Use of gene probes and an amplification method for the detection of rotaviruses in water

Persistent Link:
http://hdl.handle.net/10150/191152
Title:
Use of gene probes and an amplification method for the detection of rotaviruses in water
Author:
De Leon, Ricardo,1957-
Issue Date:
1989
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:
Rotaviruses are one of the most significant causes of diarrheal disease in the world. Their presence in groundwater and drinking water supplies constitutes a health risk to the population. The study of rotaviruses in the environment has been hampered by the lack of accessible and consistent detection methodologies. Gene probes and other molecular techniques are a novel approach for the detection of these viruses in water. The feasibility of these new techniques for the detection and study of rotaviruses in the environment has been assessed using the simian SA-11 and the culturable human Wa rotavirus strains as models. Two general approaches have been undertaken consisting of hybridization of probes with genomic RNA and hybridization with mRNA produced by the virion-incorporated transcriptase. Hybridization of gene probes with genomic dsRNA of rotaviruses in environmental concentrates resulted in the detection of 10 4 immunofoci of Wa rotavirus. In vitro transcription serves as an amplification method with sensitivity 100- to 1000-fold greater than when probing for genomic RNA. The sensitivity obtained in Wa-seeded distilled water and environmental concentrates after in vitro transcription is 2 and 20 immunofoci, respectively. Proteins in environmental concentrates decrease the efficiency of probe hybridization by 10-100 fold. Also, transcriptase-inhibiting factors found in environmental samples decrease the production of mRNA. Both proteins and transcriptase-inhibiting factors can be reduced significantly with Sephadex G-200 columns. Passage of environmental concentrate through Sephadex G-200 spun columns, followed by in vitro transcription, was used to detect rotaviruses in environmental samples. Rotaviruses were detected by this combination of techniques in eight of 20 sewage samples, one of 16 tap water samples, five of 32 ground water samples, and two of nine surface water samples. Only one of 17 samples which tested positive with Wa cDNA 4 was positive for non-specific probe binding. The probing of rotavirus mRNA, amplified by the virion-incorporated transcriptase, is a practical and feasible method for monitoring these viruses in the environment.
Type:
Dissertation-Reproduction (electronic); text
Keywords:
Hydrology.; Reoviruses.; DNA probes.; Genetic transcription.; Viruses -- Isolation.; Water -- Pollution -- Measurement.; Viral pollution of water -- Measurement.
Degree Name:
Ph. D.
Degree Level:
doctoral
Degree Program:
Microbiology and Immunology; Graduate College
Degree Grantor:
University of Arizona

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleUse of gene probes and an amplification method for the detection of rotaviruses in wateren_US
dc.creatorDe Leon, Ricardo,1957-en_US
dc.contributor.authorDe Leon, Ricardo,1957-en_US
dc.date.issued1989en_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.abstractRotaviruses are one of the most significant causes of diarrheal disease in the world. Their presence in groundwater and drinking water supplies constitutes a health risk to the population. The study of rotaviruses in the environment has been hampered by the lack of accessible and consistent detection methodologies. Gene probes and other molecular techniques are a novel approach for the detection of these viruses in water. The feasibility of these new techniques for the detection and study of rotaviruses in the environment has been assessed using the simian SA-11 and the culturable human Wa rotavirus strains as models. Two general approaches have been undertaken consisting of hybridization of probes with genomic RNA and hybridization with mRNA produced by the virion-incorporated transcriptase. Hybridization of gene probes with genomic dsRNA of rotaviruses in environmental concentrates resulted in the detection of 10 4 immunofoci of Wa rotavirus. In vitro transcription serves as an amplification method with sensitivity 100- to 1000-fold greater than when probing for genomic RNA. The sensitivity obtained in Wa-seeded distilled water and environmental concentrates after in vitro transcription is 2 and 20 immunofoci, respectively. Proteins in environmental concentrates decrease the efficiency of probe hybridization by 10-100 fold. Also, transcriptase-inhibiting factors found in environmental samples decrease the production of mRNA. Both proteins and transcriptase-inhibiting factors can be reduced significantly with Sephadex G-200 columns. Passage of environmental concentrate through Sephadex G-200 spun columns, followed by in vitro transcription, was used to detect rotaviruses in environmental samples. Rotaviruses were detected by this combination of techniques in eight of 20 sewage samples, one of 16 tap water samples, five of 32 ground water samples, and two of nine surface water samples. Only one of 17 samples which tested positive with Wa cDNA 4 was positive for non-specific probe binding. The probing of rotavirus mRNA, amplified by the virion-incorporated transcriptase, is a practical and feasible method for monitoring these viruses in the environment.en_US
dc.description.notehydrology collectionen_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.typetexten_US
dc.subjectHydrology.en_US
dc.subjectReoviruses.en_US
dc.subjectDNA probes.en_US
dc.subjectGenetic transcription.en_US
dc.subjectViruses -- Isolation.en_US
dc.subjectWater -- Pollution -- Measurement.en_US
dc.subjectViral pollution of water -- Measurement.en_US
thesis.degree.namePh. D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.committeememberGerba, Charles P.en_US
dc.contributor.committeememberHowarth, Alan J.en_US
dc.identifier.oclc213447790en_US
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