APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1993, p. 2795-28000099-2240/93/092795-06$02.00/0Copyright C 1993, American Society for Microbiology

A Simple RNA Probe System for Analysis of Listeramonocytogenes Polymerase Chain Reaction Productst

BURTON W. BLAIS* AND LUCILLE M. PHILLIPPE

Laboratory Services Division, Agrculture Canada, Ottawa, Ontario, Canada KJA 0C6

Received 26 March 1993/Accepted 17 June 1993

The synthesis of an RNA probe specific for the hlyA gene of Listeria monocytogenes by in vitro transcriptionfrom a polymerase chain reaction (PCR)-generated template incorporating bacteriophage T7 promotersequences is described. This simple method produced a high yield of RNA which hybridized specifically withhdyA PCR products on a membrane, resulting in RNA-DNA hybrids which were detected by an immunoen-zymatic assay with an anti-RNA-DNA hybrid antibody. The RNA probe hybridization system was more

sensitive in the analysis of the PCR products than was the conventional agarose gel electrophoresis method.When applied to the analysis of PCR samples from cultures of various Listeria and non-Listeria organisms, theRNA probe was reactive in the assay of 62 different L. monocytogenes isolates but not other Listeria species.Among the non-Listeria organisms tested, only Enterococcusfaecalis gave a weak positive reaction with more

than 109 cells per ml. This reactivity disappeared at lower cell densities. This strategy for the synthesis andapplication of RNA probes should facilitate the analysis ofPCR products in the detection ofL. monocytogenesand possibly other food pathogens.

Listeria monocytogenes is widely distributed throughoutthe food supply and can be isolated from a variety of raw andprocessed foods (16, 22, 25), some of which have beenimplicated in listeriosis outbreaks in humans (5). The tradi-tional approach for the detection of L. monocytogenes infoods involves its isolation and identification by culturaltechniques and biochemical tests, a process which can takeup to 10 days to complete (5, 12). To shorten the timerequired to obtain results, several investigators have devel-oped rapid tests for the direct detection ofL. monocytogenesin foods or enrichment cultures, including enzyme immu-noassays (1, 6, 15), DNA probes (3, 10, 11), and thepolymerase chain reaction (PCR) technique (14, 17, 24).Among these, PCR has shown great promise as a highlysensitive and specific method. One of the most extensivelystudied applications of the PCR technique for the detectionof L. monocytogenes in foods is the amplification of specificnucleotide sequences of the hlyA gene, which encodeslisteriolysin 0 (7, 8, 17).Most of the PCR-based procedures developed for L.

monocytogenes involve the analysis of the amplified DNAproducts by determining their size by agarose gel electro-phoresis. Although effective, this method does not easilylend itself to the large-scale screening of multiple samples byfood industry and other users, since the gels can be cumber-some to handle and can accommodate only a limited numberof samples. Furthermore, nonspecific amplifications whichmay occur and the limited sensitivity of visualizing the DNAby ethidium bromide staining can make the interpretation ofresults difficult. In general, the sensitivity and specificity ofPCR analyses can be improved by using DNA probestargeting the amplified DNA products (2). However, suchmethods usually involve labor-intensive DNA-DNA hybrid-ization procedures and require the use of a labeled probe,which can add considerably to the cost and sophistication ofthe test. As an alternative to using labeled DNA probes for

* Corresponding author.t Contribution number 93-007.

the detection of PCR products, it may be possible to use anunlabeled RNA probe which forms an RNA-DNA hybridwith target DNA immobilized on a membrane. The RNA-DNA hybrid could then be detected by an immunoenzymaticassay with an antibody recognizing such hybrids. The ad-vantages of an RNA probe can be summarized as follows: (i)RNA-DNA hybrids are more stable than DNA-DNA, per-mitting the use of higher stringency during the hybridization(18); (ii) the availability of antibodies recognizing RNA-DNAhybrid helices (20, 21) obviates the need for introducingchemical labels on the probe; and (iii) it should be possible toachieve higher signal-to-noise ratios in membrane hybridiza-tions, since carefully selected anti-RNA-DNA antibodieswill not react with nonspecifically adsorbed RNA.

