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DOI : 10.1016/j.vetmic.2007.01.026
Accepted Manuscript
Title: Rapid Detection of Escherichia coli Virulence Factor
Genes using Multiplex Real-Time TaqMan PCR Assays
S0378-1135(07)00064-8
doi:10.1016/j.vetmic.2007.01.026
VETMIC 3581
To appear in:
VETMIC
Received date:
Revised date:
Accepted date:
1-9-2006
26-1-2007
30-1-2007
Please cite this article as: West, D.M., Sprigings, K.A., Cassar, C., Wakeley, P.R.,
Sawyer, J., Davies, R.H., Rapid Detection of Escherichia coli Virulence Factor Genes
using Multiplex Real-Time TaqMan PCR Assays, Veterinary Microbiology (2007),
doi:10.1016/j.vetmic.2007.01.026
This is a PDF file of an unedited manuscript that has been accepted for publication.
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3
D. M. Westa, K. A. Sprigingsb, C. Cassarb, P. R. Wakeleya, J. Sawyera, and
R. H. Daviesb
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*Corresponding author. Tel.: +44 (0)1932 357759; fax: +44 (0)1932 357890
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E-mail: p.wakeley@vla.defra.gsi.gov.uk
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Abstract
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Three multiplex real-time TaqMan PCR assays were developed for the detection
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of E.coli virulence factor genes in veterinary samples. Target virulence factors chosen
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were the fimbriae K88 (F4), K99 (F5), F41, F17, F18 and 987p (F6) and the toxins
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LT, STa, and CDT IV. Detection of genes coding GAD were included in each assay
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as an internal control. These assays allow rapid identification of virulence factor genes
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Diagnostics; Validation
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gastrointestinal tract (Nataro and Kaper, 1998), however it is also associated with
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diarrhoea and a range of extra-intestinal diseases in both man and animals (DebRoy
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and Maddox, 2001). Farmed livestock are known to harbour not only strains
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animals and which can pass through the food chain to cause clinical disease in man,
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for example, enterohaemorrhagic E.coli such as O157 and less frequently O26
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(DebRoy and Maddox, 2001). In order to reliably differentiate pathogenic strains such
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distinguished from the normal gut flora has relied on phenotypic methods. More
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recently, the use of PCR based technology to identify virulence factor genes (Kwon et
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al., 1999) has become widely adopted. The development of gel based PCRs for
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identification of virulence factor genes (Bertin et al., 1996, Franck et al., 1998, Pass et
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al., 2000 and Toma et al., 2003) was shortly followed by the development of real-time
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PCR tests (Belanger et al., 2002, Bellin et al., 2001, Davis et al., 2003, Frydendahl et
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al., 2001, Ibekwe and Grieve, 2003, Ibekwe et al., 2002, Jinnerman et al., 2003,
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Jothikumar et al., 2002 and Sharma et al., 2003). It is these developments, which
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1. Introduction
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utilise rapid and simple methods, that have made the identification of multiple
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F41, assay 2 to detect genes coding for heat labile toxin (LT), heat stabile toxin (STa)
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and cytolethal distending toxin IV (CDT IV) and assay 3 to detect genes coding for
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the fimbriae F17, F18 and 987p. In addition, each multiplex assay includes the
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detection of genes coding for the enzyme glutamate decarboxylase (GAD), (Meiland
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et al., 2003) as an internal control. All assays were designed for use on an Mx3000P
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machine (Stratagene).
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All assays use the same PCR cycling conditions, allowing parallel testing on a
single machine. This facilitates the use of the assays and reduces the time required to
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achieve results.
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E.coli laboratory reference strains (Table 1) were selected and used for
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development of the assays; selection was on the basis of those strains typically
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received for routine testing at the Veterinary Laboratories Agency (VLA), UK. Strains
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expressing the fimbriae K88, K99, F41 and 987p and the toxins STa and LT were
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selected from a well characterised archive held at the VLA. Strains expressing F17
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fimbriae were supplied by Sigrid Van Bost at the Universit de Lige, Belgium;
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strains expressing F18 fimbriae were supplied by the Statens Serum Institut (SSI),
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Denmark and strains expressing the toxin CDT IV (28C) were supplied by Eric
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incubated overnight at 37oC. Following incubation, a single, well isolated colony was
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selected, used to inoculate a 5 % sheep blood agar plate and incubated overnight at
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37oC. Following the second incubation step a single colony was taken with an
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minutes in a water bath. 5 L of boiled culture was used as template in the PCR.
