive. Immunosensors are developed for pathogen detection at trace levels using sample enrichment, signal amplification, and new visual detection techniques.

Fluorescence in situ hybridization (FISH) in food pathogen detection

Diseases due to food borne microbial pathogens are major public health concern in the world Different methods are used to detect food borne pathogens from different types of foods to prevent from these diseases Out of those methods molecular detection methods have received more concern in the recent past Fluorescence in situ Hybridization is one of such methods used in the detection of food borne microbial pathogens The present situation of using Fluorescence in situ Hybridization to detect food borne microbial pathogens is review in this article

(Keynote) Recent Progress in Nanomaterials and Smart-Phone Based Biosensors for Biomedical, Environmental, and Food Safety Applications

In this talk, I will briefly discuss our recent progress in synthesis of functional materials based on protein cages, graphene, and nanoflowers for biosensing and bioimaging. I will discuss the development of various biosensors based on DNA assays and immunoassays for point-of care diagnostics and for cancer biomarker detection [1-3]. Nanomaterials loaded with optical or electroactive signal molecules were used for signal amplification tags for enhanced sensitivity for detection of disease biomarkers, environmental pollutants, and food pathogens. I will also discuss the recent work in 3D printing and smart-phone based biosensors for food safety applications [4-5].

Optimization of a Viability PCR Method for the Detection of Listeria monocytogenes in Food Samples.

Rapid detection of Listeria and other microbial pathogens in food is an essential part of quality control and it is critical for ensuring the safety of consumers. Culture-based methods for detecting foodborne pathogens are time-consuming, laborious and cannot detect viable but non-culturable microorganism, whereas viability PCR methodology provides quick results; it is able to detect viable but non-culturable cells, and allows for easier handling of large amount of samples. Although the most critical point to use viability PCR technique is achieving the complete exclusion of dead cell amplification signals, many improvements are being introduced to overcome this. In the present work, the yield of dead cell DNA neutralization was enhanced by incorporating two new sample treatment strategies: tube change combined with a double light treatment. This procedure was successfully tested using artificially contaminated food samples, showing improved neutralization of dead cell DNA.

Actuation of chitosan-aptamer nanobrush borders for pathogen sensing.

We demonstrate a sensing mechanism for rapid detection of Listeria monocytogenes in food samples using the actuation of chitosan-aptamer nanobrush borders. The bio-inspired soft material and sensing strategy mimic natural symbiotic systems, where low levels of bacteria are selectively captured from complex matrices. To engineer this biomimetic system, we first develop reduced graphene oxide/nanoplatinum (rGO-nPt) electrodes, and characterize the fundamental electrochemical behavior in the presence and absence of chitosan nanobrushes during actuation (pH-stimulated osmotic swelling). We then characterize the electrochemical behavior of the nanobrush when receptors (antibodies or DNA aptamers) are conjugated to the surface. Finally, we test various techniques to determine the most efficient capture strategy based on nanobrush actuation, and then apply the biosensors in a food product. Maximum cell capture occurs when aptamers conjugated to the nanobrush bind cells in the extended conformation (pH < 6), followed by impedance measurement in the collapsed nanobrush conformation (pH > 6). The aptamer-nanobrush hybrid material was more efficient than the antibody-nanobrush material, which was likely due to the relatively high adsorption capacity for aptamers. The biomimetic material was used to develop a rapid test (17 min) for selectively detecting L. monocytogenes at concentrations ranging from 9 to 107 CFU mL-1 with no pre-concentration, and in the presence of other Gram-positive cells (Listeria innocua and Staphylococcus aureus). Use of this bio-inspired material is among the most efficient for L. monocytogenes sensing to date, and does not require sample pretreatment, making nanobrush borders a promising new material for rapid pathogen detection in food.

Nucleic Acid Biosensor Synthesis of an All-in-One Universal Blocking Linker Recombinase Polymerase Amplification with a Peptide Nucleic Acid-Based Lateral Flow Device for Ultrasensitive Detection of Food Pathogens.

