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Letters in Applied Microbiology 1990, 11, 158-162

MFS/Q32

Detection of Listeria species and Listeria monocytogenes using

polymerase chain reaction

P.M. BORDER,J.J. HOWARD*,G.S. PLASTOW& K.W. SICGENSDalgety PLC, Group Research Laboratory, Station Road, Cambridge CBl 2JN, UK

Received 1 June 1990 and accepted 7 June 1990

BORDER,P.M.,

Detection of Listeria species and Listeria monocytogenes using polymerase chain

reaction. Letters in Applied Microbiology 11, 158-162.

Five oligonucleotide sequences are described that were used as primers in the poly- merase chain reaction (PCR) to amplify specific sequences from Listeria DNA. When all five primers were used in combination, three PCR products were possible; a Listeria specific product that occurs with DNA from any Listeria sp., a Listeria monocytogenes specific product that occurs only in the presence of DNA from this organism and a universal product that is found using DNA from any bacterial source. The occurrence of these PCR products was used as a diagnostic test on bacteria isolated from various food samples to detect Listeria sp. and L. mono-

cytogenes.

HOWARD,J.J.,

PLASTOW,G.S. & SIGGENS,K.W. 1990.

The detection of the human pathogen Listeria monocytogenes and other members of the genus Listeria in food products by existing methods is a time-consuming procedure, largely because of the need to isolate pure cultures before further characterization may be undertaken. Recent developments in molecular biology have raised the possibility of detecting pathogens in foods and other samples without the need for iso- lation of pure cultures using target-specific gene probes (see Walker & Dougan 1989; Wernars & Notermans 1990for recent reviews). Approaches to the detection of Listeria sp. by such methods have used standard hybridization techniques either to detect genes coding for factors thought to be involved in pathogenicity such as a-haemolysin (Datta et al. 1987, 1988), listeriolysin 0 (Mengaud et al. 1988), msp (major secreted polypeptide of L. mono- cytoyenes, Flamm et al. 1989) and L. mono- cytogenes DTH factor (Notermans et al. 1989) or to detect specific 16s ribosomal RNA (rRNA) sequences (Klinger et al. 1988; Stackenbrandt &

* Corresponding author.

disadvantages of

the methods published to date is that relatively large numbers of target cells need to be present (typically 104-105) in order to yield unam- biguous results in a background containing large numbers of non-target micro-organisms (Wernars & Notermans 1990). The recently developed technique of poly- merase chain reaction (PCR) using thermostable DNA polymerase (Saiki et al. 1988) permits the rapid amplification of specific DNA sequences by a factor of up to 10’. Polymerase chain reaction methods have been described that will detect low levels of Escherichia coli in clinical samples (Olive 1989), Pseudomonas cepacia in

Curiale 1988). One of the main

soil samples (Steffan & Atlas 1988), Mycohac- terium leprae in armadillo tissues (Hartskeerl et

al.

(Barry et al. 1990). The sensitivity of these systems typically approximates to the detection of a single bacterium. To the authors’ know- ledge there have been no published reports of DNA amplification using PCR to detect Listeria

in fish

1989) and

Aeromonas

salmonicida

sp. in food or other samples. The work reported in this paper describes the use of a number of

Detection of Listeria using PCR

159

synthetic oligonucleotide probes as primers in the PCR to amplify DNA sequences from L. monocytogenes and other Listeria sp.

Materials and Methods

maintained on Tryptose Agar (Difco) and all other strains were maintained on Nutrient Agar (Oxoid). Putative Listeria strains were isolated from a variety of sources using the method of Maclean & Lee (1988).

BACTERIAL STRAINS

The bacterial strains used in this study are detailed in Table 1. All Listeria strains were

Table 1. Origin and identity of bacterial strains used

Bacterium

Listeria grayi innocua ivanovii monacyt ogenes monorytogenes murrayi seeliyeri welshimeri Jonesia denitrijicans Aeramonas hydrophila Bacillus cereus Brochothrix thermosphacta Erwinia curotovora Escherichia coli K12

Laboratory strain

Source/reference

NCTC 10815

NCTC 11288

NCTC 11846

NCTC 10357

NCTC 11994

NCTC 10812

NCTC 11856

NCTC 11857

NCTC 10816

NCTC 8049

NCTC 11143

NCTC 10822

SCRI 193

NM522

Gemella haemolysans

NCTC 10243

Klebsiella pneumoniae

ATCC 15050

Lactobacillus

amylophilus

NCIMB 11546

helveticus

NCIMB 8652

p1antarum

NCIMB 11974

Salmonella typhimurium

NCIMB 10248

Serratia marcescens

NCIMB 2303

Staphylococcus aureus

NCIMB 9518

Enterococcus faecalis

ATCC 8043

Lactococcus lactis

subsp. lactis

NCIMB 6681

Veillunellu criceti

ATCC 8043

Yersinia enterocolitica

NCTC 11174

NCTC, National Collection of Type Cultures; NCIMB, National Collection of Industrial and

Marine

Institute;ATCC, American Type Culture Collection.

