The bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease in cattle. We previously developed the quantitative real-time PCR (qPCR) assay to measure the proviral loads of BLV using coordination of common motif (CoCoMo) degenerate primers. We here found four single mutations within the probe region of the original BLV-CoCoMo-qPCR assay, three of which have negative impact on its sensitivity in the probe sequences of the long terminal regions of the BLV-CoCoMo-qPCR-2 assay, using genomic DNA from 887 cows from 27 BLV-positive farms via a nationwide survey conducted in 2011 and 2017 in Japan. Therefore, the modified probes were designed to completely match the three BLV mutant strains identified here. Moreover, 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 naturally infected with the wild type BLV strain and three mutant strains. Finally, we successfully established an improved assay maintained the original sensitivity and reproducibility and can detect novel BLV strains.

牛白血病病毒 (BLV) 是牛地方性白血病的病原体，牛地方性白血病是牛中最常见的肿瘤疾病。我们之前开发了定量实时 PCR (qPCR) 测定法，以使用共同基序 (CoCoMo) 简并引物的协调来测量 BLV 的前病毒载量。我们在这里发现原始 BLV-CoCoMo-qPCR 测定的探针区域内有四个单突变，其中三个对其在 BLV-CoCoMo-qPCR-2 测定的长末端区域的探针序列中的敏感性产生负面影响，使用通过 2011 年和 2017 年在日本进行的一项全国性调查，来自 27 个 BLV 阳性农场的 887 头奶牛的基因组 DNA。因此，修改后的探针被设计为完全匹配此处确定的三种 BLV 突变株。而且，我们检查了与野生型混合的最佳浓度比例，并使用从自然感染野生型 BLV 菌株和三种突变菌株的牛中提取的基因组 DNA 设计了三种新的 BLV TaqMan 探针。最后，我们成功地建立了一种改进的检测方法，保持了原有的灵敏度和重现性，并且可以检测新的 BLV 菌株。