Analytical techniques for isolation and identification of food borne pathogens

Immunoassays

Fluorescent enzyme immunoassay

The assay utilizes a beta-galactosidase-murine myeloma monoclonal antibody (M467) conjugate prepared with the heterobifunctional coupling reagent, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and uses 4-methyl umbelliferyl beta-D-galactoside as a fluorogenic substrate for the enzyme.

-To test for Salmonella, Listeria, Staphylococcus Aureus

Visual Immunoassay (VIA)

Visual immunoassay is used based on two monoclonal antibodies for the semi-quantitative determination of plasma elastase levels. VIA is considered a reliable ELISA (enzyme linked immunosorbent assay) for the detection of Salmonella spp. E. coli, Campylobacter, Staphylococcus Aureus and Listeria in food samples.

High affinity "capture" antibodies specific for the pathogen tested have been adsorbed onto the surface of wells. If antigens are present in the sample, they are captured by the antibodies. All other material in the sample is washed away before the addition of enzyme labelled antibodies (conjugate) specific for the pathogen. Presence is indicated when the bound conjugate converts the substrate to a green color, detected by a colourimetric detection system. Alternatively, if the result shows absence, no green color develops.

Results can be out within 42 hours, and it is AOAC Official Methods Validated and other international approvals.

http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/volume3/mflp35_e.html

Staphylococcus Aureus Visual Immunoassay

This test employs S aureus-specific DNA probes and a colourimetric detection system for the detection of S. aureus in food samples following broth culture enrichment. A sample is considered non-reactive for the presence of S. aureus if the absorbance value obtained is less than or equal to the established cutoff value for the assay and vice versa.

Detection using DNA probe

In fluorescence in situ hybridization (FISH), a DNA probe is labeled with a fluorescent dye or a haptene. The labeled DNA is purified, concentrated, resuspended in hybridization buffer and is hybridized onto chromosomes and nuclei on slides. After overnight hybridization, the slides are rinsed in fluorescent-labeled antibodies to detect haptene-labeled DNA. The slides are mounted with antifade solution and are visualized at the fluorescent microscope, using appropriate filters. This procedure is illustrated in the figure.

This method identifies the pathogens by extracting a small amount of DNA and amplifies the target sequences without cultivation. The DNA probe allows the simultaneous analysis of thousands of gene in a short assay time providing high accuracy by using the species-specific probes. Probes made of peptide nucleic acids (PNA), which have very strong affinity for complementary DNA sequence, can further improve the specificity. Therefore, using PNA probes can more effectively discriminate the pathogens.

To detect the specific microbes, a small amount of the specific sample is necessary, with the scale-down of a DNA chip.

http://info.med.yale.edu/genetics/ward/tavi/fi16.html
http://www.biotrace.co.uk/content.php?hID=2&nhID=211

Bead immunocapture

A test using immunomagnetic beads with an antibody to test for Listeria. These beads are mixed with a sample to isolate Listeria. In this test, Listeria is bound to antibodies on microscopic magnetic beads. These beads are then isolated from the rest of food or environmental sample. Listeria is confirmed immunologically and, if desired, characterized biochemically, in about 24 hours.

http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=PubMed&list_uids=3283171&dopt=Abstract
http://seafood.ucdavis.edu/HACCP/Compendium/Chapt17.htm#Top

REVEAL for Salmonella

It is a test system that provides for the rapid recovery of Salmonella in food and allowing detection and identification of Salmonella within 21 hours.

Following selective enrichment in Rappaport-Vasiliadis broth, a portion of the sample enrichment is placed into the sample port of the Reveal Device initiating flow. The Reveal Device contains antibodies with high specificity to Salmonella antigens. These antibodies are bound to colloidal gold and, separately, to a solid support matrix. Any Salmonella antigen present will bind to the gold conjugated antibodies forming an antigen – antibody – chromogen complex. This complex flows across a lateral flow membrane and is subsequently bound by antibody immobilized on the membrane. This causes the gold conjugate to precipitate, forming a visible line and indicating a positive reaction. Proper test completion and flow is indicated by a control line which forms further up in the test window and verifies a valid test run. Absence of a control line invalidates the test. Record results at 10 minutes incubation time.

Immunodiffusion

The principle behind the technique relies on the fact that antibodies produced by the immune system are incredibly sensitive to differences in the structure of molecules they are exposed to. The antibodies of one species will react when exposed to blood serum, containing the proteins, of another. The antibodies that respond against a specific blood protein are manufactured and collected from an outgroup species. These antibodies, in the form of an antiserum are then exposed to the blood proteins of the species under investigation.

If an organism is only very distantly related to the outgroup species a strong reaction will take place between the blood serum (containing the protein) and the antibodies, and there is very tight binding between the two. The degree of relatedness between the species being tested is reflected by the strength of reaction when exposed to the antiserum. Species more closely related to the outgroup will exhibit a weaker reaction. The experiment is usually conducted on an agar gel with the test blood protein serums arranged in wells around the test antiserum, or some arrangement that will enable comparisons of the test species.

http://www.nhc.ed.ac.uk/index.php?page=236.438.445

Transia Card

For E. coli 0157, salmonella, Listeria, Staphylococcal enterotoxins.
The Transia Plate method uses only one broth, in a two-step protocol, followed by a rapid immunoassay. It uses only one broth, in a two-step protocol, followed by a rapid immunoassay. The Transia ELISA technology, with ready-to-use reagents and a microtiterplate with divisible strips, makes the kit ideal for both single tests and high-throughput automation.

http://www.diffchamb.com/website/Archive/Templates/Item/product.asp?iSecId=36

Production of Monoclonal Antibodies

Antibodies which neutralize botulinum neurotoxin serotype F are produced using biologically active botulinum neurotoxin instead of toxoid for immunization and exploiting the importance of cross reaction between various serotypes to obtain immune responses, or monoclonal antibodies, to additional serotypes of interest. Methods of preparation and uses of the neutralizing botulinum neurotoxin antibodies are described.

PCR (polymerase chain reaction)

In this technique, double-stranded target DNA is denatured to provide single-stranded templates to which specific oligonucleotide primers are hybridized, followed by primer extension with a thermostable DNA polymerase. Primer pairs complementary to opposite strands of a DNA region are chosen. Repetitive denaturation, annealing, and primer extension cycles exponentially amplify a unique DNA fragment bordered by the primers. PCR-based methods have been developed to detect foodborne pathogens, including Listeria monocytogenes, enterotoxigenic Escherichia coli, V. vulnificus, V. cholerae, Shigella flexneri, Yersinia enterocolitica, various Salmonella and Campylobacter species.

http://www.cfsan.fda.gov/~ebam/bam-28.html