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

rPCR was performed using a LightCycler 96 (Roche Molecular Systems, Pleasanton, CA, U.S.A.) to detect pol sequences according to Rola-Łuszczak, et al. [16] and the OIE terrestrial manual [23] . Reactions were modified using shorter times for denaturation and annealing according to the instructions provided with the Cycleave PCR reaction mixture (TaKaRa Bio Inc., Otsu, Japan). Briefly, 20-µl rPCR reactions comprised 10 µl of 2x Cycleave PCR reaction mixture (TaKaRa Bio), 0.08 µl each of primers (100 pmol/µl, Hokkaido System Science, 0.04 µl of probe (100 pmol/µl, Hokkaido System Science), 4.8 µl of nucleasefree water, and 5 µl of the DNA template. The cycling conditions were as follows: one cycle at 95°C for 10 sec, 50 cycles each at 94°C for 5 sec, and 60°C for 30 sec. Each amplification was performed in duplicate, and an average C T (threshold cycle) value was automatically calculated. Details of the primers and probes are shown in Table 1 .