Chemical, Biological, and Physical Methods
261
enrichment, the method could detect 2.5 cells of Salmonella serovars and 1 L. monocytogenes cell.
103
Twenty-nine salmonellae and 18 L. monocytogenes strains were employed. qPCR has been also used to
simultaneously detect stx1 and stx2 genes of E. coli.
99 A qPCR in which the SYBR Green I fluorescent
dye was used was developed for the identification of Vibrio vulnificus in oyster tissue homogenates
and Gulf waters.
155 After a 5-hour enrichment, the method detected one cell. Without enrichment, 10
2
cells could be detected in 1 g of oyster homogenate or 10 ml of Gulf water. The method targeted the
hemolysin specific gene, vvh. The entire assay could be completed within 8 hours.
Lux Gene Luminescence
Luminescence in marine bacteria such as Vibrio fischeri and V. harveyi is controlled by genes, and
the capacity to produce luminescence can be transferred to other organisms by effecting the transfer
of some of these genes. The primary genes (designated lux) for luciferase are lux A and lux B. The
former encodes the synthesis of the luciferase α-subunit and the latter the β-subunit. The other eight
genes in the bioluminescence operon of the organisms noted do not need to be transferred. In the
food microbiology application of lux phages, one starts with bacteriophages that are specific for the
bacterium of interest and thus takes advantage of the highly specific relationship that exists between
phages and their hosts (see the section on Bacteriophage Typing later in this chapter and the section on
bacteriophages in Chapter 20). If Y. enterocolitica is the bacterium of interest, one selects a phage that
will infect the widest range of strains and yet not infect closely related species. To this phage, the lux
genes are inserted by recombination methods, which amounts to about 2 kb of DNA. By themselves,
these transduced phages are not luminous because they lack all components necessary to produce light.
When added to their specific host bacteria, the lux gene-bearing phages enter and multiply, and thus
cause the host cells to luminesce by the increased production of more lux genes. The light-emitting
reaction requires the components in the following equation:
FMNH 2 + RCHO + O 2 − −−−− →
luciferase FMN + RCOOH + H 2 O + light
where FMNH 2 is reduced flavin mononucleotide and RCHO is a long-chain aliphatic aldehyde such
as dodecanal. The emitted light can be measured by luminometry as in the ATP assay. Time for results
depends on the time required for the phage to enter host cells and begin their multiplication phase;
this is typically 30–50 minutes.
The addition of lux genes to a phage genome was first described by Ulitzur and Kuhn
212 who
showed that as few as ten E. coli cells could be detected within 10 minutes. The on-line method for
the enteric bacteria in swabs from a meat-processing plant could detect 10
4 cfu/g of cm
2 .
114 A number
of studies have shown that around 100 salmonellae can be detected in about 1 hour. As few as one
S. Typhimurium cell/100 ml of water could be detected within 24 hours in one study using an MPN
method.
211 The lux gene methodology can be adapted to the detection of a wide range of bacteria
in foods by the direct addition of phage constructs. Where the initial numbers are low, enrichments
are necessary. The method does not lend itself well to Gram-positive bacteria, as light emission is
typically 100-fold less than that for Gram negatives.
195
A broad host-range reporter bacteriophage for Listeria monocytogenes has been constructed that
caries the Vibrio harveyi LuxAB protein.
130 After a 2-hour incubation, as few as 5 × 10
2 to 10
3 cells/ml
could be detected with a single-tube luminometer following an enrichment step. Less than one cell of
L. monocytogenes/g of artificially contaminated salad could be identified.
130 In meat and soft cheese,
as few as 10 cells/g could be detected. Of 348 natural food and environmental samples tested, 55 were
found positive by the lux-phage method compared to 57 by a plating method.
129 The lux-phage method
261
enrichment, the method could detect 2.5 cells of Salmonella serovars and 1 L. monocytogenes cell.
103
Twenty-nine salmonellae and 18 L. monocytogenes strains were employed. qPCR has been also used to
simultaneously detect stx1 and stx2 genes of E. coli.
99 A qPCR in which the SYBR Green I fluorescent
dye was used was developed for the identification of Vibrio vulnificus in oyster tissue homogenates
and Gulf waters.
155 After a 5-hour enrichment, the method detected one cell. Without enrichment, 10
2
cells could be detected in 1 g of oyster homogenate or 10 ml of Gulf water. The method targeted the
hemolysin specific gene, vvh. The entire assay could be completed within 8 hours.
Lux Gene Luminescence
Luminescence in marine bacteria such as Vibrio fischeri and V. harveyi is controlled by genes, and
the capacity to produce luminescence can be transferred to other organisms by effecting the transfer
of some of these genes. The primary genes (designated lux) for luciferase are lux A and lux B. The
former encodes the synthesis of the luciferase α-subunit and the latter the β-subunit. The other eight
genes in the bioluminescence operon of the organisms noted do not need to be transferred. In the
food microbiology application of lux phages, one starts with bacteriophages that are specific for the
bacterium of interest and thus takes advantage of the highly specific relationship that exists between
phages and their hosts (see the section on Bacteriophage Typing later in this chapter and the section on
bacteriophages in Chapter 20). If Y. enterocolitica is the bacterium of interest, one selects a phage that
will infect the widest range of strains and yet not infect closely related species. To this phage, the lux
genes are inserted by recombination methods, which amounts to about 2 kb of DNA. By themselves,
these transduced phages are not luminous because they lack all components necessary to produce light.
When added to their specific host bacteria, the lux gene-bearing phages enter and multiply, and thus
cause the host cells to luminesce by the increased production of more lux genes. The light-emitting
reaction requires the components in the following equation:
FMNH 2 + RCHO + O 2 − −−−− →
luciferase FMN + RCOOH + H 2 O + light
where FMNH 2 is reduced flavin mononucleotide and RCHO is a long-chain aliphatic aldehyde such
as dodecanal. The emitted light can be measured by luminometry as in the ATP assay. Time for results
depends on the time required for the phage to enter host cells and begin their multiplication phase;
this is typically 30–50 minutes.
The addition of lux genes to a phage genome was first described by Ulitzur and Kuhn
212 who
showed that as few as ten E. coli cells could be detected within 10 minutes. The on-line method for
the enteric bacteria in swabs from a meat-processing plant could detect 10
4 cfu/g of cm
2 .
114 A number
of studies have shown that around 100 salmonellae can be detected in about 1 hour. As few as one
S. Typhimurium cell/100 ml of water could be detected within 24 hours in one study using an MPN
method.
211 The lux gene methodology can be adapted to the detection of a wide range of bacteria
in foods by the direct addition of phage constructs. Where the initial numbers are low, enrichments
are necessary. The method does not lend itself well to Gram-positive bacteria, as light emission is
typically 100-fold less than that for Gram negatives.
195
A broad host-range reporter bacteriophage for Listeria monocytogenes has been constructed that
caries the Vibrio harveyi LuxAB protein.
130 After a 2-hour incubation, as few as 5 × 10
2 to 10
3 cells/ml
could be detected with a single-tube luminometer following an enrichment step. Less than one cell of
L. monocytogenes/g of artificially contaminated salad could be identified.
130 In meat and soft cheese,
as few as 10 cells/g could be detected. Of 348 natural food and environmental samples tested, 55 were
found positive by the lux-phage method compared to 57 by a plating method.
129 The lux-phage method