High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA.

The Amplified Luminescent Proximity Homogenous Assay-linked Immunosorbent Assay (AlphaLISA) is known for detecting various protein targets; however, its ability to detect nucleic acid sequences is not well established. Here, the capabilities of the AlphaLISA technology were expanded to include direct detection of DNA (aka: oligo-Alpha) and was applied to the detection of Listeria monocytogenes. Parameters were defined that allowed the newly developed oligo-Alpha to differentiate L. monocytogenes from other Listeria species through the use of only a single nucleotide polymorphism within the 16S rDNA region. Investigations into the applicability of this assay with different matrices demonstrated its utility in both milk and juice. One remarkable feature of the oligo-Alpha is that greater sensitivity could be achieved through the use of multiple acceptor oligos compared to only a single acceptor oligo, even when only a single donor oligo was employed. Additional acceptor oligos were easily incorporated into the assay and a tenfold change in the detection limit was readily achieved, with detection limits of 250 attomole of target being recorded. In summary, replacement of antibodies with oligonucleotides allows us to take advantage of genotypic difference(s), which both expands its repertoire of biological markers and furthers its use as a diagnostic tool.

Detection of Escherichia coli O157:H7 in Ground Beef Using Long-Read Sequencing.

Foodborne pathogens are a significant cause of illness, and infection with Shiga toxin-producing Escherichia coli (STEC) may lead to life-threatening complications. The current methods to identify STEC in meat involve culture-based, molecular, and proteomic assays and take at least four days to complete. This time could be reduced by using long-read whole-genome sequencing to identify foodborne pathogens. Therefore, the goal of this project was to evaluate the use of long-read sequencing to detect STEC in ground beef. The objectives of the project included establishing optimal sequencing parameters, determining the limit of detection of all STEC virulence genes of interest in pure cultures and spiked ground beef, and evaluating selective sequencing to enhance STEC detection in ground beef. Sequencing libraries were run on the Oxford Nanopore Technologies' MinION sequencer. Optimal sequencing output was obtained using the default parameters in MinKNOW, except for setting the minimum read length to 1 kb. All genes of interest (eae, stx1, stx2, fliC, wzx, wzy, and rrsC) were detected in DNA extracted from STEC pure cultures within 1 h of sequencing, and 30× coverage was obtained within 2 h. All virulence genes were confidently detected in STEC DNA quantities as low as 12.5 ng. In STEC-inoculated ground beef, software-controlled selective sequencing improved virulence gene detection; however, several virulence genes were not detected due to high bovine DNA concentrations in the samples. The growth enrichment of inoculated meat samples in mTSB resulted in a 100-fold increase in virulence gene detection as compared to the unenriched samples. The results of this project suggest that further development of long-read sequencing protocols may result in a faster, less labor-intensive method to detect STEC in ground beef.

Detection and Isolation of Campylobacter spp. from Raw Meat.

This article presents a rapid yet robust protocol for isolating Campylobacter spp. from raw meats, specifically focusing on Campylobacter jejuni and Campylobacter coli. The protocol builds upon established methods, ensuring compatibility with the prevailing techniques employed by regulatory bodies such as the Food and Drug Administration (FDA) and the U.S. Department of Agriculture (USDA) in the USA, as well as the International Organization for Standardization (ISO) in Europe. Central to this protocol is collecting a rinsate, which is concentrated and resuspended in Bolton Broth media containing horse blood. This medium has been proven to facilitate the recovery of stressed Campylobacter cells and reduce the required enrichment duration by 50%. The enriched samples are then transferred onto nitrocellulose membranes on brucella plates. To improve the sensitivity and specificity of the method, 0.45 µm and 0.65 µm pore-size filter membranes were evaluated. Data revealed a 29-fold increase in cell recovery with the 0.65 µm pore-size filter compared to the 0.45 µm pore-size without impacting specificity. The highly motile characteristics of Campylobacter allow cells to actively move through the membrane filters towards the agar medium, which enables effective isolation of pure Campylobacter colonies. The protocol incorporates multiplex quantitative real-time polymerase chain reaction (mqPCR) assay to identify the isolates at the species level. This molecular technique offers a reliable and efficient means of species identification. Investigations conducted over the past twelve years involving retail meats have demonstrated the ability of this method to enhance recovery of Campylobacter from naturally contaminated meat samples compared to current reference methods. Furthermore, this protocol boasts reduced preparation and processing time. As a result, it presents a promising alternative for the efficient recovery of Campylobacter from meat. Moreover, this procedure can be seamlessly integrated with DNA-based methods, facilitating rapid screening of positive samples alongside comprehensive whole-genome sequencing analysis.

