Vol. 30, No. 7JOURNAL OF CLINICAL MICROBIOLOGY, JUIY 1992, p. 1801-18060095-1137/92/071801-06$02.00/0Copyright C) 1992, American Society for Microbiology

Polymerase Chain Reaction for Diagnosis ofEnterohemorrhagic Escherichia coli Infection and

Hemolytic-Uremic SyndromeMICHAEL J. BRIAN,1 MIRA FROSOLONO,2 BARBARA E. MURRAY,2 ABRAHAM MIRANDA,2

EDUARDO L. LOPEZ,3 HENRY F. GOMEZ,1 AND THOMAS G. CLEARY`*Departments ofPediatrics' and Internal Medicine,2 University of Texas Medical School at Houston,

Houston, Texas 77030, and Hospital de Ninios "Ricardo Gutierrez, " Buenos Aires, Argentina3

Received 4 November 1991/Accepted 16 April 1992

Two pairs of oligonucleotide primers were designed to amplify fragments of the genes for Shiga-like toxin I(SLT-I) and SLT-II in a single reaction. A 370-bp segment and a 283-bp segment were amplified for SLT-I andSLT-II, respectively. The specificities of the polymerase chain reaction (PCR) amplification products were

confirmed by using radioactively labeled oligonucleotide probes. SLT sequences were amplified from DNAisolated from 13 previously characterized enterohemorrhagic Escherichia coli (EHEC) strains. No amplificationproduct was produced by using DNA from 20 non-EHEC strains. As little as one bacterial genome was

detectable. PCR was then applied to DNA isolated directly from stool samples. We had to remove inhibitors ofPCR that were present in lysates prepared from stool samples before amplification was achieved. First, weevaluated the sensitivity of PCR for the detection of known numbers ofEHEC added to normal stools. Second,three children with SLT in their stools were shown to have SLT sequences in their stools by PCR. Two of thesechildren had hemolytic-uremic syndrome, and a third child was asymptomatic. Stool specimens collected fromanother 26 asymptomatic children were negative by PCR for SLT sequences. PCR can be used to diagnoseEHEC infections without prior culture of stool specimens.

Shiga-like toxin (SLT; verotoxin [VT])-producing strainsor enterohemorrhagic Escherichia coli (EHEC) strains havebeen associated with hemorrhagic colitis, hemolytic-uremicsyndrome (HUS), and thrombotic thrombocytopenic pur-pura (16). Although E. coli 0157:H7 is an important EHECserotype and appears to predominate in most areas, manyserotypes produce SLTs (16). Several toxins are recognized.SLT-I is essentially identical to Shiga toxin, the cytotoxinproduced by Shigella dysenteriae type 1 (5). SLT-II isantigenically distinct but does share some areas of nucleo-tide and amino acid sequence homology with SLT-I (14). Anumber of other toxin genes homologous to SLT-II havebeen sequenced (12, 27, 32). SLT-I and SLT-II genes codefor an A subunit that is responsible for enzymatic activityand a B subunit that mediates receptor binding.SLTs are cytotoxic to cultured HeLa and/or Vero cells.

Cytotoxin detection and neutralization with specific antiserahave been used to detect EHEC infections in stool speci-mens (20). Hybridization of probes to DNA derived frombacterial growth from stool specimens has also been evalu-ated (2, 3). In addition, various forms of immunodetectionhave been developed (6). We evaluated the polymerasechain reaction (PCR) to determine its potential for thedetection of EHEC infections.

