Simultaneous detection ofListeriaspp. andListeria monocytogenesby reverse hybridization with 16S–23S rRNA spacer probes

Molecular and Cellular Probes(1995) 9, 423-432

Simultaneous detection of Listeria spp. and Listeria monocytogenes by reverse hybridization with 16S-23S rRNA spacer probes N a n c y P. Rijpens, .1 G e e r t Jannes, 2 M a r i n a Van Asbroeck, 2 Lieve M . F. H e r m a n 1 and Rudi Rossau 2

Government Dairy Research Station, Brusselsesteenweg 370, B-9090 Melle, Belgium and 21nnogenetics N.V., Industriepark Zwijnaarde 7, Box 4, B-9052 Gent, Belgium (Received 2 June 1995, Accepted 1 August 1995) Enzymatic amplification results showed that Listeria species have at least two 16S-23S rRNA spacer regions of different lengths. These spacer regions of L. monocytogenes, L. ivanovii and L. seeligeri were cloned after enzymatic amplification. Sequence analysis of the inserts revealed two spacers of 245-246 bp and 496-498 bp, respectively, of which the latter included tRNA(Ala) and tRNA(Ile) genes. One Listeria spp.-specific probe, LIS-ICG4, was deduced from the 245-bp spacer and a L. monocytogenes-specific probe, LMO-ICG5, was inferred from the 496-bp spacer. The specificity of both probes was tested in a reverse hybridization assay (Line Probe Assay, LiPA). Both LIS-ICG4 and LMO-ICG5 proved to be highly specific when hybridized to a large collection of Listeria strains and strains from other relevant taxa. The LiPA test herein described for the simultaneous detection of Listeria spp. and L. monocytogenes can be expanded to detect other foodborne pathogens. © 1995 Academic Press Limited

KEYWORDS: Listeria, Listeria monocytogenes, DNA probes, 16S-23S rRNA spacer, LiPA, PCR.

INTRODUCTION Nucleic acid probe tests have been introduced for the detection of many foodborne pathogensY ° However, over the last years foods have to be screened for an increasing number of pathogens. In this respect the use of 'multi-pathogen' tests is more cost and labour effective. Recently the line probe assay (LiPA), in which the oligonucleotides are immobilized as parallel lines on membrane strips, was developed for the simultaneous detection of cystic fibrosis mutations, bacterial pathogens in clinical samples, human leucocyte antigen typing and typing of hepatitis C virus isolates,u Herein we present the LiPA for the simultaneous detection of Listeria spp. and Listeria monocytogenes.

Over the last years the 16S-23S rRNA spacer region of eubacteria has been successfully used as a source of specific DNA probes. 1-3The 16S-23S rRNA spacer region is less conserved than the sequences of the 16S and 23S rRNA genes and therefore may be a preferred target for the development of species-specific probes for highly related organisms. The spacer region can be enzymatically amplified using primers which target quasi universally conserved sequences at the 3' end of the 16S gene and the 5" end of the 23S gene. When using this amplification product in a reverse hybridization assay4-° multiple eubacterial species can be amplified and subsequently detected at the same time. * Authorto whomcorrespondenceshouldbe addressed. 0890-8508/95/060423 + 10 $12.00/0

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The genus Listeria encompasses seven species.12 While most species are non-pathogenic to humans, L. monocytogenes has the potential to cause listeriosis. In humans, bacterial meningitis is the most common form of listeriosis while perinatal infections may result in abortion, stillbirth and infant death. L. monocytogenes-contaminated food has been the source of listeriosis outbreaks leading to severe illness and death. The presence of Listeria spp. other than L. monocytogenes in foods can be considered an indicator of unsatisfactory sanitary conditions during production. The LiPA system herein presented can be expanded to detect other foodborne pathogens.

MATERIALS AND METHODS Bacterial strains The bacterial strains used are listed in Table 1. The strains were identified by conventional means as described by Rocourt et al. ~3 or by using the Accuprobe Listeria monocytogenes culture identification test (Gen-Probe, San Diego, CA, USA).

