Combined PCR-Oligonucleotide Ligation Assay for Rapid Detection of ...

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0.5% bovine serum albumin (BSA) (Sigma, St. Louis, Mo.) ... S. derby. B. 3. S. heidelberg. B. 2. S. kiambu. B. 1. S. typhimurium ATCC 29946. B. 1. S. typhimurium.
JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1995, p. 2888–2893 0095-1137/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 33, No. 11

Combined PCR-Oligonucleotide Ligation Assay for Rapid Detection of Salmonella Serovars† GREGORY G. STONE, RICHARD D. OBERST,* MICHAEL P. HAYS, SCOTT MCVEY, AND M. M. CHENGAPPA Department of Diagnostic Medicine & Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas 66506 Received 15 June 1995/Returned for modification 19 July 1995/Accepted 9 August 1995

We have developed a rapid and sensitive assay for the detection of Salmonella serovars in veterinary clinical specimens. This method utilizes a short cultivation period followed by PCR. For detection of the amplified product, an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) was used. In this study, the PCR-OLA technique was compared with conventional culture and membrane hybridization for the detection of Salmonella bacteria. In evaluating the PCR-OLA with Salmonella serovars and non-Salmonella strains of bacteria, A490 readings for 51 Salmonella strains, representing 28 serovars, were significantly higher (P < 0.05) than those for 25 non-Salmonella bacteria. With serial 10-fold dilutions of Salmonella CFU or with known concentrations of purified chromosomal DNA from Salmonella typhimurium ATCC 29946, the PCR-OLA was able to detect >20 CFU per assay or >80 fg of chromosomal DNA (corresponding to 160 molecules of DNA). Of 102 suspect clinical specimens screened, 15 were positive for Salmonella bacteria by both culture and the PCR-OLA procedure (100% sensitivity), and 3 samples were positive only by PCR-OLA (96.6% specificity), indicating a positive predictive value of 83.3% and a negative predictive value of 100%. In all experiments, the PCR-OLA was as sensitive as membrane hybridization. These results indicate that a limited enrichment cultivation and PCR-OLA could be used as a presumptive screening test for the detection of Salmonella serovars from any sample that currently requires extensive cultivation and that this assay would be adaptable to automation. temperature. The first oligonucleotide (capture probe) is 59 biotinylated with the 39 end adjacent to the second oligonucleotide. The second oligonucleotide (reporter probe) is 59 phosphorylated and 39 end labeled with a reporter substance such as digoxigenin. The two oligonucleotides are hybridized to the target DNA and, if 100% complementarity exists, DNA ligase covalently joins the two oligonucleotides. The capture of the 59 biotinylated probe is accomplished by binding the biotin to immobilized streptavidin in a microtiter plate. If the two oligonucleotides are linked, then the reporter probe can be analyzed. We have recently described a combined cultivation and PCR-hybridization (C-PCR-H) procedure for the rapid detection of Salmonella serovars from clinical samples (23). In the present study, we (i) adapted the C-PCR-H procedure to a 96-well microtiter PCR-OLA format for detection of the 457-bp product from the invE and invA genes of Salmonella serovars and (ii) compared the sensitivity, specificity, and predictive value of this Salmonella-specific PCR-OLA with those of conventional culture techniques and C-PCR-H for detecting Salmonella serovars from veterinary clinical specimens.

The use of PCR in routine diagnostic testing has gained attention recently as a tool for rapid detection of infectious organisms. DNA amplification with PCR has been utilized for the detection of many microbial pathogens from clinical, food, and environmental samples. Listeria monocytogenes (3, 19), Shigella species (8), mycobacteria (11), Yersinia enterocolitica (29), and Salmonella serovars (2, 7, 18, 23, 26, 27) all have been targets for PCR-based detection procedures. PCR will amplify DNA molecules a thousandfold, but the presence of a specifically amplified product must be identified. A variety of methods have been employed to detect amplified products. The most common and simplest is gel electrophoresis. Unfortunately, gel electrophoresis shows only that a product has been amplified and does not show specificity of the product. DNA hybridizations with a specific probe to Southern blots of gels on nylon membranes (2, 23, 24) or dot blots (2) are means of determining specificity of the product, but these methods can take up to 48 h to complete. Other techniques include sandwich hybridization with a capture probe bound to microtiter plates (5, 9, 13, 15, 17, 20) and used of a solid-phase amplification systems (18). An alternative method for analyzing PCR products utilizes the oligonucleotide ligation assay (OLA). This procedure, first described by Landegren et al. (14) and later coupled with PCR by Nickerson et al. (16), has been used for the detection of human genetic disorders. The OLA procedure uses two adjacent oligonucleotides with approximately the same melting

