A Comparison of Centrifugal Forces to Reduce the Inhibitory Effects of Food Matrixes on Reverse Transcriptase Polymerase Chain Reaction for the Detection of Food Borne Viruses

The CDC estimated that foodborne infections resulted in approximately 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths per year in the United States (Mead, 1999). There are over 200 known diseases caused by viruses, bacteria, parasites, toxins, metals, or prions that can be transmitted through food. Of these illnesses caused by foodborne disease, the CDC estimates that 38.6 million cases are from identifiable pathogens and 30.9 million of these cases are caused by viruses. Hence, approximately 80% of foodborne illnesses of known etiology result from viral transmission (Mead, 1999). Viral gastrointestinal illness may be caused by virus families such as: enterovirus, rotavirus, calicivirus, astrovirus, or norovirus. These viruses are highly contagious and are spread through the fecal-oral route; transmission vehicles include contaminated food or beverages, infected food handlers, fomites or close contact with an infected individual (FDA Bad Bug Book, 2003). Until recently, there have been few studies concentrating on viruses found in or on foods. There are several technical difficulties that hinder progress in detecting viral agents from foods. One of these problems is the presence of matrix inhibitors. Substances responsible for matrix inhibition include humic acid, polysaccharides, myoglobins, metal ions, glycogen, and lipids (Monpoeho, 2001). These substances in foods produce smearing of the RT-PCR amplicon bands on agarose gels. Several methods to reduce inhibitory compounds utilize multiple toxic reagents in the procedure. In this study, varying centrifugal forces were tested at different steps of the virus extraction/concentration procedure to reduce matrix inhibitory effects for molecular detection of norovirus and poliovirus seeded onto food surfaces. This method incorporates the rapid detection capabilities of RT-PCR with the ability to reduce or eliminate matrix inhibitors present in food, by altering the centrifugal force. Results for both viruses showed that band intensity decreased as the viral concentration decreased and no one method was superior for all food matrices. This investigation showed that matrix specific modifications to the basic protocol are required to efficiently extract viruses from the surface of foods. Each food should be assessed to determine modifications to the standard method that would be optimal for viral concentration and extraction