Internet Journal of Food Safety, Vol.9, 2007, p.

56-62
Copyright© 2007, Food Safety Information Publishing

A Multiplex PCR for Detection of Virulence Associated Genes in

Listeria monocytogenes
Rawool, D. B.1*, Malik, S. V. S.1, Barbuddhe, S. B.2, Shakuntala, I.1 and Aurora, R.3
1
Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar 243 122 India
2
ICAR Research Complex for Goa, Ela, Old Goa 403 402, India
3
Dayalbagh Educational Institute (Deemed University), Dayalbagh, Agra 282005, India.

Abstract: Listeriosis is an important food-borne Introduction

infection caused by Listeria monocytogenes. A
multiplex PCR assay was developed for detection of Listeriosis is an important food-borne infection caused by
four virulence-associated genes of L. monocytogenes Listeria monocytogenes. L. monocytogenes has been
namely, plcA, hlyA, actA and iap. The method was used isolated from various foodstuffs including milk (Barbuddhe
to detect L. monocytogenes in milk samples with and et al., 2002). Identification of listeriae from samples
without enrichment and simultaneously comparing it contaminated with multiple species relies on selective
with conventional cultural method. The assay could enrichments and subsequent biochemical analyses. It is
detect as low as 15 cells after enrichment. Later, the laborious and requires at least five days for confirmation. In
multiplex PCR was successfully employed to detect vitro amplification of specific DNA sequences by PCR
virulence-associated genes in L monocytogenes isolated allows direct detection and identification of the pathogen
from various sources. (Herman et al. 1995). Multiple key virulence factors such as
hemolysin (hlyA), phosphatidylinositol phospholipase C
Key words: Milk, Listeria monocytogenes; multiplex (plcA), actin polymerization protein (actA) and invasive
PCR; virulence –associated genes associated protein (iap) are important in L. monocytogenes
pathogenesis (Furrer et al., 1991, Portnoy et al., 1992).
Therefore, detection of just one virulence-associated gene
by PCR is not always sufficient to identify L.
monocytogenes (Nishibori et al., 1995). In addition, it is
*
Corresponding author. mailing address: Division of plausible that some L. monocytogenes strain may lack one
Veterinary Public Health, Indian Veterinary Research or more virulence determinants because of spontaneous
Institute, Izatnagar 243 122 India, E-mail: mutations (Cooray et al., 1994). Therefore, simultaneous
drrawool@rediffmail.com detection of virulent genes in a single step will be desirable
as it reduces time, labour and it will be useful in a large
scale survey for detecting virulent strain of Listeria.
Direct detection of pathogens from foods by PCR may be
problematic due to the presence of a PCR-inhibitory factor
in the food (Fluit et al., 1993). Various sample preparation
methods have been developed to remove or to reduce the
effects of PCR inhibitors in food stuffs (Lantz et al. 1994).
A pre-enrichment method (Niederhauser et al., 1992) has
been reported to overcome the problem of inhibitors. In the
present study, we tried to find a procedure enabling the
detection of individual virulence-associated genes of L.
monocytogenes by PCR in milk samples with and without
enrichment and simultaneously comparing it with
conventional cultural method. Later, we developed a
multiplex PCR for simultaneous detection of four virulence-
associated genes of L. monocytogenes namely, plcA, hlyA,
actA and iap and employed for PCR analysis of virulence-