Scottish Crops Research

Bacteria; SCRI,

PREPARATION OF SAMPLES FOR PCR

DNA from bacteria listed in Table 1 was extracted by the method of Heath et al. (1986).

Putative Listeria strains isolated from various sources were used as crude cell lysates in the PCR. Lysates were obtained by resuspending

cells

in 50 pl of water and heating at 110°C for 5

min.

SYNTHESIS OF OLIGONUCLEOTIDE PRIMERS

Oligonucleotide primers were synthesized in the Biochemistry Department, University of Leices- ter on an Applied Biosystems 380B synthesizer using standard protocols. All reagents were sup- plied by Cruachem, Scotland. Sequences of the oligonucleotide primers used in this study are shown in Table 2.

DNA

AMPLIFICATION BY PCR

Polymerase chain reaction was performed on DNA extracts or crude cell lysates essentially as described by Saiki et al. (1988). Reactions were carried out in a final volume of 50 pl, containing 5 pl 10 x PCR buffer (100 mmol/l Tris pH 8.3, 500 mmol/l KCI, 15 mmol/l MgCI,, 0.1% gelatin; all reagents from Sigma), 5 p1 dNTP

mix (2 mmol/l each of dGTP, dTTP, dATP and

dCTP from Pharmacia Ultrapure dNTP set), 3 pl each appropriate primer (0.1 mg/ml), 0.5 pl AmpliTaq DNA polymerase (Perkin Elmer Cetus) plus 1 pl of appropriate DNA prep- aration or crude cell lysate. Tubes were overlaid with paraffin oil prior to thermal cycling. Thermal cycling was carried out using a Perkin

Table 2. Sequences of oligonucleotide primers

 

Derived

from

Sequence

Primer

+ or - strand

(5'-3')

Reference

u1

+

CAGCMGCCGCGGTAATWC

Lane et al. (1985)

u2

-

CCGTCAATTCMTTTRAGTTT

Lane et al. (1985)

LI1

-

CTCCATAAAGGTGACCCT

Stackenbrandt &

LM 1

+

CCTAAGACGCCAATCGAA

Curiale (1988) Mengaud et al. (1988)

LM2

-

AAGCGCTTGCAACTGCTC

Mengaud et al. (1988)

M denotes A or C; W denotes A or T; R denotes A or G.

160

P. M. Border et al.

Elmer Cetus DNA Thermal Cycler. Denatur- ation, annealing and extension temperatures were 95", 50" and 72°C respectively. Denatur- ation temperature was maintained for 4 min on the first cycle and for 1 min for the subsequent 29 cycles. Annealing and extension times were maintained at 2 s and 1 min respectively throughout the entire 30 cycles. After 30 cycles an additional extension period of 8 min was maintained. Tubes were then held at 4°C until the PCR products were analysed. Analysis of PCR products was by horizontal agarose gel electrophoresis using 1.5% agarose (SeaKem ME) gels run in Tris (50 mmol/l Sigma), borate (50 mmol/l Sigma), EDTA (2.5 mmol/l BDH) buffer containing 0.1 mg/ml ethidium bromide. Gels were visualized and photographed under U.V. light on a UVP inc. transilluminator.

Results and Discussion

Figure 1 shows the PCR products obtained when total genomic DNA from various bacteria was subjected to PCR using a combination of all five primers described in Table 2. The bac- teria were selected to include the type strains of all known Listeria sp. plus a wide variety of other bacterial species including some that are phylogenetically closely related to Listeria (e.g.

Brochothrix

thermosphacta,

Bacillus

cereus).

The primer sequences were designed to have dif- ferent specificities. The U1 and U2 primers were

based on 16s rRNA sequences that are essen- tially conserved throughout all bacteria (Lane et al. 1985).Hence these primers are universal and should yield a product of 408 bp derived from the 16s rRNA genes that are present in genomic DNA from any bacterial source. Reference to Fig. 1 shows that a PCR product of 408 bp was observed for each of the 26 bacterial species used in this study. The LI1 primer was also based on 16s rRNA sequence data (Stackenbrandt & Curiale 1988). When used in conjunction with the U1 primer, the LI1 primer should yield a PCR product of 938 bp. The LI1 primer was designed to be spe- cific to members of the genus Listeria as its

sequence is complementary to a 16s rRNA sequence that is highly conserved in all Listeria sp. so far studied but variable in other bacteria (Stackenbrandt & Curiale 1988).Figure 1 shows that the LI1 probe is specific to Listeria sp. since only these samples show the characteristic band at 938 bp. In contrast, none of the other bac- terial species tested showed this band amongst their PCR products. As expected Jonesia deni-

trijicans, formerly called

until its re-classification (Rocourt et al. 1987),

does not show a band corresponding to the LIl/Ul product at 938 bp. The LM1 and LM2 primers are based on sequence data of the listeriolysin 0 gene published by Mengaud et al. (1988), and are hence designed to be specific to those bacteria that carry this gene. The results in Fig. 1 show

