On-site detection of bovine leukemia virus by a field-deployable automatic nucleic extraction plus insulated isothermal polymerase chain reaction system

• Published in: Journal of virological methods 2018-09, Vol.259, p.116-121
• Main Authors: Ruggiero, Vickie J., Benitez, Oscar J., Tsai, Yun-Long, Lee, Pei-Yu Alison, Tsai, Chuan-Fu, Lin, Yu-Chun, Chang, Hsiao-Fen Grace, Wang, Hwa-Tang Thomas, Bartlett, Paul
• Format: Article
• Language: English
• Subjects: Bovine leukemia virus; Field-deployable; Insulated isothermal PCR; PCR
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2018.06.008

•On-farm performance of the field-deployable iiPCR was equivalent to lab rt-PCR.•Agreement between tests was 100% (κ = 1.0) on-site and at the laboratory.•The estimated limit of detection of the BLV iiPCR was 4 copies per reaction.•The iiPCR system could provide point-of-need detection of bovine leukemia virus. Bovine leukemia virus (BLV) is a contagious, oncogenic deltaretrovirus of cattle with a worldwide distribution. In the US, over 40% of dairy cows are infected with the virus, and evidence of its economic impact is growing. This study evaluated the performance of a field-deployable automatic nucleic acid-extraction/insulated isothermal PCR (iiPCR) system for on-site BLV-proviral DNA detection in dairy cows compared with a conventional laboratory real-time PCR (rt-PCR). Assay performance was verified in parallel tests of 36 archived blood samples with 100% agreement (κ = 1.0; n = 36) between the iiPCR and conventional rt-PCR systems, and the limit of detection of the iiPCR assay was estimated to be 4 copies (genome equivalent) per reaction. The field-deployable iiPCR system was subsequently used on-farm to test freshly collected blood samples, and showed 100% agreement (κ = 1.0; n = 32) with the laboratory rt-PCR system. Fresh blood samples were collected on a second farm and tested on both systems, also with 100% agreement (κ = 1.0; n = 34). The field-deployable iiPCR/POCKIT™ combo system performs as well as a conventional laboratory-based rt-PCR system for detection of BLV proviral DNA in whole blood and may be a useful tool for on-farm evaluation of BLV-infection status in dairy cattle.