title: Direct polymerase chain reaction from blood and tissue samples for rapid diagnosis of bovine leukemia virus infection.
abstract: Bovine leukemia virus (BLV) infection induces bovine leukemia in cattle and causes significant financial harm to farmers and farm management. There is no effective therapy or vaccine; thus, the diagnosis and elimination of BLV-infected cattle are the most effective method to eradicate the infection. Clinical veterinarians need a simpler and more rapid method of diagnosing infection, because both nested polymerase chain reaction (PCR) and real-time PCR are labor intensive, time-consuming, and require specialized molecular biology techniques and expensive equipment. In this study, we describe a novel PCR method for amplifying the BLV provirus from whole blood, thus eliminating the need for DNA extraction. Although the sensitivity of PCR directly from whole blood (PCR-DB) samples as measured in bovine blood containing BLV-infected cell lines was lower than that of nested PCR, the PCR-DB technique showed high specificity and reproducibility. Among 225 clinical samples, 49 samples were positive by nested PCR, and 37 samples were positive by PCR-DB. There were no false positive samples; thus, PCR-DB sensitivity and specificity were 75.51% and 100%, respectively. However, the provirus loads of the samples detected by nested PCR and not PCR-DB were quite low. Moreover, PCR-DB also stably amplified the BLV provirus from tumor tissue samples. PCR-DB method exhibited good reproducibility and excellent specificity and is suitable for screening of thousands of cattle, thus serving as a viable alternative to nested PCR and real-time PCR.
authors.affiliation: Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Kita-ku Kita 18- jo Nishi 9-chome, Sapporo, Hokkaido 060-0818, Japan.
authors.forename: Asami
authors.initials: A
authors.lastname: Nishimori
authors.forename: Satoru
authors.initials: S
authors.lastname: Konnai
authors.forename: Ryoyo
authors.initials: R
authors.lastname: Ikebuchi
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Okagawa
authors.forename: Ayako
authors.initials: A
authors.lastname: Nakahara
authors.forename: Shiro
authors.initials: S
authors.lastname: Murata
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ohashi
mesh_terms: D000818:Animals; D002417:Cattle / Q000097:blood / Q000821:virology; D016583:Enzootic Bovine Leukosis / Q000175:diagnosis*; D001909:Leukemia Virus, Bovine; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity