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Article Type : Original Article
Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables
Hana Vojkovska1, Iva Kubikova1, Petr Kralik1
1
Veterinary Research Institute, Department of Food and Feed Safety, Brno, Czech Republic
Correspondence Hana Vojkovska, Department of Bacteriology, Veterinary Research Institute, Hudcova 296/70, 621 00 Brno, Czech Republic. E-mail: [email protected]
Running head: Evaluation of DNA extraction methods
Significance and Impact of the study Several recent outbreaks of Listeria monocytognes have been associated with the consumption of fruits and vegetables. Real time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoilTM Microbial DNA Isolation kit can be used for extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6% and This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12367 This article is protected by copyright. All rights reserved.
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70.3%. This method is applicable to samples with bacterial loads of 103 bacterial cells per gram of L. monocytogenes.
Abstract Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice.
In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model microorganism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoilTM Microbial DNA Isolation kit, is suitable for extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6% and 70.3%. Coupled with the triplex real time qPCR assay, this DNA isolation kit is applicable to samples with bacterial loads of 103 bacterial cells per gram of L. monocytogenes.
Keywords Listeria monocytogenes, real time PCR, DNA isolation, vegetable, inhibition
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generally cannot distinguish between live and dead cells, which is fundamental from the epidemiological point of view (Ramesh et al. 2002; Liang et al. 2011).
The major complications when using PCR-based methods are caused by matrix-associated inhibitory compounds from vegetables (e. g., plant-derived polysaccharides and polyphenolic acids) that interfere with L. monocytogenes detection by PCR (Demeke and Adams 1992; Bhagwat 2003; Hargreaves et al. 2013). Moreover, vegetables are usually contaminated by soil, which serves as a source of additional inhibitory compounds (Whitehouse and Hottel 2007; Hargreaves et al. 2013). To overcome such difficulties, the application of upstream sample processing methods to concentrate the pathogen and remove the sample matrix is often necessary (Ramesh et al. 2002; Isonhood et al. 2006; Yang et al. 2007; Brehm-Stecher et al. 2009). Also, the protocol used for extraction and purification of pathogen DNA should be verified for each representative sample matrix to ensure sufficient removal of interfering substances prior to PCR analysis.
Although previous studies have analyzed the applicability of different commercial kits for the extraction of bacterial DNA from food (Heller et al. 2003; Tortajada et al. 2009; Quigley et al. 2012; Lusk et al. 2013), to our knowledge none of them has been focused on determination of the best DNA isolation procedure from vegetables. Consequently, in this study we compare the performance of seven commercially available DNA isolation kits (three column-based and four magnetic bead-based) in a PCR-based assay for the detection of L. monocytogenes from washes of carrots and cucumber peels and sectors of iceberg lettuce to determine the DNA isolation efficiency and the limit of detection of the best-performing kit for further laboratory use.
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generally cannot distinguish between live and dead cells, which is fundamental from the epidemiological point of view (Ramesh et al. 2002; Liang et al. 2011).
The major complications when using PCR-based methods are caused by matrix-associated inhibitory compounds from vegetables (e. g., plant-derived polysaccharides and polyphenolic acids) that interfere with L. monocytogenes detection by PCR (Demeke and Adams 1992; Bhagwat 2003; Hargreaves et al. 2013). Moreover, vegetables are usually contaminated by soil, which serves as a source of additional inhibitory compounds (Whitehouse and Hottel 2007; Hargreaves et al. 2013). To overcome such difficulties, the application of upstream sample processing methods to concentrate the pathogen and remove the sample matrix is often necessary (Ramesh et al. 2002; Isonhood et al. 2006; Yang et al. 2007; Brehm-Stecher et al. 2009). Also, the protocol used for extraction and purification of pathogen DNA should be verified for each representative sample matrix to ensure sufficient removal of interfering substances prior to PCR analysis.
Although previous studies have analyzed the applicability of different commercial kits for the extraction of bacterial DNA from food (Heller et al. 2003; Tortajada et al. 2009; Quigley et al. 2012; Lusk et al. 2013), to our knowledge none of them has been focused on determination of the best DNA isolation procedure from vegetables. Consequently, in this study we compare the performance of seven commercially available DNA isolation kits (three column-based and four magnetic bead-based) in a PCR-based assay for the detection of L. monocytogenes from washes of carrots and cucumber peels and sectors of iceberg lettuce to determine the DNA isolation efficiency and the limit of detection of the best-performing kit for further laboratory use.
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Results and Discussion Comparison of DNA extraction kits In the case of samples spiked with the higher concentration of bacterial suspension (1×108 CFU ml-1), five kits (PowerSoil, PowerFood, NucliSENS, Nucleospin Food, PowerMag Soil) provided positive results with an efficiency of isolation ranging between 0.1 and 91.1% (Table 1). The PrepSEQ kit and the Chemagic Food kit failed to extract bacterial DNA from all three matrices. The NucleoSpin Food kit performed successfully with carrot and cucumber, but failed to detect L. monocytogenes from iceberg lettuce.
In the case of samples spiked with the lower concentration of bacterial suspension (1×105 CFU ml-1), only two kits (PowerSoil, PowerFood) provided positive results for all vegetable matrices with an efficiency of isolation ranging between 15.4 and 52.8% (Table 1). Use of the NucleoSpin Food and the NucliSENS kits resulted in positive detection only from some of the vegetable matrices.
Generally speaking, the column-based DNA extraction kits worked better than magnetic bead-based kits. Similar observations were made in previous studies on bacterial DNA extraction from food samples (Quigley et al. 2012). This issue represents a deeper problem, as the majority of the DNA isolation kits available on the market are not intended for the isolation of bacterial DNA from such complex matrix like food. Thus the “improper” usage of DNA extraction kits will lead to reduced recovery of the bacterial DNA from the sample. However, even the PrepSeq Nucleic Acid Extraction Kit, which is primarily intended for the isolation of bacterial DNA from complex samples, performed poorly in our study. Although the kit produced the highest DNA yields (Table 1), complete PCR inhibition (IAC amplification failure) was observed. It is obvious, therefore, that this kit cannot remove all
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PCR inhibitors, e. g. plant polysaccharides, terpenoids, phenols and tannins (Tebbe and Vahjen 1993; Liu 2008; Taylor et al. 2000; Hargreaves et al. 2013). On the other hand, column-based kits seem to be more robust for the isolation of bacterial DNA from complex matrices, although they are not primarily intended for this purpose.
The PowerSoil kit provided the most consistent results for both spiking concentrations and performed with the highest overall average isolation efficiency. Therefore, the PowerSoil kit was used in subsequent optimization steps. The “recalculation coefficient” required for further quantification of L. monocytogenes in unknown samples was calculated to be 0.45, which represents a median of all isolation efficiency values (Table 1).
The majority of studies to date have dealt with leafy vegetables (Isonhood et al. 2006; Gomez et al. 2010; Sanchez et al. 2012) or sprouts (Johnston et al. 2005), in spite of the fact that other vegetable types have also been documented as sources of L. monocytogenes (Beuchat 1996). Additionally it is well known that the amplification behavior of a PCR reaction is sample-specific and highly dynamic (van Tongeren et al. 2011; Hargreaves et al. 2013). Consequently, three different kinds of vegetables (iceberg lettuce, cucumbers and carrots) were tested in this study.
Our study shows that the isolation efficiency of DNA extraction kits varies according to the vegetable type and bacterial load (Table 1). Carrot proved to be the most challenging matrix in terms of bacterial DNA isolation, while cucumber and iceberg lettuce were less problematic in this regard. In addition, the problem is much more pronounced as the washes from the peelings were used for DNA isolation. It was reported that apart from natural inhibitors (e. g. carotenoids, polysaccharides, polyphenols) carrot also contains a high amount
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of soil embedded in ruptures and cavities, which is not removed even during technological washing. Low isolation efficiencies as a result of adhesion of target DNA to soil particles in combination with the inhibitory behavior of certain compounds have been documented (Dineen et al. 2010). However, in our hands, this potential source of variability was reduced when the DNA isolation kit intended for soil was used.