MATERIALS AND METHODS Bacteria. Bacterial strains (Tables 1 and 2) were obtained from the American Type Culture Collection (Rockville, Md.) and the Department of Diagnostic Medicine & Pathobiology, Kansas State University, Manhattan. All bacterial strains were identified biochemically and serologically (6). Cultivation of Salmonella serovars. For testing specificity of the assay, Salmonella serovars were inoculated into 10 ml of selenite-cystine broth (Difco Laboratories, Detroit, Mich.) and incubated at 378C for 18 h. A 5-ml aliquot was removed and placed into a thin-walled thermocycle tube (Perkin-Elmer, Norwalk, Conn.) for PCR. Non-Salmonella bacteria were tested by growing them in 10 ml of brain heart infusion broth (Difco) for 18 h at 378C. A 5-ml aliquot also was removed and placed in a thermocycle tube for PCR. DNA amplification. Oligonucleotides used for amplification have been previously described (23). Isolation of nucleic acids and amplification of DNA were

* Corresponding author. Mailing address: Department of Diagnostic Medicine & Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506. Phone: (913) 532-4411. Fax: (913) 532-4309. Electronic mail address: [email protected]. † Contribution 95-353-J from the Kansas Agricultural Experiment Station. 2888

PCR-OLA FOR DETECTION OF SALMONELLA SEROVARS

VOL. 33, 1995 TABLE 1. Salmonella serovars examined in this study a

Salmonella serovar

Serogroup

No. of strains

S. agona S. brandenburg S. bredeney S. derby S. heidelberg S. kiambu S. typhimurium ATCC 29946 S. typhimurium S. typhimurium (copenhagen) S. saint-paul S. cholerasuis (biovar kunzendorf) S. mbandaka S. montevideo S. norwich S. tennessee S. hadar S. newport S. muenchen S. albany S. kentucky S. enteritidis S. dublin S. anatum S. muenster S. newbrunswick S. cubana S. urbana S. gera Salmonella species 45:g:z51

B B B B B B B B B B C1 C1 C1 C1 C1 C2 C2 C2 C3 C3 D1 D1 E1 E1 E2 G2 N T W

1 2 1 3 2 1 1 11 1 1 3 1 1 1 1 1 2 1 1 1 2 3 3 1 1 1 1 1 1

a Confirmed by National Veterinary Services Laboratory, Ames, Iowa. Strain ATCC 29946 was from the American Type Culture Collection; all other serovars were laboratory strains from the collection of M. M. Chengappa, Department of Diagnostic Medicine & Pathobiology, Kansas State University.

