This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oberst, R. D.
Right arrow Articles by Gillespie, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oberst, R. D.
Right arrow Articles by Gillespie, J. R.
Agricola
Right arrow Articles by Oberst, R. D.
Right arrow Articles by Gillespie, J. R.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, September 1998, p. 3389-3396, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

PCR-Based DNA Amplification and Presumptive Detection of Escherichia coli O157:H7 with an Internal Fluorogenic Probe and the 5' Nuclease (TaqMan) Assaydagger

R. D. Oberst,1,* M. P. Hays,1 L. K. Bohra,2 R. K. Phebus,2 C. T. Yamashiro,3 C. Paszko-Kolva,3 S. J. A. Flood,3 J. M. Sargeant,1 and J. R. Gillespie1

Food Animal Health & Management Center, College of Veterinary Medicine,1 and Department of Animal Sciences & Industry, College of Agriculture,2 Kansas State University, Manhattan, Kansas 66506, and Perkin-Elmer Corporation, Applied Biosystems Division, Foster City, California 944043

Received 9 February 1998/Accepted 30 June 1998

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Presumptive identification of Escherichia coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the enterohemorrhagic E. coli (EHEC) eaeA gene. In this report, we describe the development and evaluation of the sensitivity and specificity of a PCR-based 5' nuclease assay for presumptively detecting E. coli O157:H7 DNA. The specificity of the eaeA-based 5' nuclease assay system was sufficient to correctly identify all E. coli O157:H7 strains evaluated, mirroring the previously described specificity of the PCR primers. The SZ-primed, eaeA-targeted 5' nuclease detection assay was capable of rapid, semiautomated, presumptive detection of E. coli O157:H7 when >= 103 CFU/ml was present in modified tryptic soy broth (mTSB) or modified E. coli broth and when >= 104 CFU/ml was present in ground beef-mTSB mixtures. Incorporating an immunomagnetic separation (IMS) step, followed by a secondary enrichment culturing step and DNA recovery with a QIAamp tissue kit (Qiagen), improved the detection threshold to >= 102 CFU/ml. Surprisingly, immediately after IMS, the sensitivity of culturing on sorbitol MacConkey agar containing cefeximine and tellurite (CT-SMAC) was such that identifiable colonies were demonstrated only when >= 104 CFU/ml was present in the sample. Several factors that might be involved in creating these false-negative CT-SMAC culture results are discussed. The SZ-primed, eaeA-targeted 5' nuclease detection system demonstrated that it can be integrated readily into standard culturing procedures and that the assay can be useful as a rapid, automatable process for the presumptive identification of E. coli O157:H7 in ground beef and potentially in other food and environmental samples.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Enterohemorrhagic Escherichia coli O157:H7 is an important human pathogen that is predominantly associated with hemorrhagic colitis and the more severe complications of hemolytic uremic syndrome. Although human-to-human transmission of E. coli O157:H7 has been demonstrated (25), most infections have been associated with the consumption of contaminated ground beef, milk, water, produce, and apple juice products that have been improperly handled, stored, or cooked (1, 5, 7, 15, 18, 19, 25, 43). The primary reservoir is believed to be cattle (24, 27). However, a clear understanding of the farm ecology of E. coli O157:H7 is lacking, partly because of the low detected prevalence in individual cattle and herds (20, 40, 55) and the low infectious dosage required for human infections (25, 53).

Established methods for recovering and identifying E. coli O157:H7 from foods and feces have been hindered by the inability to specifically and rapidly detect small numbers of organisms from complex matrices and background microflora. Although the inclusion of preenrichment incubations and immunomagnetic separation (IMS) (6, 9, 11, 16, 22, 31, 44, 48) and additional selective subculturing or secondary enrichment incubations (12, 14, 30) have been reported to increase the detection rate of E. coli O157:H7 from foods and fecal specimens, these methods are dependent on isolating individual colonies from selective and/or indicator media and then characterizing them in immunological and biochemical/fermentation reactions. Immunological assays are used to determine if the O157 somatic and H7 flagellar antigens are present, while the biochemical/fermentation reactions determine in classical taxonomic fashion the genus and species of the isolate. Combined with the initial replication steps in the isolation process, the current E. coli O157:H7 identification process takes 5 or more days to complete. This adds considerably to the costs required to determine whether a sample contains E. coli O157:H7 and is a limiting factor in doing more E. coli O157:H7 tests.

Rapid methods for identifying E. coli O157:H7 in foods or fecal specimens have been directed at immunological or genetic targets. Antigenic targets have included the E. coli somatic (O157) or flagellar (H7) antigens (21, 50), two low-molecular-weight antigens (30, 45), and the virulence-associated Shiga-like toxin (SLT) types I and II (3, 17, 33). However, these assays are occasionally unable to distinguish certain other E. coli strains from E. coli O157:H7 strains (30, 49) and/or toxigenic from nontoxigenic E. coli O157 strains (46).

PCR-based detection procedures have been used to identify E. coli O157:H7 and have targeted the sltI and sltII genes (32, 47, 54), the enterohemorrhagic E. coli (EHEC) uidA gene (10), and a portion of a 60-MDa plasmid (23). Because similar genes are present in some nonpathogenic E. coli and in other bacteria, individual PCRs that target these genes are unable to confirm the identity of an isolate as E. coli O157:H7. Identification by PCR requires that multiple genes be targeted in separate PCRs on the DNA from a suspect organism or that the DNA from that organism be subjected to a multiplex PCR that targets the multiple genes simultaneously (10, 23, 42, 51).

Presumptive identification of E. coli O157:H7 is possible in an individual, nonmultiplexed PCR if the reaction targets the EHEC eaeA gene. In separate studies targeting two different regions of the eaeA gene, every E. coli O157:H7 reference strain evaluated demonstrated the predicted PCR product. Louie et al. (36) targeted the 3' end of the EHEC eaeA gene, whereas Meng et al. (41) amplified a 633-bp product upstream of the 5' end of the EHEC eaeA gene by using PCR primers SZ-I and SZ-II. Both reactions were limited as confirmatory PCRs for identifying E. coli O157:H7, because similar PCR products were evident with some E. coli O157:NM strains and some enteropathogenic E. coli O55:H7 and O55:NM strains (36, 41).

Although PCR can amplify DNA molecules thousands-fold, the specifically amplified product must be detected in order to prove its presence; a variety of methods have been developed for this purpose. The most commonly used research technique, gel electrophoresis, does not show the specificity of the PCR and lacks sensitivity. Southern blots or dot blot hybridizations with probes will demonstrate the specificity of the PCR, but they require multistep processing and add considerable time and expense to the detection process. Neither of these PCR detection processes is conducive to rapid, high-throughput, automated PCR detection schemes.

Recently, 5' nuclease assays (TaqMan; PE Applied Biosystems, Foster City, Calif.) that allow the automated PCR amplification, detection, and analysis of Salmonella spp. (13, 39), Listeria monocytogenes (2, 4), and SLT genes (28, 54) in various foods have been described. The 5' nuclease assay exploits the 5'right-arrow3' exonuclease activity of Thermus aquaticus (Taq) DNA polymerase (29, 37) to hydrolyze an internal TaqMan probe labeled with a fluorescent reporter dye and a quencher dye (34). For the intact probe, the quencher dye suppresses the fluorescent emission of the reporter dye because of its spatial proximity on the probe. During PCR, the probe anneals to the target amplicon and is hydrolyzed during extension by the Taq DNA polymerase. The hydrolysis reduces the quenching effect and allows for an increase in emission of the reporter fluorescence. This increase is a direct consequence of a successful PCR, whereas the emission of the quencher dye remains constant irrespective of amplification.

