Optimization of a Real Time PCR based method for the detection of Listeria
monocytogenes in pork meat

Antonietta Gattuso, Monica Virginia Gianfranceschi, Michele Sonnessa,

Elisabetta Delibato, Massimo Marchesan, Marta Hernandez, Dario De
Medici, David Rodriguez-Lazaro

PII: S0168-1605(14)00179-2
DOI: doi: 10.1016/j.ijfoodmicro.2014.04.015
Reference: FOOD 6512

To appear in: International Journal of Food Microbiology

Received date: 4 March 2014

Revised date: 8 April 2014
Accepted date: 10 April 2014

Please cite this article as: Gattuso, Antonietta, Gianfranceschi, Monica Virginia, Son-
nessa, Michele, Delibato, Elisabetta, Marchesan, Massimo, Hernandez, Marta, De
Medici, Dario, Rodriguez-Lazaro, David, Optimization of a Real Time PCR based
method for the detection of Listeria monocytogenes in pork meat, International Journal
of Food Microbiology (2014), doi: 10.1016/j.ijfoodmicro.2014.04.015

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13th February 2014

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International Journal of Food microbiology

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Brief communication

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Optimization of a Real Time PCR based method for
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the detection of Listeria monocytogenes in pork meat
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Antonietta Gattuso1*, Monica Virginia Gianfranceschi1, Michele Sonnessa1, Elisabetta

Delibato1, Massimo Marchesan2, Marta Hernandez3, Dario De Medici1 and David
Rodriguez-Lazaro3,4
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1
Istituto Superiore di Sanità, Veterinary Public Health and Food Safety Department, Viale Regina Elena
299, 00161 Rome, Italy, 2DVM, Veterinary Practitioner, 3Instituto Tecnológico Agrario de Castilla y
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León (ITACyL) Valladolid, Spain, and 4Microbiology Section, Faculty of Sciences, University of Burgos,
Plaza Misael Bauñuelos s/n, 9001 Burgos, Spain
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* Corresponding author: Tel.:+39 06 49902319

Fax: +39 06 49387101
e-mail: antonietta.gattuso@iss.it
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Abstract

The aim of this study was to optimize a real-time PCR protocol for a rapid detection of

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Listeria monocytogenes in pork meat, using reduced volumes of primary selective

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enrichment broth and times of incubation to decrease the cost and time for analysis.

Forty-five samples of pork meat were artificially contaminated with two different levels

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of L. monocytogenes (1-10 CFU per sample and 10-100 CFU per sample),

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homogenised in three different volumes of Half Fraser Broth (1:3; 1:5 and 1:10) and

incubated at 30°C ± 1°C for 5 h, 8 h and 24 h. The detection was conducted in parallel
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by real-time PCR and the ISO standard 11290-1 methods. L. monocytogenes was

detected in all the samples after 24 hours by real-time PCR method, also using reduced
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volumes of Half Fraser Broth. This represents a clear advantage as the time to final

detection and the inherent costs were significantly reduced compared to the ISO
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reference method. All samples artificially contaminated were correctly detected also
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after 8 of incubation at 30°C ± 1°C in Half Fraser Broth and 24 hours in Fraser Broth at

37°C ± 1°C using cultural method.

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Keywords: Real Time PCR, Listeria monocytogenes, pork meat, artificial

contamination.
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Listeria monocytogenes is a pathogenic bacterium that can survive and grow in food

production environments (Meyer, Fredriksson-Ahomaa, Sperner, Martlbauer, 2011;

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Norrung, 2000) and as a result, it may contaminate foods during the processing. Since

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its ubiquitous nature and its presence during the slaughtering, which is prone to cross-

contamination (Giovannacci et al., 1999), raw meat and non-heat derived products are

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considered not completely free of Listeria (Vitas, Garcia-Jalon, 2004).

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The current standard method for the detection of L. monocytogenes in food, the
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ISO 11290-1 (Anonymous, 2004), includes two consecutive enrichment steps in a

selective medium -Fraser Broth in half and full concentration, followed by culturing on

two solid selective media (PALCAM and ALOA) after each enrichment. Presumptive
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colonies have to be confirmed subsequently trough haemolysis and biochemical tests

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(Anonymous, 2004) The time for a final confirmation is more than seven days. As a

consequence, culture standard method is laborious and does not prove an effective quick
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solution to handle the pace of current food production and distribution networks
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(Rodriguez-Lazaro, Cook, Hernandez, 2013a). The availability of a rapid method is one

of the most important challenges to manage the L. monocytogenes along the food chain

(Ennaji et al., 2009; O' Grady, Sedano-Balbas, Maher, Smith, Barry, 2008; Tang et al.,

2011). There is a clear cost benefit in rapid test results allowing faster HACCP

verification and positive release of finished food product. Real Time PCR has become

the most promising technique for rapid detection of pathogens in food including L.

monocytogenes (Rodriguez-Lazaro et al., 2013a; Rodriguez-Lazaro, Hernandez, 2013b).

