Persistent Link:
http://hdl.handle.net/10150/187174
Title:
Detection of enteroviruses in marine waters using RT-PCR.
Author:
Reynolds, Kelly Anne.
Issue Date:
1995
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Human enteric viruses present in sewage may find their way into recreational marine waters, causing adverse health effects to exposed individuals. This research project focused on (i) optimization of methods for RT-PCR detection of enteroviruses in marine waters, with particular attention to the issue of sensitivity vs inhibition (ii) comparison of enterovirus detection in marine waters by cell culture and RT-PCR and (iii) development of a method for detecting infectious enteroviruses using an integrated cell culture/PCR procedure. Primary sewage effluent and marine water samples were concentrated using Viradel methods and reconcentrated by organic flocculation of beef extract V eluants. Samples were inoculated with poliovirus type 1 (strain LSc-2ab) to evaluate sample inhibition, the effectiveness of column purification and virus recovery efficiency. Samples were subsequently analyzed either by RT-PCR, cell culture or the integrated cell culture/PCR technique. Results from this research indicate: (i) Filterite filters are efficient (66%) for concentration of enteroviruses from marine waters, provided a NaCl and a double BEV elution steps are utilized. (ii) Resin column filtration aids in purifying samples from PCR inhibitory compounds, however, a trade-off exists between inhibition removal and virus recovery efficiencies. (iii) PCR may be used to evaluate large volume concentrates in marine waters, however, due to small sample size and interferences with inhibitory substances, the chance of isolating a virus is still a random chance. (iv) Viruses were isolated using a BGM cell line from primary sewage effluent, a sewage outfall, a canal, and several popular recreational beaches of Hawaii. For comparison, enteroviruses were detected, using RT-PCR, in the sewage, and the outfall only. (v) An integrated cell culture/RT-PCR technique was five times more rapid than traditional cell culture methods for detection of infectious poliovirus in distilled water and enteroviruses in sewage, without the typical problems of PCR inhibitory compounds.
Type:
text; Dissertation-Reproduction (electronic)
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Soil and Water Science; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Pepper, Ian L.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleDetection of enteroviruses in marine waters using RT-PCR.en_US
dc.creatorReynolds, Kelly Anne.en_US
dc.contributor.authorReynolds, Kelly Anne.en_US
dc.date.issued1995en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractHuman enteric viruses present in sewage may find their way into recreational marine waters, causing adverse health effects to exposed individuals. This research project focused on (i) optimization of methods for RT-PCR detection of enteroviruses in marine waters, with particular attention to the issue of sensitivity vs inhibition (ii) comparison of enterovirus detection in marine waters by cell culture and RT-PCR and (iii) development of a method for detecting infectious enteroviruses using an integrated cell culture/PCR procedure. Primary sewage effluent and marine water samples were concentrated using Viradel methods and reconcentrated by organic flocculation of beef extract V eluants. Samples were inoculated with poliovirus type 1 (strain LSc-2ab) to evaluate sample inhibition, the effectiveness of column purification and virus recovery efficiency. Samples were subsequently analyzed either by RT-PCR, cell culture or the integrated cell culture/PCR technique. Results from this research indicate: (i) Filterite filters are efficient (66%) for concentration of enteroviruses from marine waters, provided a NaCl and a double BEV elution steps are utilized. (ii) Resin column filtration aids in purifying samples from PCR inhibitory compounds, however, a trade-off exists between inhibition removal and virus recovery efficiencies. (iii) PCR may be used to evaluate large volume concentrates in marine waters, however, due to small sample size and interferences with inhibitory substances, the chance of isolating a virus is still a random chance. (iv) Viruses were isolated using a BGM cell line from primary sewage effluent, a sewage outfall, a canal, and several popular recreational beaches of Hawaii. For comparison, enteroviruses were detected, using RT-PCR, in the sewage, and the outfall only. (v) An integrated cell culture/RT-PCR technique was five times more rapid than traditional cell culture methods for detection of infectious poliovirus in distilled water and enteroviruses in sewage, without the typical problems of PCR inhibitory compounds.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineSoil and Water Scienceen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairPepper, Ian L.en_US
dc.contributor.committeememberGerba, Charles P.en_US
dc.contributor.committeememberMiller, Raina M.en_US
dc.contributor.committeememberSinclair, Norval A.en_US
dc.contributor.committeememberSonger, J. Glennen_US
dc.identifier.proquest9534680en_US
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