Molecular and Cellular Probes (1992) 6, 153-161

Polymerase chain reaction for detection of verocytotoxigenic Escherichia coli isolated from animal and food sources S. C . Read,'* R . C. Clarke,' A . Martin ; S . A. De Grandis; ) . Hii, 2 S. McEwen 3 and C. L . GyleS 3 'Agriculture Canada, Health of Animals Laboratory, 110 Stone Road West, Guelph, Ontario, Canada N1G 3W4, 2 Cangene Corporation, 3403 American Drive, Mississauga, Ontario, Canada L4V 1T4, and 3Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Received 16 August 1991, Accepted 4 October 1991)

Animals and their by-products have been implicated as important sources of verocytotoxigenic Escherichia coli (VTEC) associated with disease in humans . VTEC comprise a wide range of serotypes and produce a variety of closely related verocytotoxins (VT) . A pair of oligonucleotide primers, targeting conserved sequences found in VT1, VT2 and VTE genes, was used to develop a polymerase chain reaction (PCR) procedure to detect all types of VTEC . Supernatants of boiled broth cultures of VTEC (223 strains) isolated from ground beef, ground pork, raw milk, bovine faeces and porcine faeces ; non-VTEC E. coli (72 strains); and other enteric and food bacteria (76 strains) were tested by PCR. The verocytotoxigenicity of these strains was verified by the Vero cell assay . All 223 VTEC isolates, comprising over 50 different serotypes, were detected by the PCR procedure . Shigella dysenteriae type 1 was the only other bacterium that was positive in this assay . As little as 1 pg of VTEC DNA and as few as 17 cfu of VTEC could be detected with this method . The results indicate that these primers detect VTEC over a wide range of serotypes . This method may be applicable as a screening procedure for the detection of VTEC in samples of foods and faeces . verocytotoxigenic Escherichia coli (VTEC), polymerase chain reaction (PCR), verocytotoxins (VT), shiga-like toxins (SLT) . KEYWORDS:

INTRODUCTION Verocytotoxigenic Escherichia coli (VTEC) have been implicated as the cause of a wide spectrum of disease in humans including mild diarrhoea, haemorrhagic colitis and haemolytic uraemic syndrome (reviewed by Karmali) .' Specific serotypes of VTEC (0157 :H7 and 026:H11) associated with haemorrhagic colitis in humans have been classified as enterohaemorrhagic Escherichia coli (EHEC).' In animals, VTEC have been associated with diarrhoea in cattle,' dysentery in calves 2 and oedema disease in pigs .` Serotypes of VTEC that have been associated with human disease have also been isolated from healthy cattle' and pigs'

as well as milk,' ground beef, ground pork' and other retail meats and poultry .' Thus, animals and their byproducts may be sources of infection for humans . Verocytotoxins (VTs) 9 or Shiga-like toxins (SLTs)' 0 are a family of protein cytotoxins that are active on Vero cells and may play a role in the pathogenesis of VTEC-related disease .'" Several antigenic types of VT have been described ; VT1 (SLT I), VT2, SILT II, VTE (SLT liv), SLT-H18 19 '12,1 ' and evidence is accumulating that the number of antigenic types may expand . 7,14-16 Initially, VT2 and SLT II were considered to be the same, but it is now known that the prototype strain

Author to whom correspondence should be addressed .

0890-8508/92/020153+09 $03.00/0

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for VT2 (E32511) produces a mixture of SILT II and a closely related toxin that has been named SLT Ilc ."