In this paper we describe a simple and inexpensive methodfor the in vitro synthesis of an RNA probe by using bacte-riophage T7 RNA polymerase and a PCR-generated templatefrom hlyA incorporating bacteriophage T7 promoter se-quences. Such transcription templates have previously beenused to produce large quantities of RNA for gene sequencing(19) and in the isothermal amplification of nucleic acids (9).As an example of the applicability of such an RNA probesystem, we studied the analysis of PCR-amplified nucleotidesequences from hlyA by using an unlabeled RNA probe anda polyclonal antibody recognizing RNA-DNA hybrids in animmunoenzymatic membrane format.

MATERIALS AND METHODS

Immunoreagents and chemicals. Immunoglobulin G (IgG)from a goat antiserum to poly(A-dT) (anti-RNA-DNA IgG)was a kind gift from B. D. Stollar, Tufts University. Itspreparation has been described previously (21). Anti-goatIgG-peroxidase conjugate (no. A-3540) was obtained fromSigma Chemical Co. RNase (no. 1119 915), HindIII-digestedlambda DNA (no. 236 250), and protein-blocking reagent(no. 1096 176) were obtained from Boehringer Mannheim.dATP (no. U1201), dCTP (no. U1221), dGTP (no. U1211),and dTTP (no. U1231) were obtained from Promega. Themembrane peroxidase substrate system used in these studies

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TABLE 1. Non-Listeria organisms examined in this studyOrganism Source

Escherichia coli..................... ATCC 11775Shigella sonnei..................... ATCC 29938Enterobacter cloacae .................ATCC 13047Enterobacter aerogenes ..............ATCC 13048Klebsiella pneumoniae................ATCC 13883Proteus vulgais ..................... ATCC 13315Vibrio parahaemolyticus .............ATCC 17802Serratia marcescens...................ATCC 13880Enterococcus faecalis.................ATCC 19433Pseudomonas aeruginosa ............ATCC 10145Yersinia intennedia .................... G. Wautersa, Brussels, BelgiumYersinia kristensenii ................... G. Wautersa, Brussels, BelgiumYersinia frederiksenii.................. G. Wautersa, Brussels, BelgiumYersinia enterocolitica ................ G. Wautersa, Brussels, BelgiumYersinia mollaretii ..................... G. Wautersa, Brussels, BelgiumYersinia bercovien ..................... G. Wautersa, Brussels, BelgiumSalmonella montevideo...............ATCC 8387Salmonella typhimurium LT 2 .......ATCC 19585Bacillus subtilis ..................... ATCC 6051Bacillus cereus ..................... ATCC 14579Citrobacterfreundii....................ATCC 8090Micrococcus flavus ....................ATCC 10240Micrococcus luteus ....................ATCC 9341Staphylococcus epidermidis.........ATCC 12228Lactobacillus casei ....................ATCC 393Lactobacillus acidophilus............ATCC 4356Lactococcus lactis .....................ATCC 19257Streptococcus thennophilus.........ATCC 19258

a Universite Catholique de Louvain, Louvain, Belgium.

is a proprietary formulation based on the chromogen3,3',5,5'-tetramethylbenzidine (TMB) and an unspecifiedmembrane enhancer and was a kind gift from Ricoh Kyosan,Inc., Tokyo, Japan.

Bacterial strains and media. Bacteria used in this studyinclude two L. monocytogenes reference strains (nonhe-molytic type strain ATCC 15313 and ATCC 43256) and 60 L.monocytogenes isolates from egg, cheese, meat products,and environmental samples collected by Canadian Govern-ment inspection staff and submitted for routine microbiolog-ical analysis by Laboratory Services Division, AgricultureCanada, and the Health Protection Branch, Health andWelfare Canada. Other Listena spp. examined includedeight strains of L. innocua, two strains of L. ivanovii, threestrains of L. seeligeni, and one strain each of L. welshimen,L. grayi, and L. murrayi. Additionally, several gram-nega-tive and gram-positive organisms of other genera wereexamined (Table 1). All bacteria were routinely grown byinoculating single colonies from brain heart infusion agar(Difco) into Trypticase soy broth (BDH) and shaking for 16to 20 h at 30°C for Listeria, Bacillus, Micrococcus, Entero-bacter, and Yersinia spp. and Serratia marcescens and at37°C for all other bacteria. Viable counts were obtained byplating serial dilutions of the broth cultures on brain heartinfusion agar.

