APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1993, p. 1342-13460099-2240/93/051342-05$02.00/0Copyright C 1993, American Society for Microbiology

Rapid Detection of Salmonellae in Poultry with the MagneticImmuno-Polymerase Chain Reaction Assay

AD C. FLUIT,1.2* MYRA N. WIDJOJOATMODJO,"12 ADRIENNE T. A. BOX,"12 RUURD TORENSMA,12AND JAN VERHOEF'

Eijkman-Winkler Laboratory for Medical Microbiology, University Hospital Utrecht, Room G. 04.515,P.O. Box 85500, 3508 GA Utrecht, 1 and U-Gene Research B. V., Utrecht, 2 The Netherlands

Received 16 December 1992/Accepted 16 February 1993

Rapid detection of salmonellae in chicken meat was accomplished by using the magnetic immuno-polymerasechain reaction assay (MIPA). A direct polymerase chain reaction assay performed with chicken meat spikedwith Salmonella typhimurium resulted in poor sensitivity (approximately 107 CFU/g of meat). The use ofimmunoseparation with a Salmonella serogroup B-specific monoclonal antibody improved the sensitivity, butenrichment was required for the detection of low levels of contamination. Enrichment for 6 h in either bufferedpeptone water, lactose broth containing tergitol-7, or selenite-cystine broth resulted in the detection of an initialinoculum of 100 CFU per g of meat. Enrichment of the salmonellae present on 25 g of spiked chicken meat for24 h in either buffered peptone water or selenite-cystine broth before detection by the MIPA yielded a detectionlimit of approximately 0.1 CFU/g of meat. A detection limit of approximately 1 CFU/g of meat was obtainedwhen the spiked meat was stored at -20°C before enrichment for 24 h and analysis with the MIPA. Althoughthe MIPA was developed for S. typhimurium, a MIPA in which a panel of six monoclonal antibodies specific forSalmonella serogroups A through E was used detected the presence of 0.1 CFU of SalmoneUla enteritidis per gof chicken meat. These data indicate that the method is applicable to other commonly isolated serotypes.

Salmonellae are some of the major causes of food poison-ing in the developed world. Food products like meat, eggs,poultry, and chocolate are common sources of salmonellosis(2, 7, 12, 18). Therefore, a rapid diagnostic test is needed.Conventional methods for the isolation of Salmonella spp.require 5 days for the detection of small numbers of oftenstressed organisms (6). In recent years methods based oneither immunoassays (3, 9, 12, 14, 17, 18) or DNA probes (4,5, 15, 21) have been developed. Most prominent among theimmunoassays is the Salmonella-Tek assay (Organon-Teknika Corp., Durham, N.C.) (18). This immunoassayutilizes two monoclonal antibodies which recognize antigensfrom heat-killed salmonellae. This test, however, still re-quires 48 h to complete (4), but with enrichment in Salmo-syst broth the total time required can be reduced to 31 h (18).Several DNA probes for Salmonella spp. have been devel-oped, but only a probe that recognizes rRNA is commer-cially available (Gene-Trak Systems, Framingham, Mass.).Detection is based on the hybridization of a polydeoxyade-nylic acid-tailed capture probe and a fluorescein-labeleddetector probe for Salmonella rRNA. The resulting hybrid iscaptured on a dipstick coated with polydeoxythymidylic acidand is detected by colorimetry by using an antifluorescein-antibody conjugate with horseradish peroxidase. Neverthe-less, the earliest results are obtained on day 3 (4, 15, 21).Another major drawback of these tests is the relatively largenumber of false-positive results obtained (15).

Recently, magnetic separation technology was introducedto make the assays faster and more specific (10, 11, 19).Previously, we described (19) the development of the mag-

netic immuno-polymerase chain reaction assay (MIPA). Thisassay utilizes magnetic particles coated with monoclonalantibodies to extract bacteria from a sample. Trapped bac-teria are lysed, and the supernatant, which contains bacterial

* Corresponding author.

DNA, is then subjected to the polymerase chain reaction(PCR). A MIPA performed with a Salmonella group B-spe-cific monoclonal antibody and Salmonella-specific PCRprimers based on the origin of DNA replication was capableof detecting 10 CFU of Salmonella typhimurium in thepresence of 107 CFU of Escherichia coli (19). In this paperwe show that when the MIPA is used in combination withenrichment, as few as 0.1 CFU of Salmonella spp. per g ofpoultry meat can be detected reliably within 30 h.