In this study, a whole-course nucleic-acid-constructed biosensor that combines the all-in-one concepts of the universal blocking linker recombinase polymerase amplification (UBLRPA) and a peptide nucleic acid (PNA)-based lateral flow device (PLFD) has been developed for the ultrasensitive detection of food pathogens. Using the preamplification UBLRPA principle, a universal linker and C3 space blocker were utilized to produce the universal linker single-duplex DNA products. The developed amplification system was employed to convert duplex products to a single strand. In the signal recognition strategy, a special PNA probe was successfully employed in the portable PLFD. The UBLRPA products were identified visually using the PLFD through dual hybridization (a PNA probe on the gold nanoparticle (Au-NP) was combined with a universal linker on the end of the products; a PNA capture probe was used on the test line and a universal linker on the other end of the products). The accumulation of Au-NPs produced a characteristic red band, enabling the visual detection of a food pathogen without further testing. To demonstrate the value of the all-in-one biosensor, Salmonella enterica subsp. enterica serovar typhimurium was used as a model organism. The biosensor showed high selectivity and extraordinary repeatability using S. typhimurium, and the limit of detection was 4 CFU mL-1. Furthermore, when milk samples artificially contaminated with S. typhimurium were analyzed, the analysis was completed within 30 min without complicated instrumentation. The results exhibited good precision and recovery. This portable all-in-one biosensor demonstrates great promise for the screening of pathogens in food and environmental samples.

Functionalized MoS2 nanosheets assembled microfluidic immunosensor for highly sensitive detection of food pathogen

Two dimensional nanomaterials such as molybdenum disulfide (MoS2) has gained significant interest in designing electrochemical devices for biosensing application. In this work, we have designed cetyltrimethyl ammonium bromide (CTAB) functionalized MoS2 nanosheets (CTAB-MoS2-NS) for conjugation of protein on the microfluidics electrode for Salmonella typhimurium (S.typhimurium) detection. MoS2 nanosheets from the bulk MoS2 were synthesized using CTAB assisted exfoliation methods resulting into positive charge on the CTAB-MoS2-NS. Further, optical and electron microscopic studies of CTAB-MoS2-NS reveal, its suitability for biosensing application. These CTAB-MoS2-NS were deposited onto the patterned hydrolyzed indium tin oxide (ITO) microelelctrode, utilizing the strong electrostatic interaction with the positively charged CTAB-MoS2-NS. Then microelectrode has been integrated with polydimethylsiloxane (PDMS) microfluidic device to design a Lab on a Chip platform for S.typhimurium detection. Detection of the S.typhimurium cells have been performed using electrochemical impedance spectroscopy (EIS) technique. The fabricated microfluidic immunosensor offers highly improved biosensing parameters, as sensitivity was found to be 1.79 kΩ/CFU−1 mL cm−2 with the detection limit 1.56 CFU mL−1in the wide detection range of 101–107 CFUmL−1.

Selective and concurrent detection of viable Salmonella spp., E. coli, Staphylococcus aureus, E. coli O157:H7, and Shigella spp., in low moisture food products by PMA-mPCR assay with internal amplification control

Salmonella enterica, enterohemorrhagic Escherichia coli, and Staphylococcus aureus are major pathogens contaminating low moisture foods. E. coli and Shigella spp. may acquire significance in future since cross-contamination plays a critical role in outbreaks involving low moisture foods. This study investigated a PMA-IAC-mPCR for rapid, reliable and simultaneous detection of viable Salmonella spp., E. coli, Staphylococcus aureus, E. coli O157:H7, and Shigella spp., in low moisture foods. Propidium monoazide (PMA) was applied to detect only viable cells by eliminating PCR signal from dead cells. In addition, an internal amplification control (IAC) was included in the multiplex PCR as an indicator of false negative results arising due to inhibitors in low moisture foods. The sensitivity of the assay for viable cells with PMA treatment was 102–103 CFU/mL for all the target pathogens reflecting the non-influence of PMA treatment on sensitivity. After 10 h enrichment, the PMA-IAC-mPCR could detect 101 CFU/g of Salmonella spp., E. coli, Staphylococcus aureus, E. coli O157:H7, and Shigella spp., in artificially inoculated low moisture foods (peanut butter, chocolate, milk powder and egg powder). This PMA-IAC-mPCR assay would find its promising application in simultaneous detection of these viable target pathogens in low moisture foods.

