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Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples

Correspondence: ¹Corresponding Author: Hong Li, Animal Disease Research Unit, USDA/Agricultural Research Service, 3003 ADBF, Washington State University, Pullman, WA 99164. hli{at}vetmed.wsu.edu

Sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal disease primarily of certain ruminants, is caused by ovine herpesvirus 2 (OvHV-2). Molecular diagnosis of SA-MCF in affected animals has relied on detection of OvHV-2 DNA using a nested PCR, which has significant potential for amplicon contamination as a routine method in diagnostic laboratories. In this report, a nonnested and a previously developed real-time PCR were validated for detection of OvHV-2 DNA in samples from clinically affected animals. Three sets of blood or tissue samples were collected: 1) 97 samples from 97 naturally affected animals with evidence of clinical SA-MCF; 2) 200 samples from 8 animals with experimentally induced SA-MCF; and 3) 100 samples from 100 animals without any evidence of clinical SA-MCF. Among 97 positive samples defined by nested PCR from clinically affected animals, 95 (98%) were positive by nonnested PCR and 93 (96%) were positive by real-time PCR, respectively. One hundred percent of the samples from the animals with experimentally induced MCF were positive by real-time PCR, while 99% were positive by nonnested PCR. Neither nonnested PCR nor real-time PCR yielded a positive result on any of the 100 nested PCR-negative samples from animals without evidence of clinical MCF. The data confirmed that both nonnested and real-time PCR maintained high specificity and sensitivity for the detection of OvHV-2 DNA in clinical samples.

Key Words: Real-time PCR • sheep-associated MCF

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				H. Li, C. W. Cunha, C. J. Davies, K. L. Gailbreath, D. P. Knowles, J. L. Oaks, and N. S. Taus Ovine herpesvirus 2 replicates initially in the lung of experimentally infected sheep J. Gen. Virol., July 1, 2008; 89(7): 1699 - 1708. [Abstract] [Full Text] [PDF]