Development of a fluorescent loop-mediated isothermal amplification assay for rapid and simple diagnosis of bovine leukemia virus infection

NAGAO, Konomu; MAKINO, Ryohei; APEGO, Francis Victor; MEKATA, Hirohisa; YAMAZAKI, Wataru

Genomic DNA extraction was performed according to our previous study [9] . Briefly, among 409 blood samples, 80 seropositive and 20 seronegative samples were randomly chosen. The buffy coat was collected from the centrifuged EDTA-treated blood sample described above, and genomic DNA was extracted using a Wizard Genomic DNA Purification Kit (Promega, Fitchburg, WI, U.S.A.) in accordance with the manufacturer's instructions. Using a NanoDrop 8000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, U.S.A.), the DNA concentration was measured and adjusted to 20 ng/µl using distilled water. The genomic DNA of fetal lamb kidney cells infected with BLV (FLK-BLV) [21] was extracted in the same manner described above for the comparative determination of the LOD. Genomic DNAs were used immediately, otherwise they were stored at −20 or −80°C.