Degree Type

Thesis

Date of Award

2012

Degree Name

Master of Science

Department

Veterinary Diagnostic and Production Animal Medicine

First Advisor

Erin L. Strait

Abstract

This thesis begins with discussion of molecular diagnostics used in diagnosis of the Porcine Reproductive and Respiratory (PRRS) virus with specific emphasis on swine oral fluids are discussed through a review of literature. Risks contributing to missed detection are discussed including: virus mutation, viral degradation in clinical samples, and polymerase chain reaction (PCR) inhibition.

Chapter two reports optimization of RNA extraction and PCR protocols for swine oral fluids. The results showed marked differences among extraction protocols, PCR protocols, and combinations thereof in detecting PRRSV in the oral fluid matrix. An important finding was that PCR reactions were partially inhibited and that inhibition was reduced by use of a higher concentration of PCR enzymes. The results suggest that further optimization of PCR assays for porcine oral fluids is needed and that laboratories should not assume that methods optimized for detection of virus in serum will perform equally with porcine oral fluids.

Chapter three presents efforts to improve success rate of PRRS sequencing from PCR-positive oral fluids. Sequencing open reading frame 5 (ORF5) of the PRRS virus is a commonly used molecular diagnostic test for virus characterization and epidemiologic investigations in the United States. Attempts in recent years to obtain PRRS virus sequencing from swine oral fluids has been less successful than serum, limiting the cost effectiveness of using this specimen type in disease eradication efforts. In this study, viral RNA was extracted from swine oral fluid diagnostic submissions and tested with three different modifications of the PCR reaction; "regular" or standard method, "diluted" extract prior to PCR, and "tough" or inhibition-resistant PCR enzyme mix. Success rate of generating PCR product was evaluated along with concentration of amplified product. The "diluted method" yielded the highest cDNA concentration of the target band, resulting in a significantly higher success rate of amplifying the target than the `regular' method, and had the highest proportion of samples successfully sequence.

Copyright Owner

Wayne Alan Chittick

Language

en

File Format

application/pdf

File Size

66 pages

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