Efficiency of sample pre-processing The sample pre-processing step used in this work, which combines the agitation-connected release of bacterial cells into washings with a subsequent centrifugation step, is an option for processing of the food product itself and serves as useful alternative to culture enrichment (Isonhood et al. 2006). The average recoveries of microorganisms in other studies on vegetables or vegetable-related food ranged between 52% and 99% depending on inoculated microorganism and type of tested matrix (Johnston et al. 2005; Isonhood et al. 2006; Gomez et al. 2010; Sanchez et al. 2012).
In our study, the highest efficiency in cell detachment from vegetable surfaces was observed in the case of carrot (100.2% and 70.4% when spiked with 108 and 105 bacteria, respectively; Table 2). The lower DNA yields from iceberg lettuce (25.6% and 25.1%; Table 2) were probably caused by its complicated large-scale surface, which can reduce the proper washing of bacterial cells into rinsing solution and their follow-up concentration in the form of a pellet. Recovery efficiencies are, according to the observations of Sanchez et al. (2012) and Niemira (2003), highly dependent on the type of lettuce and pathogen. This may represent a further explanation for the lower DNA recoveries from iceberg lettuce during the sample preprocessing step. Efficiency values for cucumber which exceeded 100% (Table 2) are more likely to be related to the isolation process and PCR reaction than to the pre-processing step.
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Statistical error, which occurs as the consequence of unequal repetitive measurements may underlie this observation. Statistical error reflects the fact that the isolation procedure as well as the qPCR reaction harbors certain error rates, which are propagated to the final results (Larionov et al. 2005). If these problems occur, the calculated efficiency can be significantly biased.
Limit of detection using the optimized protocol For iceberg lettuce and cucumber peelings, the limit of detection was determined to be 1.58×103 bacterial cells g-1, whereas for carrot peelings the value was calculated to be 1.39×102 bacterial cells g-1 (Table 3). The limit of detection obtained in our study including DNA isolation and qPCR step is in agreement with previous direct PCR measurements from matrices of the food chain, where the limits of detection ranged between 103 and 102 CFU g-1 depending on the detected pathogen and type of food (Ehrs et al. 2011). This range is related to the intrinsic character of food matrices, where the combination of low levels of pathogens against a background of indigenous microflora and the complexity and diversity of a sample renders the recovery of target organisms difficult (Brehm-Stecher et al. 2009; Kawasaki et al. 2011). In summary, it can be concluded that 103 bacterial cells per gram can be detected with certainty from all three types of vegetable represented here.
Material and methods Vegetable samples Three different kinds of vegetables (iceberg lettuce, cucumbers and carrots) were purchased from local stores and processed into the form of pellets as follows: 100 g of iceberg lettuce sectors, cucumber and carrot peelings were placed into flasks and 250 ml of sterile Trisglycine beef extract (TGBE, pH 9.5) were added. After 15 min of agitation, the supernatant
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was spun at 8,000 g for 15 min at 4 °C. The supernatant was discarded and the pellet resuspended in 10 ml of buffered peptone water (BPW). Suspension aliquots (2 ml) were transferred into microtubes and spun at 5,000 g for 5 min. The residual liquid was discarded and the pellet frozen at -80 °C until use.
Bacterial cultures The CAPM 5579 strain of Listeria monocytogenes, obtained from the Collection of Animal Pathogenic Microorganisms (CAPM) at the Veterinary Research Institute, Brno, Czech Republic, was chosen as a model L. monocytogenes strain for this study. The bacterium was grown on blood agar (Oxoid) at 37 °C for 24 h. L. monocytogenes was reconstituted in TrisEDTA (TE) Buffer (pH 8.0, Amresco) and carrier DNA solution (salmon sperm DNA, 50 ng μl-1, Serva) to a concentration of approximately 1×108 CFU ml-1 (determined by optical density measurement at 600 nm and standard plate counting) for the spiking experiments. The bacterial suspension was serially diluted prior to inoculation to a concentration of approximately 1×105 CFU ml-1.
Sample spiking To assess the DNA isolation efficiency of all kits, triplicates of pelleted washes from iceberg lettuce, carrots and cucumbers were spiked with 100 μl of bacterial suspension of two different concentrations: 1×108 CFU ml-1 and 1×105 CFU ml-1. For further determination of the limit of detection of the selected DNA isolation protocol, 100 μl of L. monocytogenes suspension covering three log10 concentrations (from 1×106 CFU ml-1 to 1×104 CFU ml-1) were applied directly to the pelleted washes of all representative vegetable matrices. All samples were processed in biological triplicates. To evaluate the efficiency of the sample preprocessing step, which was included to detach bacterial cells from the vegetable surface, an
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Larionov, A., Krause, A. and Miller, W. (2005) A standard curve based method for relative real time PCR data processing. Bmc Bioinformatics 6.
Le Monnier, A., Abachin, E., Beretti, J.-L., Berche, P. and Kayal, S. (2011) Diagnosis of Listeria monocytogenes meningoencephalitis by real-time PCR for the hly gene. J Clin Microbiol 49, 3917-3923.
Liang, N., Dong, J., Luo, L. and Li, Y. (2011) Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR. J Food Sci 76, M234-M237.
Lianou, A. and Sofos, J.N. (2007) A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments. J Food Protect 70, 2172-2198.
Liu, D. (2008) Preparation of Listeria monocytogenes specimens for molecular detection and identification. Int J Food Microbiol 122, 229-242.
Low, J.C. and Donachie, W. (1997) A review of Listeria monocytogenes and listeriosis. Vet J 153, 9-29.
Lusk, T.S., Strain, E. and Kase, J.A. (2013) Comparison of six commercial DNA extraction kits for detection of Brucella neotomae in Mexican and Central American-style cheese and other milk products. Food Microbiol 34, 100-105.
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complete disruption of bacterial cells and higher DNA yield. The mechanical homogenization was performed using a MagNA Lyser instrument at 6,400 rpm for 60 s. The DNA yield was determined spectrophotometrically (NanoDrop 2200c Spectrophotometer, Thermo Fisher Scientific, Inc., USA).
Primer and probe design To quantify the L. monocytogenes cells, primers amplifying a 117-bp fragment of the prs gene, encoding a putative phosphoribosyl pyrophosphate synthetase and a 71-bp fragment of the hly gene, encoding listeriolysin O, a thiol-activated pore-forming cytolysin (Kayal and Charbit 2006) were used (Table 4). The primers and the probe for the hly gene were adapted from Le Monnier et al. (2011) and the prs gene-targeting primers were designed using the AlleleID software, version 7 (Premier Biosoft, Palo Alto, USA). Two independent targets in the L. monocytogenes genome were selected in order to increase the specificity of L. monocytogenes detection.
To monitor false negative results due to PCR inhibition, a 141-bp internal amplification control was added to each reaction. The internal amplification control was constructed as an artificial DNA sequence consisting of a part of the StTS1 gene from potato (Solanum tuberosum) flanked with hybrid primers derived partly from the Nepenthes endochitinase gene and partly from the f57 gene of Mycobacterium avium subsp. paratuberculosis (MAP). This sequence is not even remotely similar to any known organism, and thus this IAC can be used with any target or sample without the risk of false positive results (Slana et al. 2008; Table 4).
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PCR conditions PCR was performed in a total volume of 20 μl containing 2x LightCycler® 480 Probes Master (Roche Applied Science, Manheim, Germany), 250 nM of each primer, 500 nM of each probe, 1×105 copies of IAC, and 5 μl of the isolated DNA. The reaction was run on a LightCycler 480 II (Roche) with the following protocol: pre-incubation at 95 °C for 7 min, followed by 45 amplification cycles consisting of 95 °C for 10 s, 60 °C for 30 s (fluorescence acquisition), and 72 °C for 1 s.