DNA extractions Whole-cell DNA was extracted as described by Flamm et al. TM Crude cell lysates were prepared by adding 100 I~1 0.1 M NaOH, 0.25% SDS to the pellet obtained from 2 ml pure bacterial culture and subsequent heating for 17 min at 90°C.

al.

the 16S gene and the 5' end of the 23S gene. For cloning purposes the primers P1 (TGG GGT GAA GTC GTA ACA AGG TA) and P2 (GCA TGC GGC CGC CCT TTC CCT CAC GGT ACT GGT) were used, located about 50 bp from the 3' end of the 16S rRNA gene and about 500bp from the 5' end of the 23S rRNA gene, respectively. A recognition site for the Notl restriction enzyme was added to the primer P2 to facilitate cloning of the obtained PCR products. For reverse hybridization purposes on LiPA strips the same primers were used except that these were biotinylated at their 5' ends. PCR was performed in a total volume of 50 p.I using 2 U AmpliTaq DNA Polymerase (Perkin Elmer, Norwalk, CT, USA), 50 mM KCI, 2"0 mM MgCI2, 10 mM Tris-HCI (pH 8"3), 0.01% gelatine, 0-5% Tween-20, 200ram of each deoxynucleoside triphosphate, 50 pmol of each primer and 10-50 ng DNA or 1 ILl of crude cell lysate. The mixture was subjected to 30 cycles of amplification in a thermal cycler (Techne PHC-2; Techne, Cambridge, UK). The first cycle was preceeded by an initial denaturation of 5 min at 95°C, each cycle consisted of a denaturation of 1 min at 95°C, an annealing of 1 min at 50°C and an extension of 1 min at 72°C. The last cycle was followed by a final extension of 10 min at 72°C. For cloning purposes the PCR product was loaded on a 1% (w/v) GTG agarose gel (FMC Bioproducts, Rockland, ME, USA) and electrophoresed for about 90 min at 8 Vcm -1. The gel portion containing the amplified DNA fragment was removed and the DNA extracted using the Geneclean Kit (Bio 101, La Jolla, CA, USA).

Cloning Oligonucleotide synthesis, purification and labelling The oligonucleotides to be used as primers or probes were synthesized by the phosphite-triester method on an ABI 392 DNA synthesizer (Applied Biosystems, Foster City, CA, USA). After deprotection and precipitation with ethanol they were redissolved in distilled H20 and used without further purification. Oligonucleotides were biotinylated at the 5' end by adding a biotin-phosphoramidite during synthesis. Efficiency of biotinylation was checked by reversed phase HPLC.

The gel-purified PCR fragment was digested with Notl to enable the directional cloning of the amplification product in EcoRV-Notl digested pBluescript SK÷ vector (Stratagene, La Jolla, CA, USA). After overnight ligation at 15°C the recombinant plasmid was transformed in competent Escherichia coli DH5c( (Life Technologies, Gaithersburg, MD, USA) and the cell suspension was plated on LB agar containing carbenicilline [501~g carbenicilline (ml LB)-t], XGAL and IPTG following a standard protocol, is White colonies were subjected to clone analysis as described by Birnboim and Doly. 16

PCR amplification

Sequencing of the cloned PCR products

The 16S-23S rRNA spacer was amplified by the PCR using quasiuniversal primers located at the 3' end of

Plasmid DNA was prepared using the Qiagen method (Qiagen, Chatsworth, CA, USA). The inserts

LiPA detection of Listeria

Table 1

425

Bacterial strains used and hybridization results with probes LIS-ICG4 and LMO-ICG5

Species

L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. rnonocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. rnonocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. monocytogenes L. ivanovii L. ivanovii L. ivanovii L. ivanovii L. ivanovii L. ivanovii L. ivanovii L. Ivanovii L. Ivanovii L. ivanovii L. seeliger! L. seeligerl L. seeligen L. seeligerl L. seeligerl L. seeligerl L. seeligerl L. seeligerl L. seeligen L. seeligen L. seeligerl