The aim of this study was to evaluate the effect of using lower amounts of primary

selective enrichment Half Fraser Broth (HFB) and shorter incubation times coupled to a
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Real Time PCR assay previously published (Rodriguez-Lazaro, Jofre, Aymerich,

Hugas, Pla, 2004; Rodriguez-Lazaro, Pla, Scortti, Monzo, Vazquez-Boland, 2005) for

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the detection of L. monocytogenes in pork meat.

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Three L. monocytogenes strains (strains ISS A210 and ISS A267, serotype 1/2c,

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isolated by pork meat and strain ISS A1080, serotype 1/2a, isolated from meat

processing plant) were used to spike pork meat samples. Forty five pork meat sections,

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immediately after slaughter, were stored at 2°C ± 2°C for 6 days before experimental
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inoculation, in order to allow the growth of meat natural background microbiota. A

subset of each meat section was tested for the presence of L. monocytogenes using the

ISO 11290-1 (Anonymous, 2004). From the pork meat sections, 25 g-samples were
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prepared as follows: (i) 15 samples were homogenized with 225 mL of HFB (dilution
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1:10 w/v); (ii) 15 samples were homogenized with 100 mL of HFB (1:5 w/v); and 15

samples were homogenized with 50 mL of HFB (1:3 w/v). Five samples for each
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dilution (1:3, 1:5 and 1:10) were inoculated with approximately 1-10 (specifically 8.4
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CFU/sample) and 10-100 (specifically 84 CFU/sample) CFUs of L. monocytogenes, and

5 samples for each dilution were also used as negative controls (samples not spiked with

L. monocytogenes). Samples for each dilution and inoculation level, were incubated at

30°C ± 1°C for 5, 8 and 24 hours. After the primary selective enrichment, samples were

analysed by both Real Time PCR and by ISO standard methods.

In addition to evaluate the reduction of the secondary selective enrichment

incubation time, Fraser Broth was streaked onto ALOA and PALCAM agar plates after

24h and 48h of incubation at 37°C (Rijpens, Herman, 2004).

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After the primary selective enrichment step, 1 mL of the sample was taken and

transferred into a new clean micro-centrifuge tube, and centrifuged for 5 min at

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10,000g at 4ºC. The supernatant was carefully removed and the pellet was washed

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with 1 mL of PBS, and centrifuged for 5 min at 10,000g at 4ºC. Afterwards, the pellet

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was re-suspended in 200 µL of 6% Chelex® 100 (BioRad, Richmond, CA, USA) (De

Medici et al., 2003).

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All samples and controls were analysed in triplicates. Three l of DNA extract
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was used as template for the L. monocytogenes-specific real-time PCR detection assay.

Positive (DNA from L. monocytogenes ATCC7644) and negative controls (PCR grade
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water) were added in each PCR run. The final reaction volume (25 μL) for the duplex

detection assay consisted of 1X Multiplex Real time Master Mix of QIAGEN

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(QuantiTect Multiplex PCR NoRox Master Mix – Qiagen Biotechnology), 300 nM hly

gene primers (hlyF/R), IAC consisted of a 104-bp DNA fragment, 100 nM hly-probe
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labeled FAM, 100 nM IAC-probe labeled HEX (Rodriguez-Lazaro et al., 2004;

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Rodriguez-Lazaro et al., 2005). The amplification was performed using an initial hot-

start step at 95°C for 15 min, followed by 45 cycles at 95°C for 40 s of denaturation and

60°C for 60 s of annealing/extension. Real Time PCR was performed using

Stratagene™ Mx3005p spectro-fluorometric thermal cycler (Agilent, USA) using

MxPro PCR software.

Statistical analysis was performed using GraphPad Prism software version 6.0

(GraphPad Software, Inc., La Jolla, CA, USA) with default parameters. Results were

analysed using the one-way analysis of variance (ANOVA) test and Bonferroni’s
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Multiple Comparison Test for statistical significance; the mean difference was

considered significant at p < 0.05. The null hypothesis (H0) was that the dilutions rate

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for each spiked levels have same means; the null hypothesis was rejected with a P value

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lower than 0.05.

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The results obtained by Real Time PCR and cultural method (streaking the

primary selective enrichment broth directly onto ALOA and PALCAM agar plates) are

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shown in Table 1. The samples not spiked were negative with both Real-Time PCR
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method and culture-based method (data not shown).