Determination of the DNA isolation efficiency and limit of detection The amount of the L. monocytogenes cells in the sample was quantified according to a plasmid standard curve prepared by cloning the hly gene target sequence into the pDrive vector (Qiagen, Hilden, Germany) and propagation in E. coli cells. The purified plasmid was subsequently diluted in a range from 2×107 to 2×101 copies μl-1 in TE buffer with 100 ng μl-1 of fish sperm DNA (Serva) and used for the quantification of L. monocytogenes cells in tested samples. The other target, prs gene, was used as the confirmatory target and was positive in all analyses.
For the purposes of statistical evaluation, the DNA isolation was performed three times and each sample was analysed in duplicate using triplex qPCR. The DNA isolation efficiency for each dilution was calculated as the quotient of the experimentally determined number of L. monocytogenes after DNA isolation and theoretical input multiplied by 100. The theoretical and experimental number of L. monocytogenes cells was separately calculated according to the respective regression equation from the actual run; subsequently, mean and standard deviations were calculated. From both dilutions the mean value of DNA isolation efficiency for the respective L. monocytogenes dilution was calculated. In order to obtain the
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“recalculation coefficient” for the subsequent L. monocytogenes quantification in unknown samples, the median of the mean DNA isolation efficiency values was determined. The LOD for the triplex qPCR assay was determined as the lowest theoretical number of cells per gram of the carrot peels, cucumber or sector of the iceberg lettuce that was possible to detect in all replicates regardless of the absolute quantity.
Acknowledgements The authors would like to thank Neysan Donnelly for his English language corrections. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic LO1218 under the NPU I program and the Ministry of Agriculture of the Czech Republic (Grant No. QJ1210114).
Conflict of interest The authors have no conflict of interest to declare.
References Beuchat, L.R. (1996) Listeria monocytogenes: Incidence on vegetables. Food Control 7, 223228.
Bhagwat, A.A. (2003) Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR. Int J Food Microbiol 84, 217-224.
Brehm-Stecher, B., Young, C., Jaykus, L.-A. and Tortorello, M.L. (2009) Sample preparation: The forgotten beginning. J Food Protect 72, 1774-1789.
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Demeke, T. and Adams, R.P. (1992) The effects of plant polysaccharids and buffer additives on PCR. Biotechniques 12, 332-334.
Dineen, S.M., Aranda, R., Anders, D.L. and Robertson, J.M. (2010) An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil. J Appl Microbiol 109, 1886-1896.
Ehrs, S., Agren, P., Stephansson, O., Garbom, S. and Zumpe, A.L. (2011) Automated bacterial DNA isolation from food and feed samples - a biopreparedness design. J Food Safety 31, 225-231.
EFSA, ECDC (2013) The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011. EFSA Journal 2013 11, 250pp.
Germini, A., Masola, A., Carnevali, P. and Marchelli, R. (2009) Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR. Food Control 20, 733-738.
Gomez, P., Pagnon, M., Egea-Cortines, M., Artes, F. and Weiss, J. (2010) A fast molecular nondestructive protocol for evaluating aerobic bacterial load on fresh-cut lettuce. Food Sci Technol Int 16, 409-415.
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Hargreaves, S.K., Roberto, A.A. and Hofmockel, K.S. (2013) Reaction- and sample-specific inhibition affect standardization of qPCR assays of soil bacterial communities. Soil Biol Biochem 59, 89-97.
Heller, L.C., Davis, C.R., Peak, K.K., Wingfield, D., Cannons, A.C., Amuso, P.T. and Cattani, J. (2003) Comparison of methods for DNA isolation from food samples for detection of Shiga toxin-producing Escherichia coli by real-time PCR. Appl Environ Microb 69, 18441846.
Isonhood, J., Drake, M. and Jaykus, L.A. (2006) Upstream sample processing facilitates PCR detection of Listeria monocytogenes in mayonnaise-based ready-to-eat (RTE) salads. Food Microbiol 23, 584-590.
Johnston, L.M., Elhanafi, D., Drake, M. and Jaykus, L.A. (2005) A simple method for the direct detection of Salmonella and Escherichia coli O157:H7 from raw alfalfa sprouts and spent irrigation water using PCR. J Food Protect 68, 2256-2263.
Kawasaki, S., Fratamico, P.M., Kamisaki-Horikoshi, N., Okada, Y., Takeshita, K., Sameshima, T. and Kawamoto, S. (2011) Development of the multiplex PCR detection kit for Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7. Jarq-Jpn Agr Res Q 45, 77-81.
Kayal, S. and Charbit, A. (2006) Listeriolysin O: a key protein of Listeria monocytogenes with multiple functions. Fems Microbiol Rev 30, 514-529.
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Larionov, A., Krause, A. and Miller, W. (2005) A standard curve based method for relative real time PCR data processing. Bmc Bioinformatics 6.
Le Monnier, A., Abachin, E., Beretti, J.-L., Berche, P. and Kayal, S. (2011) Diagnosis of Listeria monocytogenes meningoencephalitis by real-time PCR for the hly gene. J Clin Microbiol 49, 3917-3923.
Liang, N., Dong, J., Luo, L. and Li, Y. (2011) Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR. J Food Sci 76, M234-M237.
Lianou, A. and Sofos, J.N. (2007) A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments. J Food Protect 70, 2172-2198.
Liu, D. (2008) Preparation of Listeria monocytogenes specimens for molecular detection and identification. Int J Food Microbiol 122, 229-242.
Low, J.C. and Donachie, W. (1997) A review of Listeria monocytogenes and listeriosis. Vet J 153, 9-29.
Lusk, T.S., Strain, E. and Kase, J.A. (2013) Comparison of six commercial DNA extraction kits for detection of Brucella neotomae in Mexican and Central American-style cheese and other milk products. Food Microbiol 34, 100-105.
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Niemira, B.A. (2003) Radiation sensitivity and recoverability of Listeria monocytogenes and Salmonella on 4 lettuce types. J Food Sci 68, 2784-2787.
Quigley, L., O'Sullivan, O., Beresford, T.P., Ross, R.P., Fitzgerald, G.F. and Cotter, P.D. (2012) A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese. J Appl Microbiol 113, 96-105.
Ramesh, A., Padmapriya, B.P., Chandrashekar, A. and Varadaraj, M.C. (2002) Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples. Mol Cell Probe 16, 307314.
Sanchez, G., Elizaquivel, P. and Aznar, R. (2012) A single method for recovery and concentration of enteric viruses and bacteria from fresh-cut vegetables. Int J Food Microbiol 152, 9-13.
Slana, I., Kralik, P., Kralova, A. and Pavlik, I. (2008) On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int J Food Microbiol 128, 250-257.
Taylor, J.I., Hurst, C.D., Davies, M.J., Sachsinger, N. and Bruce, I.J. (2000) Application of magnetite and silica-magnetite composites to the isolation of genomic DNA. J Chromatogr A 890, 159-166.
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Tebbe, C.C. and Vahjen, W. (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant-DNA from bacteria and yeast. Appl Environ Microb 59, 2657-2665.
Tortajada, M., Vicente Martinez-Culebras, P., Navarro, V., Monzo, H. and Ramon, D. (2009) Evaluation of DNA extraction methods for PCR detection of fungal and bacterial contamination in cocoa extracts. Eur Food Res Technol 230, 79-87.
van Tongeren, S.P., Degener, J.E. and Harmsen, H.J.M. (2011) Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR. Eur J Clin Microbiol 30, 1053-1061.
Whitehouse, C.A. and Hottel, H.E. (2007) Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples. Mol Cell Probe 21, 92-96.
Yang, H., Qu, L., Wimbrow, A.N., Jiang, X. and Sun, Y. (2007) Rapid detection of Listeria monocytogenes by nanoparticle-based immunomagnetic separation and real-time PCR. Int J Food Microbiol 118, 132-138.