Serotype

1/2a 1/2b 1/2c 4b 4a 4b 1a 1/2a 4b

1/2a 1/2 a 4c 4b 1/2a 1/2a 1/2 a 1/2 a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2b 1/2b 4b 4b 4b 3b 5 5 5 5 5 5 5 4b 3b 6b 1/2b 4a

1/2b 1/2b

Strain

MB1 MB38 MB39 MB40 MB41 19114 P7 Scott A P10 P11 P13 P15 E64 P18 P19 P47 V7 California Ohio 1005 1003 1019 1042 K513 L393 H 514 H 725 G608 K726 H577 Z597 Z596 L591 L705 Z611 H244 H245 K559 K289 3009 MB4 78.42 L1 Pe 3050 65 66 519 3010 3058 40126 40127 4433 40140 4268 59/6/89 265/65/90 4007 100100 LS 1 LT 36 B

Hybridization

Source*

RZS IHE IHE IHE IHE ATCC RZS INRA RZS RZS RZS RZS FIL RZS RZS RIVM INRA INRA INRA RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS RZS WSLC RZS CIP INRA WSLC BFM BFM SLU WSLC WSLC WSLC WSLC WSLC WSLC WSLC IHE IH E WSLC CIP INRA INRA

LIS-ICG4

LMO-ICG5

+ + + + + + + + + + + + + + + + + + + + + + -I-I+ + + + -I-t-I+ + + -I+ + + + -I-I+ + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + -t-

~L

+ + + + + + + + + + -I-I-I+

i,

--

426 Table 1

N.P. Rijpens et aL continued

Species

Serotype

Strain

Source*

Hybridization LIS-ICG4

L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. welshimeri L. innocua L. innocua L. innocua L. Innocua L. innocua L. innocua L. Innocua L. innocua L. Innocua L. innocua L. innocua L. innocua L. innocua L. innocua L. innocua L. innocua L. innocua L. Innocua L. Innocua L. innocua L. Innocua L. murrayi L. murrayi L. murrayi L. grayi L. grayi Brochothrix thermosphacta Brochothrix campestris Bacillus cereus Bacillus cereus Bacillus cereus Bacillus brevis Bacillus brevis Bacillus coagulans Bacillus pumilis Bacillus macerans Bacillus lentus Bacillus firmus Bacillus firmus Bacillus subtilis Bacillus subtilis Bacillus megantum Enterococcus faecalis Enterococcus faecium Enterococcus durans Lactobacillus bulgaricus

6b

6a 1/2b 6b 6a 1/2b

6a 6b 6b 6a 6b 6b 6b 6a 6a 6a 6a 6b 6b 6b 6a 6a 6a 6b 6b 6a 6b

81.49 LIV MB3 5339 7624 5008 50146 50149 390 383 382 394 4OO 469 19 40 41 58 61 62 64 71 80.11 $9 23 108 117 KT 67 2011 2021 2023 2012 989 1026 76.124 103213 5990 103321 68.18 20171 4712 $1 $17 $10 $12 $18 $13 $19 $21 MB7 MB8 7125 151 $20 Sll EFS 1

EFM 1 EDS 1

CIP INRA RZS BFM BFM WSLC WSLC WSLC IFM IFM IFM IFM IFM IFM IFM IFM IFM IFM IFM IFM IFM IFM CIP BFM BFM BFM BFM BFM BFM WSLC WSLC WSLC WSLC SLU SLU ClP CIP CCM CIP CIP DSM DSM RZS RIVM RZS RZS RZS RZS RZS RZS RZS RZS LMG BR RZS RZS INRA INRA INIA RZS