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All strains used in development and validation of these assays had been previously
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well characterised by phenotypic methods. FIMBREX Test Kits (VLA) were used for
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the detection of the fimbriae K88, K99 and 987p and the tests were carried out
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according to the manufacturers instructions. Briefly, the method involves adding live
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E.coli culture to latex beads coated with monoclonal antibody specific to the fimbriae
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displayed no agglutination. A FIMBREX test was also used for detection of F41 for
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strains received up until the year 2000, after this point strains were tested for F41
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using an ELISA. Detection of LT was carried out using a Verocell assay, based on the
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observed. At the time of development, phenotypic tests for STa, CDT IV, F17 and
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Probes and primers for the detection of the genes coding K88, K99, F41, LT, CDT
IV, F17, F18 and 987p were novel designs. Nucleotide sequence data found on the
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NCBI nucleotide database were aligned for genes coding each virulence factor using
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M15362, M15361, M57244 and V00275), 1 sequence for CDT IV (accession number;
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and M61713) and 2 sequences for 987p (accession numbers; M35257 and U50547).
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TaqMan probes and primers were designed against homologous regions of each
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alignment using Primer Express software, Version 2.0 (ABI) where the parameters of
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Probes and primers for genes coding STa and GAD were as previously published
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oligonucleotide primers and HPLC purified dual labelled fluorescent probes were
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DNA for amplification was released from whole bacteria by boiling at 100oC for 15
minutes. PCRs were carried out in a total volume of 50 L and included; 5 L of
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of each dNTP, 0.5 U of Taq DNA polymerase in storage buffer A (all PCR reagents
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were supplied by Promega) and 1 L of each primer and TaqMan probe appropriate
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for the assay (Table 2). The final volume was made up to 50 L with nuclease free
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water (Sigma).
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Mx3000P real-time PCR machine. Data was collected at the 72oC extension step.
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Strains that produced a Ct value and amplification plot were considered to positive
for specific virulence genes.
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In order to assess the specificity of each probe and primer, sequences were screened
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against those held in the NCBI database using the BLAST N (NCBI; Wisconsin,
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strains (Table 1) and a negative control panel of other bacterial species were tested.
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The panel of negative control bacteria species included; Yersinia species, Salmonella
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positive strains.
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To experimentally test the specificity of the PCRs the panel of positive reference
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with the appropriate multiplex assay. In addition, the efficiency of each assay was
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A panel of 137 field strains, derived from various livestock species and disease
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syndromes, that had been previously well characterised using phenotypic methods,
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were prepared using methods as described for the reference panel. All strains were
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order to compare the historically acknowledged phenotypic test results with the
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3. Results
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3.1. Specificity
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Using the BLAST N database to confirm the specificity of TaqMan probes and
primers, only those for the detection of GAD showed 100% homology with genes
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from another bacteria, Shigella flexneri. All other TaqMan probes and primers were
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specific to the target for which they had been designed. Alignment of the complete
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gadA and gadB gene sequences of E.coli (Accession numbers M84024 and M84025)
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and the gadB gene sequence of S.flexneri (Accession number AE016984, position
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62527-63927) showed that E.coli gadA and gadB genes share 97.4 % homology. In
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addition, the S.flexneri gadB gene shares 98 % homology with the E.coli gadB gene
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Testing the negative control panel of other bacterial species with each multiplex
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TaqMan assay showed that these were negative for all virulence factors. S.flexneri
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Tests to determine the specificity of TaqMan probes and primers for virulence
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genes using the positive control panel of reference strains showed that all samples
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Sequence data was produced (between 3 and 21 strains per virulence factor) and
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added to alignments used for the design of TaqMan probes and primers. All isolates
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for any particular gene showed good homology in the area where the TaqMan assay
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was designed.
