title: Validation of nonnested and real-time PCR for diagnosis of sheep-associated malignant catarrhal fever in clinical samples.
abstract: 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.
authors.affiliation: Animal Disease Research Unit, USDA-Agricultural Research Service, 3003 ADBF, Washington State University, Pullman, WA 99164, USA.
authors.forename: Donald L
authors.initials: DL
authors.lastname: Traul
authors.forename: Naomi S
authors.initials: NS
authors.lastname: Taus
authors.forename: J
authors.initials: J
authors.lastname: Lindsay Oaks
authors.forename: Donal
authors.initials: D
authors.lastname: O'Toole
authors.forename: Fred R
authors.initials: FR
authors.lastname: Rurangirwa
authors.forename: Timothy V
authors.initials: TV
authors.lastname: Baszler
authors.forename: Hong
authors.initials: H
authors.lastname: Li
mesh_terms: D000818:Animals; D016164:Bison / Q000821:virology; D002417:Cattle; D003670:Deer / Q000821:virology; D006564:Herpesviridae / Q000302:isolation & purification; D008304:Malignant Catarrh / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis* / Q000821:virology*