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Journal of Clinical Microbiology, May 2000, p. 2001-2004, Vol. 38, No. 5
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Multiplex PCRs for Identification of Escherichia coli
Virulence Genes
M. A.
Pass,1,*
R.
Odedra,2,
and
R.
M.
Batt2,
Department of Physiology and Pharmacology,
The University of Queensland, St. Lucia, Queensland, 4072, Australia,1 and Department of Small
Animal Medicine and Surgery, The Royal Veterinary College, North
Mymms, Hatfield, Hertfordshire, AL9 7TA, United
Kingdom2
Received 3 August 1999/Returned for modification 8 October
1999/Accepted 13 December 1999
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ABSTRACT |
PCRs were developed to detect 11 Escherichia coli
virulence genes. Primers amplified the respective genes without
cross-reaction with other genes. Specificity was maintained in
multiplex reactions; excellent amplification of target genes was
possible with a minimum of four multiplex reactions. These reactions
successfully identified genes in E. coli from the feces of
four dogs.
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TEXT |
The virulence mechanisms that
characterize Escherichia coli are genetically coded for by
chromosomal, plasmid, and bacteriophage DNAs and include heat-labile
(LTI, LTIIa, and LTIIb) and heat-stable (STI and STII) toxins,
verotoxin types 1, 2, and 2e (VT1, VT2, and VT2e, respectively),
cytotoxic necrotizing factors (CNF1 and CNF2), attaching and effacing
mechanisms (eaeA), enteroaggregative mechanisms (Eagg), and
enteroinvasive mechanisms (Einv). With the advent of PCR, it has become
possible to identify these genes in bacterial isolates, offering the
possibility of rapid diagnosis of the mechanisms operating in specific
E. coli infections. PCR methods using single primer sets
have been reported (1, 2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15), but screening of bacterial isolates for the myriad of
virulence genes requires a large number of individual PCRs if single
primer sets are used in separate reactions. To reduce the number of
tests needed for screening E. coli for virulence factor
genes, we have developed multiplex PCR systems that can identify 11 genes.
Primers for amplifying segments of the VT1, VT2, VT2e, CNF1, CNF2, LTI,
STI, STII, eaeA, Einv, and Eagg genes were designed so that
the PCR products from the genes were sufficiently different in size to
be distinguishable by agarose gel electrophoresis (Table 1). Comparisons with gene sequences in
the EMBL gene bank ensured that the genes for the PCR products lacked
homology with genes other than the target genes.
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TABLE 1.
Sequences of PCR primers, product sizes, and accession
numbers in the EMBL gene bank from which the sequences were
obtained
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DNA standards were extracted from bacteria known to contain the
relevant genes. Bacteria containing LTI and STII were O147:K89:K88ac and O147:KT:K88 isolates, respectively, obtained from the Central Veterinary Laboratory, Ministry of Agriculture, Fisheries and Food,
Addlestone, United Kingdom, and the bacterium containing VT2e was an
O141:H9 isolate obtained from the Public Health Laboratory Service,
London, United Kingdom. Genes in other isolates had been identified by
DNA hybridization in the laboratory of one of us (R.M.B.) or in
laboratories of colleagues. The bacteria were grown in Luria broth or
on MacConkey agar plates at 37°C. Fecal swabs from dogs were cultured
on MacConkey agar plates, and up to 100 colonies from the plates were
combined for PCR analysis.
The cell resuspension solution, cell lysis solution, neutralization
solution, and minicolumns for DNA isolation were purchased as a Wizard
Miniprep DNA Purification System from Promega. DNA was extracted from
centrifuged bacterial pellets or bacterial colonies after suspension in
200 µl of resuspension solution. Cell lysis solution (200 µl) was
added, and the sample was vortexed for 20 to 30 s. The sample was
vortexed for 10 s after the addition of 200 µl neutralization
solution and then centrifuged at 12,000 × g for 5 min.
