Chemical, Biological, and Physical Methods
265
Amplified Fragment Length Polymorphism
This is a PCR-based fingerprinting technique that requires small amounts of pure dsDNA—10–100
ng from 1–3 bacterial colonies.
89 The DNA is digested with two restriction enzymes (such as EcoRI
and MseI). Double-stranded oligonucleotide adaptors are applied, and an aliquot is subjected to 1- to
2-PCR amplifications under highly stringent conditions with adapter-specific primers. The primer
that spans the average frequency restriction site is fluorescently labeled. After subjecting to PAGE
(polyacrylamide gel electrophoresis), from 40 to 200 bands are obtained, and it has been found to be
more reproducible than RAPD.
19
In a study of 147 strains of bacteria from nine species, AFLP analysis clustered all strains within
their respective species.
89 The method was shown to produce results consistent with DNA homology
data when the genomic DNA of 98 Aeromonas strains were digested with ApaI and TaqI.
85 Using
an automated method with two restriction endonucleases, 25 Campylobacter isolates from poultry
farms in the Netherlands grouped in three C. jejuni clusters that were different from a C. coli cluster.
48
A fluorescent-AFLP (FAFLP) employs fluorophore-labeled primers, and it has been employed to
discriminate between 30 phage type 4 (PT 4) strains of S. Enteritidis from different sources. When
AFLP was used with XbaI, 73% of the PT 4 strains were classified as a single type.
43 However, when
FAFLP was employed using EcoRI + O and MseI + C, 23 profiles (with 1–61 amplified-fragment
differences) were obtained with a discriminatory power of 0.98 compared to 0.47 for PFGE.
43 Genetic
diversity of clinical and environmental isolates of Vibrio cholerae was investigated by using ApaI and
TaqI, and 20–30 bands each were generated from the genomic DNA of V. cholerae serotypes 01 and
0139 strains.
97 Of the 79 strains tested, 26 of 01 showed identical patterns and were represented by the
01 El Tor strain of the seventh pandemic. For more information on this technique, see reference 179.
Multilocus Enzyme Electrophoresis Typing
This technique may be employed to estimate the overall genomic relationships among strains of organisms within species by determining the relative electrophoretic mobilities of a set of water-soluble
cellular enzymes. The variation in electrophoretic mobility can be related to allelic variation and to
levels of genetic variation within populations of a species. Typically, 15–25 enzymes are tested for
on starch gels. Because some of the enzymes may have different mobilities (be polymorphic) among
strains of the same species, multilocus enzyme electrophoresis (MEE) typing can be used to characterize strains for epidemiologic purposes in the same general way as serotyping or phage type. The basic
technique has been described in detail and reviewed.
183 Some applications are summarized as follows:
1. After examining 175 isolates of L. monocytogenes for allelic variation among 16 enzymes,
45 allelic profiles or MEE types were distinguished. The above could be divided into two primary
phylogenetic divisions with all 4a, 4b, and 1/2b serovars in the same division.
2. A study of 245 strains of L. monocytogenes from a variety of sources in Denmark revealed
33 MEE types with 73% of strains belonging to one of two MEE types.
148 One MEE type was
found most frequently among food isolates. In a related study, 47 clinical isolates and 72 fish
isolates were found by MEE not to constitute two distinct lineages but that they belonged to two
separate populations.
20
3. When compared to restriction fragment length polymorphism (RFLP; see later in this chapter)
on 141 strains of L. monocytogenes, the two methods were in substantial agreement on recurrent
strains in certain food products.
77
265
Amplified Fragment Length Polymorphism
This is a PCR-based fingerprinting technique that requires small amounts of pure dsDNA—10–100
ng from 1–3 bacterial colonies.
89 The DNA is digested with two restriction enzymes (such as EcoRI
and MseI). Double-stranded oligonucleotide adaptors are applied, and an aliquot is subjected to 1- to
2-PCR amplifications under highly stringent conditions with adapter-specific primers. The primer
that spans the average frequency restriction site is fluorescently labeled. After subjecting to PAGE
(polyacrylamide gel electrophoresis), from 40 to 200 bands are obtained, and it has been found to be
more reproducible than RAPD.
19
In a study of 147 strains of bacteria from nine species, AFLP analysis clustered all strains within
their respective species.
89 The method was shown to produce results consistent with DNA homology
data when the genomic DNA of 98 Aeromonas strains were digested with ApaI and TaqI.
85 Using
an automated method with two restriction endonucleases, 25 Campylobacter isolates from poultry
farms in the Netherlands grouped in three C. jejuni clusters that were different from a C. coli cluster.
48
A fluorescent-AFLP (FAFLP) employs fluorophore-labeled primers, and it has been employed to
discriminate between 30 phage type 4 (PT 4) strains of S. Enteritidis from different sources. When
AFLP was used with XbaI, 73% of the PT 4 strains were classified as a single type.
43 However, when
FAFLP was employed using EcoRI + O and MseI + C, 23 profiles (with 1–61 amplified-fragment
differences) were obtained with a discriminatory power of 0.98 compared to 0.47 for PFGE.
43 Genetic
diversity of clinical and environmental isolates of Vibrio cholerae was investigated by using ApaI and
TaqI, and 20–30 bands each were generated from the genomic DNA of V. cholerae serotypes 01 and
0139 strains.
97 Of the 79 strains tested, 26 of 01 showed identical patterns and were represented by the
01 El Tor strain of the seventh pandemic. For more information on this technique, see reference 179.
Multilocus Enzyme Electrophoresis Typing
This technique may be employed to estimate the overall genomic relationships among strains of organisms within species by determining the relative electrophoretic mobilities of a set of water-soluble
cellular enzymes. The variation in electrophoretic mobility can be related to allelic variation and to
levels of genetic variation within populations of a species. Typically, 15–25 enzymes are tested for
on starch gels. Because some of the enzymes may have different mobilities (be polymorphic) among
strains of the same species, multilocus enzyme electrophoresis (MEE) typing can be used to characterize strains for epidemiologic purposes in the same general way as serotyping or phage type. The basic
technique has been described in detail and reviewed.
183 Some applications are summarized as follows:
1. After examining 175 isolates of L. monocytogenes for allelic variation among 16 enzymes,
45 allelic profiles or MEE types were distinguished. The above could be divided into two primary
phylogenetic divisions with all 4a, 4b, and 1/2b serovars in the same division.
2. A study of 245 strains of L. monocytogenes from a variety of sources in Denmark revealed
33 MEE types with 73% of strains belonging to one of two MEE types.
148 One MEE type was
found most frequently among food isolates. In a related study, 47 clinical isolates and 72 fish
isolates were found by MEE not to constitute two distinct lineages but that they belonged to two
separate populations.
20
3. When compared to restriction fragment length polymorphism (RFLP; see later in this chapter)
on 141 strains of L. monocytogenes, the two methods were in substantial agreement on recurrent
strains in certain food products.
77