title: Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of bovine ephemeral fever virus.
abstract: A rapid, sensitive, and specific assay, RAPID-BAP assay, was developed to detect and quantify the G protein-encoding gene of bovine ephemeral fever virus (BEFV). This new technique uses a nested PCR and magnetic bead-based DNA probing assay. The optimal conditions for the assay were examined. By applying a nested PCR, a minimum of 1 copy/mul of the BEFV plasmid DNA could be detected by the assay. The optimal hybridization conditions at 50 degrees C in 5x SSC and 0.5% SDS with a 20-min incubation allowed clear discrimination between negative and positive controls. The assay was also highly specific as all negative controls failed to show any positive detection. The diagnostic sensitivity of the RAPID-BAP assay, real-time RT-PCR, and conventional RT-PCR in the detection of 34 clinical blood samples suspected to have BEFV infections were 72.73, 36.36, and 18.18%, respectively. The results indicated that the RAPID-BAP assay developed in this study was more sensitive than the conventional RT-PCR and real-time RT-PCR assays for the detection of BEFV. The novel RAPID-BAP assay is an excellent diagnostic tool with high sensitivity, specificity, and fast turnaround time.
authors.affiliation: Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Yao-Ching
authors.initials: YC
authors.lastname: Hsieh
authors.forename: Shih-Hui
authors.initials: SH
authors.lastname: Chen
authors.forename: Chin-Sheng
authors.initials: CS
authors.lastname: Chou
authors.forename: Hsu-Wen
authors.initials: HW
authors.lastname: Hsiao
authors.forename: Shu-Zhen
authors.initials: SZ
authors.lastname: Chen
authors.forename: Yen-Feng
authors.initials: YF
authors.lastname: Lee
authors.forename: Hung-Jen
authors.initials: HJ
authors.lastname: Liu
mesh_terms: D000818:Animals; D002417:Cattle; D015342:DNA Probes; D018115:Ephemeral Fever Virus, Bovine / Q000235:genetics / Q000302:isolation & purification*; D015336:Molecular Probe Techniques; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000032:analysis; D012680:Sensitivity and Specificity; D019914:Taq Polymerase
chemical_list: D015342:DNA Probes; D012367:RNA, Viral; D019914:Taq Polymerase