MATERIALS AND METHODS

Media. Most of the media used were obtained from Oxoid,Basingstoke, United Kingdom; the exceptions were sodiumhydrogen selenite, which was purchased from Merck, Darm-stadt, Germany, and tergitol-7, which was bought fromSigma Chemical Co., St. Louis, Mo.

Bacteria. The Salmonella serotypes used belong to Salmo-nella choleraesuis subsp. choleraesuis. The two strains usedare referred to below as S. typhimurium and Salmonellaententidis. Unless otherwise indicated, S. typhimurium wasused.Immunomagnetic separation. Magnisort M magnetic chro-

mium dioxide particles coated with goat immunoglobulinsspecific for murine immunoglobulin G (IgG) and IgM (Du-Pont, Wilmington, Del.) were incubated with 75 RI of hybri-doma culture supernatant for 5 min at room temperaturewith continuous shaking. The magnetic particles were recov-ered by magnetic force, and the supernatant was discarded.Bacteria from a fresh culture were suspended in saline andthen added to the magnetic particles. After 15 min at roomtemperature with continuous shaking, the magnetic particleswere recovered by magnetic force, washed three times withsaline, and finally resuspended in 20 ,ul of water for use in thePCR or in saline for plating onto Giston agar or salmonella-shigella agar plates. The plates were incubated overnight at37°C, and the colonies were counted. Unless otherwise

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indicated, 5 p,l of Magnisort M and monoclonal antibody71.40 were used.Monoclonal antibodies. The generation of serogroup-spe-

cific monoclonal antibodies has been described by Torensmaet al. (16). The following six monoclonal antibodies wereused in this study: monoclonal antibody 47.15 (IgM), specificfor serogroup A; monoclonal antibody 71.40 (IgGl), specificfor serogroup B; monoclonal antibody 72.4 (IgG3), specificfor serogroup Cl; monoclonal antibody 35.71 (IgGl), spe-cific for serogroup C2; monoclonal antibody 41.11 (IgGl),specific for serogroup D; and monoclonal antibody 42.77(IgG3), specific for serogroup E.PCR. The resuspended beads were heated to 95°C for 5

min to lyse the bound bacteria. The beads were recovered bycentrifugation, and the resulting supernatant was utilized inthe PCR. The PCR was carried out as described by Widjo-joatmodjo et al. (19), except that a total volume of 50 p,l wasused instead of 100 ,ul. Briefly, the PCR mixture contained 10mM Tris-HCI (pH 8.3), 50 mM KCI, 1.5 mM MgCl2, 0.01%gelatin, deoxynucleoside triphosphates (dNTP) (each at aconcentration of 100 p,M), primers (each at a concentrationof 0.5 pM), and 1.25 U of Taq polymerase (Cetus, Em-eryville, Calif.). The primers were selected in the bacterialorigin of chromosome DNA replication (onG). Primer 1(5'-TTATTAGGATCGCGCCAGGC) was located betweenbp 60 and 79, and primer 2 (5'-AAAGAATAACCGTTGTTCAC) was located between bp 204 and 223 of the previouslypublished sequence of Salmonella onC (8). Amplificationwas carried out with a Thermocycler apparatus (Perkin-Elmer Instruments, Norwalk, Conn.) for 35 cycles and wasfollowed by a final 10-min extension at 72°C. Each cycleconsisted of 1 min at 94°C, 1 min at 58°C, and 1 s at 72°C. ThePCR incubation mixtures were analyzed for product forma-tion on an ethidium bromide-stained agarose gel. Confirma-tion was accomplished by Southern hybridization with adigoxigenin-labeled probe as described by Widjojoatmodjoet al. (19). Briefly, the probe for Southern analysis waslabeled with digoxigenin-11-dUTP (Boehringer Mannheim,Mannheim, Germany) by the PCR. The PCR reaction mix-ture was analogous to the PCR mixture described above,except that the PCR mixture contained different dNTPconcentrations (100 p,M dATP, 100 p.M dCTP, 100 p.MdGTP, 90 p.M dTTJ7P, and 10 p.M digoxigenin-11-dUTP). Thenumber of cycles was 24. S. typhimurium DNA was used asthe target DNA. The identity of the probe was confirmed byrestriction enzyme analysis. Hybridization and detectionwere performed as specified by Boehringer Mannheim.

