258 Modern Food Microbiology
grown on the duplicate plate are lysed directly on the membrane to release nucleic acid and to convert
DNA into single strands. Some of the DNA is transferred to nitrocellulose filters, where hybridization is
carried out by applying a labeled DNA or RNA probe. A modification of the traditional DNA colony hybridization technique has been made such that 60 filters with up to 48 organisms per filter can be used.
107
The colony hybridization method developed by Grunstein and Hogness
71 ) has been employed
successfully to detect Listeria monocytogenes, enterotoxigenic E. coli, and Yersinia enterocolitica.
In one study, synthetic polynucleotide probes were constructed that were homologous to a region of
the ST enterotoxin gene of E. coli and applied for the detection of strains produced by DNA colony
hybridization.
80 For the latter, colonies were placed on paper filters to free and denature cellular DNA,
hybridized overnight at 40
◦ C, and exposed to autoradiograms. By this procedure, as few as 10
5 STproducing cells could be detected. In an earlier study from the same laboratory, colony hybridization
was used to detect E. coli in artificially contaminated food without enrichment, and the method could
detect 100–1,000 cells per gram, or about 1–10 cells per filter.
79 More information on nucleic acid
probes can be obtained from the review by Wolcott.
225
Polymerase Chain Reaction
This method is fast becoming the most widely used of all molecular genetic methods for detecting
and identifying bacteria and viruses in foods. Its increasing use is due to its high sensitivity, specificity,
its availability in a number of formats, and the commercial availability of PCR-based methods in kitlike formats.
This technique, first outlined in 1971 by Kleppe et al.
112 is applicable more to the identification
of foodborne organisms than to their enumeration. The currently used methodology is that further
developed by scientists at the Perkin Elmer-Cetus Corp.
177,197 among others. For his efforts in the
development of PCR, K.B. Mullis was co-winner of the Nobel Prize in chemistry in 1993.
A general outline of a PCR test is as follows. When the starting genomic material is dsDNA, it is
heated to ca. 95
◦ C to separate the strands. When the starting material is RNA (e.g. RNA viruses), it
is converted to dsDNA by use of reverse transcriptase (RT-PCR). After heating to separate the DNA
strands, they are cooled to ca. 55
◦ C in the presence of oligonucleotide primers, which anneal to the single DNA strands. DNA polymerase plus dATP, dCTP, dTTP, and dGTP are added resulting in the synthesis of complementary strands (the “d” is deoxynucleotide; A = adenine; TP = triphosphate, etc. for
the other bases. These are precursors of DNA synthesis). When this process is repeated, the two strands
become four, the four become eight, and so on for each additional cycle, resulting in several million
copies of the original if enough cycles are run. Among commercially available kits are the following:
BAX system (Qualicon, Dupont Corp.)
Probelia (Sanofi Diagnostics Pasteur)
Foodproof (Biotecon Diagnostics)
AG-9600 Amplisensor Analyzer is an automated fluorescence-based system for detecting PCR
products. BAX is the oldest of these detection systems and its application, along with that of other
PCR-based methods, is among the synopses presented below.
1. Multiplex PCR
a. For Escherichia coli 0157:H7, the primers used were: hly 933 k, fliC h7 , stx 1, stx 2, eaeA. Could
detect ≤1 cfu/g with results obtained in 24 hours. Food and bovine fecal specimen used.
65
grown on the duplicate plate are lysed directly on the membrane to release nucleic acid and to convert
DNA into single strands. Some of the DNA is transferred to nitrocellulose filters, where hybridization is
carried out by applying a labeled DNA or RNA probe. A modification of the traditional DNA colony hybridization technique has been made such that 60 filters with up to 48 organisms per filter can be used.
107
The colony hybridization method developed by Grunstein and Hogness
71 ) has been employed
successfully to detect Listeria monocytogenes, enterotoxigenic E. coli, and Yersinia enterocolitica.
In one study, synthetic polynucleotide probes were constructed that were homologous to a region of
the ST enterotoxin gene of E. coli and applied for the detection of strains produced by DNA colony
hybridization.
80 For the latter, colonies were placed on paper filters to free and denature cellular DNA,
hybridized overnight at 40
◦ C, and exposed to autoradiograms. By this procedure, as few as 10
5 STproducing cells could be detected. In an earlier study from the same laboratory, colony hybridization
was used to detect E. coli in artificially contaminated food without enrichment, and the method could
detect 100–1,000 cells per gram, or about 1–10 cells per filter.
79 More information on nucleic acid
probes can be obtained from the review by Wolcott.
225
Polymerase Chain Reaction
This method is fast becoming the most widely used of all molecular genetic methods for detecting
and identifying bacteria and viruses in foods. Its increasing use is due to its high sensitivity, specificity,
its availability in a number of formats, and the commercial availability of PCR-based methods in kitlike formats.
This technique, first outlined in 1971 by Kleppe et al.
112 is applicable more to the identification
of foodborne organisms than to their enumeration. The currently used methodology is that further
developed by scientists at the Perkin Elmer-Cetus Corp.
177,197 among others. For his efforts in the
development of PCR, K.B. Mullis was co-winner of the Nobel Prize in chemistry in 1993.
A general outline of a PCR test is as follows. When the starting genomic material is dsDNA, it is
heated to ca. 95
◦ C to separate the strands. When the starting material is RNA (e.g. RNA viruses), it
is converted to dsDNA by use of reverse transcriptase (RT-PCR). After heating to separate the DNA
strands, they are cooled to ca. 55
◦ C in the presence of oligonucleotide primers, which anneal to the single DNA strands. DNA polymerase plus dATP, dCTP, dTTP, and dGTP are added resulting in the synthesis of complementary strands (the “d” is deoxynucleotide; A = adenine; TP = triphosphate, etc. for
the other bases. These are precursors of DNA synthesis). When this process is repeated, the two strands
become four, the four become eight, and so on for each additional cycle, resulting in several million
copies of the original if enough cycles are run. Among commercially available kits are the following:
BAX system (Qualicon, Dupont Corp.)
Probelia (Sanofi Diagnostics Pasteur)
Foodproof (Biotecon Diagnostics)
AG-9600 Amplisensor Analyzer is an automated fluorescence-based system for detecting PCR
products. BAX is the oldest of these detection systems and its application, along with that of other
PCR-based methods, is among the synopses presented below.
1. Multiplex PCR
a. For Escherichia coli 0157:H7, the primers used were: hly 933 k, fliC h7 , stx 1, stx 2, eaeA. Could
detect ≤1 cfu/g with results obtained in 24 hours. Food and bovine fecal specimen used.
65