An Update on Aptamer-Based Multiplex System Approaches for the Detection of Common Foodborne Pathogens

Foodborne ailments constitute a public health challenge and pose an incredible economic burden in healthcare system around the globe. This dilemma has urged authorities and other entities working in field of food quality control and supply chain to play a pivotal role in ensuring food safety. Analytical strategies have been developed using numerous systematic evolution of ligands by exponential enrichment (SELEX) methods to assure food safety. High-affinity and high-sensitivity ssDNA and RNA aptamers against pathogens have emerged as a novel strategy, as compared to the more resource-demanding and complicated biochemical test-based approaches. Thus, this review aims to focus on some methods used in the selection of specific bare, modified, and conjugated aptamers and on the further analysis of selected aptamers using flow cytometer or post-SELEX modifications for enhanced detection of frequently diagnosed foodborne bacteria such as Bacillus sp., Campylobacter jejuni, Escherichia sp., Salmonella sp., Staphylococcus aureus, Shigella sp., Listeria monocytogenes, and Streptococcus pyogenes and/or targeting their cell components towards attaining fast, sensitive, and selective methods for the detection of pathogens in food(s) or other sources.

Magnetoelastic biosensors: Corrosion protection of an FeNiMoB alloy from alkoxide precursors

Mass-sensitive biosensors developed from magnetoelastic alloys have been increasingly used for pathogen detection in food. To protect the magnetoelastic alloys from the corrosive medium a gold layer has been used. In this paper, it was propose two different films based upon alkoxide precursors as a new alternative as coating for biosensors. The films are easily applied on the Metglas® 2826MB3 substrate by dip-coating. It was used the alkoxide precursors 1,2-Bis(triethoxysilyl)ethane (BTSE), tetraethoxysilane (TEOS) and 3-(Trimethoxysilyl)propyl methacrylate (MAP). The film developed from BTSE was thick and irregular. The film developed from the TEOS-MAP precursors combined was found to be more compact and uniform, with good adherence to the substrate, and showed a higher electrochemical performance. Therefore, the TEOS-MAP film is the suitable as a protective coating for the Metglas®2826MB3, because this film assures the integrity of the biosensor in an aggressive environment.

Label-Free Immuno-Sensors for the Fast Detection of Listeria in Food.

Foodborne diseases are a major concern for both food industry and health organizations due to the economic costs and potential threats for human lives. For these reasons, specific regulations impose the research of pathogenic bacteria in food products. Nevertheless, current methods, references and alternatives, take up to several days and require many handling steps. In order to improve pathogen detection in food, we developed an immune-sensor, based on Surface Plasmon Resonance imaging (SPRi) and bacterial growth which allows the detection of a very low number of Listeria monocytogenes in food sample in one day. Adequate sensitivity is achieved by the deposition of several antibodies in a micro-array format allowing real-time detection. This label-free method thus reduces handling and time to result compared with current methods.

Early Recovery of Salmonella from Food Using a 6-Hour Non-selective Pre-enrichment and Reformulation of Tetrathionate Broth.

Culture based methods are commonly employed to detect pathogens in food and environmental samples. These methods are time consuming and complex, requiring multiple non-selective and selective enrichment broths, and usually take at least 1 week to recover and identify pathogens. Improving pathogen detection in foods is a primary goal for regulatory agencies and industry. Salmonella detection in food relies on a series of culture steps in broth formulations optimized to resuscitate Salmonella and reduce the abundance of competitive bacteria. Examples of non-selective pre-enrichment broths used to isolate Salmonella from food include Lactose, Universal Pre-enrichment, BPW, and Trypticase Soy broths. Tetrathionate (TT) and Rappaport–Vassiliadis (RV) broths are employed after a 24-h non-selective enrichment to select for Salmonella and hamper the growth of competitive bacteria. In this study, we tested a new formulation of TT broth that lacks brilliant green dye and has lower levels of TT . We employed this TT broth formulation in conjunction with a 6-h non-selective pre-enrichment period and determined that Salmonella recovery was possible one day earlier than standard food culture methods. We tested the shortened culture method in different non-selective enrichment broths, enumerated Salmonella in the non-selective enrichments, and used 16S rRNA gene sequencing to determine the proportional abundances of Salmonella in the TT and RV selective enrichments. Together these data revealed that a 6-h non-selective pre-enrichment reduces the levels of competitive bacteria inoculated into the selective TT and RV broths, enabling the recovery of Salmonella 1 day earlier than standard culture enrichment methods.