Preparation of bacterial samples. Bacteria were lysed by anadaptation of the method of Wang et al. (23). This involvedmixing 100 ,ul of broth culture with an equal volume of 2%(wt/vol) Triton X-100 and heating at 100°C for 5 min. Thesamples were then cooled to room temperature and usedimmediately in the PCR. Whenever necessary, chromosomalDNA was purified by phenol-chloroform-isoamyl alcoholextraction by the method of Wheatcroft and Watson (26).Purified DNA was precipitated in ethanol, resuspended in

sterile deionized-distilled water at about 1 ,ug/,ul, and storedat -20°C until use.PCR. Primers for the PCR were selected from the pub-

lished nucleotide sequence of hlyA (13). For the amplifica-tion of a 730-bp fragment spanning nucleotides 602 to 1332,we used a 21-mer forward primer, 5'-CATTAGTGGAAAGATGGAATG-3' (primer A), and a 20-mer reverse primer,5'-GTATCCTCCAGAGTGATCGA-3' (primer B). Oligonu-cleotides were synthesized on a DNA synthesizer (model391, PCR-Mate-EP; Applied Biosystems) by using phos-phoramidite chemistry (Applied Biosystems) as specified bythe manufacturer. For the PCR, 10 ,u1 of bacterial lysate wasadded to 89.5 p.1 of PCR mixture containing 0.22 mM eachdeoxynucleoside triphosphate (dNTP), 1.1 p.M each primersA and B, 2.2 mM MgCl2, 55 mM KCl, 11 mM Tris-HCl (pH8.3), and 0.11% (wt/vol) Triton X-100. The mixtures werethen overlaid with mineral oil, placed in a thermal cycler(model TC 480; Perkin-Elmer Cetus), and held at 80°C for 10min before addition of 0.5 p.l containing 2 U of Taq DNApolymerase (no. 1861; Promega). The reaction mixture wasthen subjected to 30 cycles of denaturation at 94°C for 1 min,primer annealing at 55°C for 1 min, and primer extension at72°C for 2 min. An additional 2 min was allowed for thecompletion of primer extension after the last cycle. Ampli-cons were routinely analyzed by electrophoresing 3 p. ofPCR product in a 1.2% agarose gel at 80 V for about 1.5 h,followed by staining for 20 min in ethidium bromide solution(10 p.g/ml). DNA on the gels was visualized by fluorescenceunder UV light and photographed onto Polaroid 667 film.The size of the amplicon was determined by including asample of 123-bp ladder DNA molecular size marker (no.5613SA; GIBCO-BRL) in each gel.

In vitro RNA probe synthesis. A PCR-generated templateincorporating bacteriophage T7 promoter sequences wasprepared from hlyA by using purified L. monocytogeneschromosomal DNA. The template was a 321-bp fragmentspanning nucleotides 861 to 1156 of hlyA (internal to the730-bp fragment generated with primers A and B) plus anadditional 26 nucleotides corresponding to the T7 promotersequences. The 41-mer forward primer used in the PCR togenerate the template was 5'-AATTTAATACGACTCACTATAGGGATCGGCAAAGCTGTTAC-3' (primer C: the T7RNA polymerase-binding and preferred transcriptional initi-ation sites [9] are indicated in italics). The 17-mer reverseprimer was 5'-TATCGCGTAAGTCTCCG-3' (primer D).For PCR generation of the template, 10 p.l of purified L.monocytogenes chromosomal DNA (1 ng/p.l) was added to89.5 p.l of PCR mixture containing 2.2 mM each dNTP, 1.1pM each primers C and D, 2.2 mM MgCl2, 55 mM KCl, 11mM Tris-HCl (pH 8.3), and 0.11% (wt/vol) Triton X-100. Themixtures were then subjected to PCR by using 2 U of TaqDNA polymerase and the same conditions described above.PCR product was then precipitated in ethanol and resus-pended in deionized-distilled water. It was extracted twice inphenol-chloroform (1:1) and once in chloroform, and thenthe aqueous phase was subjected to ethanol precipitation.The final DNA pellet was resuspended in deionized-distilledwater at 1 p.g/p.l and stored at -20°C. The specificity of thePCR amplification and relative purity of the 321-bp fragmentwere assessed by agarose gel electrophoresis as above.RNA probe was synthesized from the template by using

the Megascript T7 RNA polymerase in vitro transcription kit(no. 1334; Ambion) as specified by the manufacturer.Briefly, 2 p.g of template DNA was combined in a transcrip-tion buffer with NTPs, placental RNase inhibitor, and 40 Uof T7 RNA polymerase. The reaction mixture was incubated