MATERIALS AND METHODS

Bacterial strains and stool specimens. Fifteen SLT-produc-ing strains, including 13 isolates from humans, 1 recombi-nant strain, and 1 S. dysenteriae type 1 strain, were studied(Table 1). Twenty SLT-negative strains were also evaluated.Four stool samples were obtained from three children withEHEC infection. Stool samples collected from another 26

* Corresponding author.

well children attending day-care centers in Houston, Tex.,were also evaluated by PCR. Stool samples from asympto-matic children in these day-care centers were being collectedweekly as part of another study. All stool specimens (frominpatients and children in day care) were placed at 4°C assoon as possible after collection and were transferred to-70°C after logging the relevant data. Culture of stool for E.coli 0157:H7 and testing of stool for SLTs by cytotoxicityassay were performed as described previously (20).PCR primers and probes. Oligonucleotides were chosen by

using published sequences (5, 14, 27, 32) and synthesized byGenosys (Houston, Tex.). Table 2 shows the sequences ofthe oligonucleotides. The SLT-I and SLT-II sequences canbe aligned on the basis of their considerable homology (14).Therefore, Table 2 gives the nucleotide position with refer-ence to one published sequence of SLT-II (14). The PCRprimers were designed to flank the region of the A subunit ofSLT genes containing the codon corresponding to glutamicacid 166 in SLT-II (amino acid 167 in SLT-I). The 5' primersfor SLT-I and SLT-II are referred to as 5'I and 5'II,respectively, and the 3' primers for SLT-I and SLT-II arereferred to as 3'I and 3'II, respectively. We used twoadditional oligonucleotides which hybridized internally tothe PCR primers to confirm the specificity of the PCR. Oneadditional oligonucleotide (Iin) was designed to hybridizespecifically to the amplified SLT-I product between primers5'I and 31, and another oligonucleotide (Ilin) was designedto hybridize specifically to the amplified SLT-II productbetween primers 5'II and 3'II. The polynucleotide probesused were an 1,142-bp fragment which contained most of thecoding region for SLT-I and an 842-bp fragment whichcontained part of the coding region for SLT-II (23).

Preparation ofDNA derived from bacterial strains and stoolspecimens for PCR analysis. Bacteria were grown in Luria-Bertani medium at 37°C. Initially, the viable count of E. coli

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TABLE 1. Bacteria used in this study

Species and serotype Strain SLt Source ortype" referenceh

Escherichia coliEnterohemorrhagic0157:H-026:H110157:H7Ountyp:H21O15:H-0157:H70157:H70157:H70157:H704:NM0125:NM0121:H190145:NM

Recombinant

Enterotoxigenic078:H12063:H-078:H11

Fecal flora088:H8075:H-ND"NDNDNDNDND

Laboratory strains

Other gram-negative bacteriaShigella dysenteriae serotype 1

Shigella flexneriSerotype 2aSerotype 2a

Shigella sonnei

Pseudomonas aeruginosa

306-7H-30212-443-12217-8E773El 018E1057E10583377-853153-863056-8575-83

I,IIIIIII,III,I,I,IIIII

C600 (933W) II

Tx432 N19123 N(H10407 N(

232-5 N(240-2 N5108 N1035 N2007-2a N4010-3a N5107-If N5201 N

C600 N(J53 NHB101 N

60R I

24 N55 N

S-88 N(

27853 N,

II Argentina19ArgentinaArgentinaArgentina

II MinnesotaMinnesota

II MinnesotaII MinnesotaII CDC

CDCCDCCDC

24

one 22one 221one 22

oneoneoneoneone[one[one[one

ArgentinaArgentinaHoustonHoustonHoustonHoustonHoustonHouston

[one[one[one

[one 1[one 1

[one Houston

[one

None

None

ATCC

Houston

Houston

" Toxin type was determined by hybridization to colony lysates (10). Eachstrain was evaluated by using the two SLT-I PCR primers, an 1,142-bp SLT-Iprobe (23), the two SLT-II PCR primers, and an 842-bp SLT-II probe (23).

" Argentina, Hospital de Niinos "Ricardo Gutierrez," Buenos Aires; Min-nesota, M. Osterholm, Minnesota Department of Health; CDC, Centers forDisease Control, Atlanta, Ga.; Houston, Division of Infectious Diseases,Department of Internal Medicine, University of Texas Medical School at

Houston; ATCC, American Type Culture Collection, Rockville, Md.' ND, not determined.