Table 1 Performance evaluation of commercial DNA extraction kits used for the detection of Listeria monocytogenes in iceberg lettuce, carrot and cucumber. The quantity of extracted bacterial DNA was assessed using qPCR.
Isolation kit PowerSoil
Type of vegetables iceberg lettuce
DNA yield (μg μl-1)
Theoretical input mean (cells g-1)
Experimental output mean (cells g-1)
Isolation efficiency mean
1.83
2.91 ± 2.03 × 105
2.04 ± 0.19 × 105
70.3 ± 6.5 %
1.55
2
1
1.40 ± 0.34 × 10
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4.96 ± 2.52 × 10
35.4 ± 18.0 %
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carrot
2.07 2.35
2.91 ± 2.03 × 105 1.40 ± 0.34 × 102
1.59 ± 0.15 × 105 3.49 ± 1.16 × 101
54.6 ± 5.1 % 24.9 ± 8.3 %
cucumber
2.04 1.93
2.91 ± 2.03 × 105 1.40 ± 0.34 × 102
1.93 ± 0.22 × 105 4.15 ± 0.58 × 101
66.5 ± 7.5 % 29.6 ± 4.1 %
2.82
1.37 ± 0.14 × 106
6.53 ± 0.83 × 105
47.6 ± 6.0 %
2.18
3
2
16.0 ± 8.6 %
iceberg lettuce
PowerFood
1.37 ± 0.14 × 10 1.98 ± 0.16 × 103
5.03 ± 0.44 × 10 3.05 ± 3.44 × 102
36.7 ± 3.2 % 15.4 ± 17.3 %
cucumber
2.59 3.34
1.37 ± 0.14 × 106 1.98 ± 0.16 × 103
5.85 ± 0.80 × 105 4.15 ± 1.99 × 102
42.6 ± 5.9 % 20.9 ± 10.0 %
8.42
2.44 ± 0.14 × 105
2.22 ± 0.24 × 105
91.1 ± 9.8 %
9.22
1
4.16 ± 6.04 × 10
0
8.8 ± 12.8 %
3
2.6 ± 0.6 % -
2.44 ± 0.14 × 10 4.72 ± 2.85 × 101
6.45 ± 1.58 × 10 -*
cucumber
9.93 12.37
2.44 ± 0.14 × 105 4.72 ± 2.85 × 101
3.35 ± 0.84 × 104 -
8.35
9.15 ± 0.91 × 105
-
3.71
4.88 ± 1.33 × 102
-
carrot
3.93 6.33
5
9.15 ± 0.91 × 10 4.88 ± 1.33 × 102
5
1.89 ± 0.32 × 10 9.02 ± 1.56 × 101
20.6 ± 3.5% 18.5 ± 32.0 %
cucumber
4.51 4.84
9.15 ± 0.91 × 105 4.88 ± 1.33 × 102
3.15 ± 0.47 × 105 2.58 ± 0.86 × 102
34.4 ± 5.2 % 52.8 ± 17.6 %
2.56
4.53 ± 0.69 × 106
1.63 ± 0.70 × 104
0.4 ± 0.2 %
3.04
4.55 ± 1.87 × 10
3
-
-
carrot
2.69 3.15
4.53 ± 0.69 × 106 4.55 ± 1.87 × 103
1.19 ± 1.14× 104 -
0.3 ± 0.3 % -
cucumber
3.35 2.62
4.53 ± 0.69 × 106 4.55 ± 1.87 × 103
2.47 ± 2.36 × 103 -
0.1 ± 0.1 % -
1.21
NA#
NA
NA
0.89
NA NA NA NA NA
NA NA NA NA NA
13.7 ± 3.4 % -
carrot
3.67 5.62
cucumber
2.54 1.31
NA NA NA NA NA
9.34
NA
NA
NA
18.68
NA NA NA NA NA
NA NA NA NA NA
NA NA NA NA NA
iceberg lettuce PrepSEQ
5
5.97 4.92
iceberg lettuce Chemagic Food
4.72 ± 2.85 × 10
carrot
iceberg lettuce PowerMag Soil
5
2.52 3.06
iceberg lettuce
Nucleospin Food
3.18 ± 1.71 × 10
6
carrot
iceberg lettuce
NucliSENS
1.98 ± 0.16 × 10
carrot
20.39 31.33
cucumber
26.85 29.72
* - Negative result for amplification of hly and prs genes. # - Failure of amplification of IAC and target genes.
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Table 2 Detachment efficiency of sample pre-processing for the three vegetable matrices. Mean concentration (n=3) Type of vegetable iceberg lettuce
carrot
Yield (%) Vegetable
Pellet
1.33 ± 0.61 × 105
5.22 ± 2.36 × 105
25.6
1.26 ± 0.26 × 102
5.03 ± 0.48 × 102
25.1
4.04 ± 0.66 × 105
4.04 ± 1.33 × 105
100.2
2
2
2.51 ± 0.73 × 10 cucumber
3.56 ± 0.81 × 10
70.4
4.77 ± 0.96 × 105
2.03 ± 0.40 × 105
235.1*
7.76 ± 3.07 × 102
7.49 ± 3.88 × 102
103.6
* DNA yield higher than 100% indicates a biased calculation.
Table 3 Limit of detection using the PowerSoilTM DNA Isolation kit for detection of Listeria monocytogenes in iceberg lettuce, carrot and cucumber. Type of vegetable
Spike concentration (cells g1 )
Positive detection (no. of samples)
iceberg lettuce
1.58 × 103
6/6
2
5/6
1
1.69 × 10
0/6
1.58 × 103
6/6
1.39 × 102
6/6
1.69 × 101
3/6
1.58 × 103
6/6
2
5/6
1
1/6
1.39 × 10
carrot
cucumber
1.39 × 10 1.69 × 10
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Table 4 Sequences of primers and probes used in the triplex real time qPCR assay. Target gene prs
Name Lprs F Lprs R Lprs P
Type Forward Reverse Probe
hly
LMhly F LMhly R LMhly P
Forward Reverse Probe
IAC
IAC F IAC R
Forward Reverse
IAC P
Probe
Sequence 5'- GCG TTA CTC ATT TTA GTG A-3' 5'- GTT CCA TTA AAT TCT GGT TTA C-3' FAM-5'- ACA TGA CAA CCA CGG ATA CTT-3'-BHQ 5'- CAT TAG TGG AAA GAT GGA ATG-3' 5'- GTA AGC CAT TTC GTC ATC AT-3' HEX-5'- TCA AGC TTA TCC AAA TGT AAG TGC AA-3'-BHQ 5'- AGA GGA CCG GGA TAT TCG AC -3' 5'- AGG TAG TCC GAG GAA AAC TCT AAA C -3' Cy5-5'- AGG CTC TTC TAT GTT CTG ACC TTG TTG GA -3'-BHQ
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Article Type : Original Article
Evaluation of DNA extraction methods for PCR-based detection of Listeria monocytogenes from vegetables
Hana Vojkovska1, Iva Kubikova1, Petr Kralik1
1
Veterinary Research Institute, Department of Food and Feed Safety, Brno, Czech Republic
Correspondence Hana Vojkovska, Department of Bacteriology, Veterinary Research Institute, Hudcova 296/70, 621 00 Brno, Czech Republic. E-mail: [email protected]
Running head: Evaluation of DNA extraction methods
Significance and Impact of the study Several recent outbreaks of Listeria monocytognes have been associated with the consumption of fruits and vegetables. Real time PCR assays allow fast detection and accurate quantification of microbes. However, the success of real time PCR is dependent on the success with which template DNA can be extracted. The results of this study suggest that the PowerSoilTM Microbial DNA Isolation kit can be used for extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6% and This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12367 This article is protected by copyright. All rights reserved.