56
associated genes in L. monocytogenes isolated from various mM KCl, 15 mM MgCl2 and 0.01% gelatin), 0.2 mM
sources. dNTP mix, 2 mM MgCl2 and 10 µM of a primer set
containing forward and reverse primers (a final
concentration of 0.1 µM of each primer), 1 unit of Taq
Materials and Methods
DNA polymerase, 5 µl of cell lysate and sterilized milliQ
water to make up the reaction volume. The cycling
Bacteria. The strains of L monocytogenes 4b (MTCC 1143),
conditions for PCR included an initial denaturation of DNA
Staphylococcus aureus (MTCC 1144), Rhodococcus equi
at 95°C for 2 min followed by 35 cycles each of 15 s
(MTCC 1135), Streptococcus faecalis (MTCC 439),
denaturation at 95°C, 30 s annealing at 60°C and 1 min 30 s
Bacillus cereus (MTCC 1272), Escherichia coli (MTCC
extension at 72°C, followed by a final extension of 10 min
443), Aeromonas hydrophila (MTCC 646) used in the study
at 72°C and hold at 4°C. All the four set of primers for
were obtained from Institute of Microbial Technology,
virulence-associated genes were amplified under the similar
Chandigarh, India. The reference strains of Listeria namely,
PCR conditions and amplification cycles. The resultant
L. monocytogenes 4b (NCTC 11994), L. monocytogenes
PCR products were further analyzed by agarose gel
1/2a (NCTC 7973), L. monocytogenes 1/2b (NCTC 10887),
electrophoresis (1.5%; low melting temperature agarose L);
L.ivanovii (NCTC 11846), L. innocua (NCTC 11288), L.
stained with ethidium bromide and visualized by a UV
seeligeri (NCTC 11856), L. grayi (NCTC 10812), L.
trans-illuminator (UVP Gel Seq Software, England).
welshimeri (NCTC 11857) were kindly provided by Prof.
K.L. Morgan, University of Liverpool, U.K. The strains of
Sensitivity of PCR. The standardized PCR was assessed
Salmonella (1117) and Vibrio cholerae (0139) were
for its sensitivity. Briefly, L. monocytogenes 4b (MTCC
procured from Division of Veterinary Public Health, Indian
1143) culture grown overnight in BHI broth was
Veterinary Research Institute, India. The strains were tested
centrifuged at 6000 rpm for 10 min. and the pellet obtained
for their purity besides morphological and biochemical
was washed once with phosphate buffered saline (PBS), pH
characteristics.
7.2. The concentration of cells was adjusted to 3 x 108/ml
Polymerase chain reaction. For standardization of PCR a using McFarland’s nephelometric tube No.1 and 10-fold
virulent strain of L. monocytogenes MTCC 1143 was grown serial dilutions were made upto 3 cell/ml of PBS. The
overnight in Brain Heart Infusion broth (BHI) at 37°C. The template DNA was prepared by subjecting 1ml aliquot of
DNA was extracted as per the methodology of Makino et each dilution to snap-chill method as described above.
al., (1995). The obtained culture (approximately 1 ml) was
centrifuged in a microcentrifuge (Sigma, USA) at 6000 rpm Specificity of PCR. The specificity of the standardized
for 10 min. The recovered pellet was resuspended in 100 µl PCR was tested by screening the standard strains of L.
of sterilized DNAse and RNAse-free milliQ water monocytogenes, Listeria species as well as some other
(Millipore, USA), heated in a boiling water bath for 10 min. commonly prevalent and cross-reacting bacterial species
and then snap chilled in crushed ice. The obtained lysate (Table 1) with the primers used in this study. The DNA
(5µl) was used as a DNA template in PCR reaction mixture. template preparation from the test organisms and other PCR
Bacterial DNA was also extracted employing DNA conditions were similar to those described earlier.
extraction kit (Mini Prep, Sigma).
PCR was standardized for the detection of individual Multiplex PCR. The multiplex PCR was standardized for
virulence associated genes namely, plcA, hlyA, actA and iap detecting four virulence associated genes of L.
using primer pairs of 5'-CTG CTT GAG CGT TCA TGT monocytogenes namely, plcA, hlyA, actA and iap in a single
CTC ATC CCC C-3' and 5'-ATG GGT TTC ACT CTC reaction tube containing all the four primer sets for these
CTT CTA C-3' specific for plcA (Notermans et al. 1991), genes. The DNA template preparation from pathogenic
5'-GCA GTT GCA AGC GCT TGG AGT GAA-3’ and 5’- strain of L. monocytogenes, PCR reaction and agarose gel
GCA ACG TAT CCT CCA GAG TGA TCG-3' for hlyA electrophoresis of the PCR products were done as per the
(Paziak-Domanska et al. 1999), 5'-CGC CGC GGA AAT methods employed for the detection of virulence genes of L.
TAA AAA AAG A-3' and 5'- ACG AAG GAA CCG GGC monocytogenes described earlier. The standardized PCR
TGC TAG - 3' for actA (Suarez and Vazquez-Boland 2001) protocol for 50 µl reaction mixture included 10 X PCR
and 5'-ACA AGC TGC ACC TGT TGC AG-3' and 5'-TGA buffer (100mM Tris-HCl buffer, pH 8.3 containing 500 mM
CAG CGT GTG TAG TAG CA-3' for iap (Furrer et al. KCl, 15 mM MgCl2 and 0.01% gelatin), 1 mM dNTP mix,
1991) of L. monocytogenes by optimizing the different 6 mM MgCl2 and 10µM of four primer sets (containing
conditions that affect the sensitivity and specificity of the forward and reverse primers a final concentration of 0.1 µM
reaction. of each primer), 4 units of Taq DNA polymerase, 5 µl of
Based on optimization trials, the standardized PCR cell lysate and sterilized milliQ water to make up the
protocol for 50 µl reaction mixture included 10 X PCR reaction volume. The cycling conditions for multiplex PCR
buffer (100 mM Tris-HCl buffer, pH 8.3 containing 500