(three separate cultures) was compared with the opticaldensity at 600 nm. On the basis of the line of least-squaresregression, the optical density was used to estimate bacterialnumber. As reported by Maniatis et al. (21), 1 optical density

TABLE 2. Sequences of oligonucleotides

Primer Sequence Location'

5I 5'-AAATCGCCATTCGTTGACTACTTCT-3' 691-71531 5'-TGCCATTCTGGCAACTCGCGATGCA-3' 1033-1057lin 5'-GTCGCATAGTGGAACCTC-3' 730-747

5'11 5'-CAGTCGTCACTCACTGGTTTCATCA-3' 691-7153'11 5'-GGATATTCTCCCCACTCTGACACC-3' 950-973Ilin 5'-GGAGTTCAGTGGTAATAC-3' 730-747

' The nucleotide position refers to the sequences published previously (14).

unit corresponds to an approximate viable count of 8 x 108bacteria per ml.DNA was isolated from bacteria by standard techniques

(28). DNA was isolated from stool specimens by two tech-niques. By the first technique, 200 mg of stool was sus-pended in 1 ml of phosphate-buffered saline (120 mmol ofNaCl, 2.7 mmol of KCl, 10 mmol of KH2PO4, 10 mmol ofNa2HPO4 in 1 liter [pH 7.4]), and a crude bacterial pellet wasprepared by using alternating high- and low-speed centrifu-gation (centrifugation method). Particulate material was re-moved from the suspension by discarding the pellet aftercentrifugation at 300 x g (Eppendorf 5415 C) for 3 min, andsoluble substances were removed by discarding the super-natant after centrifugation at 16,000 x g for 3 min. These twosteps were repeated three times. DNA was isolated from theresulting bacterial pellet by the method used for isolatingDNA from pure bacteria, and the isolated DNA was dis-solved in 500 p. of water (28). By the second technique(heat-lysis method), 100-mg samples of stool were sus-pended in 0.5 ml of 50 mM Tris-HCl-2 mM EDTA (pH 8.0)and heated to 95 to 100°C for 3 min. The supernatant wasremoved after centrifugation at 16,000 x g for 3 min andincubated with proteinase K (100 pg/ml; Boehringer Mann-heim, Indianapolis, Ind.) at 37°C for 2 h. The aqueous phasewas removed after extraction with phenol-chloroform (1:1).DNA was ethanol precipitated and dissolved in 200 p.1 ofwater. The DNA concentration was estimated by ethidiumbromide staining (4). DNA was further purified by usingsilica particles (Geneclean; Bio 101, Inc.; La Jolla, Calif.).By this technique, the DNA that is bound to silica particles(median particle size, 10 pm) is washed with bufferedethanol before being eluted into water (31).PCR. PCR was performed in a Perkin-Elmer Cetus (Nor-

walk, Conn.) thermal cycler model 480. DNA (10 ,ul) wasoverlaid with mineral oil (50 p.l) and was denatured for 5 minat 94°C prior to adding the reaction mixture (90 p.1). Theconcentrations of nucleotides and primers were 200 and 0.25p.M, respectively. The reaction buffer was 10 mM Tris-HCl(pH 8.3), 50 mM KCl, 2.75 mM MgCl2, and 0.01% gelatin.Recombinant Taq polymerase from Perkin-Elmer Cetus (2.5U per reaction) was used. Thirty-five cycles of 1 min at 60°C,15 s at 72°C, and 1 min at 94°C were used. The final step wasa 7-min incubation at 72°C. Amplification products (10 Rl)were analyzed by electrophoresis in 2% NuSieve (FMC,Rockland, Maine)-0.5% agarose (Bethesda Research Labo-ratories, Gaithersburg, Md.) gels containing ethidium bro-mide (0.5 ,ug/ml). Molecular mass markers (1-kb ladder;Bethesda Research Laboratories) were electrophoresedsimultaneously. Gels were photographed under UV illumi-nation.PCR was performed in four types of experiments. First,

DNAs isolated from known toxin-positive strains and known

Citrobacter freundii

Providencia alcalifaciens

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PCR FOR DIAGNOSIS OF EHEC INFECTION 1803

toxin-negative strains were amplified. Second, the minimumdetectable target by PCR was established by using serialdilutions of an EHEC strain (306-7) which produces SLT-Iand SLT-II. Third, known numbers of strain 306-7 wereadded to 200-mg samples of normal stool to determine theminimum target detectable by PCR. Finally, DNA wasisolated from stool samples obtained from two children withE. coli 0157:H7-associated HUS.

Detection and prevention of cross contamination. A reagentblank containing reaction mixture but no DNA was includedin each set of reactions. Multiple additional negative controlswere included with each set of samples to be tested. Nega-tive controls included stools from well children in Houstonday-care centers as well as sham stool specimens. Thesesham specimens were tubes that were processed simulta-neously and identically to stool specimens but that containedno stool. Separate rooms were used for DNA isolation, PCR,PCR analysis, and PCR product storage. Two sets of pi-pettes were dedicated to PCR. One set was used for DNAisolation and another set was used for preparation of thePCR. A Gilson pipette with disposable positive displacementtips was used to dispense DNA aliquots (Rainer InstrumentCo., Emeryville, Calif.). Care was taken when openingmicrocentrifuge tubes to minimize aerosols, and only tubescontaining the same sample were open at any one time.

Southern blotting and dot blot analysis. Alkaline transfer ofDNA from agarose gels to Hybond-N membrane (Amer-sham, Arlington Heights, Ill.) was performed according tothe manufacturer's instructions. For dot blot analysis, 10 ,ulof heat-denatured PCR product was mixed with 20 ,u1 of 3 MNaCI-0.5 M NaOH and was applied to Hybond-N mem-brane that was previously soaked in 1.5 M NaCl-0.25 MNaOH (Bio-dot manifold; Bio-Rad, Richmond, Calif.). DNAwas fixed to membranes by placing the membranes on a UVtransilluminator for 3 min. Polynucleotide probes were la-beled with [a-32P]dCTP (Amersham) by using a randomprimer kit (Stratagene, La Jolla, Calif.). Unincorporatedlabel was removed by using Bio-spin (Bio-Rad) columns.Hybridization of polynucleotide probes (105 cpm/cm2) wascarried out in 50% formamide-0.5% sodium dodecyl sulfate(SDS)-5x SSPE (lx SSPE is 0.18 M NaCl, 10 mMNaH2PO4, 1 mM EDTA, [pH 7.4])-5 x Denhardt's solution(lx Denhardt's solution is 0.02% bovine serum albumin,0.02% Ficoll, 0.02% polyvinylpyrrolidone)-denatured calfthymus DNA (final concentration, 100 ,ug/ml) at 42°C for 18to 24 h. Oligonucleotides were 5'-end-labeled with 32P byusing [_y-32P]dATP (Amersham) and T4 polynucleotide ki-nase. Unincorporated radioactivity was removed by using aNACS columns (Bethesda Research Laboratories), and 105cpm/cm2 was added to the hybridization mixture for 18 to 24h. Hybridization of 32P-labeled oligonucleotides was per-formed as described by Karch and Meyer (17). The hybrid-ization temperatures were 54°C for the SLT-I confirmatoryoligonucleotide and 50°C for the internal SLT-II oligonucle-otide. After hybridization, membranes were washed in 5xSSC (lx SSC is 150 mM NaCl plus 15 mM sodium citrate)-0.2% SDS twice at 30°C for 10 min; this was followed by athird wash for 10 min at 54 or 50°C.