Because development of fluorogenic reporter signals occurs only with a successful PCR, detection of specific DNA sequences can be based on monitoring for an increase in reporter fluorescence following PCR with a fluorometer (ABI Prism sequence detection system; PE Applied Biosystems). Interpretation of the fluorometric data can be automatically read and interpreted by using a 96-sample format and presented as a "yes" or "no" conclusion as to the presence or absence of the DNA within 15 min of the completion of the PCR.

In this report, we describe the development and evaluation of the sensitivity and specificity of a 5' nuclease assay for amplifying and presumptively detecting E. coli O157:H7 DNA. Evaluation of the 5' nuclease assay included the comparison of two DNA extraction procedures for recovering E. coli O157:H7 DNA from pure broth cultures; from pure broth cultures immediately before IMS, where the DNA was then retrieved on sorbitol MacConkey agar supplemented with cefeximine and tellurite (CT-SMAC); from pure broth cultures following IMS plus 18-h secondary enrichment, where the DNA was then retrieved on CT-SMAC; and from spiked broth cultures containing ground beef.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bacterial strains and culture conditions. The strains of bacteria that were evaluated are listed in Table 1. The E. coli O157:H7 strain MD 380-94, originally recovered from salami by the U.S. Department of Agriculture (USDA) Food Safety Inspection Service (FSIS) after an associated disease outbreak, was used as the reference strain in all optimization and sensitivity experiments. The E. coli strains were cultured in modified E. coli broth (mEC; BBL, Cockeysville, Md.) containing 20 mg of novobiocin per liter or in tryptic soy broth (TSB; Difco, Detroit, Mich.) modified (mTSB) to contain 100 mg of novobiocin, 10 mg of cefsulodin, 8 mg of vancomycin, and 0.05 mg of cefeximine per liter. All antibiotics were obtained from Sigma Chemical Co., St. Louis, Mo. Cultures were transferred to CT-SMAC plates containing sorbitol MacConkey agar (Difco) supplemented with cefeximine and tellurite (Dynal, Lake Success, N.Y.) and then incubated (18 to 24 h, 37°C).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 1.   Bacterial strains evaluated and Delta RQ values generated in PCR with the SZ-I and SZ-II primers and the SZI-97 fluorogenic probe

Developing fluorogenic probes for E. coli O157:H7. Fluorogenic probes to the eaeA gene contained within the SZ-primed amplicon (41, 42) (GenBank accession no. U32312) were synthesized as previously described (2). The efficiency of individual probes was determined by PCR using purified DNA templates (QIAamp tissue kit; Qiagen, Inc., Chatsworth, Calif.) from reference strains of E. coli and other bacteria. Approximately 150 ng of DNA (DNA DipStick kit; Invitrogen, Carlsbad, Calif.) from each reference strain of bacteria was PCR amplified by using the E. coli O157:H7-specific primers SZ-I and SZ-II (41, 42). Following the two-step PCR, the fluorescence intensities of the fluorescent reporter dye (6-carboxy-fluorescein; lambda em = 518) and the fluorescent quencher dye (6-carboxytetram-ethylrhodamine; lambda em = 582) were determined for each tube by using a luminescence spectrometer with a 96-tube reader accessory (TaqMan LS-50B PCR detection system; Perkin-Elmer). The degree of hydrolysis was calculated by using the equation Delta RQ = RQ+ - RQ- (2), where RQ+ = emission of reporter dye/emission of quencher dye and RQ- = emission of reporter dye (no DNA template)/emission of quencher dye (no DNA template).

The Delta RQ threshold for determining the presence ("yes") of E. coli O157:H7 DNA in individual MicroAmp optical tubes and caps (MOT; Perkin-Elmer) was based on a 99% confidence interval based on the standard deviation of RQ- values from no-template controls from >20 plates (three no-template controls per plate). A "yes" interpretation of a Delta RQ value obtained by using MicroAmp optical 96-well reaction plates with MicroAmp caps (MORP) (Perkin-Elmer) was determined at 99% confidence intervals, by using the standard deviation of RQ- values of three no-template controls per plate. Data were collected and analyzed with the Fluorescence Data Manager (Perkin-Elmer) and Excel spreadsheet (Microsoft Corporation, Redmond, Wash.) programs on a personal computer.

PCR conditions. The PCR amplification conditions were modified from those described by Meng et al. (41) and included fluorogenic probes. Briefly, 5 µl of sample containing the DNA template to be evaluated was added to 45 µl of PCR master mix (5 µl of 1× PCR buffer II [Perkin-Elmer], 1.5 to 4.0 mM MgCl2, 200 nM each primer [SZ-I and SZ-II], 200 µM deoxynucleoside triphosphate, 0.025 U of AmpliTaq DNA polymerase [Perkin-Elmer], 25 to 50 nM fluorogenic probe, 26 µl of water) in 200-µl capacity MOT or in individual wells of a MORP. Each set of reactions included a single tube (well) of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA) for the autozero control and triplicate tubes (wells) that were no-template controls (containing no E. coli O157:H7 DNA templates). Each assay also included DNA collected from other bacterial species: Salmonella choleraesuis, Hafnia alvei, and Citrobacter freundii, and NovaBlue, a general-purpose cloning host with the genotype endA1 hsdR17(rK- mK+) supE44 thi-1 gyrA96 relA1 lac [F' proAB lacIqZDelta M15 Tn10(Tetr)] recA1 (Novagen, Madison, Wis.). All non-E. coli organisms were cultivated in TSB (37°C, overnight). The PCR had an initial denaturization step (95°C, 5 min) followed by 35 amplification cycles of a two-step PCR (95°C, 20 s; 60°C, 60 s), with a final extension (72°C, 10 min or longer) on a thermocycler (GeneAmp PCR System 9600; Perkin-Elmer).

DNA recovery procedures. To evaluate the sensitivity of an E. coli O157:H7 5' nuclease assay system, different DNA recovery procedures were evaluated with different types of samples.

(i) QIAamp tissue kit. The ability of the QIAamp tissue kit to recover E. coli O157:H7 DNA from pure broth cultures was evaluated according to the manufacturer's instructions. Tenfold dilutions were made of E. coli O157:H7 in mTSB. A 1.0-ml aliquot from each dilution was removed and pelleted by centrifugation (16,000 × g, 10 min), and the supernatant was carefully removed and discarded. The pellets were centrifuged again (16,000 × g, 1 min), and each pellet was resuspended in 180 µl of Buffer ATL (Qiagen) and 20 µl of proteinase K stock solution (Qiagen). Tubes were vortexed and incubated in a 55°C water bath for 30 min or until the sample was lysed. Twenty microliters of RNase A (20 mg/ml) was added to each tube, and the tubes were vortexed (5 s) and incubated (room temperature, 2 min). Then 200 µl of Buffer AL (Qiagen) was added to each tube, and tubes were vortexed (5 s) and incubated (70°C in a dry heating block, 10 min). Next, 210 µl of 100% ethanol was added to each tube, and the contents were mixed by vortexing and transferred to a QIAamp spin column that was placed in a 2-ml collection tube.

The spin column and collection tube were centrifuged (5,200 × g, 1 min). The spin column then was placed on a new collection tube, and 500 µl of Buffer AW (Qiagen) was added to the column. The spin column and a new collection tube then were centrifuged (5,200 × g, 1 min), the spin column was removed and placed into a new 2-ml collection tube, and 500 µl of Buffer AW (Qiagen) was added. The spin column and a new collection tube were centrifuged (5,200 × g for 1 min, followed by 16,000 × g for 2 min), the spin columns then were placed onto marked 1.5-ml microcentrifuge tubes, and 200 µl of 70°C buffer AE (Qiagen) was added. The liquid in the collection tubes was precipitated with ethanol, placed in QIAamp spin columns, and processed according to the manufacturer's instructions. The DNA was eluted from the QIAamp spin columns by adding 200 µl of Buffer AE preheated to 70°C, incubating the columns at room temperature for 1 min, and then centrifuging the columns (6,000 × g for 1 min). The resulting elution samples were stored at -20°C or used in 5' nuclease assays.

