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Applied and Environmental Microbiology, November 1998, p. 4210-4216, Vol. 64, No. 11
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
An Automated Fluorescent PCR Method for Detection
of Shiga Toxin-Producing Escherichia coli in
Foods
Shu
Chen,1
Renlin
Xu,1
Arlene
Yee,1
Kai Yuan
Wu,2,
Chang-Ning
Wang,2
Susan
Read,3 and
Stephanie A.
De Grandis4,*
Guelph Molecular Supercentre, Laboratory
Services Division, University of Guelph, Guelph, Ontario, Canada
N1H 8J71;
Biotronics Technologies Corp.,
Lowell, Massachusetts 018512;
Health
of Animals Laboratory, Health Canada, Guelph, Ontario, Canada N1G
3W43; and
Office of Research,
University of Guelph, Guelph, Ontario, Canada N1G
2W14
Received 23 April 1998/Accepted 12 August 1998
 |
ABSTRACT |
An automated fluorescence-based PCR system (a model AG-9600
AmpliSensor analyzer) was investigated to determine whether it could
detect Shiga toxin-producing Escherichia coli (STEC). The AmpliSensor PCR assay involves amplification-mediated disruption of a
fluorogenic DNA signal duplex (AmpliSensor) that is homologous to
conserved target sequences in a 323-bp amplified fragment of Shiga
toxin genes stx1, stx2,
and stxe. Using the Amplisensor assay, we
detected 113 strains of STEC belonging to 50 different serotypes, while
18 strains of non-Shiga-toxin-producing E. coli and 68 strains of other bacteria were not detected. The detection limits of
the assay were less than 1 to 5 CFU per PCR mixture when pure cultures
of five reference strains were used and 3 CFU per 25 g of food
when spiked ground beef samples that were preenriched overnight were
used. The performance of the assay was also evaluated by using 53 naturally contaminated meat samples and 48 raw milk samples. Thirty-two
STEC-positive samples that were confirmed to be positive by the culture
assay were found to be positive when the AmpliSensor assay was used.
Nine samples that were found to be positive when the PCR assay was used
were culture negative. The system described here is an automated
PCR-based system that can be used for detection of all serotypes of
STEC in food or clinical samples.
| |
INTRODUCTION |
Infection with Shiga
toxin-producing Escherichia coli (STEC) (also called
verotoxigenic E. coli) in humans has been associated with a
spectrum of diseases, including diarrhea, hemorrhagic colitis, and
hemolytic-uremic syndrome (HUS) (14, 21). Foods that have an
animal origin, such as beef, have been identified as the main vehicles
of these food-borne pathogens. The importance of STEC transmission
through the food chain has been illustrated by many outbreaks
worldwide. For example, the 1996 outbreak in Japan was initially caused
by the consumption of school lunches contaminated with E. coli serotype O157:H7; eventually, there were 9,578 reported cases, 11 people died, and there were more than 90 diagnosed cases of
HUS (4). In 1992 and 1993 a multistate outbreak in the
United States, which was attributed to consumption of hamburgers
contaminated with E. coli O157:H7, involved more than
700 people, and there were four deaths and 51 cases of HUS
(7). The 1996 Scottish E. coli O157 outbreak
associated with meat products resulted in 490 cases of infection and 18 deaths (5).
More than 100 serotypes of STEC have been associated with human
diseases, although serotype O157:H7 is the most commonly reported serotype (1). Other commonly isolated outbreak STEC
serotypes include O111:NM, O26:H11, O26:NM, and O103:H2
(18). The strains belonging to these STEC serotypes
can produce cytotoxins, which collectively are called Shiga toxin (Stx)
(also verotoxin and Shiga-like toxin [23]). Other
virulence factors, such as intimin (17, 19), hemolysin
(30), and other virulence proteins may also be produced
by STEC strains. STEC strains can produce different immunotypes
of Stx and other virulence factors which play different roles in the
onset of disease (21).
Major social and economic consequences underline the benefit of
preventive measures that reduce or eliminate exposure to any type of
STEC. The American Gastroenterological Association (2) has
recommended that future planning for diagnosis and prevention of
hemorrhagic colitis, and HUS in the United States should include testing for non-O157 E. coli strains that produce Stx.