57
Table 1. Standard bacterial strains used to check the specificity of PCR
Virulence genes detected
Description of the organism
hlyA plcA actA iap
L. monocytogenes 4b (MTCC 1143) + + + +
L. monocytogenes 4b (NCTC 11994) + + + +
L. monocytogenes 1/2a (NCTC 7973) + + + +
L. monocytogenes 1/2b (NCTC 10887) + + + +
L. ivanovii (NCTC 11846) - + + -
L. welshimeri (NCTC 11857) - - - -
L. innocua (NCTC 11288) - - - -
L. grayi (NCTC 10812) - - - -
L. seeligeri (NCTC 11856) + - - -
Escherichia coli (0157H7) - - - -
Vibrio cholerae (0139) - - - -
Salmonella (1117) - - - -
Bacillus cereus (MTCC 1272) - - - -
Streptococcus faecalis (MTCC 439) - - - -
Escherichia coli (MTCC 443) - - - -
Staphylococcus aureus (MTCC 1145) - - - -
Rhodococcus equi (MTCC 1135) - - - -
Aeromonas hydrophila (MTCC 646) - - - -

were similar as described earlier for the detection of spiked milk samples in set 1 were centrifuged at 12000 rpm
individual virulence gene of L. monocytogenes. for 10 min. The pellet obtained was washed once with PBS
(1ml) and recovered by centrifugation at 12000 rpm for 10
Artificially contaminated milk samples. Using the above- min. The washed pellet was dissolved in sterilized milliQ
mentioned PCR setting we tried to detect L. monocytogenes water (100µl), subjected to vigorous heating in boiling
suspended at serial concentrations ranging from 3 x 10 to 3 water bath for 10 minutes and then snap-chilled in crushed
× 108 cells ml-1 in commercially available pasteurized cow ice for 2 min. The cell lysate was centrifuged at 3000 rpm
milk. for 10 min and the supernatant was used as DNA template
The pasteurized milk samples those turned out to be for detection of the test strain by PCR. Simultaneously, 1
culturally negative for Listeria were pooled and stored at – ml of each dilution of the spiked samples in set 2 was
20˚C till used for experimental work. Aliquots (3 ml) drawn spreaded on Dominguez-Rodriguez isolation agar
from the pooled milk sample were spiked with standard (Dominguez-Rodriguez et al. 1984) and incubated at 37°C
strain of the pathogen and processed for (with and without for 24 h. Similar procedure was adopted for detection of L.
enrichment) detection of the pathogen by cultural method monocytogenes suspended in milk after enrichment in
and PCR. Briefly, milk aliquots (3 ml) drawn from the UVM-1 by PCR as well as cultural method.
pooled milk sample were inoculated with the different
concentration of test strain ranging from 30 to 3 x 108 Listeria strains. Twenty strains of L. monocytogenes
cells/ml. Each dilution of the sample was distributed in isolated from milk (10), buffaloes with reproductive
three test tubes, 1ml/test tube designated as set 1, set 2 and disorders (5) and fresh water fishes (5) were analysed for
set 3. Accordingly 9 test tubes including negative control the presence of virulence associated genes employing the
were included in each set. Of these, two sets not subjected multiplex PCR.
to enrichment (set1 and 2) were analyzed immediately for
the detection of the test strain, i.e. one (set1) by the PCR
targeted at virulence-associated genes of L. monocytogenes Results and Discussion
and another (set 2) by cultural method. The remaining set
(set 3) was enriched in University of Vermont medium 1 Rapid isolation and confirmation methods for L.
(UVM-1) in the ratio of 1: 10 (i.e. 1 part of milk and 9 parts monocytogenes in foods are still being sought (Beumer and
of UVM-1 broth) and incubated overnight at 37°C before Hazeleger, 2003). But there are certain strains of L.
attempting detection of the test strain by PCR as well as monocytogenes which behave phenotypically quite typical
cultural method. Aliquots (1 ml) from each dilution of the and inconspicuous but are non-pathogenic (Hof and

