TwistAmp RPA Technology
TwistAmp RPA Technology

Rapid detection of bovine viral diarrhea virus using recombinase polymerase amplification combined with lateral flow dipstick assays in bulk milk

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Bovine viral diarrhea virus (BVDV) is one of the most prevalent and economically important pathogens of ruminants, and leads to significant financial losses to the livestock industry worldwide. Development of rapid and accurate diagnostic methods is of great importance for the control and eradication of BVDV infection. The aim of this study was to develop a novel isothermal recombinase polymerase amplification (RPA) method combined with a lateral flow dipstick (LFD), for rapid detection of BVDV. RPA primers and a probe targeting the specific conserved 5′-UTR of BVDV genome were designed. The RPA amplification could be finished at a constant temperature of 38 C for 15 min, and the amplification product was easily visualized on a simple LFD within 5 min. The detection limit of this assay was 20 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses, such as infectious bovine rhinotracheitis virus (IBRV), bovine enterovirus (BEV), bovine coronavirus (BcoV), bovine parainfluenza virus type 3 (BPIV-3), bovine ephemeral fever virus (BEFV) and bovine respiratory syncytial virus (BRSV). The assay performance on bulk tank milk was also evaluated, and the sensitivity and accuracy of BVDV LFD RPA was compared with real-time RT-PCR. Of 284 pool or bulk tank milk samples, 51 were found to be positive by RPA assay, whereas 52 were positive by real- time RT-PCR. The coincidence rate between LFD RPA and real-time RT-PCR was 97.54% (