BLBS102-c44 BLBS102-Simpson March 21, 2012 14:34 Trim: 276mm X 219mm Printer Name: Yet to Come
844 Part 8: Food Safety and Food Allergens+ve test –ve testPositive control line
Positive resultResult windowSample portFigure 44.2.Rapid detection of pathogens using a lateral flow kit.Latex Agglutination AssaysLatex agglutination assays use antibody coated colored latex
beads or colloidal gold particles that will clump in the presence
of a specific antigen. The test is based on the insoluble bacterial
cells clumping in the presence of the antibody. In general, for
latex agglutination assays to be accurate they require a pure
culture for the specific assay as the sensitivity of the antibodies
can be relatively low, thus resulting in cross-reactions. The test is
however relatively fast, easy to perform and visual interpretation
relatively straightforward. Latex assays have been developed for
the detection of a range of organisms includingE. coliO157:H7
and other STEC members,Salmonellaspp,Campylobacterspp.,
Listeria,Shigella,S. aureus,VibrioandYersinia. One of the
drawbacks of the latex agglutination assays is the potential for
the particles to cross-react with other members of the same genus
because of structural similarities in the lipopolysaccharide layer
(Feng 2007).
A modification of the latex agglutination assay is the reverse
passive latex agglutination (RPLA), in this assay the antibody is
used to detect a soluble component of the bacterium – the toxin.
Usually, toxins are soluble and will form a diffuse lattice network
or layer in the presence of the antibody indicating a positive test.
RPLA is typically carried out in a microplate to detect toxins in
a food extract or pure culture suspension (Feng 2007).Gel ImmunodiffusionThe gel immunodiffusion assay was developed for the rapid de-
tection ofSalmonellain a food system. The test is based on
the use an L-shaped chamber containing motility agar and an-
tibodies againstSalmonellaflagella on one side of the chamber
and on the other side, selective enrichment media for enhancing
the growth ofSalmonella. A sample is added to the enrich-
ment medium side and growth of the pathogen will occur, over
time, the pathogen will migrate through the motility agar whereit cross-reacts with the anti flagellar antibodies resulting in a
visible layer of precipitation in the motility agar. The test is rel-
atively specific and has AOAC approval for use in the detection
ofSalmonellaspecies. The test, however, is limited to motile
Salmonellaspecies and will not detect nonmotile strains. The
test is marketed as aSalmonella1-2 kit by BioControl.NUCLEIC ACID-BASED DETECTION
Pathogen detection using nucleic acid-based approaches are usu-
ally designed to target DNA, RNA, or both. In recent years, there
has been a rapid increase in the use of such approaches for not
only detection of a pathogen but also for identification and char-
acterization.Hybridization TechniquesDNA/Colony HybridizationColony hybridization is a relatively low-cost method for
pathogen detection where colonies of a suspect target are se-
lected from a plate and transferred to a solid support (e.g., mem-
brane). The cells of the sample are then lysed using an enzyme
to release the DNA. The DNA is then denatured to allow linkage
to the support membrane and then hybridized to a gene probe
with complementary sequence to the target DNA. Usually, the
gene probe is labeled with a fluorescent tag or other color marker
which allows easier detection of the pathogen. In recent years,
the use of the solid support for detection of target DNA has
changed slightly and the application of hybridization can occur
through the use of a dip stick approach where the DNA is hy-
bridized to probes embedded on a solid surface. Hybridization
can also be achieved in solution. One such system that is com-
mercially available with success in the detection of pathogens
such asL. monocytogenesandSalmonellais the Gene-Trak kit
marketed by Neogen.