Magnetic capture device for large volume sample analysis.

Immunomagnetic separation (IMS) techniques employing superparamagnetic particles can successfully isolate various components from mixtures. However, their utility can be limited for large-volume samples, viscous samples, or those containing a high density of particulate matter because of the need to generate high field gradients for particle recovery. Therefore, a new class of immunomagnetic particles was devised utilizing a single, macroscopic Pyrex spinbar conjugated with biorecognition elements to address these limitations. Advantages include an inherent capacity for effective mixing, an almost instantaneous recovery of the spinbar that can be performed without expensive equipment and with no loss of magnetic particles during processing, and reduced transfer of sample matrix. As a result, spinbars can provide an effective means for IMS with large-volume assays composed of complex matrices.

Whole Genome Sequences, De Novo Assembly, and Annotation of Antibiotic Resistant Campylobacter jejuni Strains S27, S33, and S36 Newly Isolated from Chicken Meat.

Campylobacter is a leading bacterial cause of gastrointestinal infections in humans and has imposed substantial medical and public health burdens worldwide. Among a total of 39 species in the Campylobacter genus, C. jejuni is the most important species responsible for approx. 90% of human Campylobacter illness. Most cases of the infection were acquired by ingesting undercooked poultry meat due to the high prevalence of Campylobacter in the products. Here, we reported the dataset of raw sequences, de novo assembled and annotated genomes of C. jejuni strains S27, S33, and S36 recently isolated from retail chicken by using PacBio highly accurate long-read sequencing technology combined with bioinformatics tools. Our data revealed several virulence and antibiotic resistance genes in each of the chromosomes, a type IV secretion system in the plasmid (pCjS33) of C. jejuni S33, and a type VI secretion system and a phage in the plasmid (pCjS36) of C. jejuni S36. This study not only provides new sequence data but also extends the knowledge pertaining to the genomic and functional aspects of this important foodborne pathogen, including the genetic determinants of virulence and antibiotic resistance.

Use of a commercial tissue dissociation system to detect Salmonella-contaminated poultry products.

Successful detection of bacterial pathogens in food can be challenging due to the physical and compositional complexity of the matrix. Different mechanical/physical and chemical methods have been developed to separate microorganisms from food matrices to facilitate detection. The present study benchmarked a commercial tissue digestion system that applies both chemical and physical methods to separate microorganisms from tissues against stomaching, a standard process currently utilized by commercial and regulatory food safety laboratories. The impacts of the treatments on the physical properties of the food matrix were characterized along with the compatibility of the methods with downstream microbiological and molecular detection assays. The results indicate the tissue digestion system can significantly reduce the average particle size of the chicken sample relative to processing via a stomacher (P < 0.001) without adversely affecting either real-time PCR (qPCR) or plate counting assays, which are typically used to detect Salmonella. Furthermore, inoculated chicken treated with the GentleMACS resulted in a significant increase (P < 0.003) in the qPCR's detection capabilities relative to stomached controls. Cohen kappa (κ) coefficient and McNemar's test indicate the plating assays and PCR results agree with measurements obtained via the 3 M Molecular Detection System as defined in the MLG standard (κ > 0.62; P > 0.08). Collectively, the results demonstrate that the technique enables detection of pathogens in meat at lower levels of contamination using current industry standard technologies.