RESULTS

Amplification of toxin gene sequences from isolated EHECstrains. DNA was isolated from overnight cultures of 15Shiga toxin- or SLT-producing strains (13 EHEC strains)and 20 toxin probe-negative strains. No amplification prod-ucts were seen when PCR was performed on DNA isolated

* 1 2 3 4 5 6 7 1'21314'516'7'

370 bp283 bp

FIG. 1. Sensitivity of PCR for detection of SLT sequences byusing serial dilutions of E. coli 0157:H7 strain 306-7. Amplificationproducts were visualized after electrophoresis by staining the gelwith ethidium bromide. Lanes 1 to 7, DNA isolated from strain306-7; lanes 1' to 7', DNA isolated from normal stool with strain306-7 added. SLT-I and SLT-II bands are seen. Products are visiblein lanes 1 to 3 and 1' to 3'. The approximate numbers of 306-7genomes per reaction were 9 x 104 in approximately 300 pg of DNA(lane 1), 9 x 103; (lane 2), 900 (lane 3), 90 (lane 4), 9 (lane 5), 0.9 (lane6), reagent blank (lane 7), 8 x 103 (2 x 105 bacteria per mg of stool)(lane 1'2, 8 x 102 (2 x 104 bacteria per mg of stool) glane 2'), 8 x 101(2 x 10 bacteria per mg of stool) (lane 3'), 8 (2 x 10 bacteria per mgof stool) (lane 4'), 0.8 (20 bacteria per mg of stool) (lane 5'), no 306-7(lane 6'), and reagent blank (lane 7').

from toxin-negative bacterial strains. Amplification productsof the expected size (370 bp for SLT-I and 283 bp for SLT-II)were seen in toxin-positive strains. The pattern of hybrid-ization obtained with oligonucleotides end-labeled with 32pconfirmed the agarose gel results. The toxin genotypesdetermined by PCR corresponded to the results of colonyhybridization (Table 1).

Sensitivity of PCR for detecting EHEC DNA in serialdilutions of broth cultures. PCR was performed on DNAisolated from serial dilutions of EHEC strain 306-7. TargetDNA derived from 100 to 1,000 bacteria (corresponding toabout 300 fg to 3 pg of DNA) produced amplificationproducts of the predicted sizes that were visible by UVillumination (Fig. 1). Upon hybridization with 32P-labeledpolynucleotide probes and labeled oligonucleotides, sensi-tivity was increased by 2 orders of magnitude, so that DNAderived from approximately 1 to 10 bacteria could be de-tected (Fig. 2).PCR with DNA isolated from stool to which serial dilutions

ofEHEC strain 306-7 were added. Known numbers of EHECwere added to normal stool specimens. DNA was isolatedfrom the mixture by the centrifugation method. Approxi-mately 5 ,ug of DNA per mg of stool was isolated. DNAisolated from such stool specimens by the centrifugationmethod alone had to be diluted 100-fold before PCR wassuccessful. The reaction was inhibited when less dilute DNAwas used. The absence of a primer dimer band reflected theabsence of amplification. Therefore, the minimum concen-tration of bacteria detectable in stool was 100-fold less thanthat expected from the sensitivity obtained with bacteriaalone. Following PCR, DNA derived from 1 mg of stoolcontaining about 2,000 bacteria produced visible bands onthe gel (Fig. 1). Upon hybridization with 32P-labeled poly-

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1 2 3 4 5 6 7 8 * 1 2 3 4 5X" 'S

1 1 2 l 3 1 4 l 5 I

A bp

;;

_to6

1 2 3 4 5 6

a b c d e fB

FIG. 2. Sensitivity of PCR for detection of SLT sequences byusing serial dilutions of E. coli 0157:H7 strain 306-7. DNA was