58
Rocourt, 1992). Thus, to address the pathogenic potential of the DNA marker (Fig. 1). These findings commensurate
Listeria isolates, in vivo methods namely, chick embryo and with the published work for detection of hlyA gene (Paziak-
mouse inoculation tests remain the most reliable and Domanska et al., 1999), plcA and prfA genes (Notermans et
mandatory approach in order to link these isolates with the al., 1991b), iap gene (Furrer et al., 1991) and actA gene
cases of listeriosis. However, the in vivo methods remain (Suarez and Vazquez-Boland, 2001) with respective sets of
objectionable from ethical point of view and need skilled primers giving no cross-reactions with other bacteria.
personnel to perform. In view of this situation, it has been
suggested that diagnosis of pathogenic Listeria spp. and
listeric infection should ideally be based on virulence
markers (Notermans et al., 1991). Moreover, the
importance of PCR has been investigated for detection of L.
monocytogenes from foods (Gouws and Liedemann, 2005).
Thus, in the present study we first attempted to standardize
a PCR protocol, which could detect individual virulent
associated genes (plcA, hlyA, actA and iap) of L.
monocytogenes in milk samples with and without
enrichment and simultaneously comparing it with
conventional cultural method. Later, we developed a
multiplex PCR for simultaneous detection of all the
virulence-associated genes of L. monocytogenes under
study for rapid detection of virulent strains of L.
monocytogenes.

Figure 2. Multiplex PCR of four virulence associated

genes for standard Listeria monocytogenes (MTCC
1143). M: DNA Marker (150 to 2000 bp), Lane 1:
Negative control, Lane 2: Amplified products of four
genes (plcA, actA, hlyA and iap).

Each of the primer was found to be specific to the target

gene as it specifically amplified the PCR product of that
gene. All the four genes were detected in standard strains of
L. monocytogenes, two genes namely, plcA, and actA in the
standard strain of L. ivanovii and the hlyA gene in the
standard strain of L. seeligeri whereas, none of the genes
was detected in the cultures of the remaining Listeria spp.
and other bacterial species cultures (Table 1). The
amplification of plcA gene in L. ivanovii can be explained
based on the reports that plcA and plcB genes are present in
L. monocytogenes and L. ivanovii (Vazquez-Boland et al.,
Figure 1. Virulence associated genes of standard Listeria 2001). The actA gene from L. ivanovii, i-actA, encodes a
monocytogenes (MTCC 1143). M: DNA Marker (150 to protein larger than actA from L. monocytogenes. The
2000 bp), Lane 1: plcA gene, Lane 2: actA gene, Lane 3: similarity between the two proteins is also limited (34%
hlyA gene, Lane 4: iap gene, Lane 5: Negative control. identity), however, actA and i-actA have a similar overall
structure (Gerstel et al., 1996). The amplification of
haemolysin gene (hly A) in L. seeligeri genome could be
In the present study the primer sets for all the four genes explained on the basis of homology at both the protein (86-
allowed amplification of 1484 bp (plcA), 839 bp (actA), 456 91%) and in the nucleotide (76-78%) of gene sequence of
bp (hlyA) and 131 bp (iap) PCR products, respectively, each LLO, ILO and seeligerilysin O (LSO) (Mengaud et al.,
represented by a single band in the corresponding region of 1988; Haas et al., 1992). However, our amplification

