A Recombinase Polymerase Amplification Lateral Flow Dipstick for Field Diagnosis of Bovine Leukemia Virus Infection and its Effectiveness Compared to iiPCR. and ELISA

Abstract

For better control and eradicate enzootic bovine leukosis (EBL) in Taiwan, a more sensitive but also convenient method for detecting proviral bovine leukemia virus (BLV) DNA is required. The retrovirus BLV establishes a persistent infection that can result in reduced milk production and reduced survival rates, causing substantial economic losses in the dairy industry. BLV replicates by integrating its proviral DNA into the host genome; therefore, the detection of proviral DNA is recommended for identifying BLV carriers to help establish BLV-free herds. The integration of recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) in this study for on-site BLV detection. The optimal amplification condition for the RPA was 30 min at 37 ̊C and followed by 5 min of LFD at room temperature. The sensitivity of this assay of 400 pg of total DNA and 10 copies of plasmid DNA. The method showed no cross-reaction with other tested viruses, including bovine foamy virus, bovine immunodeficiency virus, and caprine arthritis-encephalitis virus. For the detection of BLV field samples, the RPA-LFD was parallel tested with serological enzyme-linked immunosorbent assay (ELISA) and hydrolysis probe insulated isothermal PCR (iiPCR). The RPA-LFD assay exhibited a better sensitivity, with 83.5% of the 200 samples collected in Taiwan testing positive. A significant difference in the positive rates was found between the iiPCR and RPA-LFD methods, indicating that the RPA-LFD method for detecting BLV nucleic acid is sensitive at a lower limit. This RPA-LFD protocol can serve as an alternative tool to ELISA for the preliminary screening of BLV for its simplicity and portability, and is suitable for both laboratory and field application.