Chemical, Biological, and Physical Methods
267
rRNA sequence.
39 Employing three 10-mer primers, 34 banding profiles were obtained with one of
the primers on 52 strains of L. monocytogenes representing five species.
56
Although RAPD amplification does not occur with starved and viable but nonculturable cells
(VBNC), both cell types can be detected by supplying starved cells with nutrients and resuscitation
of VBNC cells by temperature upshift.
217
In an epidemiologic study of L. monocytogenes, RAPD was one of five methods compared, and all
4b strains were distributed into two RAPD and four pulsed field gel electrophoresis (PFGE) types.
122
RAPD was one of the top three discriminating methods along with PFGE and ribotyping.
Pulsed Field Gel Electrophoresis
This method entails the digestion of genomic DNA by one or more restriction enzymes, separation
of the restriction fragments by field inversion electrophoresis, and resolution of fragments in agarose
gels. In contrast to conventional electrophoresis where a gel is run in one direction, PFGE is carried
out with pulse times ramped from 1 to 100 sec over varying periods of time, which is determined by
the sizes of molecules. The alternating electrical fields force molecules to change directions, and the
electrophoretic profiles are designated pulsovars. It has been used to fingerprint foodborne outbreak
strains of several pathogens.
Using two restriction enzymes (AscI and ApaI), 176 strains of L. monocytogenes and 22 other
listerial species/strains generated 87 genomically distinct groups, with ApaI generating the largest
number of bands.
23 In another study, 42 serovar 4b strains of L. monocytogenes were divided into
at least 24 different genomic varieties using one of three restriction enzymes.
24 Although all 42
cultures could be typed using PFGE, only 89% were phage typable.
24 When serovar 4b strains of L.
monocytogenes from 279 human listeriosis cases were subjected to PFGE (along with other methods),
34 pulsovars were obtained, with 89% being pulsovar 2/1/3, the human epidemic strain.
87 Using three
restriction enzymes, the strain of L. monocytogenes that caused the 1992 human outbreak in France
was shown to be genomically closely related to those that caused outbreaks in California, Denmark,
and Switzerland.
87
In addition to L. monocytogenes, PFGE has been employed on a number of other bacteria of
importance in foods. Outbreak and sporadic strains of E. coli O157:H7 involved in the 1994 Pacific
Northwest outbreak of hemorrhagic colitis were differentiated,
102 and the close relationship of the
0139 serogroup V. cholerae to the 01 E1 Tor biotype has been substantiated by PFGE.
75 Using SmaI
digests and PFGE, the genome sizes of three staphylococcal species were extrapolated.
67 In order to
trace the source of this organism on processed cold-smoked rainbow trout, Autio et al.
6 did PFGE
analyses on 303 isolates and found that those on the final product were associated with brining and
slicing, and that those associated with raw fish were not detected on the final product.
Restriction Fragment Length Polymorphism
DNA restriction fragment length polymorphisms (RFLP) are heritable differences in the lengths of
DNA fragments that arise when DNA is digested by a restriction endonuclease. In brief, cellular DNA is
digested with a restriction enzyme, separated by electrophoresis, followed by Southern blot hybridization with a DNA probe from a given gene library of the organism in question. Along with MEE, it was
used to demonstrate the recurrence of strains of L. monocytogenes in raw milk and nondairy foods.
77
267
rRNA sequence.
39 Employing three 10-mer primers, 34 banding profiles were obtained with one of
the primers on 52 strains of L. monocytogenes representing five species.
56
Although RAPD amplification does not occur with starved and viable but nonculturable cells
(VBNC), both cell types can be detected by supplying starved cells with nutrients and resuscitation
of VBNC cells by temperature upshift.
217
In an epidemiologic study of L. monocytogenes, RAPD was one of five methods compared, and all
4b strains were distributed into two RAPD and four pulsed field gel electrophoresis (PFGE) types.
122
RAPD was one of the top three discriminating methods along with PFGE and ribotyping.
Pulsed Field Gel Electrophoresis
This method entails the digestion of genomic DNA by one or more restriction enzymes, separation
of the restriction fragments by field inversion electrophoresis, and resolution of fragments in agarose
gels. In contrast to conventional electrophoresis where a gel is run in one direction, PFGE is carried
out with pulse times ramped from 1 to 100 sec over varying periods of time, which is determined by
the sizes of molecules. The alternating electrical fields force molecules to change directions, and the
electrophoretic profiles are designated pulsovars. It has been used to fingerprint foodborne outbreak
strains of several pathogens.
Using two restriction enzymes (AscI and ApaI), 176 strains of L. monocytogenes and 22 other
listerial species/strains generated 87 genomically distinct groups, with ApaI generating the largest
number of bands.
23 In another study, 42 serovar 4b strains of L. monocytogenes were divided into
at least 24 different genomic varieties using one of three restriction enzymes.
24 Although all 42
cultures could be typed using PFGE, only 89% were phage typable.
24 When serovar 4b strains of L.
monocytogenes from 279 human listeriosis cases were subjected to PFGE (along with other methods),
34 pulsovars were obtained, with 89% being pulsovar 2/1/3, the human epidemic strain.
87 Using three
restriction enzymes, the strain of L. monocytogenes that caused the 1992 human outbreak in France
was shown to be genomically closely related to those that caused outbreaks in California, Denmark,
and Switzerland.
87
In addition to L. monocytogenes, PFGE has been employed on a number of other bacteria of
importance in foods. Outbreak and sporadic strains of E. coli O157:H7 involved in the 1994 Pacific
Northwest outbreak of hemorrhagic colitis were differentiated,
102 and the close relationship of the
0139 serogroup V. cholerae to the 01 E1 Tor biotype has been substantiated by PFGE.
75 Using SmaI
digests and PFGE, the genome sizes of three staphylococcal species were extrapolated.
67 In order to
trace the source of this organism on processed cold-smoked rainbow trout, Autio et al.
6 did PFGE
analyses on 303 isolates and found that those on the final product were associated with brining and
slicing, and that those associated with raw fish were not detected on the final product.
Restriction Fragment Length Polymorphism
DNA restriction fragment length polymorphisms (RFLP) are heritable differences in the lengths of
DNA fragments that arise when DNA is digested by a restriction endonuclease. In brief, cellular DNA is
digested with a restriction enzyme, separated by electrophoresis, followed by Southern blot hybridization with a DNA probe from a given gene library of the organism in question. Along with MEE, it was
used to demonstrate the recurrence of strains of L. monocytogenes in raw milk and nondairy foods.
77