Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

Detection and quantication of Listeria monocytogenes by 5-nuclease polymerase chain reaction targeting the actA gene
K. Oravcova1, E. Kaclkova1, K. Krascsenicsova1, D. Pangallo2, B. Brezna1, 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

Keywords uorimetry, food, Listeria monocytogenes, polymerase chain reaction, rapid method. Correspondence Tomas Kuchta, Department of Microbiology and Molecular Biology, Food Research Institute, Priemyselna 4, PO Box 25, SK82475 Bratislava 26, Slovakia. E-mail: kuchta@vup.sk

Abstract Aims: The aim of this study was to develop a 5-nuclease polymerase chain reaction (PCR) for the rapid detection and quantication of Listeria monocytogenes. Methods and Results: Specic primers and a uorogenic probe were designed, which target a specic sequence of the actA gene encoding for a protein involved in the actin lament assembly. The PCR system was highly sensitive and specic for L. monocytogenes (inclusivity, 100%; exclusivity, 100%), which was determined using 46 L. monocytogenes and 28 non-L. monocytogenes strains. Detection limits of 104 cfu ml)1 after 35 cycles and 102 cfu ml)1 after 45 cycles were achieved by PCR in both real-time and end-point uorescence measurement modes. Linear calibration lines were obtained in the range from 102 to 109 cfu ml)1 for three L. monocytogenes strains in real-time PCR with 45 cycles. Conclusions: The developed 5-nuclease PCR of the actA gene provides a new target for the rapid detection and quantication of L. monocytogenes. Signicance and Impact of the Study: In conjunction with enrichment or with an appropriate quantitative sample preparation technique, the method is suitable for food safety applications. A 5-nuclease PCR-based method for quantication of L. monocytogenes in pure cultures, water, skimmed milk and unpasteurized whole milk was developed by Nogva et al. (2000). The method targeted the hlyA gene and when coupled to quantitative-bacterial cell separation by immunomagnetic separation, it proved to be useful for the quantication of L. monocytogenes in selected food products. Hein et al. (2001) developed a method for quantication of L. monocytogenes oriented to the iap gene. In this paper, an alternative 5-nuclease PCR-based method for quantication of L. monocytogenes is presented. The method targets a specic sequence of another virulence-associated gene, namely actA, which codes for a protein involved in the actin lament assembly (Pistor et al. 1994). The data presented demonstrate that, in conjunction with an appropriate enrichment or with a quantitative sample preparation technique, the method is suitable for food safety applications.
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2005/0782: received 11 July 2005, revised and accepted 20 September 2005

doi:10.1111/j.1472-765X.2005.01793.x

Introduction Listeria monocytogenes is an important pathogenic bacterium which is frequently found as a contaminant in meat and milk products, vegetable salads and other ready-toeat food products (Farber and Peterkin 1991). A zero tolerance 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 <100 cfu g)1 has been set for L. monocytogenes in certain food products for direct consumption during the shelf life (European Parliament 2004). For quantication of L. monocytogenes, a standard microbiological method EN ISO 11290-2:1998/A1 (2004) is available. However, this method requires up to 3 days for quantication of L. monocytogenes. As a method of great potential for the rapid quantication of bacteria, real-time polymerase chain reaction (PCR) has been identied.

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

PCR for L. monocytogenes

K. Oravcova et al.

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 are available 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 diluted culture samples was determined by plate-count technique 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 boiling. A volume of 1 ml of the bacterial suspension was centrifuged at 13 000 g, the sediment was then resuspended 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 nally 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-caaagcgagaatgtggctataaatga-3), 300 nmol l)1 of the primer LMrt3Rbis (5-taatttccgctgcgctatccg-3) and 200 nmol l)1 of the
Table 1 PCR results with Listeria monocytogenes and non-L. monocytogenes strains
Species L. monocytogenes serovar 1/2a L. monocytogenes serovar 1/2b L. monocytogenes serovar 1/2c L. monocytogenes serovar 4ab L. monocytogenes serovar 4b L. monocytogenes serovar 4d L. monocytogenes R Listeria innocua Listeria ivanovii Listeria grayi Listeria seeligeri Listeria welshimeri Enterococcus faecalis Micrococcus luteus Staphylococcus aureus Staphylococcus saprophyticus Salmonella Enteritidis Number of strains 13 21 3 2 2 1 4 9 4 2 2 2 3 1 3 1 1 PCR result + + + + + + + ) ) ) ) ) ) ) ) ) )

TaqMan probe listP (5-FAM-cctggatgacgacgctccacttgTAMRA-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 polymerase and 25 ll of the DNA sample. In addition, the reaction mixture contained an internal amplication control system (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, Roskilde, 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 (denaturation of 15 s at 94C, annealing and polymerization of 60 s at 60C). The amplied product was detected by uorimetry directly in the microtubes in a Genios 96-well reader (Tecan, Grodig bei Salzburg, Austria) equipped with excitation lters optimal for FAM and JOE dyes, positivity threshold being set to the uorescence value of the no tem plate control + 2 SD (Kaclkovaet al. 2005). To determine the exclusivity, amplied products were analysed by agarose 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-prole eight-microtube strips and the uorescence was measured through optical caps. PCR was carried out in a PTC-200 thermal cycler coupled to a Chromo 4 continuous uorescence 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 uorescence signals in channel 1 (FAM/Sybr) and channel 2 (VIC/JOE) were recorded and the threshold cycle values were calculated using the internal instrument software with the baseline subtraction option selected and the threshold set manually to a uorescence value of 002. To construct a calibration line, averaged threshold cycle values were plotted 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 specic detection and quantication of L. monocytogenes was developed. A sequence

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

 K. Oravcova et al.

PCR for L. monocytogenes

of the gene actA (GenBank Accession no. AF103807) was chosen as a new target for L. monocytogenes identication 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 a theoretical melting temperature of 60C as well as a corresponding 5-nuclease (TaqMan) probe (5-FAMcttcaggatccgaccgaccagctatac-TAMRA-3) were designed using the Primer Express software (Applied Biosystems). Although the primers amplied 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 identied. The extended BLAST search, when 238 L. monocytogenes strains were compared, conrmed 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 nal 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 system was 100% with 46 strains of L. monocytogenes and exclusivity was 100% with 28 non-L. monocytogenes strains (Table 1). The sensitivity of the qualitative detection of L. monocytogenes was evaluated on the basis of the determination of the detection probability. For this purpose, 12 replicates 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 system to quantication was evaluated on the basis of the analysis of decimally-diluted cultures of three L. monocytogenes strains (strain NCTC 11994 serovar 4b, strain 294 serovar 1/2b, strain 300 serovar 1/2b). For decreasing concentrations of cultures, amplication curves with proportionally increasing threshold cycle values were recorded, with no signicant difference between individual 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. monocytogenes 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 uorescence 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 uorescence values standard error of the mean are presented.

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

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PCR for L. monocytogenes

K. Oravcova et al.

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. monocytogenes 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 calibration lines obtained (data not shown). Discussion Based on the determined analytical parameters, the developed method was suitable for the qualitative detection of L. monocytogenes in food. It was specic for L. monocytogenes 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 uorimetry 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 specic and sensitive method. The method performed identically with various L. monocytogenes strains and we assume that it can be used for quantication of the entire L. monocytogenes species. The detection limit of 102 cfu ml)1 is satisfactory for its connection to quantitative bacterial separation techniques from food (Wolffs et al. 2004). This detection limit as well as calibration line parameters were equivalent to the previously published methods (Nogva et al. 2000; Hein et al. 2001). In comparison to current microbiological culturebased methods for L. monocytogenes quantication, 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 quantication of L. monocytogenes can be completed in approximately 4 h. Applicability of the method to direct quantication 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 Slovakian State Programme of Research and Development, project Food Quality and Safety.
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