Escherichia coli 0157 :H7 is the predominant serotype implicated in outbreaks of disease in humans associated with VTEC and several methods have been described to detect this specific serotype .' A wide variety of VTEC serotypes have now been associated with disease in humans and thus methods which detect all VTEC serotypes may be more appropriate in a diagnostic setting .' Original detection methods for VTEC focused on VT assays of culture supernatants with Vero cells' and cell extracts with HeLa cells .' ° These methods are unsuitable for many diagnostic laboratories due to their lack of tissue culture facilities . Other VTEC detection procedures have been described including a colony blot assay using monoclonal antibodies against SLTs,' 4, 'e cloned DNA fragments for VT1 and VT2 genes, 1121 cloned DNA fragments for genes encoding a fimbrial antigen and outer membrane protein from enterohaemorrhagic E . coli, 21,22 oligonucleotide probes for SILT I and SLT II genes,' 1,2' a sandwich enzyme-linked immunosorbent assay (ELISA) for SLT I and SLT 11, 24 and a receptor-based ELISA assay for SLT I 25 and SLT 11 . 26 Two recent publications describe the use of synthetic oligonucleotide primers for the detection of VT1, VT2 and VTE 27,21 in the polymerase chain reaction (PCR) . Since a family of VTs exist, it may be a better strategy to screen samples with one set of primers that will detect any VT genes and to characterize further, positive samples by means of typespecific primers or probes, as has been described by Karch & Meyer . 29 The objective of this study was to evaluate a primer set, which targets conserved sequences in the A sub-unit of the VT (SLT) genes, in the PCR for detection of VTEC isolated from food and animal sources . The PCR results were compared with results from the Vero cell assay for reference cultures and VTEC isolates from food and animal sources .

MATERIALS AND METHODS Bacterial strains and culture media A complete listing of bacterial strains according to species, source and pathogenic type appears in Tables 1, 2 and 3 . The E. coli strains had been previously characterized in various laboratories for VT production by the Vero cell assay and neutralization by specific antibody . They were obtained from the culture collections of R . C . Clarke and S . C. Read, Health of Animals Laboratory, Ontario ; C . L . Gyles, Ontario Veterinary College, Ontario ; A . D . O'Brien and N . A . Strockbine, Uniformed Services University of the Health Sciences, Maryland ; and M. P. Doyle,

Table 1. strains

Serotypes and toxin types of reference VTEC

VTEC strain H19 C600(933W) E32511 B2F1 412 H18

Serotype

Toxin

026 :H11 O?Rough 0157:NM 091 :H21 0139:K82 0128 :B12

VT1 SLT II

VT2 VT2vh SLT 11v

SLT-H18

Reference 9 30 31 16 32 13

University of Wisconsin, Wisconsin . The other Gramnegative bacteria used in this study were obtained from C. L . tyles ; J . Brunton, Mount Sinai Hospital Research Institute, Ontario ; M . J . Champagne, Health of Animals Laboratory, Quebec ; U . Purvis, Health Protection Branch, Ontario; J . Lynch, Veterinary Laboratory Services, Ontario ; A . Sharpe, Health Protection Branch, Ontario ; and the culture collection of the Health of Animals Laboratory, Ontario . All the strains tested, except for Campylobacter spp ., were inoculated into Brain Heart Infusion Broth (Difco) and incubated for 18 h at 37° C . Strains of Campylobacter spp . were inoculated into Rosef's broth 33 and incubated for 24 h at 37°C under an atmosphere of 85% N2, 10% CO2 and 5% 0 2 .

Synthetic oligonucleotide primers The synthetic oligonucleotide primers used in this study were designed and supplied by Cangene Corporation, Ontario . Their nucleotide sequences are presented in Table 4 .

Sample preparation Broth culture, 1 ml, from each isolate was placed in a microcentrifuge tube and centrifuged at 12,000 x g for 1 min . The supernatant was decanted and used in the Vero cell assay as described below . The pellet was suspended in 1 ml 0 .85% NaCI and centrifuged again . The supernatant was discarded and the pellet suspended in 500 µl sterile distilled water . The samples were then placed in a boiling water bath for 10 min, centrifuged and immediately placed on ice . The supernatant was used in the PCR reaction as described below . PCR The PCR reaction was performed using the PCR Core Reagents kit (Perkin Elmer Cetus, Norwalk, Conn .) . Prepared sample supernatant (5 µp was added to a

155

Detection of VTEC by PCR Table 2.

VTEC isolates tested for VT genes in the PCR procedure Number of strains tested for each serotype and their sources

VTEC serotype

O1 :H20 02 :H29 02 :H39 04* 05* 05 :NM 06 :H34 08* O8weak :H2 O8weak :H9 08 :weak :H19 08 :H19 015 :H27 019* 022 :H8 022 :H16 026* 026 :H11 026 :NM 040 :H8 043 :H2 046 :H38 076 :H21 082 :H8 084 :H2 0103 :H2 0111* 0111 :H11 0111 :NM 0113 :H2 0113 :H21 0115 :H18 0116:H21 0117:H4 0120:NM 0121 :H10 0126 :H8 0128 :H35 0130 :K0132 :NM 0136 :H16 0138 :K81 0139 :H19 0139 :K82 0141 :K85 0145 :H16 0145:NM 0149* 0149:K91 0153 :H21 0153 :H25 0153:NM 0156 :H25 0156:NM 0157:H7 0160* 0165:NM 0171 :H2 Orough :H21