Listeria

denitrijicans

this gene. The results in Fig. 1 show Listeria denitrijicans Fig. 1. Products obtained when genomic

Fig. 1. Products obtained when genomic DNA from various bacteria were subjected to PCR using a combination of U1, U2, LI1, LM1 and LM2 primers. Lanes 1 and 28, Hae 111 digested pUC 9 standards(587 bp, 458/434 bp

and 298 bp); lane 2, Listeria grayi; lane 3, L. innocua; lane 4, L. iuanouii; lane 5, L. monocytogenes NCTC 10357; lane 6, L. monocytogenes NCTC 11994; lane 7, L. murrayi; lane 8, L. seeligeri; lane 9, L. welshimeri; lane 10, Jonesia denitrijicans; lane 11, Aeromonas hydrophila; lane 12, Bacillus cereus; lane 13, Erochothrix thermosphacta; lane 14, Erwinia carotovora; lane 15, Escherichia coli; lane 16, Gemella haemolysans; lane 17, Klebsiella pneumon- iue; lane 18, Lactobacillus amylophilus; lane 19, Lact. helueticus; lane 20, Lact. plantarum; lane 21, Sulmonella typhimurium; lane 22, Serratia marcescens; lane 23, Staphylococcus aureus; lane 24, Enterococcus faecalis; lane 25, Lactococcus lactis subsp. luctis; lane 26, Veillonella criceti; lane 27, Yersinza enterocolitica.

Detection of Listeria using PCR

161

that the two L. monocytogenes strains tested were the only bacteria that exhibited the charac- teristic LMi/LM2 product at 702 bp. This finding suggests that the listeriolysin 0 gene may be unique to L. monocytogenes and that the LMI/LM2 primer combination is therefore spe- cific to this organism. Figure 2 shows the PCR products obtained when various cell lysates from bacteria isolated from a number of sources were subjected to PCR using a combination of all five primers shown in Table 2. Cell lysates prepared from cultures of L. innocua (Fig. 2, lanes 8, 9 and 10) and L. monocytogenes (Fig. 2, NCTC 11994, lanes 11, 12 and 13 and NCTC 10357, lane 18) were also included in this experiment. All iso- lates tested showed the characteristic U1/U2 PCR product at 408 bp, clearly demonstrating the usefulness of this primer combination as a positive control for the efficiency of the PCR. The cell lysates prepared from known Listeria sp. gave the same PCR products as the corre- sponding DNA extracts in the first experiment (Fig. 1). In addition to this, several of the unknown isolates showed the characteristic

LIl/U1 band to 938 bp (Fig. 2, lanes 3, 5, 14, 16, 17, 23 and 30) indicating the presence of Listeria DNA in the cell lysate. One isolate (Fig. 2, lane 2) also showed the characteristic LMl/LM2 product at 702 bp indicating the presence of L. monocytogenes in the original sample. Sub- sequent biochemical tests confirmed the results predicted by the PCR method. The results pre- sented in this paper suggest that it may be pos- sible to devise a rapid method for the detection of Listeria sp. in foods and other products based on the PCR. The primer sequences described permit the detection of all members of the genus Listeria so far investigated, as well as the spe- cific detection of L. monocytogenes. We have demonstrated that the method works not only on bacterial DNA extracts but also on crude cell lysates which may be rapidly prepared from culture plates. Although no attempt has been made to determine the detection limits of the system, the sensitivity of PCR-based methods is such that it may prove possible to develop the method into one that will detect Listeria sp. and L. monocytogenes directly in enrichment broths. Such a method would have obvious advantages

broths. Such a method would have obvious advantages Fig. 2. Products obtained when crude cell lysates

Fig. 2. Products obtained when crude cell lysates prepared from bacteria isolated from a variety of food samples were subjected to PCR using a combination of U1, U2, LI1, LM1 and LM2 primers. Lanes 1, 20,21 and 32, Hae I11 digested pUC 9 standard (587 bp, 458/434 bp, 298 bp, 267 bp, 257 bp and 174 bp); lanes 2,4,5,6,7 and 17, unknown colonies isolated from wheat; lane 3, unknown colony isolated from cream; lanes 8, 9 and 10, colonies of Listeriu innorua; lanes 1 I, 12 and 13, colonies of L. monocytogenes NCTC 11994; lanes 14, 15 and 16, unknown colonies isolated from potatoes; lane 18, colony of L. rnonocytogenes NCTC 10357; lane 19, unknown colony isolated from cucumber; lanes 22, 23, 26 and 27, unknown colonies isolated from lettuce; lanes 24, 30 and 31, unknown colonies isolated from mushrooms;lanes 25,28 and 29, unknown colonies isolated from radish.

162

P. M. Border et al.

over existing methods used for the detection of Listeria in foods.

The authors would like to thank John Keyte of the University of Leicester, Department of Bio- chemistry, for synthesizing the oligonucleotides used in this work.

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