In the last 20 years, the Vero cell assay has been the "gold
standard" for testing for STEC (9). This method, however,
requires 4 to 5 days to complete and also requires tissue culture
facilities which are not available in many laboratories. Immunological
methods for detection of STEC are more rapid and convenient than the
Vero cell assay. However, these methods are designed to detect specific toxin types or specific E. coli serotypes, such as O157:H7.
In recent years, molecular methods based on PCR have been developed and
successfully used to detect STEC. These PCR methods have been reviewed
by Olsen et al. (24) and Scheu et al. (29) in
terms of their target genes, detection systems, detection limits, and application to foods. Most of the PCR methods have been developed to
detect particular types of Stx producers or specific serotypes of STEC
(3, 6, 10, 27, 37, 38). Several of the PCR assays have been
designed to detect all serotypes of STEC (12, 20, 25, 28).
In previously described PCR methods, however, either ethidium bromide
and gel electrophoresis or post-PCR hybridization-capture methods
are used to detect PCR products. Gel-based methods are laborious and
time-consuming, lack sensitivity and specificity, and are
difficult to automate. Post-PCR hybridization-capture methods
reportedly are more sensitive and more specific than gel-based methods
and are easy to automate, but they require multiple post-PCR signal development steps that are even more time-consuming and sometimes more laborious than gel-based methods. In addition, many of
the PCR assays have been evaluated only with pure cultures or
spiked samples, and their applicability to naturally contaminated food
or clinical samples is unknown.
New and improved PCR systems have been developed for detection of STEC
in attempts to perform homogeneous and automated direct detection
assays of PCR products without the need for gel electrophoresis. One
such system is the TaqMan PCR detection system that was developed for
detection of Stx I-producing E. coli (37) and
E. coli O157:H7 (13). This system is based on the
5'-nuclease activity of Taq DNA polymerase which hydrolyzes
an internal flurogenic probe in order to monitor amplification. Another
system is the temperature-dependent fluorescence-PCR system, in which
an intercalating dye, SYBR Green, is used to monitor amplification. The
temperature-dependent fluorescence-PCR system, in combination with the
commercially available BAX system, has been used to detect E. coli O157:H7 (35).
We have previously described an automated PCR method which
requires no post-PCR handling steps; this method, which is called the
AmpliSensor assay, is used for specific detection of
Salmonella spp. in foods (8). The AmpliSensor
assay is comprised of the following two steps: (i) an initial
asymmetric amplification performed with normal primers, which
overproduces one strand of the target; and (ii) subsequent seminested
amplification and signal detection with an AmpliSensor primer. The
AmpliSensor primer is a double-stranded signal probe labelled with
fluorescein isothiocyanate and Texas Red (36). The
seminested amplification step results in dissociation of the strands of
the AmpliSensor duplex and, consequently, disruption of the
fluorescence signal. The extent of signal disruption is proportional to
the amount of the AmpliSensor primer incorporated into the
amplification product and can be measured cycle by cycle and used for
quantification of the initial target. In this paper development of the
AmpliSensor assay for detection of all serotypes of STEC is described.
The results of studies performed to elucidate characteristics of the
assay and the applicability of the assay to food samples are presented.
| |
MATERIALS AND METHODS |
Bacterial strains, media, and culture conditions.
A total of
113 STEC strains belonging to 50 different serotypes, 18 non-Stx-producing E. coli strains, and 68 strains of other bacteria were used (Tables
1 through
3). These strains were obtained from the
collections of the Laboratory Services Division (University of Guelph),
the Health of Animals Laboratory (Health Canada), and the Sunny Brook
Health Centre (Toronto, Ontario, Canada) and from the American Type
Culture Collection. Cultures were maintained on appropriate agar plates
and stored at 4°C.
Five strains of STEC (Table
1) were used as reference strains in the
detection limit and spiking experiments. The cells were
grown at 37°C
overnight in brain heart infusion broth (Becton
Dickinson). Cells were
enumerated by plating dilutions of overnight
cultures onto MacConkey
agar (Difco) plates and incubating the
plates at 37°C overnight.