Bovine faeces

Ground beef

Milk filters

3 2 1 1 1 4 2 1 1 1 3 2 1 3 2 1 1 2 1

1 -

-

1 2 3 4 3 1 2 1 10 1 3 1 2 1 1 9 1 1 4 7 1 1 1 1

1 4 2 1 -

7 3 1 4 2 1 -

Porcine faeces

Ground pork

-

1 1

-

-

1 1 -

-

1 -

1 1 1 -

1 2 1 2 1 -

12 24 9 -

-

2 -

-

-

-

156

S . C. Read et al. Table 2 (continued) Number of strains tested for each serotype and their sources VTEC serotype

O? :Hrough O? :H2 O? :H8 O? :H19 O? :H21 O? :NM NTt

Totals

Bovine faeces

Ground beef

Milk filters

3

1 2

Porcine faeces

Ground pork

2

1 -

52

10

1 3

3

9 4 3

118

Flagellar antigen not determined

1

30

7

t NT, not serotyped .

reaction mixture containing 50 mm KCI; 10 mm TrisHCI (pH 8 . 3) ; 0-01% gelatin ; 200 µM each of dATP, dCTP, dGTP and dTTP ; 0 . 1 gm of each primer; 0 . 31 Units AmpliTaq DNA polymerase ; and 1 . 5 mm MgCl 2 for a final volume of 25 µl . The reaction mixtures were overlaid with mineral oil and processed through 35 cycles in a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, Conn .). Initially, the temperature of the sample block was increased from room temperature by a heating step at 72 ° C for 2 min . The cycle consisted of denaturation at 94 ° C for 1 min, annealing of primers at 49 ° C for 1 min and primer extension by DNA polymerase at 72°C for 30 s . To ensure complete strand extension, the reaction mixture was incubated for 5 min at 72° C after the last cycle . The amplified product was visualized by standard submarine gel electrophoresis of 5 µl of the final reaction mixture on a 2% agarose gel stained with ethidium bromide (1 µg ml -1 ) . A no-template control, in which the prepared sample supernatant was substituted with sterile distilled water, was included with each amplification run . Each sample was tested once except for samples in runs in which the no-template control was positive . In these cases, the source of contamination was eliminated and the samples were retested .

Sensitivity of detection system Six strains of VTEC (Table 1) were used as reference strains to determine the sensitivity of the PCR procedure for detection of VT genes . DNA of the reference VTEC strains was extracted using a modification of the chromosomal DNA preparation method of Marmur as outlined by Maclnnes et al ." The DNA was quantified spectrophotometrically at 260 nm . 39 Serial

10-fold dilutions of this DNA were made in sterile distilled water and used in the PCR reaction . In an additional test for sensitivity, serial dilutions of overnight broth cultures of the reference VTEC strains were made in sterile 0 . 85% NaCl and used in the PCR to quantify the number of bacterial cells detected . Portions of each dilution were plated onto TSA and grown overnight at 37 ° C to determine the number of cfu in each dilution .

Vero cell assay The Vero cell assay was used to ensure that isolates reported to be VTEC were still producing VT . Supernatant from the broth cultures (50 µp was used in the Vero cell assay as described previously .'

RESULTS PCR amplification The pair of oligonucleotide primers (Table 4) which targeted conserved sequences found in VT1, SLT II and VTE genes, 11,35 generated a 323 base-pair fragment from the A sub-unit of the VT gene in the PCR reaction as illustrated in Figs 1 and 2 . Six VTEC strains (Table 1) were selected as reference strains since they have been well characterized and the VTs they produce span the spectrum of different types that have been described . The VTEC reference strains were all positive in the PCR as shown in Fig . 1 . In addition, another 217 VTEC strains isolated from a variety of food and animal sources (Table 2) and comprising over 50 different serotypes were detected by the PCR procedure . Three of the isolates tested



Detection of VTEC by PCR Table 3. Non-VTEC bacterial strains tested for VT genes in the PCR procedure Species tested

