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Food Analytical Methods

, Volume 4, Issue 1, pp 116–120 | Cite as

A Method for the Detection of Cryptosporidium parvum Oocysts in Milk Based on Microfiltration and Real-Time Polymerase Chain Reaction

  • Jana Minarovičová
  • Janka Lopašovská
  • Ľubomír Valík
  • Tomáš Kuchta
Article

Abstract

A method for the detection of Cryptosporidium parvum oocysts in milk was developed on the basis of optimizing microfiltration and elution of the material from the filter, and using a previously developed highly sensitive downstream detection by real-time polymerase chain reaction. The method involves heating of milk to 40°C, microfiltration through a membrane microfilter made of a mixture of cellulose acetate and cellulose nitrate (pore size, 3.0 μm), elution of the material from the filter by a solution containing sodium pyrophosphate and Tween 80 in a shaker, rapid DNA extraction using a Chelex-based agent, and single-tube nested real-time PCR. The detection limit of the method is 10 C. parvum oocysts per 100 ml of milk. The developed method may be useful for specific and sensitive control of contamination of milk by C. parvum oocysts.

Keywords

Cryptosporidium parvum Milk Filtration PCR 

Notes

Acknowledgement

This work was supported by Slovak Research and Development Agency, project APVT 27-044402.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Jana Minarovičová
    • 1
  • Janka Lopašovská
    • 1
  • Ľubomír Valík
    • 2
  • Tomáš Kuchta
    • 1
  1. 1.Department of Microbiology and Molecular BiologyFood Research InstituteBratislavaSlovakia
  2. 2.Department of Nutrition and Food Assessment, Faculty of Chemical and Food TechnologySlovak Technical UniversityBratislavaSlovakia

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