transferred from agarose gels to Hybond-N membranes and hybrid-ized to a 32P-labeled, 1,142-bp SLT-I probe. (A) DNA isolated fromstrain 306-7. (B) DNA isolated from normal stool with strain 306-7added. A 370-bp SLT-I band is seen. The PCRs shown here are thesame as those shown in Fig. 1. Products are visible in lanes 1 to 6 inpanel A and in lanes 1 to 5 in panel B. In panel A the approximatenumbers of 306-7 genomes per reaction were 9 x 105 in approxi-mately 3 pg of DNA (lane 1), 9 x 104 (lane 2), 9 x 103 (lane 3), 900(lane 4), 90 (lane 5), 9 (lane 6), 0.9 (lane 7), and reagent blank (lane8). In panel B the approximate numbers of 306-7 genomes perreaction were 8 x 103 (2 x 105 bacteria per mg of stool) (lane 1), 8x 102 (2 x 104 bacteria per mg of stool) (lane 2), 8 x 10 (2 x 103bacteria per mg of stool) (lane 3), 8 (2 x 102 bacteria per mg of stool)(lane 4), 0.8 (20 bacteria per mg of stool) (lane 5), and no 306-7 (lane6).

nucleotide probe, sensitivity was increased by 2 orders ofmagnitude (Fig. 2).PCR with purified DNA isolated from patient stool samples.

Consistent amplification of SLT sequences from naturallyinfected stool samples was not achieved following dilution ofDNA isolated by the centrifugation method. DNA isolatedby the centrifugation method and DNA isolated by heat lysisfollowed by silica particle purification (Geneclean) were

compared as templates for PCR of SLT-specific sequences.The source of positive DNA was stool obtained early in thecourse of E. coli 0157:H7-associated HUS. PCR was posi-tive only 4 of 18 (22%) times by the centrifugation methodcompared with 22 of 27 (81%) times by heat lysis and silicaparticle purification. This difference between the methodswas significant (P = 0.0003 by the x2 test with Yates'correction).Samples from two children with HUS in Houston, Tex.,

were positive for SLT sequences by PCR analysis (Fig. 3).The first stool from one child (patient 1) contained SLT-IIsequences alone, while the only stool obtained from a

second child (patient 2) contained both SLT-I and SLT-IIsequences. E. coli 0157:H7 was isolated from the stool ofpatient 2. The positive stool from patient 1 was obtained 4days after the onset of diarrhea and was positive by PCR andcytotoxicity assay, while a second sample obtained 3 dayslater was negative by both assays. E. coli 0157:H7 was

isolated from the stool of the grandmother of patient 1,although the organism was not isolated from either of thestool samples from patient 1. The stool of a third child waspositive for SLT-I alone by PCR. This child had no diarrhea,but SLT-I was detected in his stool by the cytotoxicity

FIG. 3. Amplification by PCR of SLT sequences from stool. (A)Lane *, molecular mass markers; lanes 1 to 5, products of PCRperformed on a 1:10 dilution of DNA isolated from stool; lanes 1' to5', products of PCR performed on a 1:100 dilution of DNA isolatedfrom stool. The sources of stool DNA were patient 1 with HUS 3days after the onset of diarrhea (lanes 1 and 1'; approximately 75 and7.5 ng of DNA, respectively); patient 1 7 days after the onset ofdiarrhea (lanes 2 and 2'), reagent blank (lanes 3 and 3), patient 2with HUS on admission (lanes 4 and 4'; approximately 30 and 3 ngof DNA, respectively); reagent blank (lanes 5 and 5'). An SLT-IIband is seen in lanes 1 and 1', and both SLT-I and SLT-II bands areseen in lanes 4 and 4'. (B) Dot blot hybridization analysis of PCRproducts. PCR products were hybridized to 32P-end-labeled oligo-nucleotides lin (SLT-I specific, lower row) and Ilin (SLT-II specific,upper row). The sources of DNA from stool specimens producingpositive signals were patient 1 with HUS 3 days after the onset ofdiarrhea (rows a and b; 1:10 dilution [row a], 1:100 dilution [row b]);HUS patient with HUS (rows e and f; (1:10 dilution [row e], 1:100dilution [row f]); stool from asymptomatic, culture negative sibling ofpatient 1 (rows c and d; 1:10 dilution [row c], 1:100 dilution [row d]).

assay. He was attending a Houston day-care center wherechildren were being prospectively enrolled in a study ofdiarrhea.We wished to exclude rigorously the possibility of false-

positive results because of contamination of the PCRs withextraneous SLT DNA. All reagent blanks, 10 sham stoolspecimens, and 26 stool specimens from well children inHouston day-care centers were negative by gel and dot blotanalysis of PCR products. These 26 control stool specimenswere also negative by the standard cytotoxicity assay. Nonegative control was chosen for over 6 months from theday-care center from which the PCR-positive stool was

obtained.