E. coli (total)

No. of strains tested

ATCC 25922

72 1

Porcine diarrhoea Porcine ETEC* Bovine ETEC

18 28 25

Shigella spp . (total)

S. boydii S . dysenteriae, type 1 S . dysenteriae, other types S. flexneri S . sonnei Other enteric and food bacteria (total) Acinetobacter calcoaceticus Aeromonas spp . A . caviae

A . hydrophila A . sobria Campylobacter coli

9 1 2 2

2 2

67 1 6

1 2 1

Enterobacter aerogenes

1 2 1 3 1 3

E . agglomerans

4

E. cloacae Hafnia alvei Klebsiella oxytoca K . pneumoniae Morganella morganii Proteus mirabilis

2 3

C . jejuni Citrobacter diversus

C . freundii Edwardsiella tarda

1

2

Providencia stuartii

4 4 2 1

Pseudomonas aeruginosa Salmonella choleraesuis

1 3

S . enteritidis S . hadar S . heidelberg

2 2 2

S . infantis S . schwarzengrund

1 1

S . typhi S . typhimurium Serratia marcescens Vibrio parahemolyticus Yersinia enterocolitica

2

P . vulgaris

3 1 1

3

* ETEC, enterotoxigenic E . coil .

from ground pork (serotypes 0115 :H18, O? :H2 and O? :H9) had verocytotoxic activity that was not neutralized by toxin-specific antibody' (also, Read, unpublished data) . Seventy-two strains of non-VTEC E . coli (Table 3), including porcine enterotoxigenic, bovine enterotoxi-

157

genic and porcine diarrhoeal strains of E. coli, were negative when tested in the PCR . Another 76 strains of bacteria from food and animal sources (Table 3) were also negative in the PCR . Non-specific amplification products were noted with two strains each of Proteus mirabilis, Salmonella choleraesuis and Salmonella typhi. These products were easily differentiated from the 323 by product from VTEC due to their larger molecular weight on agarose gels . The Salmonella strains generated a fragment of approximately 900 bp, whereas the P . mirabilis strains generated a fragment of approximately 1200 bp . Shigella dysenteriae type 1 was the only non-VTEC organism that showed primer-specific amplification when tested in this PCR procedure (Fig . 1) . Serial dilutions of DNA from the reference VTEC strains were used to determine the limit of sensitivity of the PCR. DNA, 1 pg, was detected by PCR for all the reference strains except E32511 where the limit of sensitivity was 100 pg of DNA . In a similar fashion, serial dilutions of overnight broth cultures were made to quantify the number of bacteria detected by the PCR . For all the reference strains, 10 2 bacteria in 5 µl of sample were detected and as few as 17 cfu of strain 412, as illustrated in Fig. 2 .

Vero cell assay Other investigators have reported on VTEC strains that had lost their verocytotoxic activity upon storage ." To ensure that the VTEC strains used in this study were still verocytotoxic, culture supernatants of all isolates were tested in the Vero cell assay at the same time as the PCR procedure was conducted . One porcine and seven bovine isolates previously shown to be VTEC were negative in the PCR but were also negative in the Vero cell assay (results not shown) . All the other VTEC strains (Tables 1 and 2) and S . dysenteriae type 1 (Table 3) were positive in the Vero cell assay . Bacteria other than VTEC and S . dysenteriae type 1 can be verocytotoxic 3,1o,28 so the Vero cell assay and PCR were run in parallel on the other enteric and food strains (Table 3) used in this study . Aeromonas hydrophila and Aeromonas sobria were Vero cell assay positive . The other organisms listed in Table 3 were negative .

DISCUSSION AND CONCLUSIONS In the studies reported here, a pair of VTEC primers and the PCR were used to screen for the presence of VT genes in a culture collection comprising E. coli and other bacterial species . One of these primers, ES 151,

158

S. C . Read

et al.

