Skip to main content
SpringerLink
Log in

Nucleic Acid Sample Preparation from Dairy Products and Milk

  • Protocol
Sample Preparation Techniques for Soil, Plant, and Animal Samples

Part of the book series: Springer Protocols Handbooks ((SPH))

  • 3370 Accesses

Abstract

The isolation of RNA and DNA of sufficient quantity, of an optimal intact length (quality), and without contaminants (purity) is important for a range of research initiatives including pathogen detection during an outbreak and the study of the background microflora in dairy products. Nucleic acid isolation is a multistep procedure that may begin with the separation of cells from the matrix, followed by cell lysis, then nucleic acid extraction and recovery. Herein we compare and evaluate published methods used to extract nucleic acids from various dairy matrices. Specific topics include removal of matrix components by centrifugation, organic solvents, and Proteinase K treatment along with cell lysis accomplished by enzymatic (e.g., lysozyme), physical (e.g., freeze/thawing, boiling, bead-beating), and chemical (e.g., detergent) means. Also mentioned is the recent use of automated nucleic acid extraction procedures.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Powell H, Gooding C, Garrett S et al (1994) Proteinase inhibition of the detection of Listeria monocytogenes in milk using the polymerase chain-reaction. Lett Appl Microbiol 18(1):59–61

    Article  CAS  Google Scholar 

  2. Katcher H, Schwartz I (1994) A distinctive property of Tth DNA polymerase: enzymatic amplification in the presence of phenol. Biotechniques 16(1):84–92

    CAS  PubMed  Google Scholar 

  3. Rossen L, Nørskov P, Holmstrøm K et al (1992) Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions. Int J Food Microbiol 17(1):37–45

    Article  CAS  PubMed  Google Scholar 

  4. Dongyou L (ed) (2010) Molecular detection of foodborne pathogens. CRC, Boca Raton, FL

    Google Scholar 

  5. Tamarapu S, McKillip J, Drake M (2001) Development of a multiplex polymerase chain reaction assay for detection and differentiation of Staphylococcus aureus in dairy products. J Food Prot 64(5):664–668

    CAS  PubMed  Google Scholar 

  6. Wilson I (1997) Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63(10):3741–3751

    PubMed Central  CAS  PubMed  Google Scholar 

  7. Bickley J, Short J, McDowell D, Parkes H (1996) Polymerase chain reaction (PCR) detection of Listeria monocytogenes in diluted milk and reversal of PCR inhibition caused by calcium ions. Lett Appl Microbiol 22(2):153–158

    Article  CAS  PubMed  Google Scholar 

  8. Nielson H (2011) Chapter 2. Working with RNA. In: RNA: methods and protocols. Humana

    Google Scholar 

  9. Roder B, Fruhwirth K, Vogl C et al (2010) Impact of long-term storage on stability of standard DNA for nucleic acid-based methods. J Clin Microbiol 48(11):4260–4262

    Article  PubMed Central  PubMed  Google Scholar 

  10. Monnet C, Matijasic B (2012) Application of PCR-based methods to dairy products and to non-diary probiotic products. In: Hernandez-Rodriguez P (ed) Polymerase chain reaction. Intech. doi: 10.5772/2204

    Google Scholar 

  11. Paul M, Van Hekken DL, Brewster J (2013) Detection and quantitation of Escherichia coli O157 in raw milk by direct qPCR. Int Dairy J 32(2):53–60

    Article  CAS  Google Scholar 

  12. Bonaiti C, Parayre S, Irlinger F (2006) Novel extraction strategy of ribosomal RNA and genomic DNA from cheese for PCR-based investigations. Int J Food Microbiol 107(2):171–179

    Article  CAS  PubMed  Google Scholar 

  13. Quigley L, O’Sullivan O, Beresford T et al (2012) A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese. J Appl Microbiol 113(1):96–105

    Article  CAS  PubMed  Google Scholar 

  14. O’Mahony J, Hill C (2004) Rapid real-time PCR assay for detection and quantitation of mycobacterium avium subsp. paratuberculosis DNA in artificially contaminated milk. Appl Environ Microbiol 70(8):4561–4568