(ii) DNA-ER procedure. One of the DNA recovery procedures evaluated utilized the chelating properties of chelex resin as previously described for recovering Salmonella DNA from foods (DNA extraction reagent [DNA-ER] method; TaqMan Salmonella PCR amplification detection kit; Perkin-Elmer). Briefly, 10-fold serial dilutions of mTSB and mEC broth cultures were made. Aliquots of 500 and 1,000 µl from each dilution were centrifuged (16,000 × g, 3 min), the supernatant was decanted carefully, and the pellet was resuspended in 200 µl of thoroughly mixed DNA-ER solution (DNA Extraction Reagent, product no. N808-0087; Perkin-Elmer). The tubes were vortexed for 5 to 10 s or as long as required to resuspend the pellet. The tubes then were incubated in a water bath (56°C, 30 min), floated in boiling water (7 min), and chilled on ice (5 min). The tubes then were centrifuged (16,000 × g, 3 min), and the supernatants were carefully transferred to new microcentrifuge tubes. A 5-µl aliquot of the supernatant served as the template for each PCR amplification in the 5' nuclease assay.

Sample types. To evaluate the sensitivity of an E. coli O157:H7 5' nuclease assay, different sample types were evaluated.

(i) Pure cultures. Pure cultures of E. coli O157:H7 were grown in mTSB or mEC (35°C, 12 to 15 h). Tenfold serial dilutions of the cultures were made with broth as the diluent, and aliquots were taken for E. coli O157:H7 enumeration (CFU/milliliter) on CT-SMAC and for immediate DNA recovery.

(ii) IMS from pure cultures. E. coli O157:H7 was detected and enumerated following recovery by IMS from mTSB, by using the beads as inoculum for direct plating onto CT-SMAC, by using the beads as inoculum for secondary enrichment and subsequent plating onto CT-SMAC, or by recovering DNA from each of the previous steps and subjecting it to E. coli O157:H7 5' nuclease reactions.

Specifically, 10-fold dilutions were made of pure cultures of E. coli O157:H7 while they were in logarithmic growth (between 4 and 8 h of culture). Aliquots of broth (100 µl) were plated on CT-SMAC plates in triplicate (incubated at 37°C for 18 h). Similarly, 0.5- and 1.0-ml aliquots were collected and subjected to the DNA-ER and QIAamp tissue kit DNA extraction methods. Simultaneously, 1.0-ml aliquots were collected from each dilution and transferred to microcentrifuge tubes containing 20 µl of anti-E. coli O157 immunomagnetic beads (Dynabeads Anti-E. coli O157; Dynal). After being separated by using a stationary magnet and washed according to the manufacturer's instructions, the beads were placed in 200 µl of phosphate-buffered saline. These bead solutions then were equally divided: 100 µl was plated on CT-SMAC (incubated at 37°C for 18 h), and 100 µl was added to a tube containing 9.0 ml of mTSB for secondary enrichment culturing (37°C, 18 h). Following secondary enrichment incubation, 100-µl aliquots of each dilution were plated on CT-SMAC (37°C, 18 h), and 0.5- and 1.0-ml aliquots were collected and subjected to both the DNA-ER and QIAamp tissue kit DNA recovery procedures. DNA also was recovered from 0.5- and 1.0-ml aliquots immediately prior to IMS and following 18-h secondary enrichment of IMS beads and were then subjected to 5' nuclease assay. The resulting Delta RQ values obtained from each dilution were compared with the CT-SMAC culture results. All 5' nuclease assay reactions in the IMS study were conducted in 96-well MORP.

(iii) Ground beef samples spiked with E. coli O157:H7. The ability to detect E. coli O157:H7 in ground beef was evaluated by spiking ground beef samples with different numbers of bacteria and then recovering DNA from each spiked sample and subjecting it to the 5' nuclease detection assay. Ground beef (containing 20% fat) was obtained from the Kansas State University Meat Processing Laboratory on three different occasions and confirmed to be culture negative for E. coli O157:H7 before inclusion in the study. E. coli O157:H7 strain MD 380-94 was collected from mTSB cultures while it was in logarithmic growth (4 to 8 h), and 10-fold dilutions were made in mTSB. One milliliter of each dilution was added to tubes containing 9.0 ml of ground beef-mTSB mixture (10 g of ground beef, 90 ml of mTSB) that had been previously incubated (6 h, 37°C). Aliquots (0.5 ml) were immediately removed from each dilution for DNA recovery by the DNA-ER and QIAamp tissue kit procedures and then subjected to the 5' nuclease assay. All 5' nuclease assay reactions containing ground beef were conducted in 96-well MORP.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

E. coli O157:H7 fluorogenic TaqMan probe design, 5' nuclease assay performance, and specificity. The adaptation of a 5' nuclease fluorogenic detection process to a PCR specific for E. coli O157:H7 required the evaluation of DNA sequences upstream of the eaeA gene. Recommended guidelines for designing fluorogenic probes for 5' nuclease assays were followed (35), and four fluorogenic probes (SZI-97, SZI-107, SZII-194, SZII-200) targeting both complementary DNA strands of the SZ-primed amplicon were constructed by using standard procedures (Table 2). Unless otherwise noted, all specificity evaluations were conducted in MOT.

                              
View this table:
[in this window]
[in a new window]
  		TABLE 2.   Primers and fluorogenic probes

The efficiency of the probes was evaluated in PCRs with DNA recovered from reference strains of E. coli O157:H7, other E. coli strains, and other bacteria. In preliminary evaluations, the SZI-97 and SZI-107 fluorogenic probes generated higher Delta RQ values (Table 3). Based on these findings, the SZI-97 probe was selected as the probe for optimization and further evaluation of the 5' nuclease assay. Monitoring Delta RQ values in PCRs with various concentrations of the SZI-97 probe (25 to 50 nM) identified 35 nM as the optimal probe concentration for all subsequent PCRs (data not shown). Similarly, the MgCl2 concentration was shown to influence the Delta RQ value, and subsequent PCRs used a final concentration of 4 mM MgCl2 (Table 4).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 3.   Evaluation of probes for the E. coli O157:H7 PCR-based fluorogenic 5' nuclease assay

                              
View this table:
[in this window]
[in a new window]
  		TABLE 4.   Effect of MgCl2 concentration on Delta RQ values with the SZ primers and SZI-97 fluorogenic probe

Similar to the PCR results described by Meng et al. (41), elevated Delta RQ values were demonstrated in all PCRs that used the SZ primers, 35 nM SZI-97 probe, and ~150 ng of E. coli O157:H7 DNA (Table 1). With a Delta RQ detection threshold level determined at 99% confidence limits, all E. coli O157:H7 strains evaluated in MOT demonstrated Delta RQ values that were greater than the Delta RQ threshold level of 1.04 and therefore resulted in a "yes" conclusion for the presence of E. coli O157:H7 DNA in the sample. The Delta RQ detection threshold level indicative of a "yes" conclusion with MORP at 99% confidence limits was >= 0.34.

In evaluations of the specificity of the SZ primers and the fluorogenic SZI-97 probe for organisms other than E. coli O157:H7, Delta RQ values above the detection threshold were observed with some E. coli O157:NM strains and two enteropathogenic E. coli O55:H7 strains (Table 1). This cross-reactivity was similar to that described by Meng et al. (41, 42) using the SZ primers in single and multiplex PCRs. No elevated Delta RQ values above threshold detection limits were detected for the other reference bacteria, including H. alvei and C. freundii strains.