DISCUSSION

SLTs are a heterogeneous family. The prototype toxin isSLT-I (VT1). There are only three nucleotides and oneamino acid difference between SLT-I and Shiga toxin (5).SLT-II (VT2) has about 60% nucleotide homology to SLT-Iin the genes for both A and B subunits (57 and 60%,respectively) (14). Three additional SLT-II type genesvtx2ha and vtx2hb (VT2v-a and VT2v-b, respectively) and

A370 up

B370 bp

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PCR FOR DIAGNOSIS OF EHEC INFECTION 1805

SLT-IIc have 95 to 100% nucleotide homology to each otherin both subunit genes (12, 27). SLT-IIv (VTe) has lesshomology to the other SLT-II genes, particularly in the Bsubunit (about 80%) (32). Another toxin gene whose Bsubunit gene has 98% nucleotide homology to SLT-IIv butonly about 70% homology to the A-subunit gene of eitherSLT-IIv or SLT-II has also been sequenced (9). It has beensuggested that this gene represents a new type of SLT (30).Previous investigators have used different PCR strategies toovercome the difficulty posed by genetic heterogeneityamong SLTs. Karch and Meyer (18) used one pair ofprimers. Their 20- to 21-nucleotide primers were partlyhomologous to both SLT-I and SLT-II, with a 1- to 2-nucleotide difference between either SLT gene and theprimers. PCR with these primers produced a 224-bp frag-ment of SLT-II and a 227-bp fragment of SLT-I. They usedtwo internal oligonucleotides for hybridization analysis todistinguish between SLT-I and SLT-II. They were unable toamplify SLT sequences directly from stool specimens. An-other group has used separate primer pairs to distinguishdifferent SLT-II genes. They used three separate primerpairs from different sites in three toxin genes to producefragments of different sizes for SLT-I, SLT-II, and SLT-IIv(15, 26). Recently, the same group has described a furtherprimer pair which amplifies a common fragment of the Bsubunits of SLT-IIv, vtx2ha, and vtx2hb genes (30). Thoseinvestigators distinguished between the three variant se-quences by restriction endonuclease digestion of the ampli-fied product. Neither those primers nor our SLT-II primersare complementary to one published SLT gene sequence (9).Those investigators (9) have not reported any results of PCRapplied directly to stool specimens.Our strategy was to use a limited number of primer pairs to

detect as many toxin genes as possible in a multiplex PCR.Our oligonucleotide primers were designed to amplify ahomologous segment of the A subunit of SLT genes whichcodes for a portion of the toxins containing a glutamic acidresidue that is critical for enzymatic activity (11). This regionis relatively conserved among the SLT genes. Indeed, theSLT-II primers were exactly complementary to correspond-ing regions of SLT-II, vtx2ha, vtx2hb, SLT-IIc, and SLT-lIv. A primer pair specific for SLT-I as well as a primer pairspecific for SLT-II and variants were included in eachreaction. Several copies of different SLT-II genes have beenfound in some strains (27). Our PCR primers would beexpected to produce identically sized amplification productsfrom the multiple SLT-II genes contained by the reportedstrains. PCR might be less sensitive in the detection ofsingle-copy target sequences. A second pair of oligonucleo-tides was designed to confirm the specificities of PCRproducts. The confirmatory oligonucleotides were chosen tohybridize to the fragments of SLT-I and SLT-II which wereamplified. The SLT-II confirmatory oligonucleotide wascomplementary to SLT-II, vtx2ha, vtx2hb, SLT-IIc, andSLT-IIv. Jackson (13) described the incorporation of 32p ordigoxigenin into the actual PCR product in the hope ofavoiding a hybridization step. This method was used for thesensitive detection of the SLT-I gene in bacterial DNA, butdot blot analysis was not simplified because a hybridizationstep was still required.One objective of our study was the direct detection of SLT

