Application of an Ultracentrifugation-based Method for Detection of Feline Calicivirus (a Norovirus Surrogate) in Experi
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Application of an Ultracentrifugation-based Method for Detection of Feline Calicivirus (a Norovirus Surrogate) in Experimentally Contaminated Delicatessen Meat Samples Artur Rzeżutka & Marta Chrobocińska & Agnieszka Kaupke & Beata Mizak
Received: 7 September 2007 / Accepted: 30 October 2007 / Published online: 9 February 2008 # Springer Science + Business Media, LLC 2007
Abstract The aim of this study was the application of an ultracentrifugation-based method for detection of feline calicivirus (FCV) in experimentally contaminated meat samples of roast pork chop, salami and gammon. Virus particles were liberated from food items by washing in 0.5-M glycine containing 1% bovine albumin. Food debris were removed by slow-speed centrifugation, and viruses were subsequently sedimented by ultracentrifugation. The recovery of infectious virus particles was determined by cell culture. Furthermore, RNA was extracted from virus particles and reverse-transcription polymerase chain reaction ((RT)PCR) was performed on the nucleic acid extracts. To demonstrate the efficiency of this method in removal of inhibitors from food sample extracts, an internal amplification control was constructed and incorporated into FCV (RT) PCR. Virus recovery for smoked meat, salami and cooked ham was 12.5%, 3.4% and 5.9%, respectively. The ultracentrifugation-based method is efficient in eliminating or reducing the level of inhibitory substances and can be applied for calicivirus extraction and concentration from delicatessen meat samples. Keywords Feline Calicivirus . Meat . Virus Extraction . Ultracentrifugation
A. Rzeżutka (*) : M. Chrobocińska : A. Kaupke : B. Mizak Department of Food and Environmental Virology, National Veterinary Research Institute, Al. Partyzantów 57, 24-100 Puławy, Poland e-mail: [email protected]
Introduction The foodborne viral infections in humans are currently recognised worldwide. They are mostly caused by noroviruses (NoV) and hepatoviruses (Gerba and Kayed 2003; Atreya 2004; Sanchez et al. 2007). Water and uncooked food are considered as important sources or vehicles of virus transmission to the humans. The increasing number of cases of human gastroenteritis requires continuous development of new diagnostic methods, allowing detection of these viral agents in different foodstuffs. Methods used for the detection of foodborne viruses consist of two main stages: extraction and concentration of viruses from food samples and detection of viruses using nucleic acid amplification techniques. The extraction of viruses is often carried out by homogenisation or by washing of the food sample in the extractant solutions that allow removal of viruses from food surfaces. Each stage of the method should ensure the maximum virus recovery, especially when the samples are contaminated with low numbers of virus particles. In addition, food represents the complex analytical matrix composed of many substances, which can be released to the extractant solution during the extraction process. These substances can decrease the efficienc
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