Differential Identification of Porcine Transmissible Gastroenteritis Coronavirus and Bovine Enteric Coronavirus using Cloned Nucleic Acid Probes

Author

Linda J. Shockley

Date of Award

6-1986

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

David A. Brian

Committee Members

Riggsby, Potgieter

Abstract

Cloned bovine coronavirus (BCV) and transmissible gastroenteritis virus (TGEV) cDNAs were used as quantitative hybridization probes to detect viral RNA. Insert DNA was cleaved from the cloning vector, OO purified by electroelution, and then nick-translated with ³²P-dCTP to a g specific activity of 10⁸cpm/μg of DNA. As standards, purified quantified BCV and TGEV RNAs were denatured in high salt, formaldehyde and detergent and then blotted with a 96-well minifold dot blot apparatus onto nitrocellulose. A similar dot blot methodology was applied to cell culture supernatant fluids and fecal samples. After hybridization and autoradiography, it was observed that each probe detected only RNA from its homologous virus to a sensitivity level of 25 pg of RNA per dot. No crossreactivity between the two coronaviruses was observed, even at RNA concentrations of 10 ng per dot. The probes were specific, since they did not hybridize to a variety of heterologous virus RNAs. BCV and TGEV RNA could also be readily detected in cell culture super natant fluids and in stool samples by this procedure. The volume of sample required was small (1-10 μl), sample manipulations were few, and multiple samples could be analysed simultaneously. The probes will be useful for routine diagnosis of BCV and TGEV infection and for research into the pathogenesis of chronic disease.

Recommended Citation

Shockley, Linda J., "Differential Identification of Porcine Transmissible Gastroenteritis Coronavirus and Bovine Enteric Coronavirus using Cloned Nucleic Acid Probes. " Master's Thesis, University of Tennessee, 1986.
https://trace.tennessee.edu/utk_gradthes/13809