Conventional, Molecular, and Biosensor-Based Detection Methods ◾ 553
The DNA microarray has been used to detect Lis. monocytogenes, Sal. enterica, Esc. coli O157:H7,
and Cam. jejuni individually or in combinations from several naturally contaminated fresh meat
samples. Mass-spectrometry, such as matrix-assisted laser desorption/ionization (MALDI) time of
flight mass spectrometry (TOF MS), generates a spectral signature for individual pathogens based
on protein profiles and can be applied to detect multiple pathogens. This system also requires isolated pure cultures from food samples for analysis.
Pathogenicity Assay 26–28
Laboratory rodents, such as mice, guinea pigs, rats, and rabbits, are widely used models to study the
virulent property of foodborne pathogens. To determine whether a food isolate is pathogenic, an
infectious dose or a lethal dose is assessed in animal models. Animal models, though ideal for determination of a virulence property, is very expensive and may not be suitable for routine use. Moreover,
for ethical reasons, their use sometimes is discouraged. Alternatively, mammalian cells have been
routinely used for analysis of the pathogenic potential of foodborne pathogens. Various cancerous
immortal cell lines that are used for pathogen testing are currently available. Not all cell lines are sensitive to a pathogen; thus one has to select a cell line that has been shown to be sensitive. For example,
the pathogenic potential of Lis. monocytogenes has been determined using enterocyte-like Caco-2,
macrophage J774, fibroblasts, and hybrid lymphocyte Ped-2E9 cell lines. Vero cells (monkey kidney
cell line) have been used for Shiga toxin–producing Esc. coli cultures. A Chinese hamster ovary (CHO)
cell line and lymphocyte Ped-2E9 cells have been used for detection of Bac. cereus toxins.
Cytopathogenic effects are measured by various methods. Light and electron microscopy
have been used for qualitative analysis of cytopathic effects for characteristic membrane damage,
cell lysis, clumping, elongation, vacuole formation, flocculation, and loss of surface molecules.
Quantitative cytotoxicity assays, such as the Trypan blue exclusion test, allow staining of dead
cells (blue appearance) while viable cells with intact membranes exclude stain. Dead or viable cells
are then counted under a microscope for determination of the cytotoxicity effect. The ALP release
assay, lactate dehydrogenase release assay, and MTT (3-[4,5-dimethyl thiazolyl-2]-2, 5-diphenyltetrazolium bromide) assay (metabolic staining) are also used for determination of cytotoxicity.
Furthermore, adhesion, invasion, plaque formation, and apoptosis analysis are also used to determine the cellular mechanism of pathogenesis for foodborne pathogenic microorganisms.
Biosensors for Pathogen Detection 11,13,14,28–31
Biosensor-based tools continue to capture the imaginations of researchers for their potential in
sensitive detection of pathogens in automated or semiautomated instruments in near real time.
Biosensor-based technologies are increasingly being used in methods to detect foodborne pathogens
as an alternative to conventional methods. Biosensors are devices that detect biological or chemical
complexes in the form of antigen—antibody, enzyme—substrate, or receptor—ligand, which are
placed in close proximity to a transducer that generates an electrical, optical, or mass-change signal.
Pathogen detection by biosensor tools is centered on four basic physiological or genetic properties of microorganisms: substrate utilization, phenotypic expression analysis of virulence or
physiological or structural markers by antibodies, genetic analysis, and interaction of pathogens
with eukaryotic cells (cytopathogenic effects). Many current popular commercially available rapid
methods use conventional culture-based methods coupled with automated or semiautomated
nucleic acid–based, antibody-, or substrate utilization–based methods to obtain results in 24–72
The DNA microarray has been used to detect Lis. monocytogenes, Sal. enterica, Esc. coli O157:H7,
and Cam. jejuni individually or in combinations from several naturally contaminated fresh meat
samples. Mass-spectrometry, such as matrix-assisted laser desorption/ionization (MALDI) time of
flight mass spectrometry (TOF MS), generates a spectral signature for individual pathogens based
on protein profiles and can be applied to detect multiple pathogens. This system also requires isolated pure cultures from food samples for analysis.
Pathogenicity Assay 26–28
Laboratory rodents, such as mice, guinea pigs, rats, and rabbits, are widely used models to study the
virulent property of foodborne pathogens. To determine whether a food isolate is pathogenic, an
infectious dose or a lethal dose is assessed in animal models. Animal models, though ideal for determination of a virulence property, is very expensive and may not be suitable for routine use. Moreover,
for ethical reasons, their use sometimes is discouraged. Alternatively, mammalian cells have been
routinely used for analysis of the pathogenic potential of foodborne pathogens. Various cancerous
immortal cell lines that are used for pathogen testing are currently available. Not all cell lines are sensitive to a pathogen; thus one has to select a cell line that has been shown to be sensitive. For example,
the pathogenic potential of Lis. monocytogenes has been determined using enterocyte-like Caco-2,
macrophage J774, fibroblasts, and hybrid lymphocyte Ped-2E9 cell lines. Vero cells (monkey kidney
cell line) have been used for Shiga toxin–producing Esc. coli cultures. A Chinese hamster ovary (CHO)
cell line and lymphocyte Ped-2E9 cells have been used for detection of Bac. cereus toxins.
Cytopathogenic effects are measured by various methods. Light and electron microscopy
have been used for qualitative analysis of cytopathic effects for characteristic membrane damage,
cell lysis, clumping, elongation, vacuole formation, flocculation, and loss of surface molecules.
Quantitative cytotoxicity assays, such as the Trypan blue exclusion test, allow staining of dead
cells (blue appearance) while viable cells with intact membranes exclude stain. Dead or viable cells
are then counted under a microscope for determination of the cytotoxicity effect. The ALP release
assay, lactate dehydrogenase release assay, and MTT (3-[4,5-dimethyl thiazolyl-2]-2, 5-diphenyltetrazolium bromide) assay (metabolic staining) are also used for determination of cytotoxicity.
Furthermore, adhesion, invasion, plaque formation, and apoptosis analysis are also used to determine the cellular mechanism of pathogenesis for foodborne pathogenic microorganisms.
Biosensors for Pathogen Detection 11,13,14,28–31
Biosensor-based tools continue to capture the imaginations of researchers for their potential in
sensitive detection of pathogens in automated or semiautomated instruments in near real time.
Biosensor-based technologies are increasingly being used in methods to detect foodborne pathogens
as an alternative to conventional methods. Biosensors are devices that detect biological or chemical
complexes in the form of antigen—antibody, enzyme—substrate, or receptor—ligand, which are
placed in close proximity to a transducer that generates an electrical, optical, or mass-change signal.
Pathogen detection by biosensor tools is centered on four basic physiological or genetic properties of microorganisms: substrate utilization, phenotypic expression analysis of virulence or
physiological or structural markers by antibodies, genetic analysis, and interaction of pathogens
with eukaryotic cells (cytopathogenic effects). Many current popular commercially available rapid
methods use conventional culture-based methods coupled with automated or semiautomated
nucleic acid–based, antibody-, or substrate utilization–based methods to obtain results in 24–72