Date of Award
Winter 1999
Project Type
Dissertation
Program or Major
Microbiology
Degree Name
Doctor of Philosophy
First Advisor
Aaron B Margolin
Abstract
Many wastewater treatment plants generate more sludge than can be disposed of by conventional means. The United States Environmental Protection Agency (USEPA) has encouraged communities to dispose of sludge by land application. Sludge may contain enteric viruses that are known to survive for long periods of time in sludge-amended soil and can travel great distances, potentially contaminating surface and ground water.
Standard cell culture methods for the detection of enteric viruses are costly and results are not obtained for 30 or more days. The development of methods that provide results more quickly and with lower cost are needed.
A 32P labeled RNA probe was developed for the detection of poliovirus in sludge. The probe detected 10 fg of poliovirus RNA transcripts and 90 pfu of poliovirus type 1 (LSc). RNA probe and plaque assays were used to evaluate beef extract elution methods for the isolation of poliovirus from sludge. Homogenization of sludge after the addition of beef extract powder at a pH of 7.0 resulted in the highest recovery of seeded poliovirus. Additionally, proteinase k digestion was found to result in a greater detection sensitivity than organic extractions.
Small reaction volumes and the presence of inhibitors in environmental samples have limited the use of the highly sensitive and rapid technique of RT-PCR. A magnetic separation procedure using oligo dT paramagnetic beads was developed to capture enterovirus RNA from 900 mul of sample. This method resulted in a 90-fold sample concentration, removal of RT-PCR inhibitors from lime stabilized sludges, and a detection sensitivity of 5 pfu of poliovirus type 1 (LSc). Eight lime stabilized sludge concentrates were evaluated by RT-PCR with magnetic bead capture and by plaque assay. Enteroviruses were not detected by either method.
Results may be obtained from RT-PCR within hours and at a cost much lower than the plaque assay. This method could be useful as a rapid screening technique. The direct monitoring of pathogens, such as enteric viruses, instead of the reliance upon indicator organisms, may reduce the risks from land application of sludge and make this practice more acceptable to a greater number of communities.
Recommended Citation
Moore, Amy Elisabeth, "The development of RNA probe and RT-PCR assays for the detection of enteroviruses in sludge" (1999). Doctoral Dissertations. 2108.
https://scholars.unh.edu/dissertation/2108