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

A comparison between fLAMP and rPCR data sets is shown in Fig. 2 . The rPCR assay yielded 71 positives with 2-25,883 proviral loads (copies per 100 ng DNA), corresponding to C T values ranging from 24.94 to 39.00 (mean C T , 29.74 ± SD 3.86). In contrast to the ELISA results, the remaining nine samples were negative in the rPCR assay. A 10 −5 -dilution of the BLV reference strain FLK (genotype 1) returned a "weak" C T value (39.32) in one of two duplicate samples ( Table 3 ). The LODs of the fLAMP assay were comparable, or 1-log less sensitive, compared with those of the rPCR and tLAMP assays ( Table 3) . The difference in the LOD values may explain why nine samples were fLAMP-negative but rPCR-positive. The fLAMP assay was remarkably rapid. Two reports describe the development of a LAMP assay for BLV based on the LTR region [5] and its application to routine surveys [14] . However, these studies used a tLAMP assay, requiring amplification times (22-60 min) [5, 14] that are significantly longer compared with those reported here (8-30 min). In our present study, the tLAMP and fLAMP assays took 16-34 min and 8-25 min to amplify target DNAs, respectively (Table 3) . Further, the tLAMP assay is unable to confirm the specificity of the amplified product as judged by annealing temperatures [4, 6, 17] . For this purpose, the tLAMP assay requires an open reaction tube for electrophoresis, which may be susceptible to contamination.