Complete Genome Sequence of Campylobacter jejuni BSD5, a Multidrug-Resistant Isolate from a Poultry Processing Facility in the United States.

Raw poultry can harbor microbial pathogens. Campylobacter jejuni BSD5, isolated from a critical control point within a poultry production plant, was sequenced. Genome annotation revealed several virulence genes including antibiotic resistance genes in agreement with the phenotypic results, indicating a potential risk of this strain to public health.

Complete Genome Sequences of Multidrug-Resistant Campylobacter coli Strains YH501, YH503, and YH504, from Retail Chicken.

Campylobacter coli is an important foodborne pathogen that can cause inflammation of the intestine and diarrhea in humans. The complete genomes, including megaplasmids, of C. coli strains YH501, YH503, and YH504 from retail chicken were sequenced and de novo assembled. Whole-genome analysis revealed a number of virulence and antibiotic resistance genes, suggesting significant potential for these poultry-originating isolates to cause human disease.

Flow-Through Electrochemical Biosensor for the Detection of Listeria monocytogenes Using Oligonucleotides.

Consumption of food contaminated by Listeria monocytogenes can result in Listeriosis, an illness with hospitalization rates of 94% and mortality rates up to 30%. As a result, U.S. regulatory agencies governing food safety retain zero-tolerance policies for L. monocytogenes. However, detection at such low concentrations often requires strategies such as increasing sample size or culture enrichment. A novel flow-through immunoelectrochemical biosensor has been developed for Escherichia coli O157:H7 detection in 1 L volumes without enrichment. The current work further augments this biosensor's capabilities to (1) include detection of L. monocytogenes and (2) accommodate genetic detection to help overcome limitations based upon antibody availability and address specificity errors in phenotypic assays. Herein, the conjugation scheme for oligo attachment and the conditions necessary for genetic detection are laid forth while results of the present study demonstrate the sensor's ability to distinguish L. monocytogenes DNA from L. innocua with a limit of detection of ~2 × 104 cells/mL, which agrees with prior studies. Total time for this assay can be constrained to <2.5 h because a timely culture enrichment period is not necessary. Furthermore, the electrochemical detection assay can be performed with hand-held electronics, allowing this platform to be adopted for near-line monitoring systems.

Detection of Shiga toxin-producing Escherichia coli (STEC) in beef products using droplet digital PCR.

Many of the current accredited methods for the molecular detection of Shiga toxin-producing Escherichia coli (STEC) in foods rely on a PCR-based screen for the pathotype-specific genetic markers stx and eae. Unfortunately, these methods can inaccurately conclude the presence of E.coli containing both stx and eae because of the inability of the methods to determine if the two genes originated from a single organism as opposed to a mixture of organisms. This study was undertaken to evaluate if a droplet digital PCR (ddPCR)-based method that does not require DNA isolation could reliably identify the presence of an STEC containing eae in beef samples by confirming that both genes reside within the same cell, even when present in a mixed culture. The ddPCR system used in this study, dd-Check STEC Solution (Bio-Rad), works without the need for DNA isolation by partitioning intact cells into emulsion droplets, where they are lysed, and subsequently undergo multiplexed endpoint PCR. This enables the assay to differentiate between samples where a single organism contains both stx and eae from samples in which stx and eae reside in different organisms. Comparisons were made between the dd-Check STEC Solution, the BAX System Real-Time PCR STEC assay suite (Hygiena), and the iQ-Check STEC PCR detection kit (Bio-Rad) using 37 unique simulations of E. coli contamination in ground beef. While no single platform was consistently superior at detecting eae and stx across all pathogens tested, the results indicated that the dd-Check STEC Solution has the potential to reduce the number of inaccurately identified samples when screening for E. coli with a stx+, eae+ genotype because it can identify the co-existence of multiple virulence genes within a cell even when in the presence of a mixed microbial population containing identical genes. Ultimately, incorporation of this system could result in substantial cost savings by reducing the expenses incurred when product samples are incorrectly classified as containing E. coli with a stx+, eae+ genotype.

