detection-and-isolation-of-campylobacter-spp-from-raw-meat

Detection and Isolation of <i>Campylobacter </i> 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 ...

modified-most-probable-number-assay-to-quantify-salmonella-raw-ready

Modified Most Probable Number Assay to Quantify <em>Salmonella</em> in Raw and Ready-to-Cook Chicken Products

Salmonella is a leading cause of foodborne illness in the United States, particularly in poultry products. Traditional methods for detecting Salmonella ...

Agricultural Research Service (ARS), United States Department of Agriculture (USDA)

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.

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

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.

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

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

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.

ArticleTotal : 2	Year
Publication title Cited by 3	2024
Publication title Cited by 1	2025

Article	Year
Rapid detection of Salmonella enterica serotype Typhimurium in large volume samples using porous electrodes in a flow-through, enzyme-amplified immunoelectrochemical sensor. Analytical and bioanalytical chemistry\| PubMed ID: 31127336	2019
Detection of Shiga toxin-producing Escherichia coli (STEC) in beef products using droplet digital PCR. International journal of food microbiology\| PubMed ID: 31954209	2020
Complete Genome Sequence of Campylobacter jejuni BSD5, a Multidrug-Resistant Isolate from a Poultry Processing Facility in the United States. Microbiology resource announcements\| PubMed ID: 37255436	2023
High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA. Scientific reports\| PubMed ID: 38528005	2024

Cheryl M. Armstrong

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