Processing of chicken meat samples. Samples (10 and 25 g)of chicken meat were treated for 2.5 min in a stomacher, andthe volumes were brought up to 100 and 250 ml, respec-tively, with medium. The media used were buffered peptonewater (BPW), lactose broth containing tergitol-7 (LB'), andselenite-cystine broth (S/C). The resulting suspensions werespiked with S. typhimunum. The cultures were incubated at37°C for the times indicated below. After thorough mixing,large particles were allowed to settle for 15 min at roomtemperature. A 1-ml portion of supernatant from each sus-pension was subjected to extraction with magnetic beads.

RESULTS

Recovery of SalmoneUa spp. with monoclonal antibody-coated magnetic beads. The sensitivity of the MIPA isdependent, among other things, on the number of bacteriathat are recovered from a sample by using magnetic beadscoated with a specific monoclonal antibody. The optimal

CFU

0.1 ml 0.5 ml 2.0 mlsample size

FIG. 1. Recovery of S. typhimurium (input, 3 x 104 CFU/ml)from 0.1-, 0.5-, and 2.0-ml volumes with Salmonella serogroupB-specific monoclonal antibodies bound to 2, 5, or 10 >1± of goatanti-mouse magnetic beads (solid, cross hatched, and stippled bars,respectively). The results of a PCR performed by using 20% of therecovered bacteria are also indicated.

extraction procedure was determined by using several sam-ple sizes and different amounts of beads. Sample sizes of 0.1,0.5, and 2 ml in 96-, 24-, and 6-well plates, respectively,containing 3 x 104 CFU of S. typhimurium per ml wereincubated with 2, 5, or 10 p.l of monoclonal antibody-coatedmagnetic beads and after collection were plated onto Gistonagar plates. The best recovery was obtained with 2-mlsamples and 10 p.l of magnetic beads coated with monoclonalantibodies (Fig. 1). However, a sample size of 1 ml and 5 ,ulof monoclonal antibody-coated magnetic beads were usedsince the preparations could be handled in either 24-wellplates or 1.5-ml Eppendorf tubes.

Sensitivity of direct PCR and direct MIPA with chickenmeat. The direct PCR with spiked meat suspensions wasinhibited 100-fold compared with the PCR performed withpure cultures, resulting in a detection limit of approximately1 x 107 CFU of Salmonella sp. per g of meat. The perfor-mance of the MIPA was assessed with spiked chickensamples. Directly after treatment in a stomacher, the sam-ples were analyzed with the MIPA. The presence of 1 x 105CFU/g of meat gave a clear band, while the presence of 1 x104 CFU/g could not be detected.Enrichment ofSalmoneUla spp. Enrichment was required to

increase the sensitivity of the assay. Three media, BPW,LB', and S/C, were tested for their ability to support thegrowth of Salmonella spp. Meat suspensions in these mediawere spiked with either 1 or 100 CFU of S. typhimurium perml. At various times 100-,1u portions were plated ontosalmonella-shigella agar. Pale H2S-producing colonies werenot observed on plates containing samples which were notinoculated with Salmonella sp. Therefore, only pale andH2S-producing colonies were counted on salmonella-shigellaagar plates, and no further determination of these colonieswas made. Optimal growth of Salmonella sp. was obtainedin BPW, especially when the initial numbers of Salmonellasp. were low (Fig. 2). However, most of the results with theMIPA were negative; the only exception was the 6-h enrich-ment in S/C of an initial inoculum of 100 CFU/ml.