E. coli ATCC 25922 was used as a negative
control
strain.
Preparation of food samples.
Red meat samples which were
used in the spiking experiments were purchased from a local retail
store. Fifty-three naturally contaminated meat samples were obtained
from a study of the prevalence of STEC in 327 raw meat products and 744 ready-to-eat meat products from provincially inspected plants in
Ontario (40), and 48 naturally contaminated raw milk samples
were obtained from a survey of food-borne pathogens in 1,720 Ontario
bulk tank milk samples (33). Preenriched food samples were
prepared by homogenizing 25 g of meat in 225 ml of nutrient broth
(Becton Dickinson) or 25 ml of milk in 225 ml of universal
preenrichment broth (Difco) and then incubating the preparations at
37°C overnight. The preenriched samples were subjected to two steps
of selective enrichment with MacConkey broth and brain heart infusion
broth and then tested for the presence of STEC by using the Vero cell
assay, followed by confirmatory tests performed by the method of Clarke
et al. (9). Additional confirmatory tests were performed
with meat samples by using the hydrophobic grid membrane filter (HGMF)
method described by Yee et al. (40). The preenriched samples
were also used in the AmpliSensor assays.
In the mock-contamination experiments, only those food samples that
were confirmed to be STEC negative by both culture and
PCR methods were
used. Food samples were mock contaminated in
the following two ways:
(i) food samples (25 g) were inoculated
with 3 to 336 CFU of an STEC
strain before homogenization and
preenrichment in nutrient broth (these
food samples were designated
prespiked samples) and (ii) preenriched
food samples (250 ml)
were inoculated with 1.12 × 10
2
to 1.12 × 10
5 CFU of the target cells per ml (these
food samples were designated
postspiked
samples).
Extraction of DNA from pure cultures and from food samples.
DNA were prepared from pure cultures by using an InstaGene matrix
(Bio-Rad) as described by Chen et al. (8). Briefly, 10-µl portions of serial dilutions of an overnight culture of STEC were incubated with 200 µl of InstaGene matrix at 56°C for 20 min and then boiled for 10 min. The mixtures were placed on ice for 10 min and
then centrifuged at 16,000 × g for 5 min. The
supernatants were used for PCR.
DNA were prepared from food samples by using 1-ml pellets of
preenriched food samples and a modification of the EnviroAmp
Legionella sample preparation kit protocol (Perkin-Elmer)
(
8).
Briefly, a sample pellet was resuspended in 500 µl of
the EnviroAmp
DNA extraction reagent and boiled for 20 min. The lysate
was cooled
on ice for 5 min and centrifuged at 16,000 × g for 3 min to remove
the cell and food debris. The DNA was then precipitated
from 400
µl of the supernatant by using 400 µl of 100% isopropanol
and
washed once with 500 µl of 75% isopropanol. The DNA pellet was
resuspended in 160 µl of sterile distilled
water.
Primers and DNA signal duplex for AmpliSensor.
The
amplification target was conserved sequences in subunit A of Stx genes
stx1, stx2, and
stxe, as described by Read et al. (28). The two primers used for asymmetric amplification were the primers described by Read et al. (28), and using these
primers resulted in a 323-bp PCR product. The AmpliSensor primer
designed in this study was a signal duplex which targeted the sequence 5'-CGTTTTGTCACTGTGACAGCA-3' in the stx genes and
served as the forward primer in seminested amplifications, generating a
59-bp fragment. The oligonucleotide and complement used for the signal duplex reaction were synthesized with amino-modified
deoxyribosylthymidine residues at specific positions and then
conjugated with fluorescein isothiocyanate and Texas Red, respectively,
by using the reaction conditions recommended by the supplier (Molecular
Probes). Since the stx-specific sequences were not
completely conserved, several base degeneracies were incorporated into
all three primers to allow amplification of all types of stx genes.
Asymmetric amplification.
