Fundamental Food Microbiology, Third Edition - Fuad Fathir

564 FUNDAMENTAL FOOD MICROBIOLOGY
a microtitration plate) as per the directions of the commercial producer. Blocking
agents (bovine serum albumin) are then added to block the additional protein-binding
sites on the surface. The sample suspected of containing the antigen (pathogens or
their toxins) is prepared as per requirement and then added to the well and incubated
for the antibody–antigen reaction. After removing the unbound antigen, another
antibody labeled with a specific enzyme (such as peroxidase) is added and incubated
for its binding to the antigen to form a sandwich (antibody–antigen–antibody–enzyme).
The unbound enzyme-linked antibody is then removed. The sandwich
complex is finally detected by adding a chromogenic substrate specific for the
enzyme (such as 4-chloro-1-naphthol for peroxidase), incubating for a specified
time, and adding an enzyme inactivator to stop the reaction. The intensity of color
development can then be measured to identify the presence of a specific pathogen
or toxin. Instead of a chromogenic substrate, a specific fluorogenic substrate (such
as 3-p-hydroxyphenyl propionic acid for peroxidase) can be used and the reaction
can be measured with a fluorimeter. Automated systems for the ELISA test are
available.
5. Magnetic Immunobeads Method
The objective of the method is to immunocapture the cells (antigen) of a specific
pathogen present in a food suspension with the help of magnetic immunobeads. The
immunobeads are plastic-covered ferrous beads coated with the specific antibody.
When the beads are mixed with a sample, they capture the specific antigen (cells).
The immunocaptured beads are then removed by a magnet and tested by specific
methods for the presence of a target pathogen, such as Lis. monocytogenes.
B. Nucleic Acid Probe for Detection of Pathogens
1. Hybridization Method
In this method, a DNA probe consisting of a 20- to 4000-nucleotide base sequence
unique to a group of similar pathogens, such as Salmonella spp., is prepared. The
unique sequence can be identified in the DNA or rRNA in the cells or from the
amino acid sequence of a toxin produced by the bacterial group. The DNA probe
can then be obtained from the cell DNA or synthesized from the nucleotides. The
probes are radiolabeled with 32 P if, after hybridization, autoradiography is used for
their detection. In the nonisotopic colorimetric method, an enzyme (e.g., peroxidase)
is bound to a specific protein (e.g., streptavidin), which in turn binds to specific
ligands (e.g., biotin) on the DNA probe and can be used to produce a color reaction
to aid in measurement.
Both isotopic and nonisotopic DNA probes specific for Salmonella and Lis.
monocytogenes are commercially available and approved for use by regulatory
agencies. DNA probes for toxigenic Esc. coli and Yer. enterocolitica are also available.
The commercial companies provide step-by-step procedures for each technique.
In general, there is an initial preenrichment step, followed by an enrichment step
(which can be 6 h or more) to facilitate growth of target bacteria; lysis of cells;