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3.2. Sensitivity
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Results of the sensitivity experiments are shown in Table 3. The limit of detection
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for each assay varied between 5.2 x 105 and 8 x 105 cfu/mL of culture. There was no
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significant difference in sensitivity if a strain had genes for more than one virulence
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factor in any single multiplex assay. The efficiency of each PCR ranged from 97.0 -
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3.3. Field Strains
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137 field strains were tested using each of the multiplex TaqMan PCR assays. Of
these, 81 strains produced identical results when tested by the historically
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acknowledged phenotypic tests (FIMBREX test, Verocell assay and ELISA) and
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TaqMan PCR. For the remaining 56 strains more virulence factors were detected by
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TaqMan PCR than by phenotypic tests (Table 4). The majority of these strains
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phenotypic test.
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compare the available historically acknowledged phenotypic test results with the
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newly developed multiplex real time TaqMan PCR assays for K88, K99, F41, LT
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and 987p (Table 5). Using field and control strains it was possible to perform analysis
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on 180 strains. Compared to the phenotypic tests the sensitivity of the real time
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TaqMan PCR assays was 100% for all assays, the specificity ranged between
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95.87% and 99.44% and the efficiency ranged between 97.24% and 99.44%.
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4. Discussion
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Phenotypic methods have been previously used to identify the virulence factors
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K88, K99, F41, STa, LT and 987p. The purpose of this study was to replace some of
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these more traditional tests which can have different interpretive outcomes and be
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laborious or prone to error. Additionally, this study was designed to expand the VLA
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testing portfolio for the detection of E.coli virulence factors to include STa, CDT IV,
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F17 and F18. The introduction of these new tests provides a package of simple real-
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time TaqMan PCRs allowing rapid detection of virulence genes using a single
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In total, three multiplex assays were developed; assay 1 for the detection of K88,
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K99 and F41, assay 2 for the detection of STa, LT and CDT IV and assay 3 for the
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detection of F17, F18 and 987p. These assays provide a standardised approach to
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virulence gene testing and can be applied in both surveillance and outbreak studies as
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One of the most significant advantages of our multiplex real-time TaqMan PCRs
is that every assay has the same cycling conditions allowing all three reactions to be
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negative controls, can be tested for all 9 virulence factors in 1 hour on a single 96 well
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block machine.
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During validation of the assays, some samples produced conflicting results using
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phenotypic tests and the TaqMan PCR. To resolve the disparity, both phenotypic and
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TaqMan PCR tests were repeated and a proportion of isolates were sent to Statens
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Serum Insitut (SSI), Denmark, for independent confirmatory testing (Table 4). In all
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cases, results of the repeat and confirmatory tests correlated with the original
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TaqMan PCR result. This demonstrated that the multiplex TaqMan PCR assays
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tests.
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acknowledged phenotypic tests for five virulence factors demonstrated that the
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negative predictive value was 100 % in all cases when compared to phenotypic tests.
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Specificity and positive predictive values of the TaqMan PCRs appeared lower than
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the phenotypic tests (specificity 95.87 - 99.44%, positive predictive values 75.00-
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97.30%) due to the increased number of positive results produced by TaqMan PCR.
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For each gene, a proportion of unconfirmed positive results were either sequenced or
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testing proved in all cases that the TaqMan PCR results were correct. McNemars
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values showed that for K88, K99, F41, LT and 987p the TaqMan PCR and
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As there was no phenotypic test to validate PCR results for F17 laboratory
reference strains they were tested by a published PCR method (Bertin et al., 1996).
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Results of the TaqMan PCR and the method described by Bertin et al. (1996)
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correlated well.
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The sensitivity of each virulence factor PCR was tested by amplifying serial
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low compared to other PCRs (Tsen and Jian, 1998; Paton and Paton, 2002; Sharma
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and Dean-Nystrom, 2003). Importantly, our assays were designed specifically for
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detection of virulence genes in pure E.coli cultures where very sensitive tests are not
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required and not for direct detection from veterinary or clinical samples
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The efficiency of each assay ranged between 88.2 and 115.0 %, generally the
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accepted ranges for PCR efficiency are between 95.0 and 110.0 %, which suggests
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that the low levels of sensitivity are not due to inefficient amplification.
When a negative control panel of other bacteria species were tested no positive
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results were observed; there was no cross reaction with other bacteria in the test panel.
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Specificity of the assays for virulence genes was found to be very high (100%).
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The aim of the use of an internal control PCR for the GAD gene was to introduce a
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multiplex reaction where E.coli is present. The TaqMan primers and probe described
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by Meiland et al., (2003) used in this study were shown to be GAD specific for both
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E.coli and S.flexneri. As S.flexneri is not a veterinary pathogen (Balows and Duerden,
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1998) the assay was deemed to target a suitable gene for use as an internal control for
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veterinary isolates.
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significantly lower concentration (0.04M) than those amplifying genes coding for
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virulence factors (0.4 - 0.7M). This lead to the avoidance of competition between the
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control and target virulence genes and also reduced the sensitivity of the GAD assay.
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As a result, some strains that were positive for more than one virulence factor in a
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single multiplex assay were negative for GAD. A reaction was only deemed to have
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failed and required further investigation if a strain was negative for all of the
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virulence factors in a particular multiplex PCR and the GAD control PCR.
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In each multiplex assay the primers used to amplify genes encoding GAD had a
Since development of these assays more than 200 field strains have been routinely
tested using the assays described. They have proved invaluable as reliable and rapid
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Acknowledgements
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Woodward and Catherine Fearnley for their help and support. This project was funded
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References
310
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. and
311
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Balows, A. and Duerden, B.I., 1998. Volume 2 Systematic Bacteriology. In: Collier,
315
L., Balows, A and Sussman, M. (Eds) Topley and Wilsons Microbiology and
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317
Belanger, S.D., Boissinot, M., Menard, C., Picard, F.J. and Bergeron, M.G., 2002.
319
320
cri
p
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us
Bellin, T., Pulz, M., Matussek, A., Hempen, H.G. and Gunzer, F., 2001. Rapid
323
324
an
322
325
Bertin, Y., Martin, C., Oswald, E. and Girardeau, J-P., 1996. Rapid and specific
327
328
dM
326
pte
329
330
Davis, K.C., Nakatsu, C.H., Truco, R., Weagent, S.D., Bhunia, A.K., 2003. Analysis
331
of environmental Escherichia coli isolates for virulence genes using the TaqMan
332
333
Ac
ce
321
334
335
336
Rev. 2, 129-140.
337
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Page 14 of 26
338
Franck, S.M., Bosworth, B.T. and Moon, H.W., 1998. Multiplex PCR for
339
340
341
Frydendahl, K., Imberechts, H. and Lehmann, S., 2001. Automated 5' nuclease assay
343
for detection of virulence factors in porcine Escherichia coli. Molec.Cell. Probes. 15,
344
151-160.
cri
p
342
us
Ibekwe A.M. and Grieve, C.M., 2003. Detection and quantification of Escherichia
347
348
421-31
an
346
349
Ibekwe, A.M., Watt, P.M., Grieve, C.M., Sharma, V.K. and Lyons, S.R., 2002.
351
352
coli O157:H7 in dairy waste water wetlands. Appl. Environ. Microbiol. 68, 4853-62.
dM
350
pte
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354
Jinneman, K.C., Yoshitomi, K.J., Weagent, S.D., 2003. Multiplex real-time PCR
355
method to identify Shiga toxin genes stx1 and stx2 and Escherichia coli O157:H7/H-
356
357
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ce
345
358
359
O157:H7 with multiplex real-time PCR assays. Appl. Environ. Microbiol. 68, 3169-
360
71
361
15
Page 15 of 26
362
Konowalchuk, J., Speirs, J.I. and Stavric, S., 1977. Vero Response to a Cytotoxin of
363
364
Kwon, D., Kim, O. and Chae, C., 1999. Prevalence of genotypes for fimbriae and
366
367
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p
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Meiland R., Coenjaerts, F.E.J., Geerlings, S.E., Brouwer, E.C. & Hoepelman, A.I.M.,
370
2003. Development of a quantitative Real-Time PCR assay for the rapid detection of
371
Escherichia coli in urine Samples. Proceedings for the 43rd ICAAC (Interscience
372
an
us
369
373
375
for the assessment of diagnostic tests and inter-rater agreement. Comput. Biol. Med.