An amplification reaction mixture
(20 µl) containing the following reagents was used: amplification
buffer (50 mM Tris-HCl [pH 8.9], 40 mM KCl, 4.0 mM MgCl2,
0.1% Triton X-100, 0.05% Tween 20), 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 400 µM dUTP, 0.1 µM forward primer, 0.75 µM reverse
primer, 0.625 U of AmpliTaq DNA polymerase (Perkin-Elmer), 0.25 U of
AmpErase uracil N-glycosylase (Perkin-Elmer), and 10 µl of
template DNA. The reaction mixture was overlaid with 10 µl of mineral
oil. The mineral oil and the master mixture (containing all of the PCR
components except the template DNA) were automatically dispensed into a
96-well microtiter polycarbonate plate by using a model AG-9600
AmpliSensor analyzer (Biotronics Corporation). DNA from a pure culture
of the Stx-producing organism E. coli 933W was used as a
positive control, and DNA from E. coli ATCC 25922 and water
were used as negative controls. PCR was performed in the 96-well
microtiter plates by using a model 9600 Perkin-Elmer GeneAmp PCR system
apparatus. The cycling conditions were as follows: incubation at 50°C
for 2 min and at 95°C for 5 min; 29 cycles consisting of denaturation
at 94°C for 15 s, annealing at 49°C for 1 min, and extension
at 72°C for 30 s; incubation at 72°C for 7 min; and incubation
at 4°C until seminested amplification was performed.
Seminested amplification and data acquisition.
After 29 cycles of asymmetric amplification, 4 µl of amplification buffer
containing 7.5 ng of the signal duplex was automatically added to each
reaction mixture by using the model AG-9600 AmpliSensor analyzer, and
cycling was resumed at an annealing temperature of 60°C instead of
49°C. Data were obtained after cycle 1 (in seminested amplification)
and every third cycle thereafter for 25 cycles by directly measuring
the fluorescence of the amplification mixture with the model AG-9600
AmpliSensor analyzer. The wavelengths used were 485 nm for excitation
and 630 nm for emission. A detection index was calculated by using the
AG AmpliSensor assay program (Biotronics Corporation) and the following
equation: detection index = 1 
(Fs,x/Fn,x),
where Fs,x is the fractional decrease
in energy transfer of a sample and
Fn,x is the fractional decrease in
energy transfer of the negative control. An increase in the detection
index for each sample was monitored dynamically during seminested
amplification. A sample was considered positive if there was a slope as
expressed by detection index increase/cycle number increase and if the
increase in the detection index was 0.1 or more. The PCR end products
were also visualized by using ethidium bromide-stained 2% agarose gels.
| |
RESULTS |
Optimization of the assay.
PCR conditions were optimized for
amplification of low copy numbers of target DNA sequences. The
annealing temperature used for asymmetric amplification, between 49 and
51°C, was optimal for obtaining good yields of PCR products and
minimizing nonspecific amplification. An annealing temperature of
60°C was used for seminested amplification; this temperature did not
compromise the signal intensity of the PCR products. To detect less
than 10 target copies, it was necessary to perform 25 to 30 cycles of
asymmetric PCR in the first step for detection of the PCR product in
the second step after 15 to 20 cycles.
Specificity, sensitivity, and reproducibility of the AmpliSensor
assay.
The specificity of the assay for different STEC serotypes
was determined by using 113 STEC strains (Tables 1 and 2) and 86 non-STEC strains (Table 3). All of the reaction mixtures
containing STEC resulted in detection index values between 0.5 and
0.9. PCR products of the expected size (323 bp) were visualized by
using the agarose gels. All reaction mixtures containing non-STEC had detection index values within 0.0 ± 0.10, and no PCR products of
the expected size were observed when gel electrophoresis was performed.
The limit of detection of the assay was determined by using dilutions
of overnight pure cultures of five reference STEC strains
(Table
1).
The detection limit was 1 to 5 CFU per PCR, as shown
in Fig.
1.
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FIG. 1.
(a) Detection of STEC in pure cultures of five reference
STEC strains by the AmpliSensor PCR assay. Detection index values were
obtained after 25 cycles of the seminested PCR. Symbols:  , STEC
strain H30; +, STEC strain 933W;  , STEC strain 32511;  , STEC
strain 412; ×, STEC strain HI8. (b) Detection of STEC in a pure
culture of strain H30 by the AmpliSensor PCR assay: extent of
amplification versus cycle number and initial copy number of target
DNA. Symbols: , 0.46 CFU/PCR; +, 4.6 CFU/PCR; , 46 CFU/PCR; , 460 CFU/PCR; ×, 4,600 CFU/PCR  , 46,000 CFU/PCR.
