Development of a reliable assay protocol for identification of diseases (RAPID)-bioactive amplification with probing (BAP) for detection of bovine ephemeral fever virus.
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.
Hsieh
Yao-Ching
YC
Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan.
Chen
Shih-Hui
SH
Chou
Chin-Sheng
CS
Hsiao
Hsu-Wen
HW
Chen
Shu-Zhen
SZ
Lee
Yen-Feng
YF
Liu
Hung-Jen
HJ
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
D015342:DNA Probes; D012367:RNA, Viral; D019914:Taq Polymerase