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

Based on the sequence of LC164083, a partial sequence of pol for rPCR encompassing primer target sequences was synthesized and cloned into the pEX-K4J1 vector by Eurofins Genomics, Co., Ltd (Tokyo, Japan). The amplified sequence was used to calculate the proviral loads in clinical samples as well as to prepare serial 10-fold serial dilutions of the DNA templates for LOD determination. Further, the tLAMP assay targeting the LTR region was performed to compare the LOD according to a published method [5] . Two DNA templates were used for further comparisons of different genotypes of the FLK strain (genotype 1) and a BLV-genomic DNA obtained from a naturally BLV-infected cow in Miyazaki (JA366, genotype 3). Ten-fold serial dilutions in distilled water were prepared, and the fLAMP, tLAMP and rPCR assays were performed as described above. When a sample was positive or negative in duplicate analyses, the result was interpreted as a positive in all three assays. We retrieved 401 BLV complete and partial env sequences from GenBank to design a BLV-specific LAMP primer set. The locations and nucleotide variations of the LAMP primers representing 180 complete env sequences are shown in Fig. 1 . Although LAMP primers were derived from the conserved region of env, to maintain amplification speed and accuracy, mixed bases were incorporated into the FIP and B3 primers because of the variability of the target sequences that introduce mismatches at the crucial regions between target sequences and primers (Table 1 and Fig. 1) .