conducted as previously described (20). Briefly, 15 ml of Genereleaser (Bioventures, Murfreesboro, Tenn.) was added to a thin-walled thermocycle tube containing the 5-ml aliquot of enrichment broth and thermocycled as described previously (20) to extract PCR-inhibitory substances. Amplification was conducted by adding 30 ml of a master mixture of amplification reagents (PerkinElmer) directly to the 20-ml DNA preparation and thermocycling as previously described (20). OLA. Ligation oligonucleotides were constructed by using standard phosphoramidite chemistry as described previously (16). The capture oligonucleotide (59-GCCCGAACGTGGCGATAATT) was modified with a 59 biotin group, and the reporter oligonucleotide (59-TCACCGGCATCGGCTTCAAT) was 59 phosphorylated and 39 end labeled with digoxigenin-11-dUTP (16). The capture and reporter probes were purified by reverse-phase high-performance liquid chromatography. Ligation assays were conducted as described previously (16). Briefly, a 10-fold dilution of the PCR product was made in 0.1% Triton X-100. The ligation mixture (10 ml) consisted of 40 mM Tris-HCl (pH 8.3), 50 mM KCl, 20 mM MgCl2, 1.0 mM NAD, 0.2% Triton X-100, 200 fmol of each oligonucleotide, and 0.005 U of Ampligase (Epicentre Technologies, Madison, Wis.). The ligation mixture was placed in a thermocycle tube to which 10 ml of the diluted PCR product was added. Ligation was conducted on a DNA thermocycler (model 9600; Perkin-Elmer) programmed for 10 cycles of 938C for 30 s and 588C for 2 min. The reaction was stopped with 10 ml of a solution containing 100 mM EDTA and 0.1% Triton X-100. Samples (30 ml) were transferred to 96-well flat-bottom microtiter plates (Falcon 3912; Becton Dickinson Labware, Oxnard, Calif.) which had been coated with 50 ml of 25 mg of streptavidin (Zymed, San Francisco, Calif.) per ml at 378C for 1 h and blocked for 20 min with 200 ml of 0.5% bovine serum albumin (BSA) (Sigma, St. Louis, Mo.) in phosphate-buffered saline (pH 7.0). The biotinylated probes were captured by incubating the plates at 258C for 30 min and then washing twice with 200 ml of 0.01 N NaOH– 0.05% Tween 20. This was followed by two washes with 200 ml of Tris-saline buffer containing 100 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20. The digoxigenin probes were detected by adding 30 ml of a 1:1,000 dilution of antidigoxigenin antibodies (Boehringer-Mannheim) in 0.5% BSA. Plates were incubated at 258C for 30 min and washed five times with 200 ml of Tris-saline buffer. Thirty microliters of substrate (enzyme-linked immunosorbent assay [ELISA] amplification system; Gibco-BRL, Gaithersburg, Md.) was added to

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each well, plates were incubated for 15 min at 258C, 30 ml of amplifier then was added, and plates were incubated for an additional 15 min at 258C. The reaction was stopped with 30 ml of 0.3 M H2SO4. A490 was read on a spectrophotometer (Molecular Dynamics, Palo Alto, Calif.). A 40-mer oligonucleotide, complementary to the two 20-mer oligonucleotides used as ligation probes, was used as a template for positive control reactions. Membrane hybridization. Amplified products (10 ml) were visualized electrophoretically in ethidium bromide-stained 1.5% agarose gels. Products were transferred or dot blotted to nylon membranes (Micron Separation Inc., Westboro, Mass.) and probed with a 40-mer oligonucleotide that was complementary to the two 20-mer oligonucleotides used for the OLA procedure. The probe was 39 end labeled with digoxigenin-11-dUTP by using terminal transferase according to the manufacturer’s procedure (Boehringer-Mannheim). Prehybridization, hybridization, and detection of hybridized products were completed as previously described (24). Sensitivity of OLA. A 40-mer oligonucleotide complementary to the two 20mer oligonucleotides used in the ligation assay was used to determine the sensitivity of the OLA. Serial 10-fold dilutions of the 40-mer were made in Triton X-100 and assayed. In addition, to determine the minimal detectable concentration of Salmonella DNA and the minimal detectable number of Salmonella organisms, PCR was conducted on 10-fold serial dilutions of both purified chromosomal DNA and extracts of known counts (CFU per milliliter) of bacteria as previously described (23). The amplified products from the serial dilutions were analyzed by OLA, and the results were compared with those of membrane hybridizations and culture. Evaluation of clinical samples. A total of 102 veterinary clinical samples (Table 3) suspected of salmonellosis and submitted to the Kansas State University Department of Diagnostic Medicine & Pathobiology were screened by standard culture methods for Salmonella bacteria (6). In addition, tissue samples were processed for PCR-OLA by swabbing the tissue, placing the swab in 10 ml of selenite-cystine broth, and incubating the tube at 378C for 18 h. Following incubation, a 5-ml aliquot was removed from the tube and subjected to PCROLA. An additional 25-ml aliquot was removed, plated onto Hektoen enteric agar, and incubated for 18 h at 378C. Suspected Salmonella colonies were characterized biochemically and serologically (6). Statistical analysis. Sensitivity, specificity, and predictive values were calculated (12, 22), and the one-way analysis of variance test was used to test the association among the mean absorbance values of reactions by PCR-OLA using the SAS statistical analysis program (21).

