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O R I G I N A L A R T I C L E

Detection and quantification of Listeria monocytogenes by5-nuclease polymerase chain reaction targeting the actAgene

K. Oravcova1, E. Kaclkova1, K. Krascsenicsova1, D. Pangallo2, B. Brez na1, P. Siekel1 and T. Kuchta1

1 Department of Microbiology and Molecular Biology, Food Research Institute, Bratislava, Slovakia

2 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia

Introduction

Listeria monocytogenes is an important pathogenic bacter-

ium which is frequently found as a contaminant in meat

and milk products, vegetable salads and other ready-to-

eat food products (Farber and Peterkin 1991). A zero tol-

erance for L. monocytogenes in food products has been

applied for several years but the regulation in the EU has

been updated and recently, a quantitative limit of

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Materials and methods

Bacterial strains

Listeria monocytogenes and other bacterial strains listed in

Table 1 were obtained from culture collections or were

obtained from reference laboratories, details of which areavailable in our previous publications (Pangallo et al.

2001, 2002). Cultures were grown in brain heart infusion

broth (Merck, Darmstadt, Germany) overnight at 37C

with agitation. Bacterial concentration in decimally dilu-

ted culture samples was determined by plate-count tech-

nique on plates of brain heart infusion agar (Merck)

incubated at 37C for 24 h.

DNA extraction

DNA from bacteria was extracted by cell lysis using boil-

ing. A volume of 1 ml of the bacterial suspension was

centrifuged at 13 000 g, the sediment was then resuspend-

ed in 100 ll of 1x buffer supplied with HotStarTaq DNA

polymerase (Qiagen, Hilden, Germany), incubated at

95C for 25 min, then centrifuged at 13 000 g for 3 min

and finally the resulting supernatant containing DNA was

used as the PCR template (Abolmaaty et al. 1998).

End-point polymerase chain reaction

Each reaction sample (volume, 65 ll) contained

300 nmol l)1 of the primer LMrt3F-(5-caaagcgagaatgtg-

gctataaatga-3), 300 nmol l)1 of the primer LMrt3Rbis

(5-taatttccgctgcgctatccg-3) and 200 nmol l)1

of the

TaqMan probe listP (5-FAM-cctggatgacgacgctccacttg-

TAMRA-3; all from Qiagen Operon, Cologne, Germany),

500 lmol l)1 of each dNTP (Invitrogen, Carlsbad, CA,

USA), 2 U HotStarTaq DNA polymerase (Qiagen), 65 ll

of 10x concentrated PCR buffer supplied with the polym-

erase and 25 ll of the DNA sample. In addition, the reac-

tion mixture contained an internal amplification controlsystem (Applied Biosystems, Foster City, CA, USA; cat. no.

4308323). The concentration of Mg2+ was 45 mmol l)1.

Reactions were performed in TopYield 8-strips (Nunc, Ros-

kilde, Denmark) in a GeneAmp 9700 thermal cycler

(Applied Biosystems) using a programme consisting of the

initial denaturation of 15 min at 95C and 35 cycles (dena-

turation of 15 s at 94C, annealing and polymerization of

60 s at 60C). The amplified product was detected by fluor-

imetry directly in the microtubes in a Genios 96-well reader

(Tecan, Grodig bei Salzburg, Austria) equipped with excita-

tion filters optimal for FAM and JOE dyes, positivity

threshold being set to the fluorescence value of the no tem-

plate control + 2 SD (Kaclkovaet al. 2005). To determine

the exclusivity, amplified products were analysed by ag-

arose gel electrophoresis with ethidium bromide staining

and UV-transillumination, detecting a DNA fragment of

109 bp.

