P3-56 Development of a Human Norovirus Indicator Using Enterococcus sp. 16S rRNA and Bacteroides fragilis gyrB Gene as a Rapid Detection (Monitoring) Tool in Various Environments

Wednesday, July 31, 2013
Exhibit Hall (Charlotte Convention Center)
Jia Wei Yeap, The Ohio State University, Columbus, OH
Jiyoung Lee, The Ohio State University, Columbus, OH
Richard Linton, North Carolina State University, Raleigh, NC
Jianrong Li, The Ohio State University, Columbus, OH
Introduction: Human norovirus (HuNoV) is the cause of more than 21 million cases of acute gastroenteritis in the US and it can persist at any point from farm to fork chain. However, there has been a lack of a rapid indicating system for HuNoV contamination. It has been suggested that the ratio between the phyla Firmicutes and Bacteroidetes is significantly shifted when enteric viruses are present, including HuNoV.

Purpose: The ratio of Enterococcus sp. (16S rRNA) and human-specific Bacteroides fragilis (gyrB gene) in HuNoV-infected and non-infected human fecal samples as a means to indicate the presence of HuNoV.

Methods: Total DNA and RNA were extracted from 31 HuNoV-infected samples and 17 non-infected human fecal samples using commercial extraction kits. Reverse transcription was performed on RNA template to synthesize HuNoV-specific complimentary DNA (cDNA). Extracted DNA and synthesized cDNA were quantified using real-time Polymerase Chain Reaction (PCR) system. Primers were designed to target the VP1, 16S rRNA, and gyrB genes for HuNoV, Enterococcus sp. and B. fragilis, respectively. Threshold cycles (CT) values were plotted against the log-RNA copies for HuNoV and log-transformed cell counts [Colony Forming Unit (CFU)/g-feces]. Enterococcus sp. and B. fragilis counts were compared using Analysis of Variance.

Results: There were statistically significant differences (P < 0.05) in the CFU of Enterococcus sp. and B. fragilis between HuNoV infected and non-infected fecal samples. In infected fecal samples, Enterococcus sp. was 1.37 log CFU/g-feces higher while B. fragilis was 1.35 log CFU/g-feces lower, averagely. When normalized with B. fragilis, a linear relationship was observed between Enterococcus sp. and HuNoV (R2=0.47).

Significance: These results suggest that there is a significant change in the ratio of Enterococcus sp. and B. fragilis in HuNoV-infected fecal samples. With this newly developed, rapid PCR-based detection system, the presence of HuNoV and other enteric human viruses can be effectively monitored in broad spectrum of environments in a timely manner, such as food, water, and environmental samples.