Development of a Droplet Digital Polymerase Chain Reaction for Rapid and Simultaneous Identification of Common Foodborne Pathogens in Soft Cheese.

Dairy products can harbor various microorganisms (e.g., Campylobacter spp., Salmonella spp., Listeria monocytogenes, verocytotoxin-producing Escherichia coli) arising from animal reservoirs, and which can become important sources of foodborne illness. Therefore, early detection of food pathogens is crucial to prevent diseases. We wished to develop an accurate quantitative protocol based on a droplet digital polymerase chain reaction (ddPCR) involving eight individual TaqMan™ reactions to detect simultaneously, without selective enrichment, Listeria spp., L. monocytogenes, Salmonella spp., verocytotoxin-producing E. coli and Campylobacter spp. in cheese. ddPCR (a “third-generation PCR”) provides absolute quantification of target DNAs without requirement of a standard curve, which simplifies experimentation and data comparability. The accuracy, specificity and sensitivity of the developed ddPCR system were assessed using purified DNA from 50 reference pathogenic and non-pathogenic strains from international or Italian collections and analyzing soft cheese samples artificially contaminated with serial dilutions (from 4 × 106 to 4 × 101 CFU/g) of pure cultures from the American Type Culture Collection. Finally, the performance of our ddPCR system was compared by parallel testing with quantitative PCR: it gave higher sensitivity (102 CFU/g for the Listeria spp. assay) without the necessity of a standard curve. In conclusion, this is the first ddPCR system developed for simultaneous detection of common foodborne pathogens in cheese using a single set of amplification conditions. As such, it could become a useful strategy for high-throughput screening of microorganisms to evaluate the quality and safety of food products.

Multiplex PCR for simultaneous identification of E. coli O157:H7, Salmonella spp. and L. monocytogenes in food.

The rapid detection of pathogens in food is becoming increasingly critical for ensuring the safety of consumers, since the majority of food-borne illnesses and deaths are caused by pathogenic bacteria. Hence, rapid, sensitive, inexpensive and convenient approaches to detect food-borne pathogenic bacteria is essential in controlling food safety. In this study, a multiplex PCR assay for the rapid and simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Listeria monocytogenes was established. The invA, stx and hlyA genes specifically amplified DNA fragments of 284, 404 and 510 bp from Salmonella spp., L. monocytogenes and E. coli O157:H7, respectively. The 16S rRNA gene was targeted as an internal control gene in the presence of bacterial DNA. The specificity and sensitivity of the multiplex PCR were performed by testing different strains. The multiplex PCR assay was able to specifically simultaneously detect ten colony-forming unit/mL of each pathogen in artificially inoculated samples after enrichment for 12 h. The whole process took less than 24 h to complete, indicating that the assay is suitable for reliable and rapid identification of these three food-borne pathogens, which could be suitable in microbial epidemiology investigation.

(Invited) Electrochemical Biosensors for Food and Agricultural Applications

Economic losses to agriculture due to pest and pathogen infections are estimated at $40 billion annually and the economic losses to the health care industry due to food borne illnesses are estimated at $15 billion in the U.S. alone. Early detection of pest or pathogen infection in agricultural crops and reliable detection of harmful pathogens in food are important to minimize agricultural productivity loss, ensure food safety, improve food quality and minimize food related public health issues. There is a pressing need to develop rapid, highly selective and sensitive detection technologies for early identification of plant and human pathogens. While a variety of molecular methods are currently being used for this purpose, an inexpensive, high selective, rapid method for the detection of pathogens is highly desired. Electrochemistry biosensors offer unique advantages to this application. Electrochemical sensors have been widely explored for medical and environmental sensing applications, but not as much for food and agricultural applications. An electrochemical biosensor uses a highly selective bio-recognition element such as enzymes, antibody, aptamer or virus and is capable of detecting biding events with ultra-low detection limits. This presentation will focus on some of the recent developments in our lab in the development of electrochemical biosensors for detection of crop diseases and food pathogens.