The detection of L. monocytogenes by the Real-Time PCR assay after 5 and 8 hours of

HFB incubation was not possible, the samples were detected positive by streaking
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directly the 8 and 24 hours of incubation at 30°C ± 1°C of primary selective enrichment
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broths onto ALOA and PALCAM agar plates regardless the dilution factor used (Table

1). However the Cq values obtained were different depending of the dilution factor and
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contamination level used (Table 1). A Bonferroni’s Multiple Comparison Test results
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was assayed for three different volumes of HFB and two different inoculum levels

(One-way analysis of variance: number of groups 6, F=206, R=0.977, P<0.0001) and

for separate multiple comparisons for each level of inoculum: low (8.4 CFU per sample;

one-way analysis of variance: number of groups 3, F=234.6, R=0.975, P<0.0001) and

medium (84 CFU per sample; one-way analysis of variance: number of groups 3,

F=148.0, R=0.961, P<0.0001) (Table 2). The statistical analysis of the data does not

show a statistical significance between the two volumes of primary selective enrichment

1:5 and 1:10 at both levels of inoculum (10 and 100 CFU/sample), while showing a

statistical significance for the volume of primary selective enrichment 1:3 at both levels
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of inoculation. In this latter combination, in fact, the very small quantity of HFB creates

a gelatinous consistency matrix, that it is very difficult to withdraw forming a very

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inhomogeneous extraction volume.

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The detection of Listeria monocytogenes was possible in the sample after 5, 8 and

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24 hours primary selective enrichment, regardless the HFB volumes, when sub-cultured

in Fraser Broth for 24 at 37°C ± 1°C (data not shown). In summary, in this study two

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main parameters (dilution in HFB, and incubation time) were evaluated in order to
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reduce the time and cost for final results for Real Time PCR as well as by ISO-method.

The results obtained demonstrate that a combination of an incubation in 100 mL of

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Half-Fraser Broth for 24 h coupled to a DNA extraction and a Real-Time PCR assay

could detected down to about 8 CFU L. monocytogenes per sample in less than 27
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hours. This approach is fully compatible with the ISO standard for detection of L.

monocytogenes if a 1:10 and 1:5 dilution are used, providing results more rapidly (27
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hours vs 7 days) and cost-effectively (5 times cheaper) (Gianfranceschi et al., 2014).

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This represents an obvious advantage in order to reduce cost and time of analysis by

shorter time of incubation and smaller amounts of primary selective enrichment in

comparison to analytical reference method.

Acknowledgements

This work was supported by the EU BASELINE project. DRL acknowledges the

support by the Project RTA 2011-079-C02-01 of the Ministry of Economy and

Competitiveness, Government of Spain.

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Table 1. Results obtained by the ISO and Real Time PCR methods

Cultural
L. monocytogenes Real-Time PCR
Dilution Primary selective method
(CFU/ 25 gsample)
a rate enrichment (h)
Signal ratio hly Cq valueb IAC Cq valueb Positive ratio*

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5 0/5 n.d. 32.52 ± 0.12 2/5

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1:3 8 0/5 n.d. 32.60 ± 0.10 5/5
24 5/5 35.91 ± 0.30 33.41 ± 0.14 5/5

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5 0/5 n.d. 32.15 ± 0.17 2/5

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1-10 1:5 8 0/5 n.d. 32.12 ± 0.08 5/5
5/5

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24 5/5 29.59 ± 0.30 33.49 ± 0.14

5 0/5 n.d. 32.26 ± 0.10 2/5

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1:10 8 0/5 n.d. 32.11 ± 0.06 5/5

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24 5/5 28.56 ± 0.12 33.57 ± 0.14 5/5

5 0/5 n.d. 32.28 ± 0.12 5/5

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1:3 8 CE 0/5 n.d. 32.23 ± 0.13 5/5
24 5/5 31.66 ± 0.33 33.30 ± 0.14 5/5
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5 0/5 n.d. 32.10 ± 0.12 5/5

10-100 1:5 8 0/5 n.d. 32.52 ± 0.11 5/5

24 5/5 26.31 ± 0.35 33.26 ± 0.11 5/5

5 0/5 n.d. 32.30 ± 0.15 5/5

1:10 8 0/5 n.d. 32.22 ± 0.12 5/5

24 5/5 25.38 ± 0.07 33.66 ± 0.09 5/5
a
The actual L. monocytogenes was 8 CFUs for 1-10 CFUs level and 84 CFUs for 10-100 CFUs.
b
Mean Cq values ± standard error of the mean obtained from three PCR replicates per sample.
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Table 2. Bonferroni’s Multiple Comparison Test for evaluating the statistical difference among the mean Cq

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values obtained from the different ratio primary selective enrichment in HFB and each different inoculum or
to both. The statistical significance was p=0.05

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L. monocytogenes 1-10 10-100
(CFU/ 25 g sample)a

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1:10 1:5 1:3 1:10 1:5 1:3
1:3 Yes Yes Yes Yes Yes -
10-100 1:5 Yes Yes Yes No -

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1:10 Yes Yes Yes -
1:3 Yes Yes -
1-10 1:5 No -
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1:10 -
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The actual L. monocytogenes was 8 CFUs for 1-10 CFUs level and 84 CFUs for 10-100 CFUs.
Yes: statistically significant; No: not statistically significant
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