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

In a preliminary test, we attempted to design a LAMP primer set using well-conserved pol sequences. The pol gene was not amplified by any LAMP primer set that we designed using Primer Explorer V5 software. This may be explained by the the low GC content of pol. Therefore, we chose the conserved sequence in env to design the specific LAMP primer set. To determine primer specificities, each of the six primers representing eight distinct regions of env were analysed using BLAST to query GenBank. There were no matches between the first six bases of the eight primer-binding regions with non-BLV sequences. (Table 1) . Further, 20 samples did not yield C T values using the BLV-specific real-time PCR (rPCR) assay, and their values were significantly below the threshold of baseline absorbance in the anti-BLV antibody-ELISA. These samples were all BLV-negative in the fLAMP assay. Further, false-positive signals were not detected (Fig. 2, Table 2 ).