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70.3%. This method is applicable to samples with bacterial loads of 103 bacterial cells per gram of L. monocytogenes.
Abstract Epidemiological data indicate that raw vegetables are associated with outbreaks of Listeria monocytogenes. Therefore, there is a demand for the availability of rapid and sensitive methods, such as PCR assays, for the detection and accurate discrimination of L. monocytogenes. However, the efficiency of PCR methods can be negatively affected by inhibitory compounds commonly found in vegetable matrices that may cause false negative results. Therefore, the sample processing and DNA isolation steps must be carefully evaluated prior to the introduction of such methods into routine practice.
In this study, we compared the ability of three column-based and four magnetic bead-based commercial DNA isolation kits to extract DNA of the model microorganism L. monocytogenes from raw vegetables. The DNA isolation efficiency of all isolation kits was determined using a triplex real time qPCR assay designed to specifically detect L. monocytogenes. The kit with best performance, the PowerSoilTM Microbial DNA Isolation kit, is suitable for extraction of amplifiable DNA from L. monocytogenes cells in vegetable with efficiencies ranging between 29.6% and 70.3%. Coupled with the triplex real time qPCR assay, this DNA isolation kit is applicable to samples with bacterial loads of 103 bacterial cells per gram of L. monocytogenes.
Keywords Listeria monocytogenes, real time PCR, DNA isolation, vegetable, inhibition
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generally cannot distinguish between live and dead cells, which is fundamental from the epidemiological point of view (Ramesh et al. 2002; Liang et al. 2011).
The major complications when using PCR-based methods are caused by matrix-associated inhibitory compounds from vegetables (e. g., plant-derived polysaccharides and polyphenolic acids) that interfere with L. monocytogenes detection by PCR (Demeke and Adams 1992; Bhagwat 2003; Hargreaves et al. 2013). Moreover, vegetables are usually contaminated by soil, which serves as a source of additional inhibitory compounds (Whitehouse and Hottel 2007; Hargreaves et al. 2013). To overcome such difficulties, the application of upstream sample processing methods to concentrate the pathogen and remove the sample matrix is often necessary (Ramesh et al. 2002; Isonhood et al. 2006; Yang et al. 2007; Brehm-Stecher et al. 2009). Also, the protocol used for extraction and purification of pathogen DNA should be verified for each representative sample matrix to ensure sufficient removal of interfering substances prior to PCR analysis.
Although previous studies have analyzed the applicability of different commercial kits for the extraction of bacterial DNA from food (Heller et al. 2003; Tortajada et al. 2009; Quigley et al. 2012; Lusk et al. 2013), to our knowledge none of them has been focused on determination of the best DNA isolation procedure from vegetables. Consequently, in this study we compare the performance of seven commercially available DNA isolation kits (three column-based and four magnetic bead-based) in a PCR-based assay for the detection of L. monocytogenes from washes of carrots and cucumber peels and sectors of iceberg lettuce to determine the DNA isolation efficiency and the limit of detection of the best-performing kit for further laboratory use.
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generally cannot distinguish between live and dead cells, which is fundamental from the epidemiological point of view (Ramesh et al. 2002; Liang et al. 2011).
The major complications when using PCR-based methods are caused by matrix-associated inhibitory compounds from vegetables (e. g., plant-derived polysaccharides and polyphenolic acids) that interfere with L. monocytogenes detection by PCR (Demeke and Adams 1992; Bhagwat 2003; Hargreaves et al. 2013). Moreover, vegetables are usually contaminated by soil, which serves as a source of additional inhibitory compounds (Whitehouse and Hottel 2007; Hargreaves et al. 2013). To overcome such difficulties, the application of upstream sample processing methods to concentrate the pathogen and remove the sample matrix is often necessary (Ramesh et al. 2002; Isonhood et al. 2006; Yang et al. 2007; Brehm-Stecher et al. 2009). Also, the protocol used for extraction and purification of pathogen DNA should be verified for each representative sample matrix to ensure sufficient removal of interfering substances prior to PCR analysis.
Although previous studies have analyzed the applicability of different commercial kits for the extraction of bacterial DNA from food (Heller et al. 2003; Tortajada et al. 2009; Quigley et al. 2012; Lusk et al. 2013), to our knowledge none of them has been focused on determination of the best DNA isolation procedure from vegetables. Consequently, in this study we compare the performance of seven commercially available DNA isolation kits (three column-based and four magnetic bead-based) in a PCR-based assay for the detection of L. monocytogenes from washes of carrots and cucumber peels and sectors of iceberg lettuce to determine the DNA isolation efficiency and the limit of detection of the best-performing kit for further laboratory use.
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Results and Discussion Comparison of DNA extraction kits In the case of samples spiked with the higher concentration of bacterial suspension (1×108 CFU ml-1), five kits (PowerSoil, PowerFood, NucliSENS, Nucleospin Food, PowerMag Soil) provided positive results with an efficiency of isolation ranging between 0.1 and 91.1% (Table 1). The PrepSEQ kit and the Chemagic Food kit failed to extract bacterial DNA from all three matrices. The NucleoSpin Food kit performed successfully with carrot and cucumber, but failed to detect L. monocytogenes from iceberg lettuce.
In the case of samples spiked with the lower concentration of bacterial suspension (1×105 CFU ml-1), only two kits (PowerSoil, PowerFood) provided positive results for all vegetable matrices with an efficiency of isolation ranging between 15.4 and 52.8% (Table 1). Use of the NucleoSpin Food and the NucliSENS kits resulted in positive detection only from some of the vegetable matrices.
Generally speaking, the column-based DNA extraction kits worked better than magnetic bead-based kits. Similar observations were made in previous studies on bacterial DNA extraction from food samples (Quigley et al. 2012). This issue represents a deeper problem, as the majority of the DNA isolation kits available on the market are not intended for the isolation of bacterial DNA from such complex matrix like food. Thus the “improper” usage of DNA extraction kits will lead to reduced recovery of the bacterial DNA from the sample. However, even the PrepSeq Nucleic Acid Extraction Kit, which is primarily intended for the isolation of bacterial DNA from complex samples, performed poorly in our study. Although the kit produced the highest DNA yields (Table 1), complete PCR inhibition (IAC amplification failure) was observed. It is obvious, therefore, that this kit cannot remove all
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PCR inhibitors, e. g. plant polysaccharides, terpenoids, phenols and tannins (Tebbe and Vahjen 1993; Liu 2008; Taylor et al. 2000; Hargreaves et al. 2013). On the other hand, column-based kits seem to be more robust for the isolation of bacterial DNA from complex matrices, although they are not primarily intended for this purpose.
The PowerSoil kit provided the most consistent results for both spiking concentrations and performed with the highest overall average isolation efficiency. Therefore, the PowerSoil kit was used in subsequent optimization steps. The “recalculation coefficient” required for further quantification of L. monocytogenes in unknown samples was calculated to be 0.45, which represents a median of all isolation efficiency values (Table 1).
The majority of studies to date have dealt with leafy vegetables (Isonhood et al. 2006; Gomez et al. 2010; Sanchez et al. 2012) or sprouts (Johnston et al. 2005), in spite of the fact that other vegetable types have also been documented as sources of L. monocytogenes (Beuchat 1996). Additionally it is well known that the amplification behavior of a PCR reaction is sample-specific and highly dynamic (van Tongeren et al. 2011; Hargreaves et al. 2013). Consequently, three different kinds of vegetables (iceberg lettuce, cucumbers and carrots) were tested in this study.