    Article  PubMed Central  PubMed  Google Scholar 

  15. Thomas M, Shields M, Hahn K et al (2013) Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples. J Appl Microbiol 115(1):156–162

    Article  CAS  PubMed  Google Scholar 

  16. Psifidi A, Dovas C, Banos G (2010) A comparison of six methods for genomic DNA extraction suitable for PCR-based genotyping applications using ovine milk samples. Mol Cell Probes 24(2):93–98

    Article  CAS  PubMed  Google Scholar 

  17. Marianelli C, Martucciello A, Tarantino M et al (2008) Evaluation of molecular methods for the detection of Brucella species in water buffalo milk. J Dairy Sci 91(10):3779–3786

    Article  CAS  PubMed  Google Scholar 

  18. Foley C, O’Farrelly C, Meade K (2011) Technical note: comparative analyses of the quality and yield of genomic DNA from invasive and noninvasive, automated and manual extraction methods. J Dairy Sci 94(6):3159–3165

    Article  CAS  PubMed  Google Scholar 

  19. Mertens K, Freund L, Schmoock G et al (2014) Comparative evaluation of eleven commercial DNA extraction kits for real-time PCR detection of Bacillus anthracis spores in spiked dairy samples. Int J Food Microbiol 170:29–37

    Article  CAS  PubMed  Google Scholar 

  20. Lusk T, Strain E, Kase JA (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(1):100–105

    Article  CAS  PubMed  Google Scholar 

  21. Pinto A, Forte V, Guastadisegni M et al (2007) A comparison of DNA extraction methods for food analysis. Food Control 18(1):76–80

    Article  Google Scholar 

  22. Amagliani G, Giammarini C, Omiccioli E, Brandi G, Magnani M (2007) Detection of Listeria monocytogenes using a commercial PCR kit and different DNA extraction methods. Food Control 18(9):1137–1142

    Article  CAS  Google Scholar 

  23. 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(5):663–668

    Article  CAS  Google Scholar 

  24. Nemeth A, Wurz A et al (2004) Sensitive PCR analysis of animal tissue samples for fragments of endogenous and transgenic plant DNA. J Agric Food Chem 52(20):6129–6135

    Article  CAS  PubMed  Google Scholar 

  25. Moore D, Dowhan D (2002) Purification and concentration of DNA from aqueous solutions. Curr Protoc Mol Biol. doi:10.1002/0471142727.mb0201as59

    Google Scholar 

  26. Saunders G, Rossi J, Keer J et al (2008) Chapter 4, DNA extraction. In: Keer J, Birch L (eds) Essentials of nucleic acid analysis—a robust approach. Royal Society of Chemistry, Cambridge. ISBN 978-1-61583-359-7

    Google Scholar 

  27. Sweeney P, Walker J, Burrell M (1993) Chapter 16: proteinase K (EC 3.4.21.14). In: Burrell M (ed) Enzymes of molecular biology, vol 16, Methods in molecular biology. Humana, Totowa, NJ, pp 305–311

    Chapter  Google Scholar 

  28. Bajorath J, Hinrichs W, Saenger W (1988) The enzymatic activity of proteinase K is controlled by calcium. Eur J Biochem 176(2):441–447

    Article  CAS  PubMed  Google Scholar 

  29. Morita H, Kuwahara T, Ohshima K et al (2007) An improved DNA isolation method for metagenomic analysis of the microbial flora of the human intestine. Microbes Environ 22(3):214–222

    Article  Google Scholar 

  30. Ezaki T, Suzuki S (1982) Achromopeptidase for lysis of anaerobic gram-positive cocci. J Clin Microbiol 16(5):844–846

    PubMed Central  CAS  PubMed  Google Scholar 

  31. Vazquez-Laslop N, Lee H, Hu R et al (2001) Molecular sieve mechanism of selective release of cytoplasmic proteins by osmotically shocked Escherichia coli. J Bacteriol 183(8):2399–2404