RESULTS Specificity of PCR-OLA. Ligations of probes specific to the 457-bp amplified product from all 51 Salmonella strains tested by OLA resulted in an absorbance reading that was significantly higher (P , 0.05) than those of the negative controls and non-Salmonella bacteria (Fig. 1). None of the 25 non-Salmo-

TABLE 2. Non-Salmonella strains examined in this study Microorganisma

No. of strains

Actinobacillus pleuropneumoniae......................................................... 1 Alcaligenes faecalis ................................................................................ 1 Citrobacter freundii................................................................................ 1 Corynebacterium species....................................................................... 1 Edwardsiella tarda ATCC 15947 ......................................................... 1 Enterobacter aerogenes .......................................................................... 2 Escherichia coli ATCC 25922.............................................................. 1 E. coli ..................................................................................................... 5 Klebsiella pneumoniae........................................................................... 1 Morganella morganii.............................................................................. 1 Pasteurella multocida ............................................................................ 1 Proteus vulgaris ...................................................................................... 1 Proteus mirabilis .................................................................................... 1 Providencia rettgeri ................................................................................ 1 Pseudomonas aeruginosa ATCC 27653 .............................................. 1 Serratia marcescens ............................................................................... 1 Shigella dysenteriae ATCC 11456A ..................................................... 1 Staphylococcus aureus........................................................................... 1 Streptococcus species ............................................................................ 1 Yersinia pseudotuberculosis ATCC 29910........................................... 1 a All ATCC strains were from the American Type Culture Collection; all other organisms were laboratory strains from the collection of M. M. Chengappa, Department of Diagnostic Medicine & Pathobiology, Kansas State University.

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TABLE 3. Results for clinical samples tested by PCR-OLA Specimen type and origin

No. with positive result No. tested Culture

PCR-OLA

4 4 3 2 1

0 0 1 1 0

0 0 2 1 0

Bovine Feces Lymph node Cecum Abomasum Feed Intestine Gall bladder

11 5 1 1 1 19 3

1 2 1 1 0 4 0

1 2 1 1 0 4 0

Canine Intestine Liver Gall bladder

1 1 1

0 0 0

0 0 0

10

0

0

Ovine, intestine

1

0

0

Porcine Intestine Lymph node Lung Feces

20 4 9 1

1 2 1 0

1 2 3 0

102

15

18

Avian Intestine Liver Cecum Spleen Gizzard

Equine, feces

Total

nella strains tested had an absorbance reading that was significantly different (P , 0.05) from that of the negative controls (Fig. 1). In addition, amplified products from all Salmonella strains tested had hybridization signals on X-ray film when hybridized with an internal oligonucleotide probe, and no signals were observed with any of the non-Salmonella bacteria (data not shown). Sensitivity of PCR-OLA. To determine the minimal number of molecules of DNA that can be detected by the OLA procedure separate from PCR, a 40-mer oligonucleotide complementary to the two juxtaposed 20-mer oligonucleotides was constructed and used as a template for the OLA. Reactions with $105 molecules of DNA resulted in absorbance readings that were significantly higher (P , 0.05) than those for the negative controls (Fig. 2A). The 40-mer also was dot blotted onto nylon membranes and hybridized. The results were identical to the OLA results (Fig. 2B). In addition, serial 10-fold dilutions of a known concentration of purified chromosomal DNA and a known number of CFU from Salmonella typhimurium ATCC 29946 was amplified and analyzed by OLA. Reactions with $80 fg of purified chromosomal DNA (corresponding to 160 molecules of DNA) could be detected (Fig. 2A). The amplified products also were dot blotted onto nylon membranes and hybridized. Again, the results were identical to the OLA results (Fig. 2B). Reactions with $20 CFU per reaction mixture could be detected when known counts of bacteria were used. Cultivation and PCR-OLA of clinical samples. A total of 102 clinical specimens (Table 3) were screened for Salmonella organisms by culture, membrane hybridization, and the PCROLA procedure. Of the specimens screened, 15 were positive by culture and had absorbance readings that were significantly higher (P , 0.05) than those of the negative controls by the PCR-OLA procedure. An example of the results obtained by

