Fumian Tulio Machado, Leite José Paulo G, Marin Victor Augustus, Miagostovich Marize Pereira
Laboratory of Comparative Virology, Oswaldo Cruz Institute, Fiocruz, Rio de Janeiro, Brazil.
J Virol Methods. 2009 Jan;155(1):39-43. doi: 10.1016/j.jviromet.2008.09.026. Epub 2008 Nov 8.
Noroviruses (NoVs) are recognized as the most common agents of outbreaks of food-borne viral gastroenteritis and the efficiency of different methods for detection of NoVs from food matrices have been tested in several laboratories worldwide. The aim of this study was to develop a rapid and sensitive method for recovery of NoVs by using a filtration concentration method followed by PCR amplification for detection of NoVs from cheese and fresh lettuce. Experimentally, a fecal suspension containing different number of NoVs copies was spiked in the food surface and extracted by a direct elution using a Stomacher apparatus. An Ozone-Safe solvent Vertrel XF treatment was included for cheese samples for removing particulate matter. The watery phase was collected and the viral concentration was performed by the adsorption-elution method using negatively charged membranes with inorganic solvents in a Stericup and afterwards ultrafiltered using a Centriprep Concentrator 50 to obtain a final volume of 2ml. RNA isolation was carried out with the QIAamp Viral RNA Mini Kit available commercially and reverse transcription was carried out with a Pd(N6) random primer. Real time quantitative PCR (TaqMan) and qualitative PCR were used for molecular detection of NoVs. The recovery rate of NoVs ranged from 5.2 to 72.3% in lettuce and from 6 to 56.3% in cheese. The results indicate that this method is suitable for detection of NoVs contamination in food and will help establish the cause and source of NoVs outbreaks of food-borne illness.