The supernatant was decanted into 1 ml of resin suspension containing
1.5 g diatomaceous earth in 100 ml of 4 M guanidine
isothiocyanate, 10 mM Tris (pH 7.6), and 1 mM EDTA (pH 8). The resin
suspension was applied to a minicolumn and washed with 3 ml of a column
wash solution containing 200 mM NaCl, 29 mM Tris-HCl (pH 7.5), and 2 mM
EDTA in 55% ethanol. The filter was dried by centrifugation at
12,000 × g for 2 min, and the DNA was eluted into 100 µl of water that had been preheated to 65°C. For direct PCR of
bacteria, individual colonies from culture plates were suspended in 50 µl of water.
PCR was performed with 0.5-ml Eppendorf tubes on a Techne PHC-3 thermal
cycler with a reaction volume of 25.25 or 50.5 µl overlaid with 15 or
30 µl of paraffin oil, respectively. The DNA template (5 µl
containing 90 to 200 pg of DNA) or 5 µl of bacterial suspension was
added to a 50-µl reaction mixture containing 0.1 mM each dATP, dCTP,
dGTP, and dUTP (Bioline UK Ltd.); a buffer solution consisting of 16 mM
(NH4)2SO4, 67 mM Tris-HCl (pH 8.8 at 25°C), and 0.01% Tween 20; 3 mM MgCl2; and the PCR
primers (PE-Applied Biosystems, Warrington, United Kingdom). The
concentrations of primers that gave satisfactory amplification of genes
are given in Table 2. Taq
polymerase (2.5 U in 0.5 µl; Bioline UK Ltd.) was added. The PCR
program was 95°C for 30 s followed by 72°C for 1 min for 5 cycles; 95°C for 30 s followed by 63°C for 30 s and then
72°C for 30 s for 20 cycles; and 72°C for 5 min when isolated
DNA was amplified. The PCR program was preceded by 5 min at 95°C to
lyse the bacteria when bacterial suspensions were examined. Reaction
products were separated by agarose gel electrophoresis by adding 5 µl
of loading dye containing 0.25% bromophenol blue in 40% sucrose to a
50-µl reaction mixture and loading 5 µl onto a 3% agarose gel
(NuSeive 3:1; FMC). The buffer in the electrophoresis chamber and in
the agarose gel was 0.5× Tris-borate-EDTA (9) and contained
ethidium bromide (1 µg/ml). One hundred volts and 25 mA were applied
across the gel. DNA in the gel was visualized by exposing the gel to UV
light and was photographed on Polaroid film. The figures were produced
with a computer graphics program after computer scanning of the
Polaroid photographs.
PCR primers amplified fragments of DNA of the predicted size, and
amplification was specific for the gene in question (Fig. 1 and 2).
When primer pairs were combined in various multiplex combinations, some
primers interfered with the amplification of other genes. For instance,
CNF1 and CNF2 primer pairs could not be used in combination, as
spurious products that interfered with the interpretation of the
electrophoretic results were produced. The following four combinations
of primers gave adequate amplification of their respective target
genes: VT1, VT2, VT2e, and eaeA; CNF1 and Eagg; CNF2 and
Einv; and LTI, STI, and STII (Fig. 3).
Only the target genes were amplified (Fig. 2). These combinations were used successfully to amplify genes in E. coli cultured from
feces of four dogs (Fig. 4). It was
possible to use combinations other than those listed above to amplify
more genes in a single reaction (Fig. 5).
However, there was a tendency to lose the amplification of some genes,
particularly if all of the genes corresponding to the primer pairs were
present in the reaction. Nevertheless, it is unlikely that more than
three or four genes would be present in any one clinical sample.
Therefore, more complex combinations would be satisfactory,
particularly if several positive control samples were included in the
assay, each with a limited number of genes present.
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FIG. 1.
Agarose gel electrophoresis of the PCR products of
E. coli virulence genes. Lanes: +, DNA template with the
gene;  , control DNA without the gene; S, DNA standards.
