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United States Department of Agriculture

Agricultural Research Service

Title: Quantitation of Sheep-Associated Malignant Catarrhal Fever Viral DNA by Competitive Pcr

Authors
item Hua, Y - WASHINGTON STATE UNIV.
item Li, Hong
item Crawford, T - WASHINGTON STATE UNIV.

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 19, 1998
Publication Date: N/A

Interpretive Summary: Quantitation of viral genetic materials in diseased or carrier animals is an important tool to understand the relationship between host and virus. In this study, a competitive PCR was developed to quantitate sheep-associated malignant catarrhal fever (SA-MCF) viral DNA. The assay was evaluated and validated by several criteria, such as accuracy of competitor concentration, dose-dependent relation between target and competitor, and its reproducibility. The assay was accurate for DNA measurement within the range of 30 to 300,000 copies. Reproducibility was assessed by repetitively assaying a set of blind-coded samples from a variety of animals and tissues. Results indicated that the assay is reliable and reproducible for quantitation of SA-MCF viral DNA in samples from MCF virus carrier sheep and from animals with clinical SA-MCF.

Technical Abstract: A single-step, competitive PCR (cPCR) was developed to quantitate sheep-associated malignant catarrhal fever (SA-MCF) viral DNA. The assay employed co-amplification of a fixed quantity of target DNA with graded amounts of a competitor, generated by truncation of the target sequence lying between the two primer binding sites. The assay yielded a linear response (r=0.98) for DNA measurement within the range of 30 to 300,000 copies. Amplification efficiency analysis by co-amplification of target and competitor in equal copy numbers for varying numbers of cycles showed that the relative abundance of the co-amplified products remained constant with increasing cycle numbers up to 40. Reproducibility was assessed by repetitively assaying a set of blind-coded samples from a variety of animals and tissues. Results indicated that the assay is reliable and reproducible for quantitation of SA-MCF viral DNA in samples from asymptomatically infected sheep and from animals with clinical SA- MCF.

Last Modified: 10/1/2014