Our study shows that the isolation efficiency of DNA extraction kits varies according to the vegetable type and bacterial load (Table 1). Carrot proved to be the most challenging matrix in terms of bacterial DNA isolation, while cucumber and iceberg lettuce were less problematic in this regard. In addition, the problem is much more pronounced as the washes from the peelings were used for DNA isolation. It was reported that apart from natural inhibitors (e. g. carotenoids, polysaccharides, polyphenols) carrot also contains a high amount
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of soil embedded in ruptures and cavities, which is not removed even during technological washing. Low isolation efficiencies as a result of adhesion of target DNA to soil particles in combination with the inhibitory behavior of certain compounds have been documented (Dineen et al. 2010). However, in our hands, this potential source of variability was reduced when the DNA isolation kit intended for soil was used.
Efficiency of sample pre-processing The sample pre-processing step used in this work, which combines the agitation-connected release of bacterial cells into washings with a subsequent centrifugation step, is an option for processing of the food product itself and serves as useful alternative to culture enrichment (Isonhood et al. 2006). The average recoveries of microorganisms in other studies on vegetables or vegetable-related food ranged between 52% and 99% depending on inoculated microorganism and type of tested matrix (Johnston et al. 2005; Isonhood et al. 2006; Gomez et al. 2010; Sanchez et al. 2012).
In our study, the highest efficiency in cell detachment from vegetable surfaces was observed in the case of carrot (100.2% and 70.4% when spiked with 108 and 105 bacteria, respectively; Table 2). The lower DNA yields from iceberg lettuce (25.6% and 25.1%; Table 2) were probably caused by its complicated large-scale surface, which can reduce the proper washing of bacterial cells into rinsing solution and their follow-up concentration in the form of a pellet. Recovery efficiencies are, according to the observations of Sanchez et al. (2012) and Niemira (2003), highly dependent on the type of lettuce and pathogen. This may represent a further explanation for the lower DNA recoveries from iceberg lettuce during the sample preprocessing step. Efficiency values for cucumber which exceeded 100% (Table 2) are more likely to be related to the isolation process and PCR reaction than to the pre-processing step.
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Statistical error, which occurs as the consequence of unequal repetitive measurements may underlie this observation. Statistical error reflects the fact that the isolation procedure as well as the qPCR reaction harbors certain error rates, which are propagated to the final results (Larionov et al. 2005). If these problems occur, the calculated efficiency can be significantly biased.
Limit of detection using the optimized protocol For iceberg lettuce and cucumber peelings, the limit of detection was determined to be 1.58×103 bacterial cells g-1, whereas for carrot peelings the value was calculated to be 1.39×102 bacterial cells g-1 (Table 3). The limit of detection obtained in our study including DNA isolation and qPCR step is in agreement with previous direct PCR measurements from matrices of the food chain, where the limits of detection ranged between 103 and 102 CFU g-1 depending on the detected pathogen and type of food (Ehrs et al. 2011). This range is related to the intrinsic character of food matrices, where the combination of low levels of pathogens against a background of indigenous microflora and the complexity and diversity of a sample renders the recovery of target organisms difficult (Brehm-Stecher et al. 2009; Kawasaki et al. 2011). In summary, it can be concluded that 103 bacterial cells per gram can be detected with certainty from all three types of vegetable represented here.
Material and methods Vegetable samples Three different kinds of vegetables (iceberg lettuce, cucumbers and carrots) were purchased from local stores and processed into the form of pellets as follows: 100 g of iceberg lettuce sectors, cucumber and carrot peelings were placed into flasks and 250 ml of sterile Trisglycine beef extract (TGBE, pH 9.5) were added. After 15 min of agitation, the supernatant
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was spun at 8,000 g for 15 min at 4 °C. The supernatant was discarded and the pellet resuspended in 10 ml of buffered peptone water (BPW). Suspension aliquots (2 ml) were transferred into microtubes and spun at 5,000 g for 5 min. The residual liquid was discarded and the pellet frozen at -80 °C until use.
Bacterial cultures The CAPM 5579 strain of Listeria monocytogenes, obtained from the Collection of Animal Pathogenic Microorganisms (CAPM) at the Veterinary Research Institute, Brno, Czech Republic, was chosen as a model L. monocytogenes strain for this study. The bacterium was grown on blood agar (Oxoid) at 37 °C for 24 h. L. monocytogenes was reconstituted in TrisEDTA (TE) Buffer (pH 8.0, Amresco) and carrier DNA solution (salmon sperm DNA, 50 ng μl-1, Serva) to a concentration of approximately 1×108 CFU ml-1 (determined by optical density measurement at 600 nm and standard plate counting) for the spiking experiments. The bacterial suspension was serially diluted prior to inoculation to a concentration of approximately 1×105 CFU ml-1.
Sample spiking To assess the DNA isolation efficiency of all kits, triplicates of pelleted washes from iceberg lettuce, carrots and cucumbers were spiked with 100 μl of bacterial suspension of two different concentrations: 1×108 CFU ml-1 and 1×105 CFU ml-1. For further determination of the limit of detection of the selected DNA isolation protocol, 100 μl of L. monocytogenes suspension covering three log10 concentrations (from 1×106 CFU ml-1 to 1×104 CFU ml-1) were applied directly to the pelleted washes of all representative vegetable matrices. All samples were processed in biological triplicates. To evaluate the efficiency of the sample preprocessing step, which was included to detach bacterial cells from the vegetable surface, an
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Larionov, A., Krause, A. and Miller, W. (2005) A standard curve based method for relative real time PCR data processing. Bmc Bioinformatics 6.
Le Monnier, A., Abachin, E., Beretti, J.-L., Berche, P. and Kayal, S. (2011) Diagnosis of Listeria monocytogenes meningoencephalitis by real-time PCR for the hly gene. J Clin Microbiol 49, 3917-3923.
Liang, N., Dong, J., Luo, L. and Li, Y. (2011) Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR. J Food Sci 76, M234-M237.
Lianou, A. and Sofos, J.N. (2007) A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments. J Food Protect 70, 2172-2198.
Liu, D. (2008) Preparation of Listeria monocytogenes specimens for molecular detection and identification. Int J Food Microbiol 122, 229-242.
Low, J.C. and Donachie, W. (1997) A review of Listeria monocytogenes and listeriosis. Vet J 153, 9-29.
Lusk, T.S., Strain, E. and Kase, J.A. (2013) Comparison of six commercial DNA extraction kits for detection of Brucella neotomae in Mexican and Central American-style cheese and other milk products. Food Microbiol 34, 100-105.
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complete disruption of bacterial cells and higher DNA yield. The mechanical homogenization was performed using a MagNA Lyser instrument at 6,400 rpm for 60 s. The DNA yield was determined spectrophotometrically (NanoDrop 2200c Spectrophotometer, Thermo Fisher Scientific, Inc., USA).
Primer and probe design To quantify the L. monocytogenes cells, primers amplifying a 117-bp fragment of the prs gene, encoding a putative phosphoribosyl pyrophosphate synthetase and a 71-bp fragment of the hly gene, encoding listeriolysin O, a thiol-activated pore-forming cytolysin (Kayal and Charbit 2006) were used (Table 4). The primers and the probe for the hly gene were adapted from Le Monnier et al. (2011) and the prs gene-targeting primers were designed using the AlleleID software, version 7 (Premier Biosoft, Palo Alto, USA). Two independent targets in the L. monocytogenes genome were selected in order to increase the specificity of L. monocytogenes detection.
To monitor false negative results due to PCR inhibition, a 141-bp internal amplification control was added to each reaction. The internal amplification control was constructed as an artificial DNA sequence consisting of a part of the StTS1 gene from potato (Solanum tuberosum) flanked with hybrid primers derived partly from the Nepenthes endochitinase gene and partly from the f57 gene of Mycobacterium avium subsp. paratuberculosis (MAP). This sequence is not even remotely similar to any known organism, and thus this IAC can be used with any target or sample without the risk of false positive results (Slana et al. 2008; Table 4).
