Conventional, Molecular, and Biosensor-Based Detection Methods ◾ 549
the gene products in the form of light, fluorescence, or β-galactosidase; enzymes are made, which
are detected by a luminometer, fluorimeter, or spectrophotometer. Bacteriophages that are unable
to infect a host bacterium will not produce any signal because they do not have the required
machinery for gene expression. This approach is very efficient and was used for detection of low
concentrations of Esc. coli O157:H7, Salmonella, Lis. monocytogenes, Staphylococcus aureus, Bacillus
anthracis, Yersinia pestis, and Mycobacterium tuberculosis. The detection sensitivity was reported to
be 10 2 –10 6 cells/mL, and the assay time is only one to three hours for most pathogens; however, it
may take longer for slow-growing bacteria like Mycobacterium.
Phages for Capture and Detection Using Biosensors
Intact bacteriophages or bacteriophage tail fiber proteins (endolysin) that bind to the host bacterial surface during infection are used to capture bacterial pathogens on paramagnetic beads or on
the biosensor surface for detection. The tail fiber proteins, also called receptor binding proteins
(RBPs), immobilized on paramagnetic beads are used to capture pathogens from food suspensions
and are detected by plating on selective agar media plate or by using PCR (see below).
RBPs are also used as a substitute for antibodies to capture the target pathogen on biosensor
platforms, such as surface plasmon resonance sensor (SPR), quartz crystal microbalance (QCM),
etc. (see biosensor methods below).
Bioluminescence Methods
A bioluminescence method measures the ATP content in a sample as an indirect measurement of
microbial load. As only the viable cells retain ATP, the amount of ATP is regarded as directly related
to the microbial load in the sample. Using the luciferin-luciferase (from firefly) system in the presence of Mg 2+ , the ATP concentration in the lysed cells in a sample is measured. This method can
detect as low as 10 2–3 viable bacterial cells and about 10 yeast cells per gram or milliliter of a food. It
can also be used to determine the microbial population on equipment surfaces. The method is very
rapid, and several automated systems with methods to use them are now commercially available.
In another method, the genes encoding bacterial bioluminescence (lux gene) from luminous bacteria (e.g., Vibrio fischeri) have been cloned in some pathogenic, indicator, and spoilage bacteria important
in food. The bacterial luciferase catalyzes oxidation of a reduced flavin mononucleotide (FMNH 2 ) and
a long-chain aliphatic aldehyde by molecular O 2 and emits light. As only live cells can produce light, the
bacterial strains containing the cloned lux gene can be used to determine the effectiveness of methods
employed to kill cells or to remove them from food and food environments very rapidly. 18 It is also being
used to detect injured bacteria incapable of producing light but that are able to do so following repair of
injury. Automated systems to measure light from the reaction are now commercially available.
Many other rapid methods, automated systems, and miniatures of traditional methods are
now commercially available. Some have been discussed in more detail in the references included
and can be consulted.
Nucleic Acid-Based Methods
Hybridization Method 19,20
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
the gene products in the form of light, fluorescence, or β-galactosidase; enzymes are made, which
are detected by a luminometer, fluorimeter, or spectrophotometer. Bacteriophages that are unable
to infect a host bacterium will not produce any signal because they do not have the required
machinery for gene expression. This approach is very efficient and was used for detection of low
concentrations of Esc. coli O157:H7, Salmonella, Lis. monocytogenes, Staphylococcus aureus, Bacillus
anthracis, Yersinia pestis, and Mycobacterium tuberculosis. The detection sensitivity was reported to
be 10 2 –10 6 cells/mL, and the assay time is only one to three hours for most pathogens; however, it
may take longer for slow-growing bacteria like Mycobacterium.
Phages for Capture and Detection Using Biosensors
Intact bacteriophages or bacteriophage tail fiber proteins (endolysin) that bind to the host bacterial surface during infection are used to capture bacterial pathogens on paramagnetic beads or on
the biosensor surface for detection. The tail fiber proteins, also called receptor binding proteins
(RBPs), immobilized on paramagnetic beads are used to capture pathogens from food suspensions
and are detected by plating on selective agar media plate or by using PCR (see below).
RBPs are also used as a substitute for antibodies to capture the target pathogen on biosensor
platforms, such as surface plasmon resonance sensor (SPR), quartz crystal microbalance (QCM),
etc. (see biosensor methods below).
Bioluminescence Methods
A bioluminescence method measures the ATP content in a sample as an indirect measurement of
microbial load. As only the viable cells retain ATP, the amount of ATP is regarded as directly related
to the microbial load in the sample. Using the luciferin-luciferase (from firefly) system in the presence of Mg 2+ , the ATP concentration in the lysed cells in a sample is measured. This method can
detect as low as 10 2–3 viable bacterial cells and about 10 yeast cells per gram or milliliter of a food. It
can also be used to determine the microbial population on equipment surfaces. The method is very
rapid, and several automated systems with methods to use them are now commercially available.
In another method, the genes encoding bacterial bioluminescence (lux gene) from luminous bacteria (e.g., Vibrio fischeri) have been cloned in some pathogenic, indicator, and spoilage bacteria important
in food. The bacterial luciferase catalyzes oxidation of a reduced flavin mononucleotide (FMNH 2 ) and
a long-chain aliphatic aldehyde by molecular O 2 and emits light. As only live cells can produce light, the
bacterial strains containing the cloned lux gene can be used to determine the effectiveness of methods
employed to kill cells or to remove them from food and food environments very rapidly. 18 It is also being
used to detect injured bacteria incapable of producing light but that are able to do so following repair of
injury. Automated systems to measure light from the reaction are now commercially available.
Many other rapid methods, automated systems, and miniatures of traditional methods are
now commercially available. Some have been discussed in more detail in the references included
and can be consulted.
Nucleic Acid-Based Methods
Hybridization Method 19,20
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