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FIG. 2.
Agarose gel electrophoresis of mixtures of DNA templates
showing the specificity of the multiplex reactions. DNA templates 1 to
5 contained the following genes: template 1 contained genes for VT1,
VT2, VT2e, and eaeA; template 2 did not contain any target
gene; template 3 contained genes for CNF2 and Einv; template 4 contained genes for CNF1 and Eagg; and template 5 contained genes for
LTI and STI. The reactions used to generate products in the first five
lanes in the top half of the figure contained primers for VT1, VT2,
VT2e, and eaeA; the next 5 lanes were for CNF2 and Einv. The
reactions used to generate products in the first five lanes in the
bottom half of the figure contained primers for CNF1 and Eagg; the next
five lanes were for LTI and STI. Lane S, DNA standards. Note that for
template 3, the CNF2-positive DNA also contained the gene for CNF1.
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FIG. 3.
Agarose gel electrophoresis of the PCR products of
E. coli virulence genes from multiplex PCRs. Lanes 1, 3, 5, and 7 are from reactions containing the respective genes, and lanes 2, 4, 6, and 8 are from reactions with control DNA and no target genes.
Lane S, DNA standards.
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FIG. 4.
Agarose gel electrophoresis of products from multiplex
PCRs with DNA from E. coli isolated from the feces of four
dogs. Lanes: +, positive control DNA; , negative control DNA; 1 to 4 DNA isolated from E. coli cultured from dog feces. E. coli from dog 1 was positive for eaeA and STI; that
from dog 2 was positive for eaeA, CNF1, and STI; that from
dog 3 was positive for STI; and that from dog 4 was positive for CNF1
and CNF2. Lane S, DNA standards.
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FIG. 5.
Agarose gel electrophoresis of multiplex PCRs with DNAs
containing several virulence genes (+) and DNAs without the genes ().
The genes and PCR primers in each lane, from left to right, were VT1,
VT2, VT2e, and eaeA in the 1st lane; VT1, VT2, VT2e,
eaeA, CNF2, and Einv in the 2nd and 3rd lanes; VT1, VT2,
VT2e, eaeA, CNF1, and Eagg in the 4th and 5th lanes; VT1,
VT2, VT2e, eaeA, LTI, and STI in the 6th and 7th lanes; VT1,
VT2, VT2e, eaeA, CNF2, Einv, LTI, and STI in the 8th and 9th
lanes; and VT1, VT2, VT2e, eaeA, CNF1, Eagg, LTI, and STI in
the 10th and 11th lanes. Lane S, DNA standards.
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Amplification of the target gene segment occurred when either extracted
DNA or whole bacteria were used as the DNA template. It was necessary
to heat the sample for 5 min at 95°C to release the DNA for the PCR
when intact bacteria were used as the source of DNA. This extra step
did not interfere with PCR amplification. This result indicates that
these analyses could be used for bacteria isolated directly from
biological materials.
The present results show that it is possible to detect 11 of the major
virulence genes of E. coli with four multiplex PCRs. This
method offers a practical possibility for mass screening of samples or
for rapid identification of likely pathogenic mechanisms operating in
active infections.
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ACKNOWLEDGMENTS |
We thank staff at the Royal Veterinary College, who collected
samples for analyses.
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FOOTNOTES |
*
Corresponding author. Mailing address: Faculty of
Science, University of the Sunshine Coast, Locked Bag No. 4, Maroochydore DC, Queensland, 4558, Australia. Phone: 61 7 5430 2840. Fax: 61 7 5430 2887. E-mail: mpass{at}usc.edu.au.
Present address: Amersham Laboratories, Amersham, Buckinghamshire,
HP 7 9LL, England.
Present address: Waltham Centre for Pet Nutrition,
Waltham-on-the-Wolds, Leicestershire, LE 14 4RT, England.
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Journal of Clinical Microbiology, May 2000, p. 2001-2004, Vol. 38, No. 5
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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