BLV-CoCoMo-qPCR-3: Improved BLV-CoCoMo-qPCR for Bovine Leukemia Virus Detection by Mixing Probes Targeting all BLV Variants

Abstract Background: The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease in cattle. The proviral load (PVL) is an important index for estimating disease progression. Previously, we developed a quantitative real-time PCR (qPCR) assay to measure the PVL of BLV using coordination of common motif (CoCoMo) degenerate primers that can amplify all known BLV strains. However, mutations, which potentially affect the detection ability, have been recently reported in the probe sequences of the long terminal regions (LTRs) of the BLV-CoCoMo-qPCR-2 assay. Here, we developed a new strategy to overcome these newly generated mutations located in the probe regions of this assay.Methods: We collected genomic DNA from 887 cows from 27 BLV-positive farms, using a nationwide survey conducted in 2011 and 2017 in Japan. BLV variants were investigated by quantifying the provirus using BLV-CoCoMo-qPCR-2 targeting the BLV LTR gene and the TaKaRa Cycleave PCR system targeting the BLV tax gene. Additionally, we sequenced the partial BLV LTR gene. The modified probes were designed to completely match the three BLV variants identified here, and the modified assay was established using mixed probes.Results: We found four single mutations within the probe region of the original BLV-CoCoMo-qPCR-2 assay, three of which negatively affected its sensitivity. Furthermore, we examined the optimum ratio of the concentration to be mixed with the wild type and three new BLV TaqMan probes were designed here using genomic DNAs extracted from cattle infected with the wild-type BLV strain and cattle infected with variants. Hence, we successfully established an improved BLV-CoCoMo-qPCR-3 assay that uses mixed probes corresponding to all three BLV variants. Conclusions: To overcome the loss of assay sensitivity due to newly emerging variants, we have established the BLV-CoCoMo-qPCR-3 assay that could amplify all BLV strains using newly designed mixed probes in addition to degenerate primers that were previously designed in our original assay. Our proposed method maintained the original sensitivity and reproducibility and can detect all mutant strains; thus, it is a useful tool to prevent the spread of BLV infections, especially those caused by newly emerging variants.