Real-time polymerase chain reaction

Reaction mixtures had the same composition as for the

end-point PCR, but the total volumes were reduced to

25 ll, the amounts of the template DNA and of the Taq

polymerase remained the same. Reactions were performed

in white low-profile eight-microtube strips and the fluor-escence was measured through optical caps. PCR was car-

ried out in a PTC-200 thermal cycler coupled to a

Chromo 4 continuous fluorescence detector (MJ

Research, Waltham, MA, USA) using the same thermal

programme as for the conventional PCR with the number

of cycles increased to 45. Kinetics of the fluorescence sig-

nals in channel 1 (FAM/Sybr) and channel 2 (VIC/JOE)

were recorded and the threshold cycle values were calcu-

lated using the internal instrument software with the

baseline subtraction option selected and the threshold set

manually to a fluorescence value of 002. To construct a

calibration line, averaged threshold cycle values were plot-

ted against the decadic logarithm of concentrations of a

series of decimally diluted cultures. For the qualitative

detection, a threshold cycle value of lower than 35 was

taken as an indicator of positivity.

Results

A PCR system suitable for the specific detection and quan-

tification of L. monocytogenes was developed. A sequence

Table 1 PCR results with Listeria monocytogenes and non-L. mono-

cytogenes strains

Species Number of strains PCR result

L. monocytogenes serovar 1/2a 13 +

L. monocytogenes serovar 1/2b 21 +

L. monocytogenes serovar 1/2c 3 +

L. monocytogenes serovar 4ab 2 +

L. monocytogenes serovar 4b 2 +

L. monocytogenes serovar 4d 1 +

L. monocytogenes R 4 +

Listeria innocua 9)

Listeria ivanovii 4 )

Listeria grayi 2 )

Listeria seeligeri 2 )

Listeria welshimeri 2 )

Enterococcus faecalis 3 )

Micrococcus luteus 1 )

Staphylococcus aureus 3 )

Staphylococcus saprophyticus 1 )

Salmonella Enteritidis 1 )

PCR for L. monocytogenes K. Oravcova et al.

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of the gene actA (GenBank Accession no. AF103807) was

chosen as a new target for L. monocytogenes identification

in real-time PCR. Comparison of 146 L. monocytogenes

strains using the Basic Local Alignment Search Tool

(BLAST; National Center for Biotechnology Information,

Bethesda, MD, USA) revealed conserved regions,

which were used for the primer design. Primers with atheoretical melting temperature of 60C as well as a

corresponding 5-nuclease (TaqMan) probe (5-FAM-

cttcaggatccgaccgaccagctatac-TAMRA-3) were designed

using the Primer Express software (Applied Biosystems).

Although the primers amplified the region of the target

gene in all L. monocytogenes strains in the conventional

PCR, 17 of 46 strains produced false negative results after

adding the probe and performing real-time PCR. To solve

this problem, the target fragment of the actA gene from

selected positive as well as false negative strains was

sequenced and polymorphisms at positions 9 and 10 of

the sequence targeted by the probe, and a substitution of

gt for tc, was identified. The extended BLAST search,

when 238 L. monocytogenes strains were compared, con-

firmed the occurrence of such polymorphisms in two

strains. A substitution of g for t at position 9 occurred in

further 77 strains and other polymorphisms in one or

two nucleotides occurred in further 16 strains. Based on

this more detailed comparison, new conserved regions

were selected and a new reverse primer as well as a new

probe was designed to target the conservative sequences.

The final combination of the primers LMrt3F,

LMrt3Rbis and the probe listP was tested by end-point

and real-time PCR with a panel of L. monocytogenes as

well as other bacterial strains. Inclusivity of this systemwas 100% with 46 strains of L. monocytogenes and exclu-

sivity was 100% with 28 non-L. monocytogenes strains

(Table 1).

The sensitivity of the qualitative detection of L. mono-

cytogenes was evaluated on the basis of the determination

of the detection probability. For this purpose, 12 repli-

cates of a decimal dilution series of a L. monocytogenes

NCTC 11994 culture were analysed by end-point as well

as by real-time PCR. A detection probability of 100% was

achieved at 104 cfu ml)1 after 35 cycles and at

102 cfu ml)1 after 45 cycles in both measurement modes

(data not shown).