Sensitivity ofMIPA after enrichment. Direct addition of 1-,2-, or 5-p.l samples of both BPW and S/C enrichments ofchicken meat spiked with 25 CFU of Salmonella sp. per g to

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log CFU/ml

A

4 -

3- A

2 -

0 1 2 4

log CFU/ml

0 1 2 3 4 5 6 7time in h

FIG. 2. Growth curves for S. typhimurium in BPW (0), LB'(O), and S/C (*) in the presence of 10% (wt/vol) chicken meat. Theinput was either 1 CFU/ml (A) or 100 CFU/ml (B).

the PCR incubation mixtures did not produce any amplifica-tion product when the preparations were analyzed by agar-ose gel electrophoresis.The sensitivity of the MIPA in combination with a 24-h

enrichment step was examined. To ensure that the meatsamples were free of Salmonella spp., 10 25-g portions oftwo chicken brands were tested with both the MIPA and theconventional Association of Official Analytical ChemistsBacteriological Analytical Manual method (1). Three por-

tions of one brand were contaminated with Salmonella spp.

Chicken samples from a different brand were negative andwere used for subsequent studies. Chicken meat sampleswere spiked with either 1, 0.1, 0.01, 0.001, or 0 CFU of S.typhimurium per g of meat, and this was followed byenrichment for 24 h in either S/C or BPW. The presence of0.1 CFU/g of meat after enrichment in BPW was detected byusing agarose gel analysis. No amplification products were

detected after enrichment in S/C by using agarose gel anal-ysis, but after Southern hybridization the presence of 0.1CFU/g of meat was also detected.

General applicability of the MIPA for the detection ofSalmonella spp. A panel of monoclonal antibodies specific forSalmonella serogroups A through E was used to detect S.enteritidis in 25-g samples spiked with 1, 0.1, 0.01, 0.001, or

0 CFU/g of meat after enrichment in either S/C or BPW.Figure 3A shows that Salmonella sp. was detected byagarose gel analysis in the sample spiked with 0.01 CFU/g ofmeat after enrichment in S/C. In BPW the presence of 0.1CFU of Salmonella sp. per g of meat was detected. Southernhybridization of the amplification products did not alter thedetection limit for either enrichment medium (Fig. 3B).MIPA of samples containing damaged Salmonella spp.

Recovery of damaged Salmonella spp. was studied by inoc-ulating chicken meat with either 0 or 1 x i05 CFU ofSalmonella sp. per g of meat. Inoculated chicken wasfrozen, thawed the next day and subsequently prepared forenrichment. Freezing and thawing reduced the number ofviable cells approximately 10-fold. Suspensions in BPW,LB+, and S/C were incubated for 6 h at 37°C. Portions (100,ul) were plated onto Giston agar and salmonella-shigellaagar. The results showed that higher numbers of Salmonellasp. were found in BPW than in either LB' or S/C (Table 1).However, the number of non-Salmonella bacteria was alsohigher in BPW. No salmonellae were found in nonspikedmeat samples. The MIPA was also performed to determinewhether the high numbers of contaminating bacteria had anyinfluence on the detection of Salmonella sp. in the assay. Allof the samples that were spiked with Salmonella sp. gavepositive results in the MIPA (data not shown).The sensitivity of the MIPA with poultry spiked with

damaged Salmonella sp. was determined. A total of 25 g ofchicken meat was inoculated with 1 or 0.1 CFU of Salmo-nella sp. per g, frozen, thawed the next day, and treated in astomacher either in 125 ml of BPW, 125 ml of LB+, or 125 mlof S/C. After treatment another 100 ml of medium wasadded. The resulting suspensions were cultured overnight at37°C. A 1-ml sample from each culture was subjected to theMIPA; 1 CFU of Salmonella sp. per g of meat could bedetected after enrichment in either BPW or S/C. The MIPAwas negative with LB' and when the inoculum was 0.1CFU/g when the preparations were analyzed on agarosegels.

DISCUSSION

Previously, we described a MIPA for the detection ofSalmonella spp. in which monoclonal antibodies that arespecific for live Salmonella spp. and a PCR based onSalmonella-specific primers chosen in the origin of bacterialchromosome DNA replication (oriC) were combined (16,19). The use of the MIPA allows the circumvention of Taqpolymerase inhibition by components of food, enrichmentmedia, or large amounts of non-Salmonella DNA. Indeed,the direct PCR with spiked meat suspensions was inhibited100-fold compared with the PCR with pure cultures, result-ing in a detection limit of approximately 1 x 107 CFU ofSalmonella spp. per g of meat, suggesting that immunomag-netic separation can reduce inhibition of the PCR. Inhibitionof the PCR is probably due to blood products present inmeat, which have also been found to inhibit the direct PCRin blood specimens (13). A comparable improvement insensitivity was described by Widjojoatmodjo et al. (20) whenthe MIPA was used to detect Salmonella spp. in fecalsamples. In these samples inhibition was probably due tobile salts and degradation products of heme, such as biliru-bin. The inhibitors bound to proteins present in the sampleand are probably released when the sample is heated to 94°Cin the PCR. Thus, the MIPA reduces the amount of inhibi-tors present, but inhibitors which adhere to the bacteria arenot removed, resulting in residual inhibition of the PCR.