Sensitivity of the E. coli O157:H7 5' nuclease detection system. Tests of the sensitivity of the E. coli O157:H7 fluorogenic 5' nuclease detection system using DNA recovered with the QIAamp tissue kit from overnight cultures of E. coli O157:H7 grown in either mTSB or mEC and with PCRs conducted and analyzed in MOT resulted in Delta RQ values greater than the detection threshold level when >= 104 CFU/ml was present (Fig. 1). The sensitivity of the 5' nuclease assay was increased when the PCRs, detection, and analysis were conducted in MORP (Table 5). The minimum Delta RQ levels indicative of a "yes" conclusion with the MORP were recorded when E. coli O157:H7 in logarithmic growth, present at >= 103 CFU/ml, was evaluated. These detection levels were demonstrated with both the QIAamp tissue extraction kit and the DNA-ER procedure (Table 5).


View larger version (20K):
[in this window]
[in a new window]
  		FIG. 1.   Sensitivity of the fluorogenic 5' nuclease assay for detecting E. coli O157:H7 in individual MOT (Perkin-Elmer). Tenfold dilutions of E. coli O157:H7 were made in mTSB and mEC in triplicate. One-milliliter aliquots from each dilution were subjected to QIAamp tissue kit DNA recovery, and 5 µl of the recovered DNA solution was PCR amplified with the SZ-I and SZ-II primers in the presence of the SZI-97 fluorogenic probe. Detection and analysis were completed with the ABI Prism sequence detection system. All amplification and detection reactions were completed in MOT. The average Delta RQ values for each dilution were plotted against the average CFU/milliliter as determined by plating each dilution on CT-SMAC. The adjusted Delta RQ threshold value was calculated to be 1.04 (dashed line). Error bars indicate the standard deviation of the mean (n = 6).

                              
View this table:
[in this window]
[in a new window]
  		TABLE 5.   Comparison of standard culture and the E. coli O157:H7 5' nuclease assay Delta RQ values for detecting E. coli O157:H7, using two different DNA extraction procedures and different assay conditions

Sensitivity of the 5' nuclease assay to identify E. coli O157:H7 was found to be approximately 104 CFU/ml in ground beef-mTSB mixtures (Fig. 2). The Delta RQ values obtained from various dilutions of ground beef-mTSB mixtures were indicative of a "yes" conclusion (Delta RQ values of >= 0.34) when >= 104 CFU/ml was processed in 0.5-ml aliquots with the QIAamp tissue extraction kit. This sensitivity was obtained without including an IMS step. With the same dilutions, the DNA-ER procedure demonstrated similar "yes" and "no" conclusions but gave consistently lower average Delta RQ values than the QIAamp tissue extraction kit when >= 104 CFU/ml was present (Fig. 2).


View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2.   Sensitivity of the fluorogenic 5' nuclease assay for detecting E. coli O157:H7 in mTSB containing ground beef. Tenfold dilutions of E. coli O157:H7 were made in mTSB in triplicate, and 1.0 ml was added to a tube containing 9.0 ml of ground beef-mTSB mixture (10 g of ground beef, 90 ml of mTSB; incubated for 6 h at 37°C). Aliquots (0.5 ml) were collected for DNA recovery by using the DNA-ER () and QIAamp tissue kit (down-triangle) DNA extraction methods, and 5 µl of the recovered DNA solution was amplified with the SZ-I and SZ-II primers in the presence of the SZI-97 fluorogenic probe. Detection and analysis were completed with the ABI Prism sequence detection system. All amplification and detection reactions were completed in MORP. The average Delta RQ values determined from DNA recovered from both DNA extraction methods were plotted against the average CFU/milliliter determined by plating each ground beef dilution on CT-SMAC. The Delta RQ threshold value at 99% confidence limits was calculated to be 0.34. Error bars indicate the standard deviation from the mean (n = 3).

A difference was noted in the Delta RQ values when samples containing older cultures of E. coli O157:H7 were processed by the two DNA recovery methods. The DNA recovery with the QIAamp tissue extraction kit after IMS-18-h secondary enrichment demonstrated Delta RQ values interpreted as "yes" when the starting inoculum was >= 102 (9.8 × 101) CFU/ml (Table 5). This was also the limiting dilution at which E. coli O157:H7 was recovered on CT-SMAC following IMS-18-h secondary enrichment. The Delta RQ values obtained from the same dilutions but with the DNA-ER recovery process gave "yes" conclusions when the inoculum prior to IMS was >= 104 CFU/ml. However, the Delta RQ values obtained by using the DNA-ER procedure were inconsistent and resulted in numerous false "no" interpretations, as determined by plating of IMS-18-h secondarily enriched dilutions on CT-SMAC and recovering organisms from the dilutions when the starting inoculum contained >= 102 CFU/ml. Decreasing the volume of the secondary enrichment medium processed for DNA recovery did not appear to improve the efficiency of the DNA-ER procedure, because interpretations of Delta RQ values were similar whether 1.0 or 0.5 ml was processed (Table 5).

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The ability to presumptively detect E. coli O157:H7 DNA from diverse kinds of samples by using an automated amplification/detection procedure would be beneficial to food producers, food processors, food safety regulatory agencies, and clinical microbiologists. In this report, we describe the development and evaluation of a PCR-based 5' nuclease (TaqMan) assay for automatically amplifying and then detecting E. coli O157:H7 DNA. The assay was successful in detecting E. coli O157:H7 DNA, with "yes" interpretations for all reference strains of E. coli O157:H7 evaluated, for pure cultures of E. coli O157:H7 grown in mTSB and mEC, and for ground beef-mTSB mixtures spiked with E. coli O157:H7. Detection sensitivities were improved if IMS and 18-h secondary enrichments were incorporated into the process.

The data indicate that the automated eaeA-based E. coli O157:H7 5' nuclease detection assay, when integrated with an effective DNA recovery process, is capable of rapid, semiautomated, presumptive detection of E. coli O157:H7 if >= 103 CFU/ml is present in mTSB or mEC or if >= 104 CFU/ml is present in a ground beef-mTSB mixture. These data also indicate that incorporating preenrichment and secondary enrichment steps and an IMS recovery process could further improve the sensitivity of the detection procedure. Because only 1/40 of a 1.0-ml sample (concentrated to 200 µl following DNA recovery) was actually evaluated in any PCR (5-µl sample/PCR), evaluating a larger fraction of the sample per PCR or further concentrating the DNA in the sample might increase the sensitivity of the procedure. However, both steps would increase costs or add additional manipulations to the procedure.

The strategy for developing an E. coli O157:H7-specific fluorogenic probe was to target an internal region of the SZ-primed amplicon that could detect subtle sequence differences between the eaeA gene of E. coli O157:H7 and similar genes in enteropathogenic E. coli, C. freundii, and H. alvei. Specificity of the fluorogenic probe in this PCR was based on the presumptions (i) that an increase in reporter fluorescence emission from the probe would occur only if the PCR primers annealed specifically to E. coli O157:H7 DNA templates as previously described (41, 42) and, simultaneously, the fluorogenic probe annealed specifically to the same target that contained the eaeA gene of E. coli O157:H7 and (ii) that digestion of the probe would occur only if extension of the new complementary strand of DNA proceeded in a 5'right-arrow3' direction as predicted in a successful PCR. Based on our observations and the conclusions from previous studies using the SZ primers (41, 42), the SZ primers and SZI-97 fluorogenic probe appear to be highly specific for presumptively identifying E. coli O157:H7 DNA. Additional evaluations on a larger reference collection of E. coli O157:H7 will be required to confirm this hypothesis.

