Development of droplet digital PCR for quantification of bovine leukemia virus proviral load using unpurified genomic DNA

Xinyue Wu; Kousuke Notsu; Yuichi Matsuura; Shuya Mitoma; Hala El Daous; Junzo Norimine; Satoshi Sekiguchi

doi:10.1016/j.jviromet.2023.114706

Development of droplet digital PCR for quantification of bovine leukemia virus proviral load using unpurified genomic DNA

J Virol Methods. 2023 May:315:114706. doi: 10.1016/j.jviromet.2023.114706. Epub 2023 Feb 26.

Authors

Xinyue Wu¹, Kousuke Notsu², Yuichi Matsuura³, Shuya Mitoma⁴, Hala El Daous⁵, Junzo Norimine⁶, Satoshi Sekiguchi⁷

Affiliations

¹ Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki 889-1692, Japan. Electronic address: ja21008@student.miyazaki-u.ac.jp.
² Graduate School of Medicine and Veterinary Medicine, University of Miyazaki, Miyazaki 889-1692, Japan. Electronic address: kousuke_notsu@med.miyazaki-u.ac.jp.
³ Division of Infectious Animal Diseases, National Institute of Animal Health, National Agriculture and Food Research Organization, Ibaraki 305-0856, Japan. Electronic address: zrxmatsu@affrc.go.jp.
⁴ Division of Immunology, Department of Infectious disease, Faculty of Medicine, University of Miyazaki, Japan. Electronic address: shuya_mitoma@med.miyazaki-u.ac.jp.
⁵ Faculty of Veterinary Medicine, Benha University, Toukh 13736, Egypt.
⁶ Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan; Center for Animal Disease Control, University of Miyazaki, Miyazaki 889-2192, Japan. Electronic address: nori@cc.miyazaki-u.ac.jp.
⁷ Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan; Center for Animal Disease Control, University of Miyazaki, Miyazaki 889-2192, Japan. Electronic address: sekiguchi@cc.miyazaki-u.ac.jp.

PMID: 36849053
DOI: 10.1016/j.jviromet.2023.114706

Abstract

Bovine leukemia virus (BLV) is the causative agent of a B-cell tumor called enzootic bovine leukosis. Preventing BLV spreading is required to reduce economic loss related to BLV infection of livestock. To quantify proviral load (PVL) more easily and rapidly, we developed a quantification system of PVL using droplet digital PCR (ddPCR). This method uses a multiplex TaqMan assay of the BLV provirus and housekeeping gene RPP30 for the quantification of BLV in BLV-infected cells. Furthermore, we combined ddPCR with DNA purification-free sample preparation (unpurified genomic DNA). The percentage of BLV-infected cells based on unpurified genomic DNA was highly correlated with that based on purified genomic DNA (correlation coefficient: 0.906). Thus, this new technique is a suitable method to quantify PVL of BLV-infected cattle in a large sample number.

Keywords: Bovine leukemia virus; Diagnostic test; Droplet digital PCR; Enzootic bovine leukosis; Quantification; Unpurified genomic DNA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cattle
DNA
Enzootic Bovine Leukosis* / diagnosis
Genomics
Leukemia Virus, Bovine* / genetics
Polymerase Chain Reaction / methods
Proviruses / genetics

Substances

DNA