266 Modern Food Microbiology
Restriction Enzyme Analysis
By this method, chromosomal DNA of test strains is digested by use of an appropriate restriction
endonuclease. The latter class of enzymes makes double-stranded breaks in DNA at specific nucleotide
sequences. One of the most widely used restriction endonucleases is EcoRI (obtained from Escherichia
coli), which recognizes the DNA base sequence GAATTC and cleaves between GA. Another endonuclease is HhaI (obtained from Haemophilus influenzae), and it recognizes the sequence GTPyPuAC
(Py = any pyrimidine, Pu = any purine base). The cleavage site for HhaI is between PyPu, and it has
been found to be of value in studying the epidemiology of L. monocytogenes.
223
After some L. monocytogenes serovar 4b strains associated with three food-associated outbreaks
were subjected to restriction enzyme analysis (REA) using HhaI, the method was found to be valuable
as both a taxonomic tool and an epidemiologic tracer.
222 Of 32 isolates associated with the 1981 outbreak in Nova Scotia, Canada, 29 showed restriction enzyme patterns identical to the strain recovered
from coleslaw. Also, the patterns of nine clinical isolates from the 1983 Boston cases were identical
to each other. Some of the isolates from the 1985 California outbreak were subjected to REA, and
those examined from patients, suspect cheese samples, and cheese factory environmental samples
were found to be identical.
222
The combined use of REA and PCR for subtyping of L. monocytogenes has been presented.
51
Employing 133 strains of serovar 4b from a variety of sources along with 22 other serovars, PCR-REA
divided the strains into two groups, I and II, with the former containing 37 and the latter 96 strains.
Seventy-four of the 4b serovars belonged to phagovar 2389:2425: 3274:2671:47:108:340, and all fell
in the same group, II, when cleaved with the nuclease AluI.
Random Amplification of Polymorphic DNA
In essence, random amplification of polymorphic DNA (RAPD) employs the use of PCR to obtain
randomly amplified polymorphic DNA electrophoretic profiles. Briefly, cells are harvested, suspended
in water, and lysed for their DNA. The DNA, along with a specific primer such as a 10-mer (10-bp
section), is mixed with DNA and Taq polymerase. PCR is carried out at varying temperatures for 40
or more cycles, followed by electrophoresis of the products on an agarose gel. Following staining of
gel (typically with ethidium bromide), the bands are photographed and analyzed. Purified genomic
DNA is not needed for RAPD, nor is there a need for prior sequence data. By using a capillary air
thermal cycler, which was able to complete 30 cycles in <1.0 hour, RAPD results could be obtained
in 3 hours starting with colony growth.
18
RAPD analysis has been used to fingerprint outbreak strains of L. monocytogenes by a number
of investigators. When 289 strains from a poultry-processing environment were subjected to RAPD
using a 10-mer primer, 18 profiles were identified with 64% of strains displaying a single profile.
121
Using the same 10-mer primer, 29 strains of L. monocytogenes from raw milk yielded seven profiles,
which were specific for milk isolates.
122 In the latter study, RAPD in combination with serotyping
allowed for a higher level of differentiation than either alone. RAPD was found to be more rapid and
less labor-intensive than restriction fragment length polymorphism, and pure DNA was not needed.
122
When RAPD analysis was compared to phage typing on 104 strains of L. monocytogenes from
six different outbreaks, a 98% agreement was found and RAPD was suggested as an alternative to
phage typing.
135 RAPD was found to be far better than 16S rRNA sequence data in discriminating
between strains of L. monocytogenes, and it showed differentiation even in strains with the same 16S
Restriction Enzyme Analysis
By this method, chromosomal DNA of test strains is digested by use of an appropriate restriction
endonuclease. The latter class of enzymes makes double-stranded breaks in DNA at specific nucleotide
sequences. One of the most widely used restriction endonucleases is EcoRI (obtained from Escherichia
coli), which recognizes the DNA base sequence GAATTC and cleaves between GA. Another endonuclease is HhaI (obtained from Haemophilus influenzae), and it recognizes the sequence GTPyPuAC
(Py = any pyrimidine, Pu = any purine base). The cleavage site for HhaI is between PyPu, and it has
been found to be of value in studying the epidemiology of L. monocytogenes.
223
After some L. monocytogenes serovar 4b strains associated with three food-associated outbreaks
were subjected to restriction enzyme analysis (REA) using HhaI, the method was found to be valuable
as both a taxonomic tool and an epidemiologic tracer.
222 Of 32 isolates associated with the 1981 outbreak in Nova Scotia, Canada, 29 showed restriction enzyme patterns identical to the strain recovered
from coleslaw. Also, the patterns of nine clinical isolates from the 1983 Boston cases were identical
to each other. Some of the isolates from the 1985 California outbreak were subjected to REA, and
those examined from patients, suspect cheese samples, and cheese factory environmental samples
were found to be identical.
222
The combined use of REA and PCR for subtyping of L. monocytogenes has been presented.
51
Employing 133 strains of serovar 4b from a variety of sources along with 22 other serovars, PCR-REA
divided the strains into two groups, I and II, with the former containing 37 and the latter 96 strains.
Seventy-four of the 4b serovars belonged to phagovar 2389:2425: 3274:2671:47:108:340, and all fell
in the same group, II, when cleaved with the nuclease AluI.
Random Amplification of Polymorphic DNA
In essence, random amplification of polymorphic DNA (RAPD) employs the use of PCR to obtain
randomly amplified polymorphic DNA electrophoretic profiles. Briefly, cells are harvested, suspended
in water, and lysed for their DNA. The DNA, along with a specific primer such as a 10-mer (10-bp
section), is mixed with DNA and Taq polymerase. PCR is carried out at varying temperatures for 40
or more cycles, followed by electrophoresis of the products on an agarose gel. Following staining of
gel (typically with ethidium bromide), the bands are photographed and analyzed. Purified genomic
DNA is not needed for RAPD, nor is there a need for prior sequence data. By using a capillary air
thermal cycler, which was able to complete 30 cycles in <1.0 hour, RAPD results could be obtained
in 3 hours starting with colony growth.
18
RAPD analysis has been used to fingerprint outbreak strains of L. monocytogenes by a number
of investigators. When 289 strains from a poultry-processing environment were subjected to RAPD
using a 10-mer primer, 18 profiles were identified with 64% of strains displaying a single profile.
121
Using the same 10-mer primer, 29 strains of L. monocytogenes from raw milk yielded seven profiles,
which were specific for milk isolates.
122 In the latter study, RAPD in combination with serotyping
allowed for a higher level of differentiation than either alone. RAPD was found to be more rapid and
less labor-intensive than restriction fragment length polymorphism, and pure DNA was not needed.
122
When RAPD analysis was compared to phage typing on 104 strains of L. monocytogenes from
six different outbreaks, a 98% agreement was found and RAPD was suggested as an alternative to
phage typing.
135 RAPD was found to be far better than 16S rRNA sequence data in discriminating
between strains of L. monocytogenes, and it showed differentiation even in strains with the same 16S