550 ◾ Fundamental Food Microbiology
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, after hybridization, if
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 the 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, it involves an initial preenrichment step followed by an
enrichment step (which can be just six hours or more) to facilitate growth of target bacteria; then
lysis of cells; hybridization of the DNA probe with cell DNA or rRNA; capture of the hybridized
DNA containing the probe; and measurement of either radioactivity or color production, depending upon the probe (e.g., if it is isotopic or nonisotopic). The methods are specific and relatively
more rapid than the traditional methods.
Polymerase Chain Reaction (PCR) 21–23
The most widely used DNA-based method is the PCR. A gene segment specific for a pathogen
is amplified several-fold by using a pair of specific primers (a small piece of DNA of about 20
base pair long). PCR steps include denaturation of double-stranded DNA, primer annealing,
elongation of strands with a heat-stable Taq polymerase, and amplification (Figure 42.5). After
amplification for 30–35 cycles, a single gene can be amplified to millions of copies, which are
then separated in an agarose gel for visual identification. Multiplex PCR has been developed
to increase the specificity of an assay by targeting multiple genes in a single reaction tube. The
method has been developed for the detection of pathogens, such as Lis. monocytogenes, Sal.
enterica, pathogenic Esc. coli, Campylobacter, Yer. enterocolitica, Sta. aureus, Clo. botulinum, Bac.
cereus, and Vib. vulnificus.
A real-time quantitative PCR (Q-PCR) is becoming increasingly popular because it has
some degree of automation. In this system, a fluorescent dye-labeled probe is allowed to hybridize with the target DNA; as the primer extension takes place, the endonuclease activity of the
Taq DNA polymerase removes the probe, and the reporter dye is released, and the amplified
PCR products are quantified by a built-in laser detector (Figure 42.6). The probe (~100 bp)
is labeled with both a fluorescent reporter (R) and a quencher (Q) dye, and because of their
proximate association, fluorescent activity is quenched under normal conditions. When the
reporter molecule is physically removed from the quencher molecule, such as during primer
extension (resulting from endonuclease activity), there is a fluorescent signal in the well. One
of the major drawbacks of these PCR-based methods is that they cannot differentiate live cells
from dead cells. To overcome this problem, a reverse transcriptase (RT)-PCR method has been
developed in which messenger RNA (mRNA) that is transcribed only in the viable cells is used
for amplification. Complementary DNA (cDNA) is synthesized from mRNA using a reverse
transcriptase enzyme and then amplified using a standard PCR method. RT-PCR is also used
to detect RNA viruses, such as Norovirus, rotavirus, and some other enteric viruses, responsible
for food poisoning.
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, after hybridization, if
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 the 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, it involves an initial preenrichment step followed by an
enrichment step (which can be just six hours or more) to facilitate growth of target bacteria; then
lysis of cells; hybridization of the DNA probe with cell DNA or rRNA; capture of the hybridized
DNA containing the probe; and measurement of either radioactivity or color production, depending upon the probe (e.g., if it is isotopic or nonisotopic). The methods are specific and relatively
more rapid than the traditional methods.
Polymerase Chain Reaction (PCR) 21–23
The most widely used DNA-based method is the PCR. A gene segment specific for a pathogen
is amplified several-fold by using a pair of specific primers (a small piece of DNA of about 20
base pair long). PCR steps include denaturation of double-stranded DNA, primer annealing,
elongation of strands with a heat-stable Taq polymerase, and amplification (Figure 42.5). After
amplification for 30–35 cycles, a single gene can be amplified to millions of copies, which are
then separated in an agarose gel for visual identification. Multiplex PCR has been developed
to increase the specificity of an assay by targeting multiple genes in a single reaction tube. The
method has been developed for the detection of pathogens, such as Lis. monocytogenes, Sal.
enterica, pathogenic Esc. coli, Campylobacter, Yer. enterocolitica, Sta. aureus, Clo. botulinum, Bac.
cereus, and Vib. vulnificus.
A real-time quantitative PCR (Q-PCR) is becoming increasingly popular because it has
some degree of automation. In this system, a fluorescent dye-labeled probe is allowed to hybridize with the target DNA; as the primer extension takes place, the endonuclease activity of the
Taq DNA polymerase removes the probe, and the reporter dye is released, and the amplified
PCR products are quantified by a built-in laser detector (Figure 42.6). The probe (~100 bp)
is labeled with both a fluorescent reporter (R) and a quencher (Q) dye, and because of their
proximate association, fluorescent activity is quenched under normal conditions. When the
reporter molecule is physically removed from the quencher molecule, such as during primer
extension (resulting from endonuclease activity), there is a fluorescent signal in the well. One
of the major drawbacks of these PCR-based methods is that they cannot differentiate live cells
from dead cells. To overcome this problem, a reverse transcriptase (RT)-PCR method has been
developed in which messenger RNA (mRNA) that is transcribed only in the viable cells is used
for amplification. Complementary DNA (cDNA) is synthesized from mRNA using a reverse
transcriptase enzyme and then amplified using a standard PCR method. RT-PCR is also used
to detect RNA viruses, such as Norovirus, rotavirus, and some other enteric viruses, responsible
for food poisoning.