University of New Hampshire
Mentor: Dr. Aaron Margolin, Department of Microbiology
Rapid Detection of Enteric Viruses in Water Through Cell Suspension and Integrated Cell Culture Real Time RT-PCR
Enteric viruses are found in sewage, septic systems, recreational waters, storm runoff, and both treated and untreated waters. The potential health hazards associated with enteric viruses encompass a wide range of symptoms, from diarrhea to aseptic meningitis to paralysis. Therefore, water quality is vital to public health. Water quality is accessed by the U.S. Environmental Protection Agency and enforced by the Safe Water Drinking Act. As mandated by this act, if a monitoring protocol cannot adequately detect the presence of a wide range of pathogenic viruses, a treatment process is employed instead. Currently, treatment processes are in effect to safeguard against pathogenic organisms. The standard method for the growth and detection of infectious viruses is cell culture. Cell culture is costly, labor intensive and time-consuming. Polymerase chain reaction (PCR) is a rapid and sensitive assay used for viral detection, but it does not detect infectivity. The proposed research will investigate the use of both these assays; cell culture in combination with PCR, known as integrated cell culture-polymerase chain reaction (ICC-PCR). Poliovirus will be propagated in Buffalo Green Monkey Kidney cells (BGMK) and then detected by the traditional plaque assay and by ICC-PCR assay. To enhance recovery of the virus, three different cell lines, BGMK cells, Rhesus Monkey Kidney cells (MA104), and Caucasian Colon Adenocarcinoma cells (CaCo) will be used for the ICC-PCR technique, while BGMK cells will be used for the plaque assay. Various concentrations of poliovirus will be incubated for different times to determine the minimum incubation time and concentration of virus that can be subsequently detected. The results of this research will elucidate the minimum amount of incubation time necessary for detection of low levels of virus found in contaminated water using the ICC-PCR technique.