FIG. 1. Specificity of PCR-OLA for the identification of Salmonella serovars. A490 readings for several Salmonella serovars and non-Salmonella bacteria are shown. Sample 1, negative control (no streptavidin); sample 2, positive control template (40-mer) for OLA; samples 3 through 16, Salmonella heidelberg, S. agona, S. brandenburg, S. derby, S. anatum, S. mbandaka, S. choleraesuis (kunzendorf), S. typhimurium, S. typhimurium (copenhagen), S. dublin, S. montevideo, S. urbana, S. newport, and S. muenchen, respectively; samples 17 through 29, Staphylococcus aureus, Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Escherichia coli, Actinomyces pyogenes, Pasteurella multocida, Citrobacter freundii, Proteus mirabilis, Morganella morganii, Actinobacillus pleuropneumoniae, Enterobacter aerogenes, Klebsiella pneumoniae, and Shigella dysenteriae, respectively; sample 30, negative control (no template) for PCR; sample 31, negative control (no template) for OLA; sample 32, negative control (streptavidin only). All values are means 6 standard errors for triplicate values. Absorbances for Salmonella isolates were significantly higher than those for non-Salmonella isolates and negative controls (P , 0.05).

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FIG. 2. Sensitivity of PCR-OLA procedure for detection of minimal numbers of molecules of DNA (40-mer) and known concentrations of Salmonella chromosomal DNA. (A) A490 readings for serial 10-fold dilutions of 40-mer and chromosomal DNA. The 40-mer template contained 5 pmol of DNA at a dilution of 100 and 800 pg of Salmonella chromosomal DNA at a dilution of 1023. Neg, negative control (no template for ligation). All values are means 6 standard errors for six replicate values. Absorbances from reaction mixtures with more than 105 molecules of 40-mer template and absorbances from reaction mixtures with more than 80 fg of chromosomal DNA were significantly higher than those from negative controls (P , 0.05). (B) Membrane hybridization of dot-blotted serial 10-fold dilutions of 40-mer and amplified products obtained by PCR-OLA. Lanes correspond to the log 10 dilutions in panel A.

PCR-OLA is displayed in Fig. 3A. Three additional specimens were negative by culture but had absorbance readings that were significantly higher (P , 0.05) than those of the negative controls by PCR-OLA (data not shown). The results obtained by PCR-OLA were also compared with results obtained by hybridization on nylon membranes with an internal oligonucleotide probe. Results from membrane hybridization were identical to the PCR-OLA results (Fig. 3B). An example of the membrane hybridization results is displayed in Fig. 3B.

DISCUSSION The use of PCR has increased the rapidity and sensitivity of diagnosing a number of infectious diseases. Many studies have described PCR-based procedures for detecting pathogenic organisms, including L. monocytogenes (3, 19), Mycobacterium tuberculosis (11), and Salmonella serovars (2, 5, 7, 18, 23, 24). We have reported previously a C-PCR-H procedure for the detection of Salmonella serovars from clinical samples (23). The presence study was conducted to develop an ELISA-based assay for detecting the Salmonella PCR products from clinical specimens and to compare its sensitivity, specificity, and predictive values with those of the C-PCR-H and traditional culture techniques. Our goal was to validate a manual procedure that eventually could be automated for the rapid, routine iden-