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Sowmya N, Thakur M, Manonmani H (2012) Rapid and simple DNA extraction method for the detection of enterotoxigenic Staphylococcus aureus directly from food samples: comparison of PCR and LAMP methods. J Appl Microbiol 113(1):106–113

    Article  CAS  PubMed  Google Scholar 

  33. Mounier J, Le Blay G et al (2010) Application of denaturing high-performance liquid chromatography (DHPLC) for yeasts identification in red smear cheese surfaces. Lett Appl Microbiol 51(1):18–23

    CAS  PubMed  Google Scholar 

  34. Monnet C, Ulvé V et al (2008) Extraction of RNA from cheese without prior separation of microbial cells. Appl Environ Microbiol 74(18):5724–5730

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  35. Odumeru J (2001) Use of the bead beater for preparation of Mycobacterium paratuberculosis template DNA in milk. Can J Vet Res 65(4):201–205

    PubMed Central  CAS  PubMed  Google Scholar 

  36. Li F, Hullar M, Lampe J (2007) Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut microbiota. J Microbiol Methods 68(2):303–311

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  37. Rantakokko-Jalava K, Jalava J (2002) Optimal DNA isolation method for detection of bacteria in clinical specimens by broad-range PCR. J Clin Microbiol 40(11):4211–4217

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Bai Y, Song M, Cui Y, Shi C, Wang D, Paoli G, Shi X (2013) A rapid method for the detection of foodborne pathogens by extraction of a trace amount of DNA from raw milk based on amino-modified silica-coated magnetic nanoparticles and polymerase chain reaction. Anal Chim Acta 787:93–101

    Article  CAS  PubMed  Google Scholar 

  39. Peirson S, Butler J (2007) RNA extraction from mammalian tissues. In: Ezio R, Walker J (eds) Circadian rhythms: methods and protocols, Methods in molecular biology. Humana, Totowa, NJ, pp 315–327. doi:10.1007/978-1-59745-257-1_22

    Google Scholar 

  40. Garcia-Nogales P, Serrano A, Secchi S, Gutierrez S, Aris A (2010) Comparison of commercially-availably RNA extraction methods for effective bacterial RNA isolation from milk spiked samples. Electronic Journal Biotechnol 13 (5). doi:10.2225/vol13-issue5-fulltext-10

    Google Scholar 

  41. Calles-Enriquez M, Ladero V, Fernandez M, Cruz Martin M, Alvarez M (2010) Extraction of RNA from fermented milk products for in situ gene expression analysis. Anal Biochem 400(2):307–309

    Article  CAS  PubMed  Google Scholar 

  42. Ablain W, Soulier S, Causeur D et al (2009) A simple and rapid method for the disruption of Staphylococcus aureus, optimized for quantitative reverse transcriptase applications: application for the examination of Camembert cheese. Dairy Sci Technol 89(1):69–81

    Article  CAS  Google Scholar 

  43. Derzelle S, Grine A, Madic J, de Garam C, Vingadassalon N, Dilasser F, Jamet E, Auvray F (2011) A quantitative PCR assay for the detection and quantification of Shiga toxin-producing Escherichia coli (STEC) in minced beef and dairy products. Int J Food Microbiol 151(1):44–51

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Microbiology, Office of Regulatory Science, US FDA, 5100 Paint Branch Parkway, HFS-711, College Park, MD, 20740, USA

    Julie Ann Kase Ph.D. & Tina Lusk Pfefer

Authors
  1. Julie Ann Kase Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tina Lusk Pfefer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tina Lusk Pfefer .

Editor information

Editors and Affiliations

  1. Department of Engineering Design Technology, Cerritos College, Norwalk, California, USA

    Miodrag Micic

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Kase, J.A., Pfefer, T.L. (2016). Nucleic Acid Sample Preparation from Dairy Products and Milk. In: Micic, M. (eds) Sample Preparation Techniques for Soil, Plant, and Animal Samples. Springer Protocols Handbooks. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3185-9_16

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3185-9_16

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3184-2

  • Online ISBN: 978-1-4939-3185-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation