Author manuscript, published in "Veterinary Microbiology 122, 3-4 (2007) 323"

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

peer-00532201, version 1 - 4 Nov 2010

Authors: D.M. West, K.A. Sprigings, C. Cassar, P.R. Wakeley,

J. Sawyer, R.H. Davies
PII:
DOI:
Reference:

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
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Rapid Detection of Escherichia coli Virulence Factor Genes using

Multiplex Real-Time TaqMan PCR Assays

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D. M. Westa, K. A. Sprigingsb, C. Cassarb, P. R. Wakeleya, J. Sawyera, and

R. H. Daviesb

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Technology Transfer Unita and Department of Food and Environmental Safetyb,

Veterinary Laboratories Agency, Woodham Lane, New Haw, Addlestone, Surrey,

KT15 3NB. United Kingdom

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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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using identical cycling conditions on an Mx3000P Real-Time PCR machine with

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the capacity to test up to 20 strains for 9 virulence genes in 1 hour.

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Keywords: Escherichia coli; Virulence factors; Real-time PCR; Multiplex PCR;

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Diagnostics; Validation

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Escherichia coli is well recognised as a commensal inhabitant of the

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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pathogenic to animals but also strains which cause asymptomatic infections of

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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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as those categorised as enterotoxigenic, enteropathogenic, enterohaemorrhagic or

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necrotoxigenic from normal flora, the presence of virulence characteristics needs to be

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identified (Nataro and Kaper, 1998; DebRoy and Maddox, 2001).

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Identification of the virulence factors that allow pathogenic strains to be

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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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virulence factors possible in a routine testing environment.

In this study we describe the development and use of three multiplex real-time
TaqMan PCR assays. Assay 1 to detect genes coding for the fimbriae K88, K99 and

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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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2. Material and methods

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2.1. Reference bacterial strains and growth conditions

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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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Oswald at the Ecole Nationale Vtrinaire de Toulouse, France and Martina

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Bielaszewska at Universittsklinikum Mnster, Germany.

To obtain single colonies each sample was cultured on 5 % sheep blood agar plates,

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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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inoculating loop, used to inoculate a room temperature 3 mL LB-G broth and

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incubated statically overnight at 37oC. Cultures were inactivated by boiling for 15

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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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2.2. Phenotypic Tests

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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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of interest. Positive samples displayed macroagglutination and negative samples

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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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method described by Konowalchuk et al., (1977) in which cytopathic effect was

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observed. At the time of development, phenotypic tests for STa, CDT IV, F17 and

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F18 were not employed at the VLA.

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2.3. TaqMan Probes and Primers

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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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the Lasergene, MegAlign software, Version 5 (DNASTAR Inc.). There were 21

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sequences for K88 (accession numbers; AJ616256, AJ616237, AJ616238, AJ616239,

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AJ616240, AJ616241, AJ616242, AJ616243, AJ616244, AJ616245, AJ616246,

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AJ616247, AJ616248, AJ616249, AJ616250, AJ616251, AJ616252, AJ616253,

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AJ616254, AJ616255, and AJ616236), 1 sequence for K99 (accession number;

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M35282), 2 sequences for F41 (accession numbers; X14354 and M21788), 8

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sequences for LT (accession numbers; AB011677, K01995, M35581, S60731,

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M15362, M15361, M57244 and V00275), 1 sequence for CDT IV (accession number;

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AY162217), 7 sequences for F17 (accession numbers; AF055306, AF055307,

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AF055308, AF055309, AF022140, L14318 and L77091), 8 sequences for F18

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(accession numbers; L26236, L26104, L26105, L26106, L26107, L26108, AY569976

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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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the software were set to the manufacturers default.

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Probes and primers for genes coding STa and GAD were as previously published

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(Meiland et al., 2003, Frydendahl et al., 2001). Desalted and deprotected

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oligonucleotide primers and HPLC purified dual labelled fluorescent probes were

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supplied by Sigma Genosys.

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2.4. DNA amplification

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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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DNA template, 5 L of 10X enzyme reaction buffer, 5 L of 25 mM MgCl2, 20 mM

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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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Reactions were subjected to 1 cycle of 94oC for 2 minutes, followed by 28 cycles of

94oC for 20 seconds, 55oC for 20 seconds, 72oC for 30 seconds on a Stratagene

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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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2.5. Specificity and Sensitivity

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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USA) programme (Altschul et al., 1997).