tification of Salmonella serovars from any sample that currently requires extensive cultivation. Many of the procedures for detecting PCR products described in the literature are time-consuming or laborious or involve the use of radioisotopes (26–28). In addition, many of the procedures do not easily lend themselves to automation. The most common way to detect an amplified product is by gel electrophoresis. Although multiple samples can be assayed by electrophoresis, it does not show specificity of the PCR product, lacks sensitivity, and is not adapted easily for automation in most laboratories. Similarly, Southern blots (2, 23, 24) or dot blot hybridizations with probes will demonstrate specificity of the PCR, but they require multistep processing and add considerable time to the detection process. Advantages that PCR-OLA has over other DNA detection assays are its ability to detect and differentiate single-nucleotide variations and its adaptability to automation. These have been demonstrated in detecting a number of human genetic disorders, such as cystic fibrosis and sickle-cell anemia (16). The PCR-OLA procedure also has the potential to target multiple sequences simultaneously in a multiplex PCR. With salmonellae, it is feasible to design genus-specific PCR primers in the lipopolysaccharide-coding region and then design ligation probes that recognize different factors specific to serogroups or serovars (25). In addition to detecting Salmonella bacteria, the procedure could be adapted to detect other pathogens from the same sample in a multiplexed PCR with primers and OLA probes to genetic sequences specific to a particular target analyte. In the present study, the OLA procedure was used to detect a genus-specific amplified product from the invE and invA

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FIG. 3. Detection of Salmonella serovars from several clinical samples by PCR-OLA. (A) A490 readings of reaction mixtures containing clinical samples. Sample 1, negative control (no streptavidin); sample 2, positive control for OLA (40-mer); sample 3, positive control for PCR (Salmonella-spiked sample); sample 4, porcine intestine; sample 5, porcine intestine; sample 6; porcine lung; sample 7, porcine intestine; sample 8, bovine lymph node; sample 9, bovine abomasum; sample 10, porcine lung; sample 11, porcine lung, sample 12, porcine lung; sample 13, porcine intestine; sample 14, porcine intestine; sample 15, porcine lung; sample 16, porcine intestine; sample 17, porcine intestine; sample 18, porcine intestine; sample 19, porcine intestine; sample 20, bovine intestine; sample 21, bovine intestine; sample 22, bovine feces; sample 23, bovine intestine; sample 24, ovine intestine; sample 25, negative control for PCR (unspiked sample); sample 26, negative control for PCR (no template); sample 27, negative control for OLA (no template); sample 28, negative control (streptavidin only). All samples were run in triplicate; values are means 6 standard errors. Absorbances from samples 8 through 12, 14, and 22 were significantly higher from than those from negative controls (P , 0.05). (B) Membrane hybridization of amplified products obtained by PCR-OLA. Lanes correspond to the sample numbers in panel A.

genes of Salmonella serovars. All Salmonella strains screened by PCR-OLA resulted in absorbance readings that was significantly higher than those of negative controls. This was confirmed by visualization of the 457-bp amplified product in ethidium bromide-stained gels and in C-PCR-H with an oligonucleotide probe specific to the PCR product. None of the non-Salmonella bacteria had absorbance readings that were significantly different from those of the negative controls or showed positive signals by membrane hybridization. Of 102 varied clinical specimens submitted to the Kansas State University Department of Diagnostic Medicine & Pathobiology and suspected of having salmonellosis, 15 were positive by both culture and the PCR-OLA procedure (100% sensitivity) and 3 were positive only by PCR-OLA (96.6% specificity), indicating a positive predictive value of 83.3% and negative predictive value of 100% (19). No attempts were made to reculture the three samples that were positive only by PCROLA. In the present study, $105 molecules of DNA (40-mer) was detected with the OLA procedure. When coupled with PCR, the OLA procedure was able to detect $80 fg (corresponding