Impacts of Clarification Techniques on Sample Constituents and Pathogen Retention.

Determination of the microbial content in foods is important, not only for safe consumption, but also for food quality, value, and yield. A variety of molecular techniques are currently available for both identification and quantification of microbial content within samples; however, their success is often contingent upon proper sample preparation when the subject of investigation is a complex mixture of components such as foods. Because of the importance of sample preparation, the present study employs a systematic approach to compare the effects of four different separation techniques (glass wool, 50 µm polypropylene filters, graphite felt, and continuous flow centrifugation (CFC)) on sample preparation. To define the physical effects associated with the use of these separation methods, a multifactorial analysis was performed where particle size and composition, both pre- and post- processing, were analyzed for four different food matrices including lean ground beef, ground pork, ground turkey and spinach. Retention of three important foodborne bacterial pathogens (Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes) was also examined to evaluate the feasibility of the aforementioned methods to be utilized within the context of foodborne pathogen detection. Data from the multifactorial analysis not only delineated the particle size ranges but also defined the unique compositional profiles and quantified the bacterial retention. The three filtration membranes allowed for the passage of bacteria with minimal loss while CFC concentrated the inoculated bacteria. In addition, the deposition and therefore concentration of food matrix observed with CFC was considerably higher for meat samples relative to spinach. However, filtration with glass wool prior to CFC helped clarify meat samples, which led to considerably lower amounts of solids in the CFC vessel post processing and an increase in the recovery of the bacteria. Overall, by laying a framework for the deductive selection of sample preparation techniques, the results of the study can be applied to a range of applications where it would be beneficial to scientifically guide the pairing of the criteria associated with a downstream detection method with the most advantageous sample preparation techniques for complex matrices such as foods.

Rapid Detection of Salmonella enterica via Directional Emission from Carbohydrate-Functionalized Dynamic Double Emulsions.

Reliable early-stage detection of foodborne pathogens is a global public health challenge that requires new and improved sensing strategies. Here, we demonstrate that dynamically reconfigurable fluorescent double emulsions can function as highly responsive optical sensors for the rapid detection of carbohydrates fructose, glucose, mannose, and mannan, which are involved in many biological and pathogenic phenomena. The proposed detection strategy relies on reversible reactions between boronic acid surfactants and carbohydrates at the hydrocarbon/water interface leading to a dynamic reconfiguration of the droplet morphology, which alters the angular distribution of the droplet's fluorescent light emission. We exploit this unique chemical-morphological-optical coupling to detect Salmonella enterica, a type of bacteria with a well-known binding affinity for mannose. We further demonstrate an oriented immobilization of antibodies at the droplet interface to permit higher selectivity. Our demonstrations yield a new, inexpensive, robust, and generalizable sensing strategy that can help to facilitate the early detection of foodborne pathogens.

Rapid detection of Salmonella enterica serotype Typhimurium in large volume samples using porous electrodes in a flow-through, enzyme-amplified immunoelectrochemical sensor.

Foodborne illness is a common yet preventable public health concern generating significant costs for the healthcare system, making systems to accurately detect this pathogen a topic of current research. Enzyme-based immunoassays are highly desirable because they offer shorter response times compared to traditional culture-based methods. Biosensors employing the electrochemical and optical detection of a substrate oxidized by horseradish peroxidase (HRP) have been used to successfully detect biomolecules; however, their inability to handle large sample volumes severely limits their application to food safety despite their accuracy and reliability. Here, we describe a biosensor with the capacity to process a large sample volume by utilizing an Ag/AgCl reference electrode, a platinum counter electrode, and a porous working electrode made from graphite felt coated with antibodies specific for Salmonella common structural antigens. This design allows samples to flow-through the electrode while capturing target pathogens. Following sample exposure, HRP-conjugated antibodies facilitate pathogen detection that culminates in an oxidation reaction with the output analyzed via Osteryoung square wave voltammetry. Detection limits of 1000 Salmonella enterica serotype Typhimurium cells were achieved using this newly devised flow-through, enzyme-amplified, electrochemical biosensor in samples as large as 60 mL. The low cost of the sensor allows for incorporation into disposable detection devices while its design not only broadens its applicability in sample processing but also permits the detection of various microbes by simply exchanging the antibodies.

Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA.

Amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) is comprised of a bead-based immunoassay that is used for small molecule detection. In this study, a novel AlphaLISA was developed and optimized for the detection of Shiga-toxin 2 (Stx2). Efficacy and sensitivity trials showed the AlphaLISA could detect ≥0.5 ng/mL of purified Stx2, which was comparable to the industry-standard enzyme-linked immunosorbent assay (ELISA) tests for Stx2 detection. In addition, evaluation of Shiga toxin-producing Escherichia coli (STEC)-inoculated Romaine lettuce and ground beef samples demonstrated that both the AlphaLISA and the ELISA were able to discern uninoculated samples from 1× and 10× diluted samples containing ~10 CFU/mL of STEC enriched in modified tryptic soy broth with mitomycin C for 16 h. Overall, the increased signal-to-noise ratios indicated a more robust signal was produced by the AlphaLISA compared to the ELISA and the delineation of higher toxin concentrations without the need for sample dilution implied a greater dynamic range for the AlphaLISA. Implementation of the newly developed AlphaLISA will allow for more rapid analysis for Stx2 with less manual manipulation, thus improving assay throughput and the ability to automate sample screening while maintaining detection limits of 0.5 ng/mL.

Highly sensitive detection of HER2 extracellular domain in the serum of breast cancer patients by piezoelectric microcantilevers.

Rapid and sensitive detection of serum tumor biomarkers are needed to monitor cancer patients for disease progression. Highly sensitive piezoelectric microcantilever sensors (PEMS) offer an attractive tool for biomarker detection; however, their utility in the complex environment encountered in serum has yet to be determined. As a proof of concept, we have functionalized PEMS with antibodies that specifically bind to HER2, a biomarker (antigen) that is commonly overexpressed in the blood of breast cancer patients. The function and sensitivity of these anti-HER2 PEMS biosensors was initially assessed using recombinant HER2 spiked into human serum. Their ability to detect native HER2 present in the serum of breast cancer patients was then determined. We have found that the anti-HER2 PEMS were able to accurately detect both recombinant and naturally occurring HER2 at clinically relevant levels (>2 ng/mL). This indicates that PEMS-based biosensors provide a potentially effective tool for biomarker detection.

Label-free Growth Receptor-2 Detection and Dissociation Constant Assessment in Diluted Human Serum Using a Longitudinal Extension Mode of a Piezoelectric Microcantilever Sensor.

We have investigated real-time, label-free, in-situ detection of human epidermal growth factor receptor 2 (Her2) in diluted serum using the first longitudinal extension mode of a lead zirconate-lead titanate (PZT)/glass piezoelectric microcantilever sensor (PEMS) with H3 single-chain variable fragment (scFv) immobilized on the 3-mercaptopropyltrimethoxysilane (MPS) insulation layer of the PEMS surface. We showed that with the longitudinal extension mode, the PZT/glass PEMS consisting of a 1 mm long and 127 µm thick PZT layer bonded with a 75 µm thick glass layer with a 1.8 mm long glass tip could detect Her2 at a concentration of 6-60 ng/ml (or 0.06-0.6 nM) in diluted human serum, about 100 times lower than the concentration limit obtained using the lower-frequency flexural mode of a similar PZT/glass PEMS. We further showed that with the longitudinal mode, the PZT/glass PEMS determined the equilibrium H3-Her2 dissociation constant K(d) to be 3.3±0.3 × 10(-8) M consistent with the value, 3.2±0.28 ×10(-8) M deduced by the surface plasmon resonance method (BIAcore).

Label free detection of white spot syndrome virus using lead magnesium niobate-lead titanate piezoelectric microcantilever sensors.