Analytical Applications of Nanomaterials in Monitoring Biological and Chemical Contaminants in Food.

The detection of food pathogens is an important aspect of food safety. A range of detection systems and new analytical materials have been developed to achieve fast, sensitive, and accurate monitoring of target pathogens. In this review, we summarize the characteristics of selected nanomaterials and their applications in food, and place focus on the monitoring of biological and chemical contaminants in food. The unique optical and electrical properties of nanomaterials, such as gold nanoparticles, nanorods, quantum dots, carbon nanotubes, graphenes, nanopores, and polydiacetylene nanovesicles, are closely associated with their dimensions, which are comparable in scale to those of targeted biomolecules. Furthermore, their optical and electrical properties are highly dependent on local environments, which make them promising materials for sensor development. The specificity and selectivity of analytical nanomaterials for target contaminants can be achieved by combining them with various biological entities, such as antibodies, oligonucleotides, aptamers, membrane proteins, and biological ligands. Examples of nanomaterial-based analytical systems are presented together with their limitations and associated developmental issues.

Applying graphene oxide nano-film over a polycarbonate nanoporous membrane to monitor E. coli by infrared spectroscopy

Nano-biosensors are excellent monitoring tools for rapid, specific, sensitive, inexpensive, in-field, on-line, and/or real-time detection of pathogens in foods, soil, air, and water samples. A variety of nano-materials (metallic, polymeric, and/or carbon-based) were employed to enhance the efficacy, efficiency, and sensitivity of these nano-biosensors, including graphene-based materials, especially graphene oxide (GO)-based materials. GO bears many oxygen-bearing groups, enabling ligand conjugation at the high density critical for sensitive detection. We have fabricated GO-modified nano-porous polycarbonate track-etched (PCTE) membranes that were conjugated to an Escherichia coli-specific antibody (Ab) and used to detect E. coli. The random distribution of nanopores on the PCTE membrane surface and the bright coating of the GO onto the membrane were confirmed by scanning electron microscope. Anti-E. coli β-gal Abs were conjugated to the GO surface via 1-ethyl-3,3-dimethylaminopropyl carbodiimide hydrochloride–N-hydroxysuccinimide chemistry; antibody coating was confirmed by the presence of a characteristic IR peak near 1600cm−1. A non-corresponding Ab (anti-Pseudomonas) was used as a negative control under identical conditions. When E. coli interacted anti-E.coli β-gal with Ab-coated GO-nano-biosensor units, we observed a clear shift in the IR peak from 3373.14 to 3315cm−1; in contrast, we did not observe any shift in IR peaks when the GO unit was coated with the non-corresponding Ab (anti-Pseudomonas). Therefore, the detection of E. coli using the described GO-nano-sensor unit is highly specific, is highly selective and can be applied for real-time monitoring of E. coli with a detection limit between 100μg/mL and 10μg/mL, similar to existing detection systems.

Identification of Shiga toxin producing E. Coli from raw Meat

Emergence of Shiga toxin genes in Shiga toxin producing E. coli (STEC), all over the world, has become problematic and causes GIT illnesses in human originating in food of animals mainly from poultry. The aim of this study was to render rapid diagnostics of Shiga toxin producing E. coli (STEC) from raw meat. During a 4 month period from May to August a total of 200 samples were collected from beef (100) and chicken (100) and analyzed for Shiga toxin producing E. coli. Of the total of 200 samples of raw meat only chicken (2%) and beef (1%) were detected as PCR positive. Over the past decade many improvements have been made both in conventional and modern technique for detection of bacterial pathogens in food that include sample preparation, plating techniques, counting and identification kits but polymerase chain reaction technique is increasingly used which is considered more specific, sensitive, rapid and cost effective. Raw meat could be a source of Shiga toxin producing E. coli which indicates that possible risks of infections to people could be transferred by the consumption of raw meat and their rapid diagnostics could be made possible by the use of rapid diagnostic technique polymerase chain reaction.