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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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species, Proteus species, Staphylococcus species, Taylorella species, Streptococcus

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species, Shigella flexneri, Hafnia alvei, Enterobacter cloacae, Escherichia fergusonii,

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Pseudomonas aeruginosa, Klebsiella pneumoniae, Pasturella multocida, Oligella

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urethralis, Arcanobacterium pyogenes, Enterococcus faecalis and Bacteroides

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ureolyticus. In addition, specificity was confirmed by sequencing a proportion of

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positive strains.

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The sensitivity of each assay was determined by preparing serial dilutions of a

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To experimentally test the specificity of the PCRs the panel of positive reference

selection of positive control reference strains (nine in total) followed by amplification

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with the appropriate multiplex assay. In addition, the efficiency of each assay was

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determined by the Mx3000P software.

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2.6 Field Strains

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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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tested using each multiplex PCR.

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2.7 Statistical Analysis

Statistical analysis was performed using 2 x 2 box analysis (MacKinnon, 2000) in

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order to compare the historically acknowledged phenotypic test results with the

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TaqMan PCR assay results.

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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and 96.5 % homology with the E.coli gadA 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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was positive for GAD.

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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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(100%) were positive for the appropriate genes.

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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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111.0 %, as determined by the Mx3000P software.

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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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expressed virulence factors for which there was no historically acknowledged

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phenotypic test.

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3.4. Statistical analysis

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Statistical analysis using 2 x 2 box analysis (Mackinnon, 2000) was performed to

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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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proportion of strains were tested by other methods (Table 4).

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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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platform and a common approach.

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For virulence factors where no historically acknowledged test was available a

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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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throughput is high and results are produced rapidly.

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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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performed in a single PCR plate simultaneously. Up to 20 strains, plus positive and

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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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have improved performance compared to the historically acknowledged phenotypic

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tests.

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Statistical analysis to compare the TaqMan PCR assays to the historically

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acknowledged phenotypic tests for five virulence factors demonstrated that the

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TaqMan PCR assays produced equivalent or improved results. Sensitivity and

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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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sent to an independent laboratory for confirmatory testing (Table 4) . Confirmatory

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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

dilutions of a selection of positive reference strains. The sensitivity was found to be

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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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phenotypic tests were comparable with respect to performance.

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amplification reaction specific for E.coli producing a positive result in every

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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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tests to help characterise E.coli isolates.

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Acknowledgements

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Our thanks to Louise Finch, Felicity Clifton-Hadley, Ernesto Liebana, Martin

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Woodward and Catherine Fearnley for their help and support. This project was funded

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through the VLA internal Test Development Programme.

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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

conference on antimicrobial agents and chemotherapy) Chicago, USA.

an

us

369

373

Mackinnon, A., 2000. A spreadsheet for the calculation of comprehensive statistics

375

for the assessment of diagnostic tests and inter-rater agreement. Comput. Biol. Med.

376

30, 127-134

pte

377

dM

374

378

Nataro, J.P. and Kaper, J.B., 1998. Diarrheagenic Escherichia coli. Clin. Microbiol.

379

Rev. 11, 142-201

380

Ac
ce

peer-00532201, version 1 - 4 Nov 2010

368

381

Pass, M.A., Odedra, R and Batt, R.M., 2000. Multiplex PCRs for identification of

382

Escherichia coli virulence Genes. J Clin. Microbiol. 38, 2001-2004

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

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386

Microbiol. 40, 271-274

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

intimin and Shiga toxins. Vet. Microbiol. 93, 247-60

cri
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

Escherichia coli. J Clin. Microbiol. 41, 2669-2671

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

and II genes of Escherichia coli in water. J. Appl. Microbiol. 84, 585-592.

dM

396

399

pte

400

Ac
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peer-00532201, version 1 - 4 Nov 2010

388

17

Page 17 of 26

400

Table 1. E.coli Laboratory Reference Strains

Identification

Origin

Virulence Factors Expressed as

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
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peer-00532201, version 1 - 4 Nov 2010

Code

K88, LT
K88, LT
K88, LT

18

Page 18 of 26

Identification

Origin

Virulence Factors Expressed as

Bovine

K99

EC458/04

Quality Assurance Strain

987p

EC371/03

Quality Assurance Strain

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

F17, Others Unknown

1560S89

Bovine

F17, Others Unknown

1561S89

Bovine

F17, Others Unknown

40KH90

Bovine

F17, Others Unknown

217KH90

Bovine

F17, Others Unknown

1630S89

Bovine

F17, Others Unknown

EC808/04

K88, STa
STa

us

K99, STa
F41, STa

an

K88, LT, STa

dM

Ac
ce

EC940/04

EC912/04

cri
p

Determined Phenotypically

pte

peer-00532201, version 1 - 4 Nov 2010

Code

STa
K99, STa
K88, LT

401
19

Page 19 of 26

Identification

Origin

Virulence Factors Expressed as

Determined Phenotypically

Bovine

F17, Others Unknown

99AP236

Avian

F17, Others Unknown

C161-94

Unknown

F18, Others Unknown

C155-94

Unknown

F18, Others Unknown

C904-90

Unknown

F18, Others Unknown

C820-90

Unknown

F18, Others Unknown

28C (Oswald)