By utilizing the SZ-I and SZ-II primers in PCR in the presence of the fluorogenic SZI-97 probe (in MOT), it was possible to presumptively determine the presence of E. coli O157:H7 DNA in broth cultures of mTSB and mEC with similar sensitivities (>= 104 CFU/ml). The sensitivity was increased to >= 103 CFU/ml by using MORP and was comparable to the previously reported sensitivity of the SZ-primed eaeA-based PCR as determined by visually interpreting ethidium bromide-stained agarose gels (41). We attribute the difference in sensitivities between MOT and MORP to improved reproducibility of the optical characteristics of MORP, resulting in more consistent Delta RQ- values, which, in turn, reduced the Delta RQ detection threshold level. Regardless, all E. coli O157:H7 strains evaluated demonstrated Delta RQ values above the detection threshold levels and resulted in "yes" interpretations at 99% confidence levels.

Elevated Delta RQ values above the detection threshold were also observed with some E. coli O157:NM strains and two enteropathogenic E. coli O55:H7 strains. These findings were not unexpected, because similar cross-reactivity has been demonstrated in assays using the SZ primers in single and multiplex PCRs (41, 42). In most circumstances, cross-reactivity in a PCR would be sufficient to limit the usefulness of those primers for detection purposes; however, several points suggest that the SZ-primed, eaeA-based 5' nuclease detection system would be useful as a rapid, sensitive, semiautomated, presumptive detection process for E. coli O157:H7 in food and environmental samples. First, the eaeA-based 5' nuclease assay system was able to correctly identify all E. coli O157:H7 strains evaluated and mirrored the previously described specificity of the primers (41, 42). Second, all cross-reactivity was limited to organisms that would be considered human pathogens and undesirable in foods. Specifically, E. coli O157:NM has been increasingly isolated from hemolytic uremic syndrome patients in Europe (8, 26), and the enteropathogenic E. coli O55:H7 is associated with worldwide outbreaks of infantile diarrhea (52). Phylogenetic analyses have also suggested that E. coli O157:H7 may have originated from an E. coli O55:H7 clone (52).

Regardless, definitive confirmation could be completed by standard culture techniques on all presumptive "yes" samples identified by the SZ-primed 5' nuclease assay. Similarly, all presumptive "yes" samples could be confirmed by PCR amplification/detection approaches targeting the eaeA and the sltI and sltII genes as previously described by Meng et al. (41, 42). The latter approach would eliminate the need to complete biochemical or immunological analyses to correctly confirm the identity of E. coli O157:H7 isolates. Once organisms were identified by the presumptive eaeA-based 5' nuclease assay, efforts to recover viable organisms from the broth culture could be initiated by using standard techniques. For example, once sorbitol-non-fermenting colonies were identified on CT-SMAC, confirmatory E. coli O157:H7 genetic testing could be initiated on colonies by using fluorogenic 5' nuclease detection systems that individually targeted the eaeA and the sltI and sltII genes with specific fluorogenic probes (28, 41, 42).

Perhaps the most important attribute of the eaeA-based 5' nuclease assay for presumptively detecting E. coli O157:H7 is that the entire identification process (disregarding culture incubation times) takes approximately 2.5 h (about 20 min for DNA recovery, <2 h for PCR preparation and thermal cycling, and <15 min for PCR product detection and analysis).

Sample processing is an important component of any DNA-based detection system. To optimize sample preparation, we compared the abilities of two extraction procedures to recover E. coli O157:H7 DNA for the 5' nuclease detection system. When E. coli O157:H7 cultures in logarithmic growth were evaluated, the DNA-ER and QIAamp tissue kit DNA extraction methods were equally effective in demonstrating "yes" Delta RQ values (read in MORP) if >= 103 CFU/ml was present. However, when older cultures (IMS plus 18-h secondary enrichment) were evaluated, the efficiency of the DNA-ER procedure, as determined by Delta RQ values, demonstrated differences that could be associated only with the DNA recovery processes. When DNA was recovered with the DNA-ER procedure, Delta RQ values of samples containing older cultures were highly variable. This variability hindered interpretation of the endpoint sensitivity of the assay and, more importantly, resulted in false-negative interpretations when DNA-ER results were compared to CT-SMAC culture results. When the QIAamp tissue kit was evaluated in assays using the same dilutions, the Delta RQ values and "yes" interpretations were identical to the IMS-18-h secondary enrichment culture results on CT-SMAC, indicating a detection capability when >= 102 CFU/ml was present in the original dilution.

The 5' nuclease detection system was able to detect E. coli O157:H7 when >= 103 CFU/ml was present in pure culture in mTSB or mEC and yielded results comparable to those that Meng et al. (41) obtained by visually interpreting agarose gels. However, incorporating an IMS step, followed by a secondary enrichment culturing step and QIAamp tissue kit DNA recovery, improved the detection threshold to >= 102 CFU/ml in the original sample. This detection level is closer to the suggested minimum infectious dosage for humans (25, 53).

Surprisingly, the sensitivity of CT-SMAC culturing immediately after IMS was such that identifiable colonies resulted only if >= 104 CFU/ml was present in the original sample. Several factors might be involved in creating these false-negative culture results. First, the detection limit of the IMS procedure using Dynabeads Anti-E. coli O157 beads (Dynal) is approximately 102 organisms/ml of preenriched sample, indicating that E. coli O157:H7 concentrations of <102 CFU/ml could go undetected. Also, the successful isolation of some strains of E. coli O157:H7 could be affected adversely by plating the strains onto CT-SMAC. A recent study indicated that plating unstressed, laboratory-reared E. coli O157 isolates on CT-SMAC adversely affected their isolation by delaying their growth, resulting in false-negative conclusions (38). Surprisingly, we were able to detect organisms in dilutions that originally contained >102 and <104 CFU/ml only when they were subjected to IMS and an additional 18-h secondary enrichment prior to being plated on CT-SMAC. To overcome this delay in growth and improve the recovery for some E. coli O157 strains, others have suggested reducing the levels of antibiotics in preenrichment broths prior to IMS and plating on CT-SMAC (9, 38). Regardless, additional research will be required to address the inhibitory effect of CT-SMAC and the effects that such inhibition would have on accurately detecting E. coli O157:H7 in food and environmental samples.

To demonstrate the applicability of a presumptive E. coli O157:H7 5' nuclease detection system in food production and processing facilities, throughput capability of the system must be enhanced by integrating automated liquid handling capability into the process, automating the DNA recovery process, and linking all phases of the integrated detection process with a computer-based system that would allow increased retrieval and storage of data. As demonstrated in this study, presumptive pathogen detection systems using 5' nuclease assay components can be integrated readily into standard culture/detection procedures to reduce the time required to presumptively detect E. coli O157:H7 or other microbial contaminants. Cost and time savings could also be realized by reducing the need for complete bacterial isolation procedures and biochemical/immunological characterizations on every sample, unless suggested by an eaeA-based 5' nuclease assay presumptive "yes" result.

    ACKNOWLEDGMENTS

This research was funded in part by USDA Special Research Grant 96-34359-2593, Kansas State University's Food Animal Health & Management Center, with additional support provided by the State Research Extension Educational Service, USDA, under agreement 92-34211-8362, Food Safety Consortium.