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B

1 2 3 4 5 6 .8 9 10 11 12 13 14FIG. 3. (A) Agarose gel analysis of MIPA products of S. enteritidis-spiked chicken meat after overnight culture in S/C (lanes 1 through

5) and BPW (lanes 6 through 10). The inocula used were 1 CFU/g of meat (lanes 1 and 6), 0.1 CFU/g (lanes 2 and 7), 0.01 CFU/g (lanes 3 and8), 0.001 CFU/g (lanes 4 and 9), and 0 CFU/g (lanes 5 and 10). Lane 11 contained a negative MIPA control. Lane 12 contained a phage lambdaPstI digest as a marker. Lane 13 contained a positive PCR control, and lane 14 contained a negative PCR control. (B) Southern blot of theagarose gel shown in panel A, obtained with a digoxigenin-labeled probe specific for the amplified product.

Adherence of bacteria to the large particulate matterwhich was allowed to settle also lowers the level of recov-ery. Enrichment is required to obtain improved sensitivity.A culture of Salmonella spp. in BPW showed the highestlevel of recovery, but contaminating flora grew equally wellin this medium. The selective media S/C and LB' produced

lower numbers of Salmonella spp., but also lower numbersof contaminating flora. Since LB' performed more poorly inthe MIPA, this enrichment medium was not fully investi-gated. Direct PCR with samples of BPW and S/C enrich-ments of Salmonella-spiked chicken meat did not result inthe detection of 1 CFU/g, which was probably due to the

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TABLE 1. Growth of S. typhimunium damaged by freezing andthawing in BPW, LB', and S/C in the presence of 10% (wt/vol)

chicken meat'

No. of CFU/ml 4 h No. of CFU/ml 6 hMedium after thawing after thawing

BPW 5 x 106 1 x 108LB+ 3 x 105 3 x 107S/C 2 x 105 5 x 105a The input was 105 CFU/g of meat.

presence of PCR-inhibitory substances. This indicates theneed for immunomagnetic separation. Indeed, with theMIPA the presence of 0.1 CFU of Salmonella spp. per gcould be detected by using 25-g meat samples. A similarsensitivity was obtained when a panel of six monoclonalantibodies specific for serogroups A through E was used todetect S. enteritidis. This demonstrates the applicability ofthis method for other commonly isolated Salmonella sero-types. Even damaged salmonellae were detected after stor-age of contaminated meat at -20'C. Our results show thatLB' is ineffective, whereas both BPW and S/C had adetection limit of 1 CFU/g. This detection limit is compara-ble to those reported for the Salmonella-Tek and Gene-Trakassays in which enrichment cultures are used. These assaysrequire 48 h, but Van Poucke (18) demonstrated that with an

improved medium (Salmosyst) the detection time for theSalmonella-Tek assay could be reduced to 31 h. The MIPAdescribed here requires a total time of 30 h. Given thenecessary short enrichment incubation, determination in 1work day is not yet feasible, but the samples can be collectedand prepared for overnight culture in enrichment broth. Thenext day MIPA and the detection of amplification productscan be carried out on several samples simultaneously. TheMIPA in combination with a 24-h enrichment step over-comes the disadvantages of both enzyme assays and thePCR alone. Non-Salmonella bacteria, which are eitherbound nonspecifically to the beads or cross-react specificallywith the monoclonal antibodies (16), are not detected in thePCR when Salmonella-specific primers are used. Further-more, material which inhibits the Taq polymerase is re-moved during immunomagnetic separation. This approachresults in the detection of 2.5 CFU of Salmonella spp. per 25g of meat and 25 CFU/25 g of meat after the salmonellae aredamaged by freezing and thawing.

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