Porcine

28C

Porcine

us

cri
p

1650S89

CDT IV

an

CDT IV

(Bielaszewska)

pte

dM

402

Ac
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peer-00532201, version 1 - 4 Nov 2010

Code

20

Page 20 of 26

rip
t

Table 2

ggttcagtgaaagtcaatgcatct
ccccgtccgcagaagtaac
Cy5 - ccacctctccctaacacaccggcat BHQ2
gctattagtggtcatggcactgtag

FAM attttaaactaaaaccagcgcccggca - TAMRA

F41

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

HEX tgatgtaatttcaccaccaataataatgtcacctg TAMRA

Accession

Number

35

tttgttttcgctaggcagtcatta

Assay

Concentration (M)

ted

K99

sc

Working

nu

K88

Oligonucleotide Sequence (5'-3')*

Ma

Target

Ac
ce
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peer-00532201, version 1 - 4 Nov 2010

Oligonucleotide primers and TaqMan Probes used in Multiplex assays

319-338

30

406-382

343-377

70

This study

80

This study

88

This study

21

Page 21 of 26

Number

K01995

20

HEX tagcaggtttcccaccggatcacc - TAMRA

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

FAM cgttccagtattccagatatactcattcattgggata - TAMRA

6489-6453

gcagaaaattcaatttatcctt

20

Cy5 ggcggctgcgtcatcttctgct BHQ2

Reference

(bp)

20

tattgtaccgtcataagcaagc

Product Size

320-294

20

aagctgctgtggacggctat

Position

20

ted

aacggaacgtgaatttcgtaca

rip
t

Number

sc

Concentration (M)

gaatccagggttcttctctccaa

FAM ttacctcccgtcatgttgtttcacggat - TAMRA

F17

Accession

20

acagaaataaaaattgccaacattagc

Cdt IV

Assay

nu

Sta

Working

ccggcagaggatggttacag

Ac
ce
p

peer-00532201, version 1 - 4 Nov 2010

LT

Oligonucleotide Sequence (5'-3') *

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

FAM tttcggttaactgcccgctccaagt - TAMRA

ccaaagtattccactgcaagca

20

gccgtaactccaccgtttgt

M61713

U50547

agcaacagttcagcaaagtcca

ted

ROX cattatgtgtcgtcgcggcttcgaa - BHQ2

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

HEX - acatcggaaccaccacagggaatcct - TAMRA

GAD

rip
t
Accession

sc

Assay

nu

987p

Working

ttgtgcttccttgtccaataaaac

1068-1093
1, 2, &

M84024

1263-1281

M84025

1330-1309

al. (2003)

1283-1307

* Fluorophores and quenchers are shown for TaqMan Probes.

Ac
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peer-00532201, version 1 - 4 Nov 2010

F18

Oligonucleotide Sequence (5-3) *

Ma

Target

23

Page 23 of 26

Table 3. Limits of detection and efficiency of each assay.

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
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peer-00532201, version 1 - 4 Nov 2010

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

3 sequenced, 3 tested by SSI

STa

69/ND

151/ND

6 sequenced

CDT IV

2/ND

218/ND

F17

21/ND

dM

2 sequenced

199/ND

4 sequenced, 8 tested by published

4/ND

t
cri
p

9 sequenced

us

7 sequenced

5 sequenced, 4 tested by SSI

21 sequenced

an

pte

F18

Confirmatory Tests

216/ND

PCR (Bertin et al, 1996)

3 sequenced

ND = Not Determined

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strains.

26

Page 25 of 26

Table 5. Statistical analysis of TaqMan PCR compared to the historically

acknowledged phenotypic tests.

Values for each Gene

Sensitivity

100 %

100 %

Specificity

95.87 %

97.04 %

Efficiency

97.24 %

97.79 %

Positive Predictive Value

92.31 %

92.00 %

Negative Predictive Value

100 %

Probability of False Positive Result

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

Probability of false Negative Result

F41

K99

cri
p

K88

Ac
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peer-00532201, version 1 - 4 Nov 2010

Analysis Term

27

Page 26 of 26