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The reproducibility of the assay was studied on different days by using
three replicates of an overnight culture of strain
933W. Figure
2 shows the relationship between the log
number of
cells and the detection index values when strain 933W was
used.
The interassays were reproducible. All of the subsequent spiking
experiments were performed in duplicate.
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FIG. 2.
Detection of STEC in a pure culture of strain 933W by
the AmpliSensor PCR assay. Detection index values were obtained after
22 cycles of the seminested PCR. Symbols: , run 1; +, run 2; ,
run 3.
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Detection of STEC in spiked ground beef.
To test
the limit of detection of the assay for STEC in
foods, samples of ground beef were prespiked or postspiked with target cells. Figure 3 shows the
cycle-dependent accumulation of the PCR products for the
prespiked samples tested. The detection limit was 3 CFU/25 g.
In samples that exhibited cycle-dependent accumulation of the PCR
product, a DNA band at 323 bp was detected by agarose gel
electrophoresis (results not shown). Figure
4 shows the relationships between the log
numbers of cells and the detection index values in a
cycle-dependent manner for postspiked ground beef samples. The
detection limit was 112 CFU/ml (equivalent to 8.6 CFU/PCR).
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FIG. 3.
Detection of STEC in prespiked ground beef by the
AmpliSensor PCR assay: extent of amplification versus cycle number and
initial copy number of target DNA. Symbols: , no STEC; +, 3 CFU/25 g; , 6 CFU/25 g; , 33 CFU/25 g; ×, 66 CFU/25 g; , 336 CFU/25 g.
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FIG. 4.
Detection of STEC in postspiked ground beef by the
AmpliSensor PCR assay: extent of amplification versus cycle number and
initial copy number of target DNA. Symbols: , no STEC; +, 112 CFU/ml; , 1,120 CFU/ml; , 11,200 CFU/ml; , 112,000 CFU/ml.
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Detection of STEC in naturally contaminated foods.
To evaluate
the applicability of the assay to naturally contaminated foods, 53 meat
samples and 48 raw milk samples were tested by using the AmpliSensor
assay. The results are summarized in Tables
4 through
6. All of the detection index values fell
into the following two groups: higher than 0.4 and within 0.0 ± 0.10. Of the 101 samples, 32 (17 meat samples and 15 raw milk
samples) were positive by both the culture and AmpliSensor PCR
methods, 59 (28 meat samples and 31 raw milk samples) were negative by both methods, 9 (7 meat samples and 2 raw milk samples) were
positive by the AmpliSensor PCR method alone, and 1 was positive by the culture method alone. One sample which was positive by the culture method alone was examined further by using the HGMF method and the PCR method with less inhibitory selective enrichment broth instead
of preenrichment broth. The sample remained negative, and culture
contamination was suspected.
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TABLE 4.
Detection of STEC in naturally contaminated raw milk
samples by the AmpliSensor PCR assay and the culture method
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TABLE 5.
Detection of STEC in naturally contaminated meat samples
by the AmpliSensor PCR assay and the culture method: samples
positive for STEC by the PCR assay or the culture method
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TABLE 6.
Detection of STEC in naturally contaminated meat samples
by the AmpliSensor PCR assay and the culture method: samples
negative for STEC by the PCR assay or the culture method
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| |
DISCUSSION |
Specificity and sensitivity of the AmpliSensor assay.
The
results obtained in our study demonstrate the specificity of the
primers based on the tests performed with 113 strains of STEC
belonging to 50 common serotypes and 86 non-STEC strains. This
indicates that the degenerate primers did not compromise the
specificity of the assay at a measurable level. The degeneracy of
the primers may be compensated for because three primers are used in the system. The specificity of the two primers used
for asymmetric amplification was demonstrated previously in the study of Read et al. (28), in which all 223 STEC strains tested
and 2 of the 148 non-STEC strains tested were identified as STEC. The only two positive non-STEC strains in the study of Read et al. were Shigella dysenteriae type 1 strains. However,
detection of S. dysenteriae type 1 in foods was considered
advantageous (28).
