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 100 bovine clinical blood samples, comprising 80 ELISA-positive and 20 ELISA-negative samples, to evaluate the performance of the BLV specific fLAMP assay. The results were compared with those of the rPCR assay [16, 23] . As shown in Table 2 and Fig. 2 , among the 80 ELISA-positive samples, 62 and 71 were positive and 18 and 9 were negative in the fLAMP and rPCR assays, respectively. The 20 ELISA-negative samples were negative in both assays. Compared with the ELISA results, the fLAMP assay achieved 77.5% (62/80) sensitivity and 100% (20/20) specificity and the rPCR assay showed 88.8% (71/80) sensitivity and 100% (20/20) specificity. Compared with a conventional real-time PCR (rPCR) assay, the fLAMP assay achieved 87.3% (62/71) sensitivity and 100% (29/29) specificity. The rPCR assay required 65 min from the beginning of reaction to the final extension step. In contrast, the fLAMP assay yielded 62 positive results between 7 min, 15 sec and 28 min (mean Tp 10 min 8 sec ± SD 3 min 23 sec). Among the 13-samples that were low proviral loads (2-39 copies per 100 ng DNA), corresponding to weak rPCR positives (C T range, 35.37-39.00), the fLAMP assay generated four true-positives and nine false-negatives ( Table 2 , Fig. 2 , and Table S1 ). The fLAMP assay did not detect BLV sequences in 29 samples comprising nine rPCR-negative/ELISA-positive and 20 rPCR-and ELISA-negative samples ( Table 2) .