期刊
JOURNAL OF VIROLOGICAL METHODS
卷 297, 期 -, 页码 -出版社
ELSEVIER
DOI: 10.1016/j.jviromet.2021.114264
关键词
Bovine leukemia virus; Real-time PCR; Proviral load; Mixing probe; Degenerating primers; LTR
资金
- MAFF
- Projects of the NARO Bio-oriented Technology Research Advancement Institution (the special scheme project on regional developing strategy) [16817983]
The study identified four single mutations within the probe region of the original BLV-CoCoMo-qPCR assay, three of which decreased sensitivity. Modified probes were designed to match the mutant strains, resulting in an improved assay with maintained sensitivity and reproducibility.
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.
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