Agric. Chem. Biotechnol. 47(2), 102-105 (2004)
PCR Detection of Listeria monocytogenes Using
Specific Virulence Factor Genes
Hyun-Joong Kim, Sang-Hoon Jung, Jong-Hyun Park1, Geun-Eog Ji2 and Hae-Yeong Kim*
Department of Food Biotechnology, Kyung Hee University, Suwon 449-701, Korea
1Department of Food and Bioengineering, Kyungwon University, Songnam 461-701, Korea
2Department of Food Science and Nutrition, Seoul National University, Seoul 151-742, Korea
Received February 23, 2004; Accepted April 7, 2004
To investigate the novel specific genes of Listeria monocytogenes, six genes related with virulence factor
of L. monocytogenes were selected from database. Primer sets were constructed from the selected six
genes to evaluate the specificity of L. monocytogenes with other Listeria species. The primers were tested
with 12 strains of Listeria species: L. monocytogenes (7 strains), L. welshimeri, L. innocua, L. seeligeri, L.
ivanovii, and L. grayi. All primer sets showed specificity to L. monocytogenes but not with other Listeria
species. These genes could be used as target genes for the detection of L. monocytogenes.
Key words: Listeria monocytogenes, polymerase chain reaction (PCR), food-borne pathogen, detection,
virulence factor gene.
Listeria monocytogenes causes severe infections in humans,
including neonatal listeriosis, endocarditis, and meningitis,
with a fatality rate as high as 20 to 40%.1) Therefore, interest in
developing a fast, economical, and specific tests for the
detection of L. monocytogenes in various types of food has
been increasing. However, one of the major problems involved
in L. monocytogenes detection is the inability to distinguish L.
monocytogenes from other non-pathogenic Listeria species,
particularly L. innocua which may predominate in food
samples. Conventional methods for detecting L. monocytogenes
involve multiple selective enrichment steps followed by
biochemical and serotyping. However, these methods are
time-consuming and generally require more than 2 days.2)
The advantages of polymerase chain reaction (PCR) with
respects to its specificity, reliability, and rapidity helps in the
detection of food-borne pathogens. Detection methods of
Listeria monocytogenes using PCR have been reported using
one or two target genes including hly (listeriolysin O),3-7) iap
(60-kDa secreted invasion-associated protein p60),8-10) inlA
(internalin A),11,12) and inlB (internalin B).12) However, this
method of detection of Listeria monocytogenes using one
target gene has the problems of specificity. The sequence
analyses by using hly and iap as target gene of L.
monocytogenes, showed strain-specific nucleotide differences
among L. monocytogenes strains.13) These problems can be
addressed using several target genes for PCR detection.
Analysis of PCR products can offer more precise results in the
detection of L. monocytogenes. Therefore, selection of
specific genes of L. monocytogenes among Listeria species is
needed for PCR detection. The sequences of virulence factor
genes such as prfA, plcA, hly, mpl, actA, and plcB in L.
monocytogenes have been reported.14) However, as such there
are no reports regarding the specificity of these genes in
In this study, specific genes of L. monocytogenes were
selected to find unique genes for novel target of PCR. Primers
from selected genes were prepared and evaluated in PCR
assays for the detecting L. monocytogenes with genomic DNA
extracted from pure cultured Listeria species.
Materials and Methods
Korean Collection for Type Cultures (KCTC) and Korean
Culture Center of Microorganisms (KCCM). Seven strains of L.
monocytogenes and 5 of non-pathogenic Listeria species were
collected as outlined below: L. monocytogenes (ATCC 19111),
L. monocytogenes (ATCC 19115), L. monocytogenes (ATCC
19114), L. monocytogenes (ATCC 7644), L. monocytogenes
(ATCC 15313), L. monocytogenes (ATCC 19113), L.
monocytogenes (ATCC 19118), L. welshimeri (ATCC 35897),
L. innocua (ATCC 33090), L. seeligeri (ATCC 35967), L.
ivanovii (ATCC 19119), and L. grayi (ATCC 25401). Listeria
strains were grown in brain heart infusion (BHI) broth medium
(Difco Laboratories) at 37oC for 20 h.
Preparation of genomic DNA.
was harvested in a microtube, and genomic DNA was
extracted using genomic DNA isolation kit (Nucleogen,
Korea) according to the manufacturer's manual. DNA
concentration was measured by UV-spectrophotometer
Listeria species were purchased from
Each grown bacterium
Phone: +82-31-201-2660; Fax: +82-31-204-8116
Abbreviations: PCR, polymerase chain reaction.
PCR Detection of Listeria monocytogenes
(Model 220s, Hitachi, Japan) at 260 nm. The extracted DNAs
were then stored at 4oC.
Selection of target genes.
monocytogenes and target gene sequences were obtained from
National Center for Biotechnology Information (NCBI, http://
www.ncbi.nlm.nih.gov). The target genes, which are related
with virulence factor of L. monocytogenes, were selected
based on the previous reports.14-17) The DNA sequences of
virulence factor genes were compared using BLAST program
(Basic Local Alignment Search Tool) with nr database of
NCBI. Six genes showed low homology results with nr
database were selected as target genes.
Oligonucleotide primers were designed from the
selected target genes. Primer information is listed in Table 1.