    FOOTNOTES

* Corresponding author. Mailing address: Food Animal Health & Management Center, College of Veterinary Medicine, Kansas State University, 1800 Denison Ave., VCS Bldg., Manhattan, KS 66506. Phone: (785) 532-4411. Fax: (785) 532-4288. E-mail: oberst{at}vet.ksu.edu.

dagger Contribution no. 98-291-J from the Kansas Agricultural Experiment Station.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Alexander, E. R., J. Boase, M. Davis, L. Kirchner, C. Osaki, T. Tanino, M. Samadpour, P. Tarr, M. Goldoft, S. Lankford, J. Kobyashi, P. Stehr-Green, P. Bradley, B. Hinton, P. Tighe, B. Pearson, G. R. Flores, S. Abbott, R. Bryant, S. B. Werner, and D. J. Vugia. 1995. Escherichia coli O157:H7 outbreak linked to commercially distributed dry-cured salami---Washington and California, 1994. Morbid. Mortal. Weekly Rep. 44:157-159[Medline].
2. Bassler, H. A., S. J. A. Flood, K. J. Livak, J. Marmaro, R. Knorr, and C. A. Batt. 1995. Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes. Appl. Environ. Microbiol. 61:3724-3728[Abstract].
3. Basta, M., M. Karmali, and C. Lingwood. 1989. Sensitive receptor-specified enzyme-linked immunosorbent assay for Escherichia coli verocytotoxin. J. Clin. Microbiol. 27:1617-1622[Abstract/Free Full Text].
4. Batt, C. A. 1997. Molecular diagnostics for dairy-borne pathogens. J. Dairy Sci. 80:220-229[Abstract/Free Full Text].
5. Belongia, E. A., K. L. MacDonald, G. L. Parham, K. E. White, Korlath, M. N. Lobato, S. M. Strand, K. A. Casale, and M. T. Osterholm. 1991. An outbreak of Escherichia coli O157:H7 colitis associated with consumption of precooked meat patties. J. Infect. Dis. 164:338-343[Medline].
6. Bennett, A. R., S. MacPhee, and R. P. Betts. 1996. The isolation and detection of Escherichia coli O157 by use of immunomagnetic separation and immunoassay procedures. Lett. Appl. Microbiol. 22:237-243[Medline].
7. Besser, R. E., S. M. Lett, J. T. Weber, M. P. Doyle, T. J. Barrett, J. G. Wells, and P. M. Griffin. 1993. An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia coli O157:H7 in fresh-pressed apple cider. JAMA 269:2217-2220[Abstract/Free Full Text].
8. Bitzan, M., K. Ludwig, M. Klemt, H. Konig, J. Buren, and D. E. Muller-Wiefel. 1993. The role of Escherichia coli O157 infections in the classical (enteropathic) haemolytic uraemic syndrome: results of a Central European, multicenter study. Epidemiol. Infect. 110:183-196[Medline].
9. Bolton, F. J., L. Crozier, and J. K. Williamson. 1996. Isolation of Escherichia coli O157 from raw meat products. Lett. Appl. Microbiol. 23:317-321[Medline].
10. Cebula, T. A., W. L. Payne, and P. Feng. 1995. Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shiga-like toxin type by mismatch amplification mutation assay-multiplex PCR. J. Clin. Microbiol. 33:248-250[Abstract].
11. Chapman, P. A., D. J. Wright, and C. A. Siddons. 1994. A comparison of immunomagnetic separation and direct culture for the isolation of verocytotoxin-producing Escherichia coli O157 from bovine faeces. J. Med. Microbiol. 40:424-427[Abstract/Free Full Text].
12. Chapman, P. A., A. T. Cerdan Malo, C. A. Siddons, and M. Harkin. 1997. Use of commercial enzyme immunoassays and immunomagnetic separation systems for detecting Escherichia coli O157 in bovine fecal samples. Appl. Environ. Microbiol. 63:2549-2553[Abstract].
13. Chen, S., A. Yee, M. Griffiths, C. Larkin, C. T. Yamashiro, R. Behari, C. Paszko-Kolva, K. Rahn, and S. A. De Grandis. 1997. The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. Int. J. Food Microbiol. 35:239-250[Medline].
14. Coleman, D. J., K. J. Nye, K. E. Chick, and C. M. Gagg. 1995. A comparison of immunomagnetic separation plus enrichment with conventional salmonella culture in the examination of raw sausages. Lett. Appl. Microbiol. 21:249-251[Medline].
15. Como-Sabetti, K., S. Reagan, S. Allaire, K. Parrott, C. M. Simonds, S. Hrabowy, B. Ritter, W. Hall, J. Altamirano, R. Martin, F. Downes, G. Jennings, R. Barrie, M. F. Dorman, N. Keon, M. Kucab, A. Al Shab, B. Robinson-Dunn, L. Moshur, L. Reese, J. Smith, K. Wilcox, J. Tilden, G. Wojtala, J. D. Park, M. Winnett, L. Petrilack, L. Vasquez, S. Jenkins, E. Barrett, M. Linn, D. Woolard, R. Hackler, H. Martin, D. McWilliams, B. Rouse, S. Willis, J. Rullan, G. Miller, S. Henderson, J. Pearson, J. Beers, R. Davis, and D. Saunders. 1997. Outbreaks of Escherichia coli O157:H7 infection associated with eating alfalfa sprouts---Michigan and Virginia, June-July 1997. Morbid Mortal. Weekly Rep. 46:741-744.
16. Cubbon, M. D., J. E. Coia, M. F. Hanson, and F. M. Thomson-Carter. 1996. A comparison of immunomagnetic separation, direct culture, and polymerase chain reaction for the detection of verocytotoxin-producing Escherichia coli O157 in human faeces. J. Med. Microbiol. 44:219-222[Abstract/Free Full Text].
17. Donohue-Rolfe, A., D. W. K. Acheson, A. V. Kane, and G. T. Keusch. 1989. Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and the production of cross-reactive monoclonal antibodies. Infect. Immun. 57:3888-3893[Abstract/Free Full Text].
18. Dorn, C. R. 1993. Review of foodborne outbreak of Escherichia coli O157:H7 infection in the western United States. J. Am. Vet. Med. Assoc. 203:1583-1587[Medline].
19. Doyle, M. P. 1991. Escherichia coli O157:H7 and its significance in foods. Int. J. Food Microbiol. 12:289-302[Medline].
20. Faith, N. G., J. A. Shere, R. Brosch, K. W. Arnold, S. E. Ansay, M.-S. Lee, J. B. Luchansky, and C. S. Kaspar. 1996. Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin. Appl. Environ. Microbiol. 62:1519-1525[Abstract].
21. Farmer, J. J., and B. R. Davis. 1985. H7 antiserum-sorbitol fermentation medium for detecting Escherichia coli O157:H7 associated with hemorrhagic colitis. J. Clin. Microbiol. 22:620-625[Abstract/Free Full Text].
22. Fratimico, P. M., F. J. Schultz, and R. L. Buchanan. 1992. Rapid isolation of Escherichia coli O157:H7 from enrichment cultures of foods using an immunomagnetic separation method. Food Microbiol. 9:105-113.
23. Fratimico, P., K. S. Solomon, M. Wiedmann, and M. Y. Deng. 1995. Detection of Escherichia coli O157:H7 by multiplex PCR. J. Clin. Microbiol. 33:2188-2191[Abstract].
24. Garber, L. P., S. J. Wells, D. D. Hancock, M. P. Doyle, J. Tuttle, J. A. Shere, and T. Zhao. 1995. Risk factors for fecal shedding of Escherichia coli O157:H7 in dairy calves. J. Am. Vet. Med. Assoc. 207:46-49[Medline].
25. Griffin, P., and R. V. Tauxe. 1991. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13:60-98[Free Full Text].