Table 4 . Nucleotide sequence of primers compared with the nucleotide sequences of VT1 and comparable sequences in Shiga toxin, SLT II, VTE, VT2vh and SLT-HI8 Base nucleotide sequence (5'-3')* Primer/VT gene Position 611-631t ES 151

gag

ES 149 Shiga toxin, VT1 35,36 SLT II, VT2vh'6,35

--a

VTE 32 SLT-H18"

--a --a

cga

aat

Position 912-933 aat

tta

tat

gt cga

aat

ccc

ctc

tgt

t-t t-t --g

---

--c

att

tgc

C

--c --t

--g g-

* Only nucleotides different from those in the primer have been noted in corresponding sequences . t Numbered positions correspond to the position of the bases in the published nucleotide sequences of SLT I ."

a 0

b

C

d

e

t

9

b

C

d

e

h

Fig. 1 . Electrophoretic profile of VT gene fragments (323 bp) amplified by the PCR . Lanes: (a) 123 by ladder (Gibco/BRL, Gibco Canada Inc ., Burlington, Ontario) ; (b) VT1 reference strain H19 (026 :H11); (c) SLT II reference strain C600(933W) (O?Rough); (d) VT2 reference strain E32511 (0157 :NM) ; (e) VT2vh reference strain B2F1 (091 :H21); (f) SLT llv reference strain 412 (0139 :K82); (g) SLT-H18 reference strain HI8 (0128 :812) ; (h) VT negative E . coli (ATCC 25922) ; (i) Shiga toxin (Shigella dysenteriae type 1) .

Sensitivity of the PCR protocol in detecting cells strain 412 (SLT IIv) . Lanes : (a) 123 by ladder ; (b) 1 . 7 x 103 cfu ; (c) 1 . 7 x 10 1 cfu ; (d) 1 . 7 X 101 cfu ; (e) 1 . 7 cfu . Fig. 2.

of E . coli

We were able to detect 102 bacterial cells from the majority of our reference strains and a minimum of 17 cfu from strain 412, by visualization of the amplified product on an ethidium bromide stained gel . This corroborates the data of Karch & Meyer on the

was essentially the same as the complement of MK2

sensitivity of PCR to detect bacterial cells with VT

described by Karch & Meyer. 29 This primer was used

genes ." Other researchers have demonstrated sensit-

to flank a region upstream from the second primer in our study as opposed to downstream from the

ivity of the PCR for detection of VT genes using .27 .28 Our sensitivity extracted DNA as the template

second primer in the study by Karch . The PCR

limit for extracted DNA varied from 1 pg to 100 pg

protocol outlined in this study was sensitive, specific,

depending on the reference strain used, but was the

simple and required very little technical time com-

same order of magnitude that has been demon-

pared with other methodologies for the detection of

strated in these previous studies . The diversity

VTEC . There was complete correlation between the

observed among strains may be explained by variable

results of the PCR and the Vero cell assay for both

copy number of the VT genes and/or heterogeneity

reference and wild VTEC strains from a variety of

in the VT gene sequence among strains .

sources . In addition, 146 non-VTEC enteric and food bacteria were negative in the PCR .

As expected, the two strains of S .

dysenteriae

type

1 that were tested were positive in the PCR . The



Detection of VTEC by PCR

nucleotide sequence of Shiga toxin from S . dysenteriae type 1 has been shown to be almost identical to VT1, 36 and is identical to VT1 in the area targeted by the primers used in this study . Other methods for the detection of VTEC have failed to differentiate Shiga toxin of S . dysenteriae type 1 from VT . 18 '' 9 'Z .26 ' 28 Primers and a PCR protocol have been described recently that differentiate the genes for Shiga toxin from the genes for VT1 40 by targeting the promoter region for sit IA . However, the detection of S . dysenteriae type 1 in foods would be an advantage . Other specific primers have been described for the detection of VT1, VT2 and VTE genes in the PCR . 27,28 The use of two or more pairs of primers to detect different types of toxin in a single PCR reaction may lead to false results as one may amplify more readily than the other. In addition, it can be quite costly to run PCR on a large group of samples when using two or more pairs of primers . It may be better to use a general screening PCR method to detect VTEC and subsequently examine positive samples with specific primer sets designed to identify the various types of VT genes . A number of methods have been described for detection of VTEC using cloned DNA fragments 14-21 or oligonucleotide DNA probes .", " These probe techniques use radiolabelled isotopes as their reporter molecule and are inappropriate in a diagnostic setting. Oligonucleotide DNA probes with non-radioactive labels have been reported to be as sensitive as the radiolabelled probes for the detection of enterotoxigenic E. coli,42 however, to date none have been described for VTEC . ELISA techniques for the detection of VT1 and VT2 must be run separately for each toxin type .' 4,1824-28 In addition, these methods lack the ability to identify all members of the VTEC family and are restricted to the detection of only moderate and high level toxin producers . Low level toxin production occurs frequently among porcine VTE-producing strains of E . coli 4' 24 Our PCR protocol positively identified all 63 VTEC strains of porcine origin . O'Brien et al. have demonstrated low levels of neutralizable SLT activity in cell extracts from non-VTEC strains of bacteria, including Vibrio cholerae, Vibrio parahaemolyticus, Salmonella typhimurium and several isolates of Campylobacter jejuni ." Strains of V. parahaemolyticus, S . typhimurium and C . jejuni were tested in this study and were all found to be negative in both the Vero cell assay and the PCR . This is in agreement with results from other studies where low level toxin producers were investigated .","," Further work is needed to clarify the source of the low cytotoxic activity noted by O'Brien et al . in the strains they examined .

