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Evaluation of DNA Extraction Methods for Culture-Independent Real-Time PCR-Based Detection of Listeria monocytogenes in Cheese

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Abstract

Listeria monocytogenes, the causative agent of listeriosis, is a foodborne pathogen with significant public health and economic impacts. The control of pathogen presence in food requires rapid and sensitive methods. Real-time PCR is considered to be a fast and accurate tool for the detection of foodborne pathogens. A crucial step for the success of a culture-independent PCR-based detection is the template DNA extraction. Two open-formula extraction procedures and five kits were used to extract DNA prior to detection of L. monocytogenes in cheese. The extraction procedures were evaluated in quantitative PCR analyses of the total extracted bacterial DNA from cheese samples artificially contaminated with L. monocytogenes. The results of the study suggested that all seven extraction procedures can be used to obtain intact and amplifiable bacterial DNA, although with different L. monocytogenes detection limits in four types of cheese. PowerFood Microbial DNA Isolation Kit and DNeasy Mericon Food Kit performed the best with detection limits of 2.1 × 102 to 4.7 × 102 CFU/g in all analysed cheese samples. No differences in PCR detection limits of low numbers were found when different L. monocytogenes serotypes (1/2a, 1/2b, 1/2c and 4b) were used, despite the relatively lower DNA extraction efficiency observed in L. monocytogenes serotype 1/2c strain. Six out of seven evaluated methods demonstrated quantitative response with linear calibration lines and quantification limits of 4.6 × 102 to 9.3 × 103 CFU/g. The results demonstrated that culture-independent sample preparation and total DNA extraction, followed by target-specific DNA amplification have a potential to direct determination of low L. monocytogenes numbers in cheese within hours.

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References

  1. Abdelhai MH, Hassanin HAM, Sun X (2016) Comparative study of rapid DNA extraction methods of pathogenic bacteria. Am J Biosci Bioeng 4:1–8

  2. Ausubel FM, Brent R, Kingston RE et al (2000) In: Current protocols in Molecular Biology, 1. John Wiley and Sons Inc., New York, USA

  3. Bhunia AK (2014) One day to one hour: How quickly can foodborne pathogens be detected? Future Microbiol 9:935–946

  4. Bonilauri P, Bardasi L, Leonelli R et al (2016) Detection of food hazards in foods: Comparison of real time polymerase chain reaction and cultural methods. Ital J Food Safety 5:37–40

  5. Cabicarová T, Kaclíková E, Mader A et al (2016) Improvement of the detection sensitivity for Staphylococcus aureus in spices and herbs. Food Anal Methods 9:1980–1984

  6. Cheng HR, Jiang N (2006) Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol Lett 28:55–59

  7. Comission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs (2005) Off J Eur Union, L 338:1–26

  8. Dalmasso M, Bolocan AS, Hernandez M et al (2014) Comparison of polymerase chain reaction methods and plating for analysis of enriched cultures of Listeria monocytogenes when using the ISO11290-1 method. J Microbiol Methods 98:8–14

  9. De Medici D, Croci L, Delibato E et al (2003) Evaluation of DNA extraction methods for use in combination with SYBR Green I real-time PCR to detect Salmonella enterica serotype Enteritidis in poultry. Appl Environ Microbiol 69:3456–3461

  10. EFSA (2018) The European Union summary report on trends and sources of zoonoses, zoonotic agents and foodborne outbreaks in 2017. EFSA Journal 2018 16(12):5500. https://doi.org/10.2903/j.efsa.2018.5500

  11. Elizaquivel P, Aznar R (2008) A multiplex RTi-PCR reaction for simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus on fresh, minimally processed vegetables. Food Microbiol 25:705–713

  12. Fusco V, Quero GM (2014) Culture-dependent and culture-independent nucleic-acid-based methods used in the microbial safety assessment of milk and dairy products. Compr Rev Food Sci F 13:493–537

  13. Healey A, Furtado A, Cooper T, Henry J (2014) Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species. Plant Methods 10:21–28

  14. Heuer H, Hartung K, Wieland G, Kramer I, Smalla K (1999) Polynucleotide probes that target a hypervariable region of 16S rRNA genes to identify bacterial isolates corresponding to bands of community fingerprints. Appl Environ Microbiol 65:1045–1049

  15. Hoorfar J (2011) Rapid detection, characterization, and enumeration of foodborne pathogens. APMIS 119(Suppl.133):1–24

  16. ISO 11290-1:2017 Microbiology of the food chain – Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. – Part 1: Detection method, International Organization for Standardization, Geneva.

  17. ISO 11290-2:2017 Microbiology of the food chain – Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. – Part 2: Enumeration method, International Organization for Standardization, Geneva.

  18. ISO 4832: 2006. Microbiology of the food and animal feeding stuffs – Horizontal method for the enumeration of coliforms – Part 1: Colony count technique, International Organization for Standardization, Geneva.

  19. ISO 4833-1:2013. Microbiology of the food chain – Horizontal method for the enumeration of microorganisms – Part 1: Colony count at 30 °C by the pour plate technique, International Organization for Standardization, Geneva.