sequences in stool specimens. It is apparent from the liter-ature that it can be difficult to apply PCR to the directdetection of pathogens in stool specimens. DNA isolatedfrom stool specimens has been reported by some investiga-tors to contain inhibitors of PCR. Olive (25) used a chro-

matographic DNA purification step prior to PCR to detectenterotoxigenic E. coli infection by using DNA isolated fromstool specimens. However, using a routine DNA isolationtechnique, Frankel et al. (8) amplified Shigella invasion-associated locus sequences from Shigella and enteroinvasiveE. coli isolates as well as normal stool spiked with Shigellaflexneri. They were also able to amplify sequences specificfor the heat-stable and heat-labile toxins of enterotoxigenicE. coli and an invasion-associated locus of Shigella speciesfrom stools of children in Mexico (7). They mentioned nodifficulty with stool inhibitors. Wilde et al. (33) have system-atically evaluated the presence of PCR inhibitors in stoolspecimens. They successfully used cellulose CF11 extrac-tion to remove inhibitors from DNA in stool specimens priorto reverse transcriptase-PCR detection of rotavirus nucleicacid in stool specimens. We introduced an analogous methodwhereby the DNA was bound to silica particles and washedwith buffered ethanol to remove inhibitors. The DNA wasthen eluted for the purposes of PCR. This allowed amplifi-cation of SLT sequences from the stools of two children withEHEC-related disease and the stool of a third asymptomaticchild with SLT-I in his stool.

In our study, the stool of one child with HUS (patient 1)was positive for SLT-II sequences by PCR 4 days after theonset of diarrhea but negative 3 days later. It is known thatthe chances of isolating EHEC decrease with time after theonset of symptoms. Tarr et al. (29) presented data on 57patients with HUS over a 3-year period. The majority hadpreceding bloody diarrhea. E. coli 0157:H7 was isolatedfrom 33 of the 52 patients with diarrhea (63%) and closecontacts of another 4 of these 52 patients. E. coli 0157:H7was isolated from all 13 patients whose stools were collectedwithin 2 days of the onset of diarrhea, 11 of 12 patientswhose stools were submitted within 3 to 6 days of the onsetof diarrhea, and only 9 of 27 patients whose stools werecollected 7 days or more after the onset of diarrhea. Thestool of one child with HUS (patient 1) was positive forSLT-II by PCR but was negative for E. coli 0157:H7 byculture, although a family contact had a positive stoolculture. There was no reason to suspect that this specimenhad been compromised in any way, but it is reasonable toexpect PCR to be able to detect SLT-producing organismswhen culture techniques fail, because PCR should detectnonviable organisms and viable organisms in low numbers.By using the primers evaluated in this study, amplification

of SLT sequences by PCR distinguished between SLT-positive E. coli and SLT-negative bacterial strains. PCRidentified SLT-positive E. coli regardless of serotype and isan advantage in areas where serotypes other than 0157:H7are prevalent. PCR resulted in amplified SLT sequences thatwere visible after agarose gel electrophoresis by using DNAderived from about 100 to 1,000 SLT-positive E. coli. WhenPCR was applied to DNA isolated from stool specimens towhich SLT-positive E. coli was experimentally added, thesensitivity level corresponded to the detection of at least2,000 bacteria per mg of stool. The demonstration of SLTsequences in stool by using PCR makes possible the detec-tion of EHEC infections without the need for isolation of thecausative strain. However, consistent amplification of SLTsequences from naturally infected stool specimens was pos-sible only by using silica-purified DNA.

ACKNOWLEDGMENTS

This study was funded in part by Public Health Service grantHD-13021 and Advanced Technology Program of the State of Texasgrant 011618-060.

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We acknowledge Rory Van for isolation and identification of E.coli 0157:H7.

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