We have investigated rapid, label free detection of white spot syndrome virus (WSSV) using the first longitudinal extension resonance peak of five lead-magnesium niobate-lead titanate (PMN-PT) piezoelectric microcantilever sensors (PEMS) 1050-700 µm long and 850-485 µm wide constructed from 8 µm thick PMN-PT freestanding films. The PMN-PT PEMS were encapsulated with a 3-mercaptopropyltrimethoxysilane (MPS) insulation layer and further coated with anti-VP28 and anti-VP664 antibodies to target the WSSV virions and nucleocapsids, respectively. By inserting the antibody coated PEMS in a flowing virion or nucleocapsid suspension, label free detection of the virions and nucleocapsids were respectively achieved by monitoring the PEMS resonance frequency shift. We showed that positive label free detection of both the virion and the nucleocapsid could be achieved at a concentration of 100virions(nucleocapsids)/ml or 10 virions(nucleocapsids)/100 µl, comparable to the detection sensitivity of polymerase chain reaction (PCR). However, in contrast to PCR, PEMS detection was label free, in situ and rapid (less than 30 min), potentially requiring minimal or no sample preparation.

Label-free, all-electrical, in situ human epidermal growth receptor 2 detection.

Using 3-mercaptopropyltrimethoxysilane (MPS)-coated (PbMg 1/3 Nb 2/3 O3)0.63-(PbTiO3)0.37 (PMN-PT)/tin and lead zirconate titanate/glass piezoelectric microcantilever sensors (PEMSs) with single-chain variable fragment (scFv) immobilized on the MPS surface, we have demonstrated real-time, label-free detection of human epidermal growth factor receptor 2 (Her2) in a background of 1 mg/ml bovine serum albumin. Coupled with a scFv with a KD of 3.4 x 10(-8)M, the MPS-insulated PMN-PT/tin PEMS 560 microm long and 720 microm wide exhibited a Her2 concentration sensitivity of 5 ng/ml in a background of 1 mg/ml BSA.

3-mercaptopropyltrimethoxysilane as insulating coating and surface for protein immobilization for piezoelectric microcantilever sensors.

We have examined coating (PbMg(13)Nb(23)O(3))(0.63)-(PbTiO(3))(0.37) (PMN-PT)/tin and lead zirconate titanate (PZT)/glass piezoelectric microcantilever sensor (PEMS) with 3-mercaptopropyl-trimethoxysilane (MPS) by a simple solution method to electrically insulate the PEMS for in-water applications. In contrast to earlier methytrimethoxysilane insulation coating, the MPS coating also facilitated receptor immobilization on the sensor surface via bonding of its sulhydryl group to a bifunctional linker, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. We showed that a MPS coating of 21 nm in thickness is sufficient to electrically insulate and provide immobilization surface to the PEMS for in-liquid electrical self-excitation and self-sensing. The in-phosphate buffered saline solution resonance spectra were stable with Q values ranging from 41 to 55. The mass detection sensitivities were determined to be 5x10(-11) and 8x10(-12) gHz for the MPS-insulated PZT-glass and PMN-PT/tin PEMSs, respectively.

Therapist self-disclosure to child patients.

The primary aim of this study was to investigate therapists' use of self-disclosure with their child patients. A sample of 126 mental health professionals with an average of 20 years of clinical experience completed the Therapist-to-Child Disclosure Inventory (TCDI), a 42-item Likert-type measure created for this study. Therapist self-disclosure was examined using three principle dimensions: the mean frequency of specific disclosures, the degree to which child patients solicit these disclosures, and the extent to which specific self-disclosures are seen as advancing treatment aims for child patients. Results indicate that therapists reveal personal information to their child patients infrequently, that children almost never solicit personal disclosures from their therapists, and that therapists perceive self-disclosure as seldom advancing treatment aims for child therapy. Future studies, the authors suggest, should examine the differences between therapists' use of self-involving disclosures to children and their use of factual self-disclosures.