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

LMO-ICG5

LiPA detection of Listeria

427

Table 1 continued Species

Serotype

Strain

Source*

Hybridization LIS-ICG4

Streptococcus thermophilus Lactococcus lactis Lactococcus lactis Lactobacillus case'f Leuconostoc lactis Escherichia coli Hafnia halvei Agrobacterium tumefaciens Mycoplana dimorpha Clostridium tyrobutyricum Clostridium peffringens Clostridium sporogenes Clostridium acetobutyricum Brucella abortus Brucella suis Brucella melitensis Staphylococcus aureus Salmonella typhimurium Salmonella enteritidis Yersinia enterocolitica

MRZ 1076 1363 MB57 774 MB60 711 196 4961 620B 12224 126B 5711 TULYA P30 P34 P35 P95

RZS INRA LMG RZS INIA RZS INRA LMG LMG INRA LMG INRA LMG NIDO NIDO NIDO RZS RZS RZS RZS

LMO-ICG5

D

D

D

D

R

m

m

B

m

m

B

D

* Abbreviationsare: ATCC,AmericanType Culture Collection, USA; BFM, Bundesanstaltf0r Milchforschung,Institutf0r Hygiene, Germany; CCM, Czech Collection of Microorganisms,Czech Republic;CIP,Collection de Bactdriesde I'lnstitut Pasteur,France; DSM, DeutscheSammlungyon Mikroorganismenund Zeikulturen,Germany; FIL, FederationInternationalde Laiterie, France; IFM, Institute for Milchhygiene,Austria; IHE, Institutefor Hygieneand Epidemiology,Belgium; INIA, Instituto Nacional de InvestigacionesAgrarias, Spain; INRA, lnstitut Nationalede RecherchesLaiti~res,France; LMG, CultureCollection of the Laboratoryof Microbiology, Ghent, Belgium; NIDO, National Institutefor VeterinaryResearch,Belgium; RIVM, Rijksinstituutvoor Voedingsmiddelentechnologie , The Netherlands; RZS,GovernmentDairy ResearchStation, Belgium; SLU, SwedishUniversityof Agricultural Sciences,Sweden;WSLC, WeihenstephanListeriaCollection, Germany.

of the recombinant plasmids were sequenced using the dideoxy chain termination method of Sanger et aL lz Sequencing reactions were performed using the reagents from the Deaza G/A~ Sequencing Mixes Kit (Pharmacia, Uppsala, Sweden) and cz-3ssdATP (Amersham, Buckinghamshire, UK), and were analysed on the Macrophor sequencing system (Pharmacia). Alternatively, sequencing reactions were performed using the Taq Dye Primer Cycle Sequencing Kit or the Taq Dye Deoxy Terminator Cycle Sequencing Kit (Applied Biosystems) and were analysed on an automatic DNA sequencer (ABI 373 A). The sequencing primers used were T7 a promotor/ primer adjacent to the multiple cloning site of pBluescript SK + and P3 (GTC CTT CTT CGG CTC CT) a primer allocated 40 bp from the 5' end of the 23S rRNA gene of Listeria spp. Sequence alignment was done using the PC/GENE software provided by Intelligenetics Inc. and Genofit SA.

Preparation of LiPA strips and hybridization Oligonucleotide probes (LIS-ICG4 and LMO-ICG5) were dT-tailed and fixed onto nitrocellulose strips as described by Stuyver etaL 11The strips were incubated with alkaline-denatured biotinylated PCR product (101~1) in 1 ml hybridization buffer (3 x SSC, 20% deionized formamide, 0.5% blocking reagent (Boehringer Mannheim, Mannheim, Germany), 0.1% N•lauroylsarcosinate) for 1 h at 50°C in a shaking waterbath. The strips were washed twice at room temperature (R'I') for 1 min with 1 ml wash buffer (3 x SSC, 20% deionized formamide), followed by a stringent wash at 50°C for 15 rain. After a brief rinse in 1 ml rinse solution (Inno-LiPA kit, Innogenetics, Antwerp, Belgium) at RT, the strips were incubated with 1 ml conjugate solution (streptavidine coupled to alkaline phosphatase, Innogenetics) for 30 rain at RT. Then the strips were washed three times with 1 ml rinse solution and once with 1 ml substrate diluent (lnno-LiPA kit).

