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

We used Primer Explorer V5 software (Fujitsu System Solutions Ltd., Tokyo, Japan) to design a new primer set based on BLV env sequences. We used Clustal omega for multiple alignment of 180 BLV complete env sequences (https://www.ebi.ac.uk/Tools/ msa/clustalo/), as well as Jalview to identify conserved env nucleotide sequences in an alignment of 401 BLV partial env gene sequences [22] . Details of the primers are shown in Table 1 . The predicted specificities of the six primers were determined using the Basic Local Alignment Search Tool (BLAST) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The fLAMP assay was performed using a Genie III (OptiGene, Horsham, U.K.). Amplification was performed at 67°C for 30 min, followed by inactivation of enzymatic activity at 98°C for 2 min, and cooling to 80°C for annealing analysis with ramping at 0.05°C/sec. The 25-µl fLAMP reaction comprised 15 µl of an isothermal master mix (ISO-002, Optigene), 0.4 µl each of FIP and BIP primers (100 pmol/µl), 0.2 µl each of LF and LB primers (100 pmol/µl), 0.05 µl each of F3 and B3 primers (100 pmol/µl), 3.7 µl of nuclease-free water, and 5 µl of the DNA template. All LAMP primers were produced using column-grade purification methods by Hokkaido System Science (Sapporo, Japan). When the fluorescence intensity reached 20,000 within a 30-min amplification, and the annealing temperature (Ta) value ranged between 89.0 and 92.0°C, the results were interpreted as positive. Time of positivity (Tp) was automatically calculated by the Genie III.