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at 37°C for 5 h, and then the DNA template was subjected todegradation by further incubating the mixture with 4 U ofRNase-free DNase I at 37°C for 15 min. The reaction wasthen stopped by dilution with diethylpyrocarbonate-treateddeionized-distilled water and addition of ammonium acetatestop solution. The mixture was extracted once with phenol-chloroform (1:1) and once with chloroform. RNA was etha-nol precipitated from the aqueous phase, and the pellet wasresuspended in diethylpyrocarbonate-treated 10 mM Tris-HCl (pH 8.0)-i mM EDTA (TE). RNA was determined bymeasuring the A260 of the solution and calculating theconcentration as described by Sambrook et al. (18). ThisRNA probe stock was stored at -70°C, although we havefound it to be stable at -20°C for at least 6 months.RNA probe hybridization and immunoenzymatic detection

on membranes. Samples of PCR product from bacteriallysates amplified with primers A and B were heated at 100°Cfor 10 min, and 3-,J aliquots were pipetted onto a 5- by 5-cmsheet of nylon membrane (no. 1209 299; Boehringer Mann-heim), which could accommodate a total of 24 samplesspotted at ca. 1-cm intervals. The spots were allowed to airdry, and the DNA was cross-linked to the membrane by a5-min exposure to short-wave UV light. The membrane wasthen saturated with DEPC-treated hybridization solution(HS; 5x SSC [lx SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate], 1% [wt/vol] protein-blocking reagent, 0.1% [wt/vol]N-lauroylsarcosine, 0.02% [wt/vol] sodium dodecyl sulfate),placed in a sealed 50-ml Falcon tube, and hybridized at 80°Cfor 1 h with 5 ml of RNA probe suspended at 0.5 ,ug/ml in HSin a hybridization incubator (model 410; Robbins Scientific).After hybridization, the RNA probe solution was removedand the membrane was washed twice for 5 min with 0.01 Mphosphate-buffered 0.85% NaCl (PBS) containing 0.05%Tween 20 (PBST). All subsequent steps were carried out atroom temperature. RNA-DNA hybrids formed on the mem-brane were detected by incubation for 30 min with 5 ml ofgoat anti-RNA-DNA IgG at 5 ,ug/ml in PBST containing0.2% (wt/vol) protein-blocking reagent (PBST-B). The mem-brane was then washed twice for 3 min and once for 5 min inPBST and incubated for 30 min with 5 ml of anti-goatIgG-peroxidase conjugate diluted 1:2,000 in PBST-B. Themembrane was washed as before, and bound peroxidase wasassayed by incubation for 20 min with 5 ml of tetramethyl-benzidine (TMB) membrane peroxidase substrate system.

RESULTS

Synthesis of an RNA probe. A simple method for synthe-sizing an RNA probe specific for the hlyA gene of L.monocytogenes was developed by using a PCR-generatedDNA template incorporating bacteriophage T7 promotersequences and T7 RNA polymerase in an in vitro transcrip-tion system. A 321-bp PCR-generated template was preparedby amplifying sequences corresponding to positions 861 to1156 of hlyA from purified L. monocytogenes DNA with aset of primers (primers C and D) in which bacteriophage T7promoter sequences were attached to the 5' end of theforward primer. The expected product of transcription initi-ating at the T7 promoter sequences on this template was a295-nucleotide transcript. Figure 1 shows that the product ofthe transcription reaction (lane 2) migrated at a slightly largerapparent molecular size than the 321-bp DNA template (lane1), possibly because of the single stranded RNA nature ofthe transcript. The RNA nature of the transcription productwas confirmed in two ways: (i) the material visualized in lane2 was resistant to the DNase I treatment used in the in vitro

m 1 23

FIG. 1. Characterization of RNA probe. A 321-bp PCR-gener-ated DNA template and its transcription product were electropho-resed on a 1.2% agarose gel before and after treatment with RNase.Lanes: m, 123-bp marker DNA ladder; 1, 0.3 p,g of template DNA;2, 1.5 ,ug of transcription product; 3, 1.5 p,g of transcription productafter digestion with 0.5 p,g of RNase for 15 min at 37°C.

