Environmental sampling for detection of norovirus using a real-time RT-PCR Assay: A Tool for Foodborne Outbreak Investigations

ii This project is dedicated to my supportive and loving family; my husband who always had words of encouragement and motivated me to the end, my children who were understanding when I could not take them to the pool or when " Mom " just had to work, and my parents who lovingly cared for my children when I was in class, conducting research, or studying. Thank-you! iii ACKNOWLEDGMENTS I would like to thank and acknowledge my thesis committee; Dr. Hall, your mentoring and project assistance were invaluable. Jennine Wolf, your passion and dedication to serving your community was inspiring. Thank-you all. iv ABSTRACT Noroviruses (NoVs) are a leading cause of acute gastroenteritis. They are highly infectious and can be transmitted from infected individuals to susceptible individuals via multiple routes. Epidemiological investigations of outbreaks can aid public health interventions and understanding the mechanisms of transmission can prevent additional infections. This project was designed to develop a method for the collection of environmental samples during prolonged NoV outbreak investigations, and to adapt real-time RT-PCR assays to analyze environmental samples for GI and GII noroviruses. Real-time RT-PCR assays for the detection of GI and GII NoVs were developed by adapting the State Hygienic Laboratory clinical GI and GII assays to the AB 7500 Fast platform. The performance characteristics of the tests were determined, and showed the tests to be sensitive with high amplification efficiencies. Optimum efficiencies range from 95%-105%, with a 100% efficiency indicating exponential amplification of targeted nucleic acid. Analysis of the GI assay performance yielded a slope = 3.28, R 2 = 0.999 and a calculated amplification efficiency of 102%. The GII assay yielded a slope = 3.39, R 2 = 0.999 and a calculated amplification efficiency of 97%. To develop a method for the collection of environmental samples, multiple swab types were tested to determine their ability to recover NoV from laboratory spiked environmental surfaces. It was determined that foam swabs moistened with viral transport media were most effective in recovering NoV from spiked surfaces. A field test of the environmental sampling method was conducted by sampling environmental surfaces in four restaurants in one Iowa community. NoVs were not detected in these samples, however this method may be of value in future outbreak investigations and requires further study.