428

N.P. Rijpens etaL

M

1

2

3

RESULTS PCR amplification and cloning PCR amplification of chromosomal DNA of L. monocytogenes IHE MB 41, L. ivanovii CIP 78.42 and L. seeligeriWSLC 4268 using primers P1 and P2 yielded at least two amplification products after examination on agarose gel. This result confirmed earlier observations of Listeria possessing more than one rRNA operon. ~-2° As is the case in other bacteria, these different operons may contain 1 6S-23S rRNA spacer regions of different length or sequence. 2'-2s The amplicon of 800bp had a much greater intensity than the 1100-bp amplicon when loaded on an agarose gel (Fig. 1). Both amplification products were cloned in pBluescript SK÷ for all three Listeria species mentioned above.

Fig. 1. Ethidium bromide stained agarose (4%) gel of PCR products obtained with primerset P1/P2. M; Boehringer marker VIII (Boehringer Mannheim, Mannheim, Germany); 1, L. monocytogenes IHE MB 41; 2, L. ivanovii CIP 78.42; 3, L. seeligeri WSLC 4268.

Sequencing

Colour development was achieved by adding substrate (BCIP and NBT, Innogenetics) and incubation of the strips at RT for 30 min on an orbital shaker. The colour reaction was stopped by replacing the substrate with TE-buffer.

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monocytogenes

L. L. L.

monocytogenes

ivanovii

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40

50

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ivanovii

L. seellgeri

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90

120

160

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140

.................. 180

190

CCGAGAATCATCTGAAAGTGAATCTTTCATCTGATTGGAAGTATCATCGC

22~2222222~222~22~22~222222~21222c~2Z2;2~2~ 200

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80

AAAGTTAGTAAAGTTAG CATAGATAATTTAT ..................... A. • .GG.A.

150

monocytogenes

70

TGAGAG GTTAGTACTTCT CAGTA-TGTTTGTTCTTTGAAAACTAGATAAG .......... A.T ...... TA..C .......................... ..,o..,..,To., ..... T...- ......... , ...... • .........

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C CTGTGAGTTTTCGTTCTT .........................

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For each Listeria strain one clone with an insert of 800 bp and one clone with an insert of 1100 bp wei'e analysed. Sequence analysis of these clones revealed that the spacer included in the 800-bp PCR fragment was 245 bp long for L. monocytogenes and 246 bp long for L. ivanovii and L. seeligeri (Fig. 2). No differences were found when comparing this spacer sequence of L. monocytogenes IHE MB 41 with the spacer sequence of L. monocytogenes earlier

210

220

230

240

TGATACGGAAAATCAGAAAAACAACCTTTACTTCGTAGAAGTAAATT .......A..........................A.C.......... . . . . . . A..C...T . . . . . . . . . . . . . . . . . . . . . AC ..........

Fig. 2. Sequence alignment of the 245 bp-246 bp rRNA spacer region of L. monocytogenes IHE MB 41, L. ivanovii CIP 78.42 and L. seeligeri WSLC 4268.

LiPA detection of Listeria 10 *

20 *

429

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TGAGAGGTTACTCTCTTTTATGTCAGATAAAGTATGCAAGG .................. G ............... Ii0 *

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CACTATG CT ..... GT...C

140 *

150 *

TGAAGCATCGCGCCACTACATTTTTGACGGGC CTATAGCTCAGCTGGTT~ .TGG...AA.A ........... A ........................... 160 *

170 *

180 *

190 *

200 *

GAGCGCACGCCTGATAAGCGTGAGGTCGATGGTTCGAGTCCATTTAGGCC ...... ..........o ..... ..o...o.o. .... o..oo.ooo.oo.. 210

220

230

240

CACTTTTTCTTTCTGACATAAGAAATACAAATAATCATACCCTTTTAC-.................. G ....... ---C..TTG..C.T...A...ATA 260 -GGGG

270

CCTTAGCTCAGCTGGGAGAG

310

280 CGCCTGCTTTG

320

330

290 CACGCAGGAGGTCA

340

GCGGTTCGATCCCGCTAGG CTCCACCAAAATTGTTCTTTGAAAACTAGAT oo.ooooo .... ...................................... 360

AAGAAAGTTAGTAAAGTTAG ........................ 410

370

380

390

CATAGATAATTTAT-TATTTATGACACAAG AG..G.A..AC ............... 420

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440

TAACCGAGAATCATCTGAAAGTGAATCTTTCATCTGATTGGAAGTATCAT ................................... A. • .C..C 460

470

480

.......