RNA synthesis reaction; and (ii) this material was com-pletely degraded on treatment with DNase-free RNase (lane3). The yield of RNA from the PCR-generated template inthe in vitro transcription system was substantial. A total ofabout 300 p,g of RNA was synthesized from 2 ,ug of templateDNA.

Hybridization of RNA probe. The suitability of the tran-script synthesized from the PCR-generated template as aprobe for L. monocytogenes was examined by studying thehybridization of the RNA with hlyA DNA sequences immo-bilized on a nylon membrane, followed by detection of theRNA-DNA hybrids by an immunoenzymatic assay involvingsequential reactions with an anti-RNA-DNA goat IgG and ananti-goat IgG-peroxidase conjugate. An aliquot of lysatefrom a broth culture of L. monocytogenes (ca. 106 cells) wassubjected to PCR amplification by using primers A and Bdefining a 730-bp amplicon of hlyA within which was locatedthe 295-bp sequence corresponding to the RNA probe. ThePCR product was then serially diluted in PCR buffer, and asample of each dilution was spotted on a nylon membrane.The membrane was hybridized with the RNA probe and thenassayed by the immunoenzymatic method with anti-RNA-DNA antibody. For the purpose of comparison, the PCRproduct dilutions were also analyzed by agarose gel electro-phoresis followed by ethidium bromide staining. The 730-bpamplicon could be visualized on the gel to a maximumdilution of 1:32 (Fig. 2A), whereas analysis of the samesamples by hybridization with the RNA probe permitted thedetection of the PCR product diluted as much as 1:256 (Fig.2B). Thus, the RNA probe hybridization system gave almost10-fold-higher sensitivity in the detection of hlyA PCR prod-ucts than the agarose gel electrophoresis method did. Theinclusion of 1 ,ug of HindIII-digested lambda DNA on themembrane as a negative control and its failure to give areaction on the blot (Fig. 2B) confirm the specificity of theRNA probe for the hlyA sequences and that of the anti-RNA-DNA IgG for the hybrids.The sensitivity of the RNA probe hybridization system in

the detection of hlyA amplicons was further examined by

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mi 2 3 4 5 6 7

0

* .1*tS

FIG. 2. Comparative sensitivity of the RNA probe hybridizationsystem in the detection of the PCR product. A 730-bp amplicon fromhlyA was prepared by PCR with primers A and B and then seriallydiluted and analyzed by agarose gel electrophoresis (A) and RNAprobe hybridization (B). In panel A, lane m contains the 123-bpmarker DNA ladder and lanes 1 to 7 contain PCR product dilutionsas follows: lane 1, undiluted; lane 2, 1:2; lane 3, 1:4; lane 4, 1:8; lane5, 1:16; lane 6, 1:32; lane 7, 1:64. In panel B, PCR product dilutionsare as follows: spot 1, 1:2, spot 2, 1:4; spot 3, 1:8; spot 4, 1:16; spot5, 1:32; spot 6, 1:64; spot 7, 1:128; spot 8, 1:256; spot 9, 1:512; spot10, 1 ,ug of HindIII-digested lambda DNA.

studying its detectability limit when applied to the analysis ofPCR products from lysates of serially diluted L. monocyto-genes cells and purified chromosomal DNA. A broth cultureof L. monocytogenes was serially diluted in TSB or PCRbuffer to give different cell densities. A purified chromo-somal DNA preparation from this organism was also seriallydiluted in PCR buffer to give different DNA concentrations.Samples of these dilutions were subjected to PCR withprimers A and B, and the amplicons were then analyzed bythe agarose gel electrophoresis and RNA probe hybridiza-tion methods described above. In this application, the RNAprobe hybridization system was at least as sensitive as theagarose gel electrophoresis method in terms of the minimumquantity of sample (cell lysate or purified DNA) which couldbe detected by the PCR (Table 2). In this case, the limitingfactor for the sensitivity of L. monocytogenes detectionappears to be the performance of the PCR amplification,rather than the RNA probe, since the sensitivity of the RNAprobe hybridization system in the detection of the PCRproduct itself was much greater than that of the agarose gelelectrophoresis method (see above). The sensitivity of thePCR system appeared to improve when the L. monocyto-genes cells were serially diluted in PCR buffer instead ofbroth (Table 2).

Specificity of the combined PCR-RNA probe system. Thespecificity of the combined hlyA PCR-RNA probe hybridiza-

TABLE 2. Comparative sensitivity of the PCR analyticalmethods for L. monocytogenes

Sample (quantity) Detectability by:Gel electrophoresis RNA probe hybridization

Lysate (cells)bA 1,170 ± 216 580 ± 130B 270 51 170 35

DNA (pg) 8 0 4 0a Minimum quantity of cells or DNA subjected to PCR that could be

visualized on analysis of the PCR products by the agarose gel electrophoresisand RNA probe hybridization methods. Results are reported as mean +standard deviation (n = 3).

b Cells were serially diluted in TSB (A) or PCR buffer (B).

tion detection system for L. monocytogenes was examinedby its application to various Listena and non-Listeria organ-isms. Broth cultures of the bacteria containing ca. 109 cellsper ml were lysed and then subjected to PCR with primers Aand B. The PCR products were then analyzed by the agarosegel electrophoresis and RNA probe hybridization methodsas above. Table 3 shows that all of the L. monocytogenesisolates tested (including two ATCC strains, one of which isnonhemolytic) and none of the other Listeria species gavepositive results in the PCR when analyzed by both methods.With the exception of the Enterococcusfaecalis strain, noneof the non-Listeria organisms tested (Table 1) gave a positivePCR result by either method. Although no evidence of a730-bp amplicon could be seen on analysis of the E. faecalisPCR sample by agarose gel electrophoresis, a weak reaction(faint-colored spot) was visible on the blot with the RNAprobe (data not shown). This weak reactivity persisted evenwhen the blot was processed in the absence of the RNAprobe. However, when suspensions of this organism con-taining fewer than 109 cells per ml were subjected to PCR, novisible reaction occurred on the blot with or without theRNA probe. Therefore, it can be surmised that this weakreactivity of E. faecalis at high cell densities is due to somefactor contributed by the broth culture (perhaps high levelsof endogenous RNA-DNA complexes) rather than being anartifact of the PCR and appears to be a unique feature of thisorganism.

DISCUSSION

We have demonstrated a simple procedure for the in vitrosynthesis of an RNA probe and its application in the analysisof PCR products for the detection of L. monocytogenes.

TABLE 3. Specificity of the combined PCR-RNA probe systemfor various Listeria organisms

No. positive/total no. tested by':Listeria species

Gel electrophoresis RNA probe hybridization

L. monocytogenes 62/62 62/62L. ivanovii 0/2 0/2L. innocua 0/8 0/8L. grayi 0/1 0/1L. murrayi 0/1 0/1L. welshimeri 0/1 0/1L. seeligeri 0/3 0/3

a Number of different isolates giving a positive response on analysis of thePCR products by the agarose gel electrophoresis and RNA probe hybridiza-tion methods.

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Because this system is based on the use of an antibody forthe detection of RNA-DNA hybrids formed between theprobe and DNA samples immobilized on a membrane, theneed for costly chemical or enzymatic methods of introduc-ing labels on the probe has been eliminated. This willfacilitate the standardization of different probe batches,since the batch-to-batch variations which may occur in theincorporation of labels by chemical or enzymatic means areavoided. Furthermore, this RNA probe system, which usesthe anti-RNA-DNA hybrid antibody in the immunoenzy-matic detection, eliminated the need for an extensive prehy-bridization step, which is necessary to block nonspecificbinding sites on the membrane when using labeled probes.This procedure could be further shortened by directly con-jugating the anti-RNA-DNA antibody to the indicator en-zyme, thus reducing the number of immunoreaction stepsrequired in the assay.On the basis of the limited number of strains tested, the

RNA probe hybridization system for the analysis of the hlyAPCR product seemed to be specific for L. monocytogenes,except for the weak reactivity observed with high celldensities of E. faecalis. It may be possible to eliminate thisreactivity by treatment of the cell lysates with RNase,RNase H, or a combination of the two, to degrade possibleendogenous RNA-DNA complexes prior to performing thePCR. Alternatively, the PCR product could be denaturedunder conditions which selectively hydrolyze RNA, such aswith strong alkali, prior to blotting on the membrane. How-ever, this matter was not pursued further since this reactivitywas very weak compared with that of L. monocytogenes atsimilar cell densities and did not occur with broth cultures ofE. faecalis containing fewer than 109 cells per ml.

It is interesting that the present PCR system producedpositive results with the nonhemolytic L. monocytogenestype strain (ATCC 15313) by both analytical methods. This isin agreement with the findings of Mengaud et al., whoobserved hybridization of an hlyA-specific DNA probe withthe same strain (13). Their explanation, i.e., that the gene ispresent in this strain but not detectably expressed, seemslikely.The sensitivity of this PCR method could be improved by

diluting the L. monocytogenes cells in PCR buffer instead ofthe enrichment broth (Table 2). This may be due in part tothe possible presence of inhibitory substances in the broth.However, in practice it would be more convenient to sampleenrichment broth directly for the PCR, especially when largenumbers of samples must be processed. It should be possibleto achieve sufficient cell densities by culture enrichment offood or environmental samples to meet the sensitivity limitof the present method. Although in principle this methodsuccessfully detected L. monocytogenes cells in pure cul-tures, it has yet to be tested on actual contaminated food andenvironmental samples.The present method of synthesizing large quantities of the

RNA probe by in vitro transcription of a PCR productincorporating a bacteriophage promoter is much simplerthan the traditional approach involving cloning of the tem-plate into a transcription vector and its propagation in abacterial host (4). Provided that the nucleotide sequence ofinterest is known, the synthesis of an RNA probe specific forvirtually any segment of DNA should be possible by using asuitable pair of primers for PCR generation of the template,without the need for specific restriction endonuclease sitesas required in the recombinant DNA approach. Thus, thestrategy for this RNA probe hybridization system should beapplicable to the analysis of PCR products in a wide variety

of applications, such as the detection of food pathogens,bacterial and viral disease diagnosis, and basic research.

ACKNOWLEDGMENTS

We thank Y. Trottier and L. Laflamme, Laboratoire d'hygieneveterinaire et alimentaire, Agriculture Canada; D. Christensen andM. Perlette, Laboratory Services Division West, Agriculture Can-ada, and M. Burzynski, Laboratory Services Division, AgricultureCanada, for providing cultures of L. monocytogenes food isolates.We also thank G. Wauters, Universite Catholique de Louvain,Brussels, Belgium, for providing the Yersinia strains.

REFERENCES1. Bhunia, A. K., and M. G. Johnson. 1992. Monoclonal antibody

specific for Listeria monocytogenes associated with a 66-kilodalton cell surface antigen. Appl. Environ. Microbiol. 58:1924-1929.

2. Brooks, J. L., A. S. Moore, R. A. Patchett, M. D. Collins, andR. G. Kroll. 1992. Use of the polymerase chain reaction andoligonucleotide probes for the rapid detection and identificationof Carnobacterium species from meat. J. Appl. Bacteriol.72:294-301.

3. Chenevert, J., J. Mengaud, E. Gormley, and P. Cossart. 1989. ADNA probe specific for L. monocytogenes in the genus Listeria.Int. J. Food Microbiol. 8:317-319.

4. Digweed, M., and U. Gunthert. 1989. Recombinant selection bymicroinjection: a simple cDNA cloning procedure for produc-tion of exclusively sense RNA transcripts. Gene 83:147-152.

5. Farber, J. M., and P. I. Peterkin. 1991. Listeria monocytogenes,a food-borne pathogen. Microbiol. Rev. 55:476-511.

6. Farber, J. M., and J. I. Speirs. 1987. Monoclonal antibodiesdirected against the flagellar antigens of Listeria species andtheir potential in EIA-based methods. J. Food Prot. 50:479-484.

7. Fitter, S., M. Heuzenroeder, and C. J. Thomas. 1992. A com-bined PCR and selective enrichment method for rapid detectionof Listeria monocytogenes. J. Appl. Bacteriol. 73:53-59.

8. Furrer, B., U. Candrian, C. Hoefelein, and J. Luethy. 1991.Detection and identification of Listeria monocytogenes incooked sausage products and in milk by in vitro amplification ofhaemolysin gene fragments. J. Appl. Bacteriol. 70:372-379.

9. Guatelli, J. C., K. M. Whitfield, D. Y. Kwoh, K. J. Barringer,D. D. Richman, and T. R. Gingeras. 1990. Isothermal, in vitroamplification of nucleic acids by a multienzyme reaction mod-eled after retroviral replication. Proc. Natl. Acad. Sci. USA87:1874-1878.

10. King, W., S. Raposa, J. Warshaw, A. Johnson, D. Halbert, andJ. D. Klinger. 1989. A new colorimetric nucleic acid hybridiza-tion assay for Listenia in foods. Int. J. Food Microbiol. 8:225-232.

11. Kohler, S., M. Leimeister-Wachter, T. Chakraborty, F. Lott-speich, and W. Goebel. 1990. The gene coding for protein p60 ofListeria monocytogenes and its use as a specific probe forListeria monocytogenes. Infect. Immun. 58:1943-1950.

12. McLauchlin, J., and P. N. Pini. 1989. The rapid demonstrationand presumptive identification of Listeria monocytogenes infood using monoclonal antibodies in direct immunofluorescencetest (DIFT). Lett. Appl. Microbiol. 8:25-27.

13. Mengaud, J., M. Vicente, J. Chenevert, J. M. Pereira, C.Geoffroy, B. Gicquel-Sanzey, F. Baquero, J. Perez-Diaz, and P.Cossart. 1988. Expression in Escherichia coli and sequenceanalysis of the listeriolysin 0 determinant of Listeria monocy-togenes. Infect. Immun. 56:766-772.

14. Niederhauser, C., U. Candrian, C. Hofelein, M. Jermini, H.-P.Buhler, and J. Luthy. 1992. Use of polymerase chain reactionfor detection of Listeria monocytogenes in food. Appl. Environ.Microbiol. 58:1564-1568.

15. Olapedo, D. K., A. A. G. Candlish, and W. H. Stimson. 1992.Detection of Listeria monocytogenes using a polyclonal anti-body. Lett. Appl. Microbiol. 14:26-29.

16. Rohrbach, B. W., F. A. Draughon, P. M. Davidson, and S. P.Olivier. 1992. Prevalence of Listeria monocytogenes, Campylo-bacter jejuni, Yersinia enterocolitica and Salmonella in bulk

VOL. 59, 1993

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 01

Jan

uary

202

2 by

184

.22.

55.8

1.

2800 BLAIS AND PHILLIPPE

tank milk: risk factors and risk of human exposure. J. FoodProt. 55:93-97.

17. Rossen, L., K. Hoistrom, J. E. Olsen, and 0. F. Rasmussen.1991. A rapid polymerase chain reaction (PCR)-based assay forthe identification of Listeria monocytogenes in food samples.Int. J. Food Microbiol. 14:135-145.

18. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecularcloning: a laboratory manual, 2nd ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

19. Stoflet, E. S., D. D. Koeberl, G. Sarkar, and S. S. Sommer. 1988.Genomic amplification with transcript sequencing. Science 239:491-494.

20. Stollar, B. D. 1980. The experimental induction of antibodies tonucleic acids. Methods Enzymol. 70:70-85.

21. Stollar, B. D., and A. Rashtchian. 1987. Immunochemical ap-proaches to gene probe assays. Anal. Biochem. 161:387-394.

22. Varabioff, Y. 1990. Incidence and recovery of Listeria from

APPL. ENVIRON. MICROBIOL.

chicken with a preenrichment technique. J. Food Prot. 53:555-557.

23. Wang, R. F., W. W. Cao, and M. Johnson. 1992. 16S rRNA-based probes and polymerase chain reaction method to detectListeria monocytogenes cells added to foods. Appl. Environ.Microbiol. 58:2827-2831.

24. Wernars, K., C. J. Heuvelman, T. Chakraborty, and S. H. W.Notermans. 1991. Use of the polymerase chain reaction fordirect detection of Listeria monocytogenes in soft cheese. J.Appl. Bacteriol. 70:121-126.

25. Westoo, A., and M. Peterz. 1992. Evaluation of methods fordetection of Listeria monocytogenes in foods: NMKL collabo-rative study. J. Assoc. Off. Anal. Chem. Int. 75:46-52.

26. Wheatcroft, R., and R. J. Watson. 1988. A positive strainidentification method for Rhizobium meliloti. Appl. Environ.Microbiol. 54:574-576.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 01

Jan

uary

202

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.22.

55.8

1.