A Method for the Detection of Cryptosporidium parvum Oocysts in Milk Based on Microfiltration and Real-Time Polymerase Chain Reaction |
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| Authors: | Jana Minarovi?ov?? Janka Lopa?ovsk?? ?ubom??r Val??k Tom??? Kuchta |
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| Affiliation: | (1) Department of Microbiology and Molecular Biology, Food Research Institute, P.O. Box 25, 82475 Bratislava, Slovakia;(2) Department of Nutrition and Food Assessment, Faculty of Chemical and Food Technology, Slovak Technical University, Radlinsk?ho 9, 812 37 Bratislava, Slovakia; |
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| 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. |
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