490

CGCTGATACGGAAAATCAGAAAAACAACCTTTACTTCGTAGAAGTAAATT ......... A..C. • .T ..................... AC ..........

Fig. 3. Sequencealignment of the 496 bp-498 bp rRNA spacer region of L. monocytogenes IHE MB 41 (top) and L. seeligeri WSLC 4268 (bottom). Sequencescoding for the tRNA"e and tRNAa'agenes are underlined. submitted to the EMBL databank under accession number L05172 by Emond et al. 24 We found very high homology values (~94%) when comparing the spacers of the three species examined. The spacer included in the 1100-bp PCR fragment was 496 bp for L. monocytogenes and 498 bp for L. seeligeri (Fig. 3). The sequence of this spacer for L. ivanovii was not completely determined. Again the homology values between the species were high (~90%). When comparing the two spacers of different lengths of one species it is striking that except for an insertion of about 250bp both spacers are nearly identical. This is exemplified for L. monocytogenes in Fig. 4. In both L. monocytogenes and L. seeligeri this insertion contained the sequences for the two tRNA genes: tRNA(Ala) and tRNA(Ile). When the tRNA genes are excluded, the lowest degree of homology between two species is found in this 250 bp insertion.

Sensitivity and specificity of the probes Several oligonucleotide probes were deduced from the 245-bp spacer. None of these probes showed absolute species specificity when hybridized with amplified DNA from different Listeria species (data not shown). One probe deduced from the 245-bp spacer, LISICG4, hybridized with amplified DNA from all Listeria species giving an acceptable intensity. One L. monocytogenes probe, LMO-ICG5, was deduced from the sequence of the 496-bp spacer and proved to be species specific when hybridized to different Listeria species. Both probes (LIS-ICG4 and LMO-ICG5) were subjected to extensive sensitivity and specificity testing. This was done in a reverse hybridization format on LiPA strips. On these strips both oligonucleotide probes were immobilized as parallel lines, after which

430

N.P. Rijpens et al. i0

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120

TGAAGCATCGCG

130

140

CCACTACATTTTTGACGGG

160

170

150

CCTATAGCTCAGCTGGTTA

180

190

200

GAGCGCACGCCTGATAAGCGTGAGGTCGATGGTTCGAGTCCATTTAGGCC .................................................. 210 *

220 *

230 *

240 *

250 *

CACTTTTTCTTTCTGACATAAGAAATACAAATAATCATACCCTTTTACGG .................................................. 260

270

280

290

300

GGCCTTAG CTCAGCTGGGAGAGCGCCTGCTTTG CACG CAGGAGGTCAG .................................................. 310

320

330

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340

350

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370

380

390

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Sequence alignment of the 496 bp (top) and the 245 bp (bottom) rRNA spacer region of L. monocytogenes IHE

MB 41.

a bcde

control LIS-ICG-4

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Fig. 5. Examples of hybridization results with the LiPA strips, a, L. ivanovii INRA L1Pe; b, L. seeligeri WSLC 40126; c, L. monocytogenes ATCC 19114; d, L. monocytogenes INRA Ohio; e, L. monocytogenes RIVM P47; f, L. monocytogenes FIL E64; g, L. monocytogenes INRA Scott A; h, L. monocytogenes RZS L705; i, B. cereus RZS $1; j, 13. thermosphacta DSM 20171; k, L. lactis LMG 1363; I, PCR blank. these strips were hybridized with biotinylated amplification product from a large collection of bacterial strains (see Table 1). The presence of the expected amplicons was checked by electrophoresis of 8 pl of

the amplification mixture on a 1-5% agarose gel and visualization after ethidium bromide staining. Some representative hybridization results on LiPA strips are shown in Fig. 5.