376
30, 127-134
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Nataro, J.P. and Kaper, J.B., 1998. Diarrheagenic Escherichia coli. Clin. Microbiol.
379
380
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ce
368
381
Pass, M.A., Odedra, R and Batt, R.M., 2000. Multiplex PCRs for identification of
382
383
384
Paton, A.W., and Paton, J.C., 2002. Direct detection and characterization of shiga
385
toxigenic Escherichia coli by multiplex PCR for stx1, stx2, eae, ehxA and saa. J. Clin.
16
Page 16 of 26
386
387
Sharma, V.K. and Dean-Nystrom, E.A., 2003. Detection of enterohemorrhagic
389
Escherichia coli O157:H7 by using a multiplex real-time PCR assay for genes coding
390
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p
391
Toma, C., Lu, Y., Higa, N., Nakasone, N., Chinen, I., Baschkier, A., Rivas, M. and
393
Iwanaga, M., 2003. Multiplex PCR assay for identification of human diarrheagenic
394
an
395
us
392
Tsen, H.-Y. and Jian, L.-Z., 1998. Development and use of a multiplex PCR system
397
for the rapid screening of heat labile toxin I, heat stabile toxin II and shiga-like toxin I
398
dM
396
399
pte
400
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388
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Page 17 of 26
400
Origin
Porcine
K88
EC762/03
Porcine
K88, LT
EC763/03
Porcine
EC914/03
Porcine
EC1113/03
Porcine
EC1986/03
Porcine
EC2142/03
Porcine
EC2213/03
Porcine
EC92/04
Porcine
EC439/04
Porcine
EC37/03
Bovine
K99, F41
EC261/03
Ovine
K99, F41
Bovine
K99, F41
Bovine
K99, F41
Ovine
K99, F41
EC46/04
Bovine
K99, F41
EC378/03
Bovine
K99, F41
EC55/04
Bovine
K99, F41
EC767/04
Bovine
K99, F41
EC998/04
Bovine
K99, F41
EC316/03
EC345/03
K88, LT
K88, LT
us
K88, LT
K88, LT
dM
an
K88, LT
pte
EC296/03
EC251/02
cri
p
Determined Phenotypically
Ac
ce
Code
K88, LT
K88, LT
K88, LT
18
Page 18 of 26
Identification
Origin
Bovine
K99
EC458/04
987p
EC371/03
987p
EC32/94
Porcine
EC367/94
Porcine
EC370/94
Porcine
EC421/94
Porcine
EC438/94
Porcine
EC443/94
Porcine
EC509/94
Unknown
EC90/03
Porcine
EC239/03
Porcine
K88, LT
EC591/03
Porcine
K88, LT
EC600/03
Porcine
K88, LT
Porcine
LT
Porcine
K88, LT
1648S89
Bovine
1560S89
Bovine
1561S89
Bovine
40KH90
Bovine
217KH90
Bovine
1630S89
Bovine
EC808/04
K88, STa
STa
us
K99, STa
F41, STa
an
dM
Ac
ce
EC940/04
EC912/04
cri
p
Determined Phenotypically
pte
Code
STa
K99, STa
K88, LT
401
19
Page 19 of 26
Identification
Origin
Bovine
99AP236
Avian
C161-94
Unknown
C155-94
Unknown
C904-90
Unknown
C820-90
Unknown
28C (Oswald)
Porcine
28C
Porcine
us
cri
p
1650S89
CDT IV
an
CDT IV
(Bielaszewska)
pte
dM
402
Ac
ce
Code
20
Page 20 of 26
rip
t
Table 2
ggttcagtgaaagtcaatgcatct
ccccgtccgcagaagtaac
Cy5 - ccacctctccctaacacaccggcat BHQ2
gctattagtggtcatggcactgtag
ctgctgattggacggaaggt
ccagtcttccatagccatttaacag
Number
20
AJ616236
Position
430-453
499-481
456-479
1
M35282
241-265
35
320-297
269-295
1
X14354
Product Size
Reference
(bp)
20
30
Accession
Number
35
tttgttttcgctaggcagtcatta
Assay
Concentration (M)
ted
K99
sc
Working
nu
K88
Ma
Target
Ac
ce
p
319-338
30
406-382
343-377
70
This study
80
This study
88
This study
21
Page 21 of 26
Number
K01995
20
gcaaaatccgtttaactaatctcaaa
20
680-699
73
This study
91
Frydendahl
752-730
701-725
M25607
230-255
et al.
257-284
(2001)
AY162217
6426-6447
6510-6491
6489-6453
gcagaaaattcaatttatcctt
20
Reference
(bp)
20
tattgtaccgtcataagcaagc
Product Size
320-294
20
aagctgctgtggacggctat
Position
20
ted
aacggaacgtgaatttcgtaca
rip
t
Number
sc
Concentration (M)
gaatccagggttcttctctccaa
F17
Accession
20
acagaaataaaaattgccaacattagc
Cdt IV
Assay
nu
Sta
Working
ccggcagaggatggttacag
Ac
ce
p
LT
Ma
Target
AF055306
3-28
20
79-56
33-54
84
This study
77
This study
22
Page 22 of 26
Concentration (M)
Number
Number
20
ctcccccttgattagcaaaacc
20
ccaaagtattccactgcaagca
20
gccgtaactccaccgtttgt
M61713
U50547
agcaacagttcagcaaagtcca
ted
Product Size
Reference
(bp)
390-413
82
This study
72
This study
68
Meiland et
472-451
1045-1066
1116-1097
accgacatcgtggtgatgc
Position
449-424
20
rip
t
Accession
sc
Assay
nu
987p
Working
ttgtgcttccttgtccaataaaac
1068-1093
1, 2, &
M84024
1263-1281
M84025
1330-1309
al. (2003)
1283-1307
Ac
ce
p
F18
Ma
Target
23
Page 23 of 26
Virulence
Limit of
Assay used
Factors Isolate
Target
Detection
for Test
Efficiency
cri
p
(cfu/ml)
K88/LT/STa
K88
6.1 x 105
97.80%
912/04
K99/STa
K99
6.2 x 105
109.10%
1771/02
K99/F41
K99
8 x 105
110.60%
1771/02
F41/K99
F41
762/03
LT
LT
92/04
LT/STa/K88
LT
92/04
STa/LT/K88
STa
367/94
STa
912/04
STa/K99
28C
CDT IV
371/03
987p
1648S89
100.10%
6 x 105
88.20%
an
8 x 105
6 x 105
108.20%
6 x 105
104.70%
dM
STa
7 x 105
109.50%
STa
6 x 105
97.50%
CDT IV
9.35 x 105
93.30%
987p
1.8 x 105
90.60%
F17
F17
8.6 x 104
115%
F18
F18
6.6 x105
103%
pte
C161-94
us
92/04
Ac
ce
Positive For
Isolate
PCR
25
Page 24 of 26
Table 4. Summary of phenotypic and TaqMan PCR results for reference and field
Virulence
n PCR Positive /
n PCR Negative /
Factor
n Phenotypically
n Phenotypically
Positive
Negative
K88
65/60
116/121
K99
50/46
131/135
F41
37/36
142/143
LT
55/52
126/129
987p
4/3
176/177
STa
69/ND
151/ND
6 sequenced
CDT IV
2/ND
218/ND
F17
21/ND
dM
2 sequenced
199/ND
4/ND
t
cri
p
9 sequenced
us
7 sequenced
an
pte
F18
Confirmatory Tests
216/ND
ND = Not Determined
Ac
ce
strains.
26
Page 25 of 26
Sensitivity
100 %
100 %
Specificity
95.87 %
97.04 %
Efficiency
97.24 %
97.79 %
92.31 %
92.00 %
100 %
4.13%
987p
100 %
99.31 %
97.67%
99.44 %
99.44 %
98.34%
99.44 %
97.30 %
94.55%
75 %
100 %
100 %
100%
100 %
2.96%
0.69%
2.33%
0.56%
an
us
100%
0.00%
0.00%
0.00%
0.00%
0.00%
0.0736
0.1336
1.00
0.2482
1.00
pte
McNemars Test
LT
100 %
dM
F41
K99
cri
p
K88
Ac
ce
Analysis Term
27
Page 26 of 26