26. Gunzer, F., H. Bohm, H. Russman, M. Bitzan, S. Aleksic, and H. Karch. 1992. Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-uremic syndrome. J. Clin. Microbiol. 30:1807-1810[Abstract/Free Full Text].
27. Hancock, D. D., T. E. Besser, M. K. Kinsel, P. I. Tarr, D. H. Rice, and M. G. Paros. 1994. The prevalence of Escherichia coli O157:H7 in dairy and beef cattle in Washington State. Epidemiol. Infect. 113:199-207[Medline].
28. Ho, M., S. Flood, E. Schreiber, M. Matsuura, and C. Paszko-Kolva. 1997. Rapid and specific fluorogenic PCR-based system for the detection of Shiga-like toxins I and II producing E. coli, abstr. P-17, p. 439. In Abstracts of the 97th General Meeting of the American Society for Microbiology 1997. American Society for Microbiology, Washington, D.C.
29. Holland, P. M., R. D. Abramson, R. Watson, and D. H. Gelfand. 1991. Detection of specific polymerase chain reaction product by utilizing the 5' to 3' exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88:7276-7280[Abstract/Free Full Text].
30. Johnson, R. P., R. J. Durham, S. T. Johnson, L. A. MacDonald, S. R. Jeffrey, and B. T. Butman. 1995. Detection of Escherichia coli O157:H7 in meat by an enzyme-linked immunosorbent assay, EHEC-Tek. Appl. Environ. Microbiol. 61:386-388[Abstract].
31. Karch, H., C. Janetzki-Mittmann, S. Aleksic, and M. Datz. 1996. Isolation of enterohemorrhagic Escherichia coli O157 strains from patients with hemolytic-uremic syndrome by using immunomagnetic separation, DNA-based methods, and direct culture. J. Clin. Microbiol. 34:516-519[Abstract].
32. Karch, H., and T. Meyer. 1989. Single primer pair for amplifying segments of distinct Shiga-like toxin genes by polymerase chain reaction. J. Clin. Microbiol. 27:2751-2757[Abstract/Free Full Text].
33. Kleanthous, H., N. K. Fry, H. R. Smith, R. J. Gross, and B. Rowe. 1988. The use of sorbitol-MacConkey agar in conjunction with a specific antiserum for the detection of Vero cytotoxin-producing strains of Escherichia coli O157. Epidemiol. Infect. 101:327-335[Medline].
34. Lee, L. G., C. R. Connell, and W. Bloch. 1993. Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res. 21:3761-3766[Abstract/Free Full Text].
35. Livak, K. J., S. J. A. Flood, J. Marmaro, W. Giusti, and K. Deetz. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 4:357-362[Medline].
36. Louie, M., J. De Azavedo, R. Clarke, A. Borczyk, H. Lior, M. Richter, and J. Brunton. 1994. Sequence heterogeneity of the eae gene and detection of verotoxin-producing Escherichia coli using serotype-specific primers. Epidemiol. Infect. 112:449-461[Medline].
37. Lyamichev, V., M. A. D. Brow, and J. E. Dahlberg. 1993. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science 260:778-783[Abstract/Free Full Text].
38. MacRae, M., T. Rebate, M. Johnston, and I. D. Ogden. 1997. The sensitivity of Escherichia coli O157 to some antimicrobials by conventional and conductance assays. Lett. Appl. Microbiol. 25:135-137[Medline].
39. Matsuura, M., C. Yamashiro, S. Flood, and C. Paszko-Kolva. 1997. Detection of Salmonella in food using a fluorogenic 5' nuclease assay. Am. Environ. Lab. March:24-25.
40. Mechie, S. C., P. A. Chapman, and C. A. Siddons. 1997. A fifteen month study of Escherichia coli O157:H7 in a dairy herd. Epidemiol. Infect. 118:12-25.
41. Meng, J., S. Zhao, M. P. Doyle, S. E. Mitchell, and S. Kresovich. 1996. Polymerase chain reaction for detecting Escherichia coli O157:H7. Int. J. Food Microbiol. 32:103-113[Medline].
42. Meng, J., S. Zhao, M. P. Doyle, S. E. Mitchell, and S. Kresovich. 1997. A multiplex PCR for identifying Shiga-like toxin-producing Escherichia coli O157:H7. Lett. Appl. Microbiol. 24:172-176[Medline].
43. O'Brien, A. D., A. R. Melton, C. K. Schmitt, M. L. McKee, M. L. Batts, and D. E. Griffin. 1993. Profile of Escherichia coli O157:H7 pathogen responsible for hamburger-borne outbreak of hemorrhagic colitis and hemolytic uremic syndrome in Washington. J. Clin. Microbiol. 31:2799-2801[Abstract/Free Full Text].
44. Okrend, A. J. G., B. E. Rose, and C. P. Lattuada. 1992. Isolation of Escherichia coli O157:H7 using O157 specific antibody coated magnetic beads. J. Food Prot. 55:214-217.
45. Padhye, N. V., and M. P. Doyle. 1991. Production and characterization of a monoclonal antibody specific for enterohemorrhagic Escherichia coli O157:H7 and O26:H11. J. Clin. Microbiol. 29:99-103[Abstract/Free Full Text].
46. Park, C. H., N. M. Vandel, and D. L. Hixon. 1996. Rapid immunoassay for detection of Escherichia coli O157 directly from stool specimens. J. Clin. Microbiol. 34:988-990[Abstract].
47. Paton, A. W., J. C. Paton, P. N. Goldwater, and P. A. Manning. 1993. Direct detection of Escherichia coli Shiga-like genes in primary fecal cultures by polymerase chain reaction. J. Clin. Microbiol. 31:3063-3067[Abstract/Free Full Text].
48. Sanderson, M. W., J. M. Gay, D. D. Hancock, C. C. Gay, L. K. Fox, and T. E. Besser. 1995. Sensitivity of bacteriologic culture for detection of Escherichia coli O157:H7 in bovine feces. J. Clin. Microbiol. 33:2616-2619[Abstract].
49. Sernowski, L. P., and S. C. Ingham. 1992. Frequency of false presumptive positive results obtained using a commercial ELISA kit to screen retail ground beef for Escherichia coli O157:H7. J. Food Prot. 55:846.
50. Vanderzant, C., and D. F. Spittstoesser. 1992. Compendium of methods for microbiological examination of foods, 3rd ed. Edward Brothers, Ann Arbor, Mich.
51. Venkateswaran, K., Y. Kamijoh, E. Ohashi, and H. Nakanishi. 1997. A simple filtration technique to detect enterohemorrhagic Escherichia coli O157:H7 and its toxins in beef by multiplex PCR. Appl. Environ. Microbiol. 63:4127-4131[Abstract].
52. Whittam, T. S., M. L. Wolfe, I. K. Wachsmuth, F. Ørskov, I. Ørskov, and R. A. Wilson. 1993. Clonal relationships among Escherichia coli strains that cause hemorrhagic colitis and infantile diarrhea. Infect. Immun. 61:1619-1629[Abstract/Free Full Text].
53. Willshaw, G. A., J. Thirlwell, A. P. Jones, S. Parry, R. L. Salmon, and M. Hickey. 1994. Vero cytotoxin-producing Escherichia coli O157 in beefburgers linked to an outbreak of diarrhea, haemorrhagic colitis and haemolytic uraemic syndrome in Britain. Lett. Appl. Microbiol. 19:304-307[Medline].
54. Witham, P. K., C. T. Yamashiro, K. J. Livak, and C. A. Batt. 1996. A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef. Appl. Environ. Microbiol. 62:1347-1353[Abstract].
55. Zhao, T., M. P. Doyle, J. Shere, and L. Garber. 1995. Prevalence of enterohemorrhagic Escherichia coli O157:H7 in a survey of dairy herds. Appl. Environ. Microbiol. 61:1290-1293[Abstract].