The applicability of the developed real-time PCR sys-

tem to quantification was evaluated on the basis of the

analysis of decimally-diluted cultures of three L. monocy-

togenes strains (strain NCTC 11994 serovar 4b, strain

294 serovar 1/2b, strain 300 serovar 1/2b). For

decreasing concentrations of cultures, amplification curves

with proportionally increasing threshold cycle values were

recorded, with no significant difference between individ-

ual strains (Fig. 1). Threshold cycle values were plotted

against bacterial concentrations with practically identical

calibration lines being obtained. These were linear

(r2 0995) over the range from 102 to 109 cfu ml)1

(Fig. 2).

Figure 1 A cumulative record of a real-time 5 -nuclease PCR with

decimal dilutions of Listeria monocytogenes NCTC 11994, L. monocy-

togenes 294 (serovar 1/2b) and L. monocytogenes 300 (serovar 1/2b)

showing curves for 109 cfu ml)1 (9), 108 cfu ml)1 (8), 107 cfu ml)1

(7), 106 cfu ml)1 (6), 105 cfu ml)1 (5), 104 cfu ml)1 (4), 103 cfu ml)1

(3) and 102 cfu ml)1 (2); ranges of fluorescence values at individual

measurement points are depicted by short horizontal lines.

Figure 2 A cumulative calibration line (y )351x + 4808; r2

0999) of the real-time 5 -nuclease PCR with decimal dilutions of

Listeria monocytogenes NCTC 11994, L. monocytogenes 294 (serovar

1/2b) and L. monocytogenes 300 (serovar 1/2b); mean fluorescence

values standard error of the mean are presented.

K. Oravcova et al. PCR for L. monocytogenes

2005 The Authors

Journal compilation 2005 The Society for Applied Microbiology, Letters in Applied Microbiology 42 (2006) 1518 17

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The interference of related bacteria with the developed

real-time PCR system was investigated on the basis of the

analysis of a decimally diluted culture of L. monocyto-

genes NCTC 11994 with a background of L. innocua 79

(106 cfu ml)1) or Staphylococcus aureus CCM 3958

(106 cfu ml)1). Presence of these considerably high

amounts of competing bacteria had no effect on the calib-ration lines obtained (data not shown).

Discussion

Based on the determined analytical parameters, the devel-

oped method was suitable for the qualitative detection of

L. monocytogenes in food. It was specific for L. monocyto-

genes and appropriately sensitive to be connected to

enrichment, as the detection limit after the number of

cycles decreased to 35, as recommended for routine

microbiological analyses to avoid false positive artefacts

(Rijpens and Herman 2002), was 104 cfu ml)1 both for

the real-time and end-point versions. The latter technical

alternative employing fluorimetry in a 96-well reader was

included to suit laboratories that are not equipped with

real-time thermal cyclers.

Concerning quantitative applications, the presented

real-time 5-nuclease PCR proved to be a highly specific

and sensitive method. The method performed identically

with various L. monocytogenes strains and we assume that

it can be used for quantification of the entire L. monocy-

togenes species. The detection limit of 102 cfu ml)1 is sat-

isfactory for its connection to quantitative bacterial

separation techniques from food (Wolffs et al. 2004). This

detection limit as well as calibration line parameters wereequivalent to the previously published methods (Nogva

et al. 2000; Hein et al. 2001).

In comparison to current microbiological culture-

based methods for L. monocytogenes quantification, the

presented real-time PCR is considerably faster. While

several days are required to obtain results by methods

based upon the growth of typical colonies, real-time

PCR-based quantification of L. monocytogenes can be

completed in approximately 4 h. Applicability of the

method to direct quantification of L. monocytogenes in

food safety and technological hygiene is, however,

dependent on the development of quantitative methods

for the separation of bacterial cells that should be

adapted to various sample types (Benoit and Donahue

2003; Wolffs et al. 2004).

Acknowledgements

This research was performed in the framework of the Slo-

vakian State Programme of Research and Development,

project Food Quality and Safety.

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