Letters in Applied Microbiology 1990, 11, 158-162 MFS/Q32

Detection of Listeria species and Listeria monocytogenes using

polymerase chain reaction

P . M . B O R D E RJ,. J . H O W A R D *G, . S . PLASTOW

& K . W . SICGENS
Dalgety PLC,
Group Research Laboratory, Station Road, Cambridge C B l 2 J N , U K

Received 1 June 1990 and accepted 7 June 1990

BORDER,P.M., HOWARD,J.J., PLASTOW, G.S. & S I G G E N SK.W.

, 1990.
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.

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 1990 for 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.
Curiale 1988). One of the main 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
soil samples (Steffan & Atlas 1988), Mycohac-
terium leprae in armadillo tissues (Hartskeerl et
al. 1989) and Aeromonas salmonicida in fish
(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
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 maintained on Tryptose Agar (Difco) and all
the PCR to amplify DNA sequences from L. other strains were maintained on Nutrient Agar
monocytogenes and other Listeria sp. (Oxoid). Putative Listeria strains were isolated
from a variety of sources using the method of
Materials and Methods Maclean & Lee (1988).

BACTERIAL STRAINS P R E P A R A T I O N OF S A M P L E S F O R P C R

The bacterial strains used in this study are DNA from bacteria listed in Table 1 was
detailed in Table 1. All Listeria strains were extracted by the method of Heath et al. (1986).
Putative Listeria strains isolated from various
Table 1. Origin and identity of bacterial strains used sources were used as crude cell lysates in the
Bacterium Source/reference PCR. Lysates were obtained by resuspending
cells in 50 pl of water and heating at 110C for 5
Listeria
grayi NCTC 10815 min.
innocua NCTC 11288
ivanovii NCTC 11846 S Y N T H E S I S OF O L I G O N U C L E O T I D E P R I M E R S
monacyt ogenes NCTC 10357
monorytogenes NCTC 11994 Oligonucleotide primers were synthesized in the
murrayi NCTC 10812 Biochemistry Department, University of Leices-
seeliyeri NCTC 11856 ter on an Applied Biosystems 380B synthesizer
welshimeri NCTC 11857
Jonesia denitrijicans NCTC 10816 using standard protocols. All reagents were sup-
Aeramonas hydrophila NCTC 8049 plied by Cruachem, Scotland. Sequences of the
Bacillus cereus NCTC 11143 oligonucleotide primers used in this study are
Brochothrix thermosphacta NCTC 10822 shown in Table 2.
Erwinia curotovora SCRI 193
Escherichia coli K12 Laboratory strain
NM522 D N A AMPLIFICATION BY PCR
Gemella haemolysans NCTC 10243
Klebsiella pneumoniae ATCC 15050 Polymerase chain reaction was performed on
Lactobacillus DNA extracts or crude cell lysates essentially as
amylophilus NCIMB 11546 described by Saiki et al. (1988). Reactions were
helveticus NCIMB 8652 carried out in a final volume of 50 pl, containing
p 1antarum NCIMB 11974
Salmonella typhimurium NCIMB 10248 5 pl 10 x PCR buffer (100 mmol/l Tris pH 8.3,
Serratia marcescens NCIMB 2303 500 mmol/l KCI, 15 mmol/l MgCI,, 0.1%
Staphylococcus aureus NCIMB 9518 gelatin; all reagents from Sigma), 5 p1 dNTP
Enterococcus faecalis ATCC 8043 mix (2 mmol/l each of dGTP, dTTP, dATP and
Lactococcus lactis
subsp. lactis NCIMB 6681
dCTP from Pharmacia Ultrapure dNTP set), 3
Veillunellu criceti ATCC 8043 pl each appropriate primer (0.1 mg/ml), 0.5 pl
Yersinia enterocolitica NCTC 11174 AmpliTaq DNA polymerase (Perkin Elmer
NCTC, National Collection of Type Cultures; Cetus) plus 1 pl of appropriate DNA prep-
NCIMB, National Collection of Industrial and aration or crude cell lysate. Tubes were overlaid
Marine Bacteria; SCRI, Scottish Crops Research with paraffin oil prior to thermal cycling.
Institute; ATCC, American Type Culture Collection. 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 &
Curiale (1988)
LM 1 + CCTAAGACGCCAATCGAA 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 72C 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 4C 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 Listeria denitrijicans
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

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 P C R
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
161
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

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 MCCLEAN,D. & LEE, W.H. 1988 Development of
Listeria in foods. USDA/FSIS method for the isolation of Listeria
rnonocytogenes from raw meat and poultry. Journal
of the Association of Analytical Chemistry 71, 660-
The authors would like to thank John Keyte of 664.
the University of Leicester, Department of Bio- MENGAUD, J., VICENTE, M.F., CHENEVERT, J., PEREIRA,
chemistry, for synthesizing the oligonucleotides J.M., GEOFFREY, C., GICQUEL-SANZEY, B., BAQUERO,
F., PEREZ-DIAZ,J.C.& COSART, P. 1988 Expres-
used in this work. sion in Escherichia coli and sequence analysis of the
listeriolysin 0 determinant of Listeria mono-
cytogenes. Infection and Immunity 56, 766772.
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