1 59

Some non-specific amplification occurred with S . choleraesuis, S. typhi and P . mirabilis but the products from these samples were easily differentiated from the primer-specific product by size on the gel electrophoresis . The PCR protocol outlined here may require further modifications to eliminate this nonspecific amplification if methods other than gel electrophoresis are used to detect the product . The Vero cell assay has been used as the standard detection method for VT but its use in a diagnostic setting is limited by the requirement for tissue culture facilities . Another drawback is that cytotoxicity for Vero cells is not specific for VT, and positive samples must be verified by other methods such as neutralization assays with VT-specific antibody or hybridization with DNA probes for VT. The neutralizations can be difficult to interpret and may give variable results .", " In some cases, E . coli may be identified that are verocytotoxic but are not neutralized by antiserum' or may be probe-negative ." The cytotoxicity of three E . coli isolated from ground pork and used in this study was not neutralized by specific antiserum' (also, Read, unpublished finding), but all three isolates were positive in the PCR . It is likely that these strains produce VTs that are new additions to the growing family of VT-related toxins . Numerous methods for the detection of VTEC are restricted to identification of only E . coil 0157 :H7. 1 Although E . coli 0157 :H7 is the most common serotype associated with outbreaks of disease in humans, over 55 different VTEC serotypes have been isolated from humans .' The other VTEC methodologies described above have evolved around the specific detection of VT1 and VT2, and their differentiation from VTE which is associated with disease in pigs . We chose reference strains representative of all the different types of VT that have been described . These strains were all positive in the PCR with the VTEC primers . Brown et al . have speculated on the presence of VTE (SLT llv) genes in isolates of VTEC from human disease . 23 VTE is more closely related to SLT II than is VT1 32,35 and there is evidence accumulating that a large family of VT2-related toxins exist," - " many of which have been associated with disease in humans . The failure to screen for all serotypes of VTEC, antigenic types of VT and levels of cytotoxin production may lead to bias regarding the prevalence of a specific group of VTEC . The PCR has the potential to amplify a single copy of a gene ." Thus, by concentrating the original sample and optimizing the PCR protocol, the possibility exists to test samples with little or no prior enrichment, reducing test turnaround time to less than a day . However, large numbers of bacteria and substances in stool filtrates may interfere with the

160

S. C . Read et al.

PCR reaction 29,44 and sample preparation and the PCR protocol described here will require further refinement before this method will be applicable directly to food and faecal samples . PCR technology applied to the detection of VTEC from these samples will aid in the monitoring of food products for VTEC and also in epidemiological investigations into the source and extent of outbreaks of VTEC-related disease . ACKNOWLEDGEMENTS Some of the strains used in this study were kindly provided by M . J . Champagne, M . Doyle, J . Lynch, J . McKenzie, A . O'Brien, U . Purvis, A . Sharpe and N . Strockbine. Serotyping of the Canadian isolates was kindly provided by H . Lior, Laboratory Centre for Disease Control, Ottawa . We thank J . B . Brunton for critical discussions and review of this manuscript . The assistance of Agriculture Canada and Cangene is gratefully acknowledged .

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Polymerase chain reaction for detection of verocytotoxigenic Escherichia coli isolated from animal and food sources.

Animals and their by-products have been implicated as important sources of verocytotoxigenic Escherichia coli (VTEC) associated with disease in humans...
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