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PCR conditions PCR was performed in a total volume of 20 μl containing 2x LightCycler® 480 Probes Master (Roche Applied Science, Manheim, Germany), 250 nM of each primer, 500 nM of each probe, 1×105 copies of IAC, and 5 μl of the isolated DNA. The reaction was run on a LightCycler 480 II (Roche) with the following protocol: pre-incubation at 95 °C for 7 min, followed by 45 amplification cycles consisting of 95 °C for 10 s, 60 °C for 30 s (fluorescence acquisition), and 72 °C for 1 s.
Determination of the DNA isolation efficiency and limit of detection The amount of the L. monocytogenes cells in the sample was quantified according to a plasmid standard curve prepared by cloning the hly gene target sequence into the pDrive vector (Qiagen, Hilden, Germany) and propagation in E. coli cells. The purified plasmid was subsequently diluted in a range from 2×107 to 2×101 copies μl-1 in TE buffer with 100 ng μl-1 of fish sperm DNA (Serva) and used for the quantification of L. monocytogenes cells in tested samples. The other target, prs gene, was used as the confirmatory target and was positive in all analyses.
For the purposes of statistical evaluation, the DNA isolation was performed three times and each sample was analysed in duplicate using triplex qPCR. The DNA isolation efficiency for each dilution was calculated as the quotient of the experimentally determined number of L. monocytogenes after DNA isolation and theoretical input multiplied by 100. The theoretical and experimental number of L. monocytogenes cells was separately calculated according to the respective regression equation from the actual run; subsequently, mean and standard deviations were calculated. From both dilutions the mean value of DNA isolation efficiency for the respective L. monocytogenes dilution was calculated. In order to obtain the
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“recalculation coefficient” for the subsequent L. monocytogenes quantification in unknown samples, the median of the mean DNA isolation efficiency values was determined. The LOD for the triplex qPCR assay was determined as the lowest theoretical number of cells per gram of the carrot peels, cucumber or sector of the iceberg lettuce that was possible to detect in all replicates regardless of the absolute quantity.
Acknowledgements The authors would like to thank Neysan Donnelly for his English language corrections. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic LO1218 under the NPU I program and the Ministry of Agriculture of the Czech Republic (Grant No. QJ1210114).
Conflict of interest The authors have no conflict of interest to declare.
References Beuchat, L.R. (1996) Listeria monocytogenes: Incidence on vegetables. Food Control 7, 223228.
Bhagwat, A.A. (2003) Simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella strains by real-time PCR. Int J Food Microbiol 84, 217-224.
Brehm-Stecher, B., Young, C., Jaykus, L.-A. and Tortorello, M.L. (2009) Sample preparation: The forgotten beginning. J Food Protect 72, 1774-1789.
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Demeke, T. and Adams, R.P. (1992) The effects of plant polysaccharids and buffer additives on PCR. Biotechniques 12, 332-334.
Dineen, S.M., Aranda, R., Anders, D.L. and Robertson, J.M. (2010) An evaluation of commercial DNA extraction kits for the isolation of bacterial spore DNA from soil. J Appl Microbiol 109, 1886-1896.
Ehrs, S., Agren, P., Stephansson, O., Garbom, S. and Zumpe, A.L. (2011) Automated bacterial DNA isolation from food and feed samples - a biopreparedness design. J Food Safety 31, 225-231.
EFSA, ECDC (2013) The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2011. EFSA Journal 2013 11, 250pp.
Germini, A., Masola, A., Carnevali, P. and Marchelli, R. (2009) Simultaneous detection of Escherichia coli O175:H7, Salmonella spp., and Listeria monocytogenes by multiplex PCR. Food Control 20, 733-738.
Gomez, P., Pagnon, M., Egea-Cortines, M., Artes, F. and Weiss, J. (2010) A fast molecular nondestructive protocol for evaluating aerobic bacterial load on fresh-cut lettuce. Food Sci Technol Int 16, 409-415.
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Hargreaves, S.K., Roberto, A.A. and Hofmockel, K.S. (2013) Reaction- and sample-specific inhibition affect standardization of qPCR assays of soil bacterial communities. Soil Biol Biochem 59, 89-97.
Heller, L.C., Davis, C.R., Peak, K.K., Wingfield, D., Cannons, A.C., Amuso, P.T. and Cattani, J. (2003) Comparison of methods for DNA isolation from food samples for detection of Shiga toxin-producing Escherichia coli by real-time PCR. Appl Environ Microb 69, 18441846.
Isonhood, J., Drake, M. and Jaykus, L.A. (2006) Upstream sample processing facilitates PCR detection of Listeria monocytogenes in mayonnaise-based ready-to-eat (RTE) salads. Food Microbiol 23, 584-590.
Johnston, L.M., Elhanafi, D., Drake, M. and Jaykus, L.A. (2005) A simple method for the direct detection of Salmonella and Escherichia coli O157:H7 from raw alfalfa sprouts and spent irrigation water using PCR. J Food Protect 68, 2256-2263.
Kawasaki, S., Fratamico, P.M., Kamisaki-Horikoshi, N., Okada, Y., Takeshita, K., Sameshima, T. and Kawamoto, S. (2011) Development of the multiplex PCR detection kit for Salmonella spp., Listeria monocytogenes, and Escherichia coli O157:H7. Jarq-Jpn Agr Res Q 45, 77-81.
Kayal, S. and Charbit, A. (2006) Listeriolysin O: a key protein of Listeria monocytogenes with multiple functions. Fems Microbiol Rev 30, 514-529.
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Larionov, A., Krause, A. and Miller, W. (2005) A standard curve based method for relative real time PCR data processing. Bmc Bioinformatics 6.
Le Monnier, A., Abachin, E., Beretti, J.-L., Berche, P. and Kayal, S. (2011) Diagnosis of Listeria monocytogenes meningoencephalitis by real-time PCR for the hly gene. J Clin Microbiol 49, 3917-3923.
Liang, N., Dong, J., Luo, L. and Li, Y. (2011) Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR. J Food Sci 76, M234-M237.
Lianou, A. and Sofos, J.N. (2007) A review of the incidence and transmission of Listeria monocytogenes in ready-to-eat products in retail and food service environments. J Food Protect 70, 2172-2198.
Liu, D. (2008) Preparation of Listeria monocytogenes specimens for molecular detection and identification. Int J Food Microbiol 122, 229-242.
Low, J.C. and Donachie, W. (1997) A review of Listeria monocytogenes and listeriosis. Vet J 153, 9-29.
Lusk, T.S., Strain, E. and Kase, J.A. (2013) Comparison of six commercial DNA extraction kits for detection of Brucella neotomae in Mexican and Central American-style cheese and other milk products. Food Microbiol 34, 100-105.
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Niemira, B.A. (2003) Radiation sensitivity and recoverability of Listeria monocytogenes and Salmonella on 4 lettuce types. J Food Sci 68, 2784-2787.
Quigley, L., O'Sullivan, O., Beresford, T.P., Ross, R.P., Fitzgerald, G.F. and Cotter, P.D. (2012) A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese. J Appl Microbiol 113, 96-105.
Ramesh, A., Padmapriya, B.P., Chandrashekar, A. and Varadaraj, M.C. (2002) Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples. Mol Cell Probe 16, 307314.
Sanchez, G., Elizaquivel, P. and Aznar, R. (2012) A single method for recovery and concentration of enteric viruses and bacteria from fresh-cut vegetables. Int J Food Microbiol 152, 9-13.
Slana, I., Kralik, P., Kralova, A. and Pavlik, I. (2008) On-farm spread of Mycobacterium avium subsp. paratuberculosis in raw milk studied by IS900 and F57 competitive real time quantitative PCR and culture examination. Int J Food Microbiol 128, 250-257.
Taylor, J.I., Hurst, C.D., Davies, M.J., Sachsinger, N. and Bruce, I.J. (2000) Application of magnetite and silica-magnetite composites to the isolation of genomic DNA. J Chromatogr A 890, 159-166.
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Tebbe, C.C. and Vahjen, W. (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant-DNA from bacteria and yeast. Appl Environ Microb 59, 2657-2665.