Mixture of PCR amplification is as follows: 2.5 µl PCR
buffer, 1 µl each primers (10 µM), 2 µl dNTP (2.5 mM), 1 µl
DNA template (25 ng/µl), 0.5 Unit EX Taq DNA Polymerase
(TaKaRa, Japan), and distilled water added to make final
volume of 25 µl. PCR mixtures were heated to 94oC for 4 min
and then amplified for 35 cycles, each consisting of 94oC for
45 s followed by annealing of each primer set of as Table 1 for
1 min, and 72oC for 1 min 30 s. A 5-min final extension at
72oC was followed by holding at 4oC using Thermal Cycler
(Model PC 808, ASTEC, Japan). Amplified products were
electrophoresed on 1% agarose gel in 0.5X TAE buffer,
stained with ethidium bromide, and photographed with digital
camera (Model DC120, Kodak, Japan) under UV-irradiation.
DNA sequencing of PCR products
were executed by GreenGene BioTech Inc. (Yongin, Korea).
PCR products of L. monocytogenes were extracted from
agarose gel using gel extraction kit (Qiagen, U.S.A.) and
sequenced by ABI PRISM 377 model (Perkin Elmer, USA).
Genomic sequences of L.
Results and Discussion
Genome sequencing of L. monocytogenes (2.9 Mb) was
carried out and L. monocytogenes strain-specific 270 genes
have been reported in comparison with genomics of Listeria
species.14) Among these candidate genes, the target genes
related with virulence factor were selected for preparing
specific probes of Listeria monocytogenes. From among 6
genes a shown in Table 1, the prfA, hly, actA, and plcB genes
were clustered on a 10-kb region of the chromosome which is
known as virulence gene cluster and this region is present in L.
ivanovii and L. seeligeri15, 16) The other two genes related with
virulence factor were inlB and lmo2067, among which inlB
gene was known as a basic surface protein of L.
Table 1. Primers of virulence genes
ProductPrimerSequence (5' to 3')
specific phospholipase C
AF497176 45 1920
M24199 Listeriolysin O 501590
Similar to conjugated bile
Fig. 1. PCR amplification products using virulence primer
sets against Listeria species. M, Maker (100 bp ladder); lane 1,
L. monocytogenes (ATCC 19111); lane 2, L. monocytogenes
(ATCC 19115); lane 3, L. ivanovii (ATCC 19119); lane 4, L.
innocua (ATCC 33090); lane 5, L. grayi (ATCC 25401); lane 6,
L. welshimeri (ATCC 35897); lane 7, L. seeligeri (ATCC
35967); lane 8, L. monocytogenes (ATCC 19114); lane 9, L.
monocytogenes (ATCC 19118); lane 10, L. monocytogenes
(ATCC 19113); lane 11, L. monocytogenes (ATCC 7644); lane
12, L. monocytogenes (ATCC 15313).
104Hyun-Joong Kim et al.
monocytogenes which is required for entry into various host
cells.18) The lmo2067 gene was known as a protein, possibly
involved in the degradation of bile salts to survive in the
mammalian gut and was preceded by a PrfA-box, suggesting
that it may encode a virulence factor.14) Among the six
selected genes, hly and inlB genes have been used as target
genes for the detection of L. monocytogenes,3-7, 12) and the other
genes were firstly evaluated in this study as specific target
genes of L. monocytogenes. The specificity of these six genes
was evaluated in Listeria strains using PCR. Six primer sets
were found to be very specific in identifying L.
monocytogenes, and PCR products were not amplified with
Listeria species except L. monocytogenes. Amplified PCR
products were confirmed by sequence analysis and showed
the same sequences with each target gene sequence of L.
monocytogenes. Table 2 summarizes the results of PCR
specificity with different primer sets used. PCR product of
inlB primer showed a specific-amplified band at 1893 bp size
in L. monocytogenes and no PCR product with other Listeria
species at expected size. PCR of plcB gene showed a specific-
amplified band at 870 bp size in L. monocytogenes. PCR
results of other four primer sets showed specific amplified
fragments at expected size in L. monocytogenes but no band
with other Listeria species. prfA and actA genes were either
reported to be missing from some Listeria species or highly
divergent.17) All L. monocytogenes strains in this study showed
positive results with these primers (Table 2). The hly gene has
been the most widely used target gene of L. monocytogenes.
PCR results showed very specific to L. monocytogenes in this
study. To increase the specificity of L. monocytogenes using
PCR, the information of unique sequences in L.
monocytogenes was selected and confirmed by PCR and DNA
sequencing. In this study, PCR detection of L. monocytogenes
using 6 primer sets was performed using Listeria species and
the selected target genes showed specificity in L.
monocytogenes. Selected target genes are related with
virulence factor of L. monocytogenes in or out of virulence
gene cluster, and prfA, actA, plcB, and lmo2067 genes were
newly evaluated by PCR detection in this study. The
sequences of 4 novel target genes showed low homology by
BLAST program of NCBI, and firstly confirmed its specificity
of L. monocytogenes using PCR detection.
The lmo2067, as virulence factor is unknown to its function
in infection cycle of L. monocytogenes. These new target
genes showed possibility for specific detection of L.
monocytogenes. Also, selected genes in Listeria species might
be useful as probes of microarray for rapid, high-throughput
screening of food-borne pathogens.19)
Acknowledgments. This work was supported by a grant of
the Korea Health 21 R&D Project, Ministry of Health &
Welfare, Republic of Korea (02-PJ1-PG1-CH08-0002).
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