A Method for the Detection of Cryptosporidium parvum Oocysts in Milk Based on Microfiltration and Real-Time Polymerase C
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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
Received: 26 February 2010 / Accepted: 31 March 2010 / Published online: 24 April 2010 # Springer Science+Business Media, LLC 2010
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
Introduction Cryptosporidium parvum is a protozoan parasite that is pathogenic to humans. It is a causative agent of cryptospoJ. Minarovičová : J. Lopašovská : T. Kuchta (*) Department of Microbiology and Molecular Biology, Food Research Institute, P.O. Box 25, 82475 Bratislava, Slovakia e-mail: [email protected] Ľ. Valík Department of Nutrition and Food Assessment, Faculty of Chemical and Food Technology, Slovak Technical University, Radlinského 9, 812 37 Bratislava, Slovakia
ridiosis, which is a self-limiting watery diarrhoea lasting up to two weeks. Contaminated water and contaminated raw food are considered important vehicles for the parasite (Laberge et al. 1996a; Deng & Cliver 1999). Because of C. parvum chlorine resistance, the microorganism is a particular threat to drinking water supplies. Recently, a foodborne cryptosporidiosis outbreak was reported for the first time (The Community Summary Report on Trends and Sources of Zoonoses 2010). C. parvum oocysts are commonly found in dairy farms, and may be transmitted to humans by raw milk and dairy products (Harp et al. 1996; Dawson et al. 2004). However, the presence of C. parvum in milk is difficult to detect because of the small numbers of oocysts present in the limited size of a sample, which requires detection methods with high sensitivity and specificity (Laberge et al. 1996a; Deng & Cliver 1999; Smith et al. 2007). Several methods have been published for C. parvum detection in milk, beverages and food samples, employing oocyst concentration and molecular detection (Laberge et al. 1996b; Di Pinto & Tantillo 2002; Frazar & Orlandi 2007; Smith & Nichols 2010). Sensitive molecular detection methods for the parasite, using real-time polymerase chain reaction (PCR), have been developed (Fontaine & Guillot 2003; Guy et al. 2003; Minarov
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