LiPA detection of Listeria

All 39 L. monocytogenes strains tested hybridized with both LIS-ICG4 and LMO-ICG5 while the 61 strains belonging to other Listeria species only hybridized to LIS-ICG4 and not to LMO-ICG5 (Table 1). None of the 40 strains belonging to other genera hybridized to LIS-ICG4 or LMO-ICG5 (Table 1). Both LIS-ICG4 and LMO-ICG5 can be considered to be highly specific probes for Listeria spp. and L. monocytogenes, respectively.

DISCUSSION Listeria monocytogenes is a frequently encountered human pathogen in food products, with the potential to cause severe disease upon consumption. Consequently its presence in food products should be strictly excluded. 2sThe situation is less clear for other Listeria species which are not considered major pathogens. However, monitoring the presence of other Listeria species in food products is advisable. The presence of Listeria in general in food is an indicator of sanitary conditions and other Listeria species, especially L. innocua may mask the presence of L. monocytogenes. 26 At the present time no DNA test exists which directly discriminates between Listeria and Listeria monocytogenes in a single test. The aim of this study was to find DNA probes specific for the species L. monocytogenes and the genus Listeria in the same DNA target. The spacer region between the 165 and 235 rRNA was explored since this region offers the possibility for simultaneous amplification of most bacterial taxa with one set of primers, and for other bacterial taxa evidence was available that DNA probes at different taxonomic level may be derived from this region. '-~ Enzymatic amplification results showed that Listeria species have at least two 165-235 rRNA spacer regions of different lengths. These spacer regions of L. monocytogenes, L. ivanovii and L. seeligeri were sequenced. This revealed two spacers of 245-246 bp and 496-498bp, respectively, of which the latter included tRNA(Ala) and tRNA(Ile) genes. Both spacers of one species are identical except for an insertion of 250 bp which contains the tRNA sequences. A Listefia spp-specific probe, (LiS-iCG4), was deduced from the 245-bp spacer. However we were not able to deduce a L. monocytogenes-specific probe from the 245-bp spacer. This result was surprising since few, but enough, sequence differences were found in the 245-246 bp spacers of L. monocytogenes, L. seeligeri and L. ivanovii to deduce a specific probe. This led us to presume that besides the two spacers we have sequenced for each species, other 165-235

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rRNA spacers with even greater similarity are present in the Listeria genomes. These spacers may be of the same length but may have a different sequence than the ones already sequenced. The existence of several rather than one spacer of about 245 bp may also explain the remarkable higher amount of 800-bp amplification product over 1100-bp amplification product. However, an L. monocytogenes-specific probe (LMO-ICG5) could be deduced from the 496 bp spacer. This spacer of L. monocytogenes showed less homology with other Listeria species than did the 245bp spacer. The region showing the highest variation between Listeria species was situated in the insertion sequence containing the tRNA sequences. Both probes, LIS-ICG4 and LMO-ICG5, were tested against a large panel of bacterial strains in a reverse hybridization assay(the LiPA), and proved to be 100% specific and sensitive. Hence this LiPA test can be used to reliably and conveniently identify Listeria in general and Listeria monocytogenes in particular, simultaneously, in food. Moreover, by using other specific spacer probes the LiPA system herein presented can be expanded to detect other foodborne pathogens. As a multipathogen test, LiPA would offer a fast, accurate, cost effective and easy to handle detection system for foodborne pathogens. Further work is being done on the development of a suitable sample preparation for the application of LiPA on food.

ACKNOWLEDGEMENTS

This work was supported by IWONL (Instituut ter bevordering van bet Wetenschappelijk Onderzoek in de Nijverheid en de Landbouw).

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