The detection limits of the AmpliSensor assay were 1 to 5 CFU/PCR
when pure cultures of five reference STEC strains that produce
different types of toxins were used and 8.6 CFU/PCR when
postspiked
ground beef samples were used. No noticeable difference in
amplification
efficiency was observed when different
stx genes were amplified.
These detection limits are similar
to the detection limit of the
5' nuclease assay for
stx1 (10 ± 5 CFU) (
37)
and 1 to 2 logs
lower than the detection limit of the gel-based PCR
assays (10
2 CFU) (
20,
28). In this study, a
2-log decrease in the detection
limit was also observed when the same
PCR products were analyzed
by the AmpliSensor assay
compared-to agarose gels (results not
shown).
Other features of the assay.
The model AG-9600 AmpliSensor
analyzer is an automated system for dispensing PCR reagents and
for detecting PCR products. For both the PCR process and PCR product
detection, only one 96-well microplate and one pipetting step (for the
addition of template DNA) are required. The simplicity of the
procedure increases the reproducibility of the assay and reduces the
chance of laboratory DNA contamination. The AmpliSensor assay also
employs uracil N-glucosylase and dUTP (22) to
minimize potential problems caused by PCR product carryover contamination.
Applicability of the AmpliSensor assay to food samples.
One
important criterion that is necessary for a rapid STEC testing method
to be applicable to food samples is that the rapid method must be as
sensitive as or more sensitive than conventional methods (which have a
theoretical level of detection of 1 CFU/25 g or 1 CFU/25 ml of
food). In this study we were able to detect 3 CFU of STEC in 25 g
of food following overnight preenrichment. In the prespiked samples the
concentration of the target cells after preenrichment was not
determined. However, the detection limit of 112 CFU/mL for the
postspiked samples suggests that overnight preenrichment is sufficient
to ensure the sensitivity of the assay because the concentration of
STEC in food samples usually is 104 CFU/ml or more
after overnight preenrichment.
The AmpliSensor assay was also successfully used with 101 naturally
contaminated food samples. When the AmpliSensor assay
was used, nine
additional STEC-positive samples (seven meat samples
and two raw milk
samples) were detected compared to the number
of positive samples
detected by the traditional culture method,
suggesting that the
AmpliSensor assay is more sensitive than the
culture method. In a
previous study, the same seven meat samples
that were positive only by
the AmpliSensor PCR method were analyzed
by using the HGMF method, a
method that was found to be more efficient
than the traditional method
for recovering STEC (
40). STEC belonging
to different
serotypes were isolated from three of the seven samples,
suggesting
that the culture method was not sensitive enough to
detect of all of
the STEC-positive samples. In addition to the
greater sensitivity of
the PCR method, the following factors may
also result in PCR-positive
and culture-negative detection: (i)
the target cells may be injured or
dead and therefore nonculturable
but detectable by the PCR method and
(ii) the presence of
S. dysenteriae type 1 could result in
positive reactions in the PCR assay (
28)
but no recovery of
STEC.
It should be noted that the prevalence of STEC in the food samples
tested in this study (32 of 101 samples [31.7%]) is not
the true
prevalence because the naturally contaminated samples
used in this
study were selected from two previous prevalence
studies (
33,
40). The samples used included all of the culture-positive
samples identified in the prevalence studies plus randomly selected
STEC-negative
samples.
| |
ACKNOWLEDGMENTS |
This research was supported by the Ontario Food Quality and
Safety Research Fund.
We are grateful to provincial food inspectors for collecting the food
samples and to M. Rozwadowski and M. Steele for performing the culture assays.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Office of
Research, University of Guelph, Reynolds Building, Guelph, Ontario,
Canada N1G 2W1. Phone: (519) 824-4120, ext. 3523. Fax: (519) 821-5236. E-mail: steph{at}ornet.or.uoguelph.ca.
Present address: Affymetrix, Santa Clara, CA 95051.
| |
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Abstract
Introduction