Applied and Environmental Microbiology, September 1998, p. 3389-3396, Vol. 64, No. 9
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Kim, H., Bhunia, A. K. (2008). SEL, a Selective Enrichment Broth for Simultaneous Growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Appl. Environ. Microbiol. 74: 4853-4866 [Abstract] [Full Text]  
  • Hsu, C.-F., Tsai, T.-Y., Pan, T.-M. (2005). Use of the Duplex TaqMan PCR System for Detection of Shiga-Like Toxin-Producing Escherichia coli O157. J. Clin. Microbiol. 43: 2668-2673 [Abstract] [Full Text]  
  • Loens, K., Ursi, D., Goossens, H., Ieven, M. (2003). Molecular Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infections. J. Clin. Microbiol. 41: 4915-4923 [Full Text]  
  • Jinneman, K. C., Yoshitomi, K. J., Weagant, S. D. (2003). Multiplex Real-Time PCR Method To Identify Shiga Toxin Genes stx1 and stx2 and Escherichia coli O157:H7/H- Serotype. Appl. Environ. Microbiol. 69: 6327-6333 [Abstract] [Full Text]  
  • Dodd, C. C., Sanderson, M. W., Sargeant, J. M., Nagaraja, T. G., Oberst, R. D., Smith, R. A., Griffin, D. D. (2003). Prevalence of Escherichia coli O157 in Cattle Feeds in Midwestern Feedlots. Appl. Environ. Microbiol. 69: 5243-5247 [Abstract] [Full Text]  
  • Koo, K., Jaykus, L.-A. (2003). Detection of Listeria monocytogenes from a Model Food by Fluorescence Resonance Energy Transfer-Based PCR with an Asymmetric Fluorogenic Probe Set. Appl. Environ. Microbiol. 69: 1082-1088 [Abstract] [Full Text]  
  • Renter, D. G., Sargeant, J. M., Oberst, R. D., Samadpour, M. (2003). Diversity, Frequency, and Persistence of Escherichia coli O157 Strains from Range Cattle Environments. Appl. Environ. Microbiol. 69: 542-547 [Abstract] [Full Text]  
  • Ibekwe, A. M., Watt, P. M., Grieve, C. M., Sharma, V. K., Lyons, S. R. (2002). Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands. Appl. Environ. Microbiol. 68: 4853-4862 [Abstract] [Full Text]  
  • Jothikumar, N., Griffiths, M. W. (2002). Rapid Detection of Escherichia coli O157:H7 with Multiplex Real-Time PCR Assays. Appl. Environ. Microbiol. 68: 3169-3171 [Abstract] [Full Text]  
  • Lindecrona, R. H., Jensen, T. K., Andersen, P. H., Moller, K. (2002). Application of a 5' Nuclease Assay for Detection of Lawsonia intracellularis in Fecal Samples from Pigs. J. Clin. Microbiol. 40: 984-987 [Abstract] [Full Text]  
  • Knutsson, R., Lofstrom, C., Grage, H., Hoorfar, J., Radstrom, P. (2002). Modeling of 5' Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica. J. Clin. Microbiol. 40: 52-60 [Abstract] [Full Text]  
  • Hristova, K. R., Lutenegger, C. M., Scow, K. M. (2001). Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR. Appl. Environ. Microbiol. 67: 5154-5160 [Abstract] [Full Text]  
  • Feder, I., Nietfeld, J. C., Galland, J., Yeary, T., Sargeant, J. M., Oberst, R., Tamplin, M. L., Luchansky, J. B. (2001). Comparison of Cultivation and PCR-Hybridization for Detection of Salmonella in Porcine Fecal and Water Samples. J. Clin. Microbiol. 39: 2477-2484 [Abstract] [Full Text]  
  • Stults, J. R., Snoeyenbos-West, O., Methe, B., Lovley, D. R., Chandler, D. P. (2001). Application of the 5' Fluorogenic Exonuclease Assay (TaqMan) for Quantitative Ribosomal DNA and rRNA Analysis in Sediments. Appl. Environ. Microbiol. 67: 2781-2789 [Abstract] [Full Text]  
  • Kimura, B., Kawasaki, S., Nakano, H., Fujii, T. (2001). Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish. Appl. Environ. Microbiol. 67: 206-216 [Abstract] [Full Text]  
  • Angen, O., Jensen, J., Lavritsen, D. T. (2001). Evaluation of 5' Nuclease Assay for Detection of Actinobacillus pleuropneumoniae. J. Clin. Microbiol. 39: 260-265 [Abstract] [Full Text]  
  • Sharma, V. K., Carlson, S. A. (2000). Simultaneous Detection of Salmonella Strains and Escherichia coli O157:H7 with Fluorogenic PCR and Single-Enrichment-Broth Culture. Appl. Environ. Microbiol. 66: 5472-5476 [Abstract] [Full Text]  
  • Takai, K., Horikoshi, K. (2000). Rapid Detection and Quantification of Members of the Archaeal Community by Quantitative PCR Using Fluorogenic Probes. Appl. Environ. Microbiol. 66: 5066-5072 [Abstract] [Full Text]  
  • Wilson, D. L., Abner, S. R., Newman, T. C., Mansfield, L. S., Linz, J. E. (2000). Identification of Ciprofloxacin-Resistant Campylobacter jejuni by Use of a Fluorogenic PCR Assay. J. Clin. Microbiol. 38: 3971-3978 [Abstract] [Full Text]  
  • Jourdan, A. D., Johnson, S. C., Wesley, I. V. (2000). Development of a Fluorogenic 5' Nuclease PCR Assay for Detection of the ail Gene of Pathogenic Yersinia enterocolitica. Appl. Environ. Microbiol. 66: 3750-3755 [Abstract] [Full Text]  
  • Vishnubhatla, A., Fung, D. Y. C., Oberst, R. D., Hays, M. P., Nagaraja, T. G., Flood, S. J. A. (2000). Rapid 5' Nuclease (TaqMan) Assay for Detection of Virulent Strains of Yersinia enterocolitica. Appl. Environ. Microbiol. 66: 4131-4135 [Abstract] [Full Text]  
  • Weller, S. A., Elphinstone, J. G., Smith, N. C., Boonham, N., Stead, D. E. (2000). Detection of Ralstonia solanacearum Strains with a Quantitative, Multiplex, Real-Time, Fluorogenic PCR (TaqMan) Assay. Appl. Environ. Microbiol. 66: 2853-2858 [Abstract] [Full Text]  
  • Kimura, R., Mandrell, R. E., Galland, J. C., Hyatt, D., Riley, L. W. (2000). Restriction-Site-Specific PCR as a Rapid Test To Detect Enterohemorrhagic Escherichia coli O157:H7 Strains in Environmental Samples. Appl. Environ. Microbiol. 66: 2513-2519 [Abstract] [Full Text]  
  • Wang, L., Rothemund, D., Curd, H., Reeves, P. R. (2000). Sequence Diversity of the Escherichia coli H7 fliC Genes: Implication for a DNA-Based Typing Scheme for E. coli O157:H7. J. Clin. Microbiol. 38: 1786-1790 [Abstract] [Full Text]  
  • Kato, T., Mizokami, M., Mukaide, M., Orito, E., Ohno, T., Nakano, T., Tanaka, Y., Kato, H., Sugauchi, F., Ueda, R., Hirashima, N., Shimamatsu, K., Kage, M., Kojiro, M. (2000). Development of a TT Virus DNA Quantification System Using Real-Time Detection PCR. J. Clin. Microbiol. 38: 94-98 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oberst, R. D.
Right arrow Articles by Gillespie, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oberst, R. D.
Right arrow Articles by Gillespie, J. R.
Agricola
Right arrow Articles by Oberst, R. D.
Right arrow Articles by Gillespie, J. R.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»