59
system does not yield DNA fragments of hlyA gene in cross-reactions with non-pathogenic and related strains can
standard strain of L. ivanovii, which can be explained on the potentially improve upon current approaches.
basis of report that the genetic information for haemolysin In the present study L. monocytogenes strains isolated
production in L. ivanovii could be divergent from L. from various sources were analyzed for the presence of
monocytogenes hemolysin gene despite high homology in virulence-associated genes employing the multiplex PCR.
partial amino acid sequence analysis (Kreft et al., 1989). Different combinations of genes were detected in different
Enrichment of food samples prior to PCR analysis isolates (data not shown). The choice of the target gene is of
overcomes most of the problems and has been utmost importance for detection of virulent strains of L.
recommended by several workers (Wang et al., 1992, Boer monocytogenes by PCR. The use of primers specific for
and Beumer 1999, Olsen 2000). In the present study, the plcA, hlyA, actA and iap seems to be reasonable and unique
sensitivity of PCR was compared for the detection of the to pathogenic Listeria (Furrer et al., 1991, Notermans et al.
pathogen in spiked milk samples before and after their 1991, Paziak-Domanska et al., 1999, Suarez and Vazquez-
enrichment in UVM-1 at 37°C for 24 h. It was possible to Boland 2001). However, it is plausible that some L.
detect as low as 3x106 cells/ml suspended in PBS by using monocytogenes strain may lack one or more virulence
each of the primer set. Compared with the detection limit determinants because of some mutation (Cooray et al.,
for bacteria suspended in PBS, the enrichment of milk 1994). Moreover, our previous studies have also revealed
samples in UVM-1 at 37°C for 24 h has increased the that using PCR assay for detection of single virulence
sensitivity of PCR from 3 x 106 cells/ml (before associated gene is neither sufficient to identify the L.
enrichment) to 3 x 103 cells/ml. With the PCR, setting monocytogenes isolates nor to reveal its true pathogenic
fewer than 15 cells could be detected after enrichment. potential as majority of L. monocytogenes isolates showed
Similar type of increase in the sensitivity of cultural method different gene profiles (Rawool et al., 2007; Shakuntala et
was also observed following 24 h enrichment from 3 x 103 al., 2006). Therefore, simultaneous detection of virulent
cells/ml (before enrichment) to 30 cells/ml of the milk genes namely plcA, hlyA, besides actA and iap genes in a
samples in UVM-1. The poor sensitivity of PCR as single step will be desirable as it reduces time, labour and
also it would be useful in a large scale survey aiming at
compared to culture method might be because only 5.0µl of
detection of virulent strain of Listeria.
the DNA was used in PCR reaction whereas 1ml of sample
(200 times more) was used for plating in cultural method.
Our results are in agreement with Cartyyvels et al., (1996)
Summary
who observed similar results of poor sensitivity of PCR for
detection of Mycobacterium tuberculosis. They explained
The multiplex PCR was successfully employed for the
that greater sensitivity of the culture method could be due to
detection of different genes in L. monocytogenes. The PCR
40-times greater sample volume used in the culture method.
method reported here could be completed in 6 h without
In the present study, the better detection of the pathogen
enrichment however since the detection level was poor
(as less as 3 x 103 cells/ml) by PCR in the enriched spiked without enrichment, the authors would like to recommend
samples compared to the detection level (3 x 106 cells/ml) the use of this multiplex PCR after enrichment for a large
in samples not subjected to enrichment in UVM-1 might be scale survey to detect of virulent strains of Listeria from
attributed to the significant increase in the number of the milk samples.
pathogen following enrichment of the samples compared to
the relatively lesser number of L. monocytogenes present in
the milk samples before enrichment. This approach is in
Acknowledgements
agreement with reported detection of L. monocytogenes by
the PCR in artificially contaminated milk samples (Cooray
We thank Director, Indian Veterinary Research Institute,
et al., 1994). Since only 5 µl of lysate was taken as DNA Izatnagar, India for providing facilities for the research
template into the PCR mixture, it indicated that fewer than work. Thanks are due to Dr. A.R. Datta, US FDA for
15 cells could be detected after enrichment. The present critically checking the manuscript
level of sensitivity in the detection appeared to be
applicable to the practical survey of milk and milk products
for L. monocytogenes. References
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