  20. Jany JL, Barbier G (2008) Culture-independent methods for identifying microbial communities in cheese. Food Microbiol 25:839–848

  21. Kuchta T, Knutsson R, Fiore A et al (2014) A decade with nucleic acid-based microbiological methods in safety control of foods. Lett Appl Microbiol 59:263–271

  22. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. John Wiley and Sons, New York, pp 115–175

  23. López-Campos G, Martínez-Suárez J, Aguado-Urda M, López-Alonso V (2012) Detection, identification, and analysis of foodborne pathogens. In: Microarray detection and characterization of bacterial foodborne pathogens. Springer, US, pp 13–32

  24. Lusk TS, Strain E, Kase JA (2013) Comparison of six commercial DNA extraction kits for detection of Brucella neotomae in Mexican and Central American-Sytoe cheese and other milk products. Food Microbiol 34:100–105

  25. Mandal P, Biswas A, Choi K, Pal U (2011) Methods for rapid detection of foodborne pathogens: as overview. Am J Food Technol 6:87–102

  26. Nguyen LT, Gillespie BE, Nam HM et al (2004) Detection of Escherichia coli O157:H7 and Listeria monocytogenes in beef products by real-time polymerase chain reaction. Foodborne Pathog Dis 1:231–240

  27. Oravcová K, Kaclíková E, Krascsenicsová K et al (2006) Detection and quantification of Listeria monocytogenes by 5′-nuclease polymerase chain reaction targeting the actA gene. Lett Appl Microbiol 42:15–18

  28. Oravcová K, Kuchta T, Kaclíková E (2007) A novel real-time PCR-based method for the detection of Listeria monocytogenes in food. Lett Appl Microbiol 45:568–573

  29. Oravcová K, Trnčíková T, Kuchta T, Kaclíková E (2008) Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua. J Appl Microbiol 104:429–437

  30. Pirondini A, Bonas U, Maestri E et al (2010) Yield and amplificability of different DNA extraction procedures for traceability in the dairy food chain. Food Control 21:663–668

  31. Pontiroli A, Travis ER, Sweeney FP et al (2011) Pathogen quantitation in complex matrices: a multi-operator comparison of DNA extraction methods with a novel assessment of PCR inhibition. PLoS One 6:e17916

  32. Quigley L, O’Sullivan O, Beresford TP et al (2012) A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese. J Appl Microbiol 113:96–105

  33. Rodriguez-Lazaro D, Gonzales-García P, Gattuso A, Gianfranceschi MV, Hernandez M (2014) Reducing time in the analysis of Listeria monocytogenes in meat, dairy and vegetable products. Int J Food Microbiol 184:98–105

  34. Rodriguez-Lazaro D, Gonzales-García P, Valero A, Hernandez M (2015) Application of the SureTect detection methods for Listeria monocytogenes and Listeria spp. in meat, dairy, fish, and vegetable products. Food Anal Methods 8:1–6

  35. Rossmanith P, Wagner M (2011) The challenge to quantify Listeria monocytogenes – a model leading to new aspects in molecular biological food pathogen detection. J Appl Microbiol 110:605–617

  36. Stefanova P, Taseva M, Georgieva T, Gotcheva V, Angelov A (2013) A modified CTAB method for the DNA extraction from soybean and meat products. Biotechnol Biotechnol Equip 27:3803–3810

  37. Välimaa AN, Tilsala-Timisjärvi A, Virtanen E (2015) Rapid identification and detection methods for Listeria monocytogenes in the food chain – A review. Food Control 55:103–144

  38. Véghová A, Minarovičová J, Kaclíková E (2017) Prevalence and trecing of persistent Listeria monocytogenes strains in meat processing facility production chain. J Food Saf 37:e12315

  39. Vojkovska H, Kubikova I, Kralik P (2014) Evaluation of DNA extractions methods for PCR-based detection of Listeria monocytogenes from vegetables. Lett Appl Microbiol 60:265–272

  40. Wang Y, Salazar JK (2016) Culture-independent rapid detection methods for bacterial pathogens and toxins in food matrices. Comp Rev. Food Sci F 15:183–205

  41. Widjojoatmodjo MN, Fluit AC, Verhoef J (1995) Molecular identification of bacteria by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S rRNA gene. J Clin Microbiol 33:2601–2606

  42. Yang Y, Xu F, Xu H, Aguilar ZP et al (2013) Magnetic nano-beads based separation combined with propidium monoazide treatment and multiplex PCR assay for simultaneous detection of viable Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in food products. Food Microbiol 34:418–424

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Acknowledgements

Authors thank Dr. T. Kuchta for fruitful discussions and valuable suggestions.

Funding

This work was supported by Slovak Research and Development Agency under the contract No. APVV-16-0119.

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Correspondence to Kaclíková Eva.

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Jana Minarovičová declares that she has no conflict of interest. Adriana Véghová declares that she has no conflict of interest. Eva Kaclíková declares that she has no conflict of interest.

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Jana, M., Adriana, V. & Eva, K. Evaluation of DNA Extraction Methods for Culture-Independent Real-Time PCR-Based Detection of Listeria monocytogenes in Cheese. Food Anal. Methods 13, 667–677 (2020). https://doi.org/10.1007/s12161-019-01686-2

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Keywords

  • Listeria monocytogenes
  • Detection
  • DNA extraction
  • Real-time PCR
  • Culture-independent
  • Cheese