to 160 molecules of DNA) of chromosomal target DNA and $20 CFU per reaction. Additionally, the OLA was as sensitive as dot blot hybridizations when either the 40-mer or the PCR product was used as a target. The sensitivity of this procedure is consistent with that of a microtiter plate format for detecting amplified products (5, 9, 17). We used an alkaline phosphatase colorimetric substrate system (ELISA amplification system [Gibco-BRL]) for detection of ligated products. The results can be determined by visualization of a color change, but determination of optical density is recommended. Cano et al. (5) reported that fluorometric substrates have greater sensitivity than colorimetric substrates, but fluorometric systems are more costly and require additional equipment to analyze the results. For a molecular diagnostic system based on PCR to be useful, it must be more rapid, reliable, and cost-effective than traditional culture methods. The OLA procedure for detecting PCR products described in this report takes less than 3 h to perform. This is a considerable reduction in time compared with other methods of detecting amplified products, such as membrane hybridizations (2, 3, 8, 11, 19, 20, 23, 24). When coupled with a limited cultivation period, the entire PCR-OLA could be completed in less than a day. With automation, it has

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been estimated from similar assays as many as 6,000 reactions could be processed in a day (16). The greatest obstacle to a successful PCR-based procedure for detecting infectious agents in clinical samples is the presence of PCR-inhibitory substances in the sample (10, 28). In these experiments overnight enrichment incubation in selenitecystine broth was used to (i) dilute PCR-inhibitory substances and (ii) replicate organisms present in the sample. The medium and incubation time could be changed and decreased to further optimize the turnaround time. Previously we have shown that selenite-cystine or brain heart infusion broth could be used in C-PCR-H to detect as few as 80 or 100 CFU of Salmonella organisms, respectively, after only 2 h of incubation (23). Another report indicates that as little as 1 h of incubation in an enrichment broth can lead to successful PCR from food samples (2), indicating that future research will be required to optimize incubation times for detection thresholds. Many procedures describe time-consuming, laborious DNA extraction protocols (10, 26–28). Instead of those, we used a commercially available reagent (Genereleaser) for extracting inhibitors to PCR from our reaction mixtures. This saved time and allowed the extraction and DNA amplification to be completed in a single tube. Rapid procedures based on molecular diagnosis for Salmonella bacteria are needed to expedite the analysis and routine surveillance requirements envisioned in hazard analysis critical control point systems (1, 4). Unfortunately, no rapid procedure will completely replace established culture procedures in the foreseeable future, because of the necessity to confirm results, assess viability of the target analyte (i.e., ‘‘gold standard’’ false negatives [PCR positive but culture negative]), and complete ancillary procedures, such as antibiograms and serotyping. Our results do indicate that a combined cultivation and manual PCR-OLA procedure could be used as a presumptive screening test for detecting Salmonella bacteria in many types of specimens and would be adaptable to automation. ACKNOWLEDGMENTS This work was supported by grants from the Kansas Racing Commission and the Kansas Agricultural Experiment Station. We thank Deborah Nickerson, University of Washington, for advice and construction of the labeled oligonucleotide probes for the OLA procedure. REFERENCES 1. Baird-Parker, A. C. 1990. Foodborne salmonellosis. Lancet 336:1231–1235. 2. Bej, A. K., M. H. Mahbubani, M. J. Boyce, and R. M. Atlas. 1994. Detection of Salmonella spp. in oysters by PCR. Appl. Environ. Microbiol. 60:368–373. 3. Bessesen, M. T., Q. Luo, H. A. Rotbart, M. J. Blaser, and R. T. Ellison III. 1990. Detection of Listeria monocytogenes by using the polymerase chain reaction. Appl. Environ. Microbiol. 56:2930–2932. 4. Blackburn, C. W. 1993. Rapid and alternative methods for the detection of salmonellas in food. J. Appl. Bacteriol. 75:199–214. 5. Cano, R. J., S. R. Rasmussen, G. Sa ´nchez Fraga, and J. C. Palomares. 1993. Fluorescent detection-polymerase chain reaction (FD-PCR) assay on microwell plates as a screening test for salmonellas in foods. J. Appl. Bacteriol. 75:247–253. 6. Carter, G. R., and M. M. Chengappa. 1991. Enterobacteriaceae, p. 150–164. In Essentials of veterinary bacteriology and mycology, 4th ed. Lea and Febiger, Philadelphia.

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