Tortajada, M., Vicente Martinez-Culebras, P., Navarro, V., Monzo, H. and Ramon, D. (2009) Evaluation of DNA extraction methods for PCR detection of fungal and bacterial contamination in cocoa extracts. Eur Food Res Technol 230, 79-87.
van Tongeren, S.P., Degener, J.E. and Harmsen, H.J.M. (2011) Comparison of three rapid and easy bacterial DNA extraction methods for use with quantitative real-time PCR. Eur J Clin Microbiol 30, 1053-1061.
Whitehouse, C.A. and Hottel, H.E. (2007) Comparison of five commercial DNA extraction kits for the recovery of Francisella tularensis DNA from spiked soil samples. Mol Cell Probe 21, 92-96.
Yang, H., Qu, L., Wimbrow, A.N., Jiang, X. and Sun, Y. (2007) Rapid detection of Listeria monocytogenes by nanoparticle-based immunomagnetic separation and real-time PCR. Int J Food Microbiol 118, 132-138.
Table 1 Performance evaluation of commercial DNA extraction kits used for the detection of Listeria monocytogenes in iceberg lettuce, carrot and cucumber. The quantity of extracted bacterial DNA was assessed using qPCR.
Isolation kit PowerSoil
Type of vegetables iceberg lettuce
DNA yield (μg μl-1)
Theoretical input mean (cells g-1)
Experimental output mean (cells g-1)
Isolation efficiency mean
1.83
2.91 ± 2.03 × 105
2.04 ± 0.19 × 105
70.3 ± 6.5 %
1.55
2
1
1.40 ± 0.34 × 10
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4.96 ± 2.52 × 10
35.4 ± 18.0 %
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carrot
2.07 2.35
2.91 ± 2.03 × 105 1.40 ± 0.34 × 102
1.59 ± 0.15 × 105 3.49 ± 1.16 × 101
54.6 ± 5.1 % 24.9 ± 8.3 %
cucumber
2.04 1.93
2.91 ± 2.03 × 105 1.40 ± 0.34 × 102
1.93 ± 0.22 × 105 4.15 ± 0.58 × 101
66.5 ± 7.5 % 29.6 ± 4.1 %
2.82
1.37 ± 0.14 × 106
6.53 ± 0.83 × 105
47.6 ± 6.0 %
2.18
3
2
16.0 ± 8.6 %
iceberg lettuce
PowerFood
1.37 ± 0.14 × 10 1.98 ± 0.16 × 103
5.03 ± 0.44 × 10 3.05 ± 3.44 × 102
36.7 ± 3.2 % 15.4 ± 17.3 %
cucumber
2.59 3.34
1.37 ± 0.14 × 106 1.98 ± 0.16 × 103
5.85 ± 0.80 × 105 4.15 ± 1.99 × 102
42.6 ± 5.9 % 20.9 ± 10.0 %
8.42
2.44 ± 0.14 × 105
2.22 ± 0.24 × 105
91.1 ± 9.8 %
9.22
1
4.16 ± 6.04 × 10
0
8.8 ± 12.8 %
3
2.6 ± 0.6 % -
2.44 ± 0.14 × 10 4.72 ± 2.85 × 101
6.45 ± 1.58 × 10 -*
cucumber
9.93 12.37
2.44 ± 0.14 × 105 4.72 ± 2.85 × 101
3.35 ± 0.84 × 104 -
8.35
9.15 ± 0.91 × 105
-
3.71
4.88 ± 1.33 × 102
-
carrot
3.93 6.33
5
9.15 ± 0.91 × 10 4.88 ± 1.33 × 102
5
1.89 ± 0.32 × 10 9.02 ± 1.56 × 101
20.6 ± 3.5% 18.5 ± 32.0 %
cucumber
4.51 4.84
9.15 ± 0.91 × 105 4.88 ± 1.33 × 102
3.15 ± 0.47 × 105 2.58 ± 0.86 × 102
34.4 ± 5.2 % 52.8 ± 17.6 %
2.56
4.53 ± 0.69 × 106
1.63 ± 0.70 × 104
0.4 ± 0.2 %
3.04
4.55 ± 1.87 × 10
3
-
-
carrot
2.69 3.15
4.53 ± 0.69 × 106 4.55 ± 1.87 × 103
1.19 ± 1.14× 104 -
0.3 ± 0.3 % -
cucumber
3.35 2.62
4.53 ± 0.69 × 106 4.55 ± 1.87 × 103
2.47 ± 2.36 × 103 -
0.1 ± 0.1 % -
1.21
NA#
NA
NA
0.89
NA NA NA NA NA
NA NA NA NA NA
13.7 ± 3.4 % -
carrot
3.67 5.62
cucumber
2.54 1.31
NA NA NA NA NA
9.34
NA
NA
NA
18.68
NA NA NA NA NA
NA NA NA NA NA
NA NA NA NA NA
iceberg lettuce PrepSEQ
5
5.97 4.92
iceberg lettuce Chemagic Food
4.72 ± 2.85 × 10
carrot
iceberg lettuce PowerMag Soil
5
2.52 3.06
iceberg lettuce
Nucleospin Food
3.18 ± 1.71 × 10
6
carrot
iceberg lettuce
NucliSENS
1.98 ± 0.16 × 10
carrot
20.39 31.33
cucumber
26.85 29.72
* - Negative result for amplification of hly and prs genes. # - Failure of amplification of IAC and target genes.
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Accepted Article
Table 2 Detachment efficiency of sample pre-processing for the three vegetable matrices. Mean concentration (n=3) Type of vegetable iceberg lettuce
carrot
Yield (%) Vegetable
Pellet
1.33 ± 0.61 × 105
5.22 ± 2.36 × 105
25.6
1.26 ± 0.26 × 102
5.03 ± 0.48 × 102
25.1
4.04 ± 0.66 × 105
4.04 ± 1.33 × 105
100.2
2
2
2.51 ± 0.73 × 10 cucumber
3.56 ± 0.81 × 10
70.4
4.77 ± 0.96 × 105
2.03 ± 0.40 × 105
235.1*
7.76 ± 3.07 × 102
7.49 ± 3.88 × 102
103.6
* DNA yield higher than 100% indicates a biased calculation.
Table 3 Limit of detection using the PowerSoilTM DNA Isolation kit for detection of Listeria monocytogenes in iceberg lettuce, carrot and cucumber. Type of vegetable
Spike concentration (cells g1 )
Positive detection (no. of samples)
iceberg lettuce
1.58 × 103
6/6
2
5/6
1
1.69 × 10
0/6
1.58 × 103
6/6
1.39 × 102
6/6
1.69 × 101
3/6
1.58 × 103
6/6
2
5/6
1
1/6
1.39 × 10
carrot
cucumber
1.39 × 10 1.69 × 10
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Accepted Article
Table 4 Sequences of primers and probes used in the triplex real time qPCR assay. Target gene prs
Name Lprs F Lprs R Lprs P
Type Forward Reverse Probe
hly
LMhly F LMhly R LMhly P
Forward Reverse Probe
IAC
IAC F IAC R
Forward Reverse
IAC P
Probe
Sequence 5'- GCG TTA CTC ATT TTA GTG A-3' 5'- GTT CCA TTA AAT TCT GGT TTA C-3' FAM-5'- ACA TGA CAA CCA CGG ATA CTT-3'-BHQ 5'- CAT TAG TGG AAA GAT GGA ATG-3' 5'- GTA AGC CAT TTC GTC ATC AT-3' HEX-5'- TCA AGC TTA TCC AAA TGT AAG TGC AA-3'-BHQ 5'- AGA GGA CCG GGA TAT TCG AC -3' 5'- AGG TAG TCC GAG GAA AAC TCT AAA C -3' Cy5-5'- AGG CTC TTC TAT GTT CTG ACC TTG TTG GA -3'-BHQ
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