Fundamental Food Microbiology, Third Edition - Fuad Fathir

DETECTION OF MICROORGANISMS IN FOOD AND FOOD ENVIRONMENT 563
which are automated, have been developed to detect microbial loads, foodborne
pathogens, and their toxins. 1,4,5 Some of these methods have been approved by
regulatory agencies. In addition to being rapid, they are quite specific, sensitive,
relatively accurate, and less labor intensive. However, their effectiveness in food
systems with a low level of contamination needs further evaluation. Many new
methods are constantly being developed, which differ mainly in technique. The
principles, procedures, and applications in food of some of these methods are briefly
presented. Exact methods and steps are provided by commercial firms.
A. Immunoassays for Rapid Detection of Pathogens
Several rapid and automated methods have been developed that rely on the specific
antigen–antibody reaction and production of agglutination, color formation from
chromogenic substrate, formation of an immunoband, or fluorescence.
1. Immunofluorescence Method
In this method, commercially available specific fluorescence-conjugated antibodies
(against somatic or flagellar antigens of a pathogen) are mixed with an enriched
medium suspected to contain the specific pathogen, such as Salmonella (antigen),
on a glass slide. Following incubation and removal of reagents, the slide is examined
under a fluorescence microscope for cells showing fluorescence on the cell wall or
flagella, or both.
2. RPLA Method
This method has been developed to detect toxins of several foodborne pathogens,
such as Sta. aureus, Clo. perfringens, Bac. cereus, Vib. cholerae, and enteropathogenic
Esc. coli. The antibody of a specific toxin is immobilized on latex particles
that are then mixed with a sample preparation suspected to contain toxin (antigen)
in wells of microtitration plates. If the specific toxin is present in the sample, a
diffused pattern will appear in the bottom; in its absence, a ring or button will appear.
3. Immunoimmobilization Method
This method was developed for motile pathogens such as Salmonella and Esc. coli
O157:H7. The motile cells and antibody against flagellar antigen are applied on soft
agar to enable them to diffuse in the same direction from opposite sides. When the
bacterial cells and specific antibody meet, they form a visible arc due to the immobilization
of cells.
4. Enzyme Immunoassay (EIA) or ELISA Methods
These methods were commercially developed and used to detect several foodborne
pathogens and their toxins, namely, Salmonella, Lis. monocytogenes, and Cam.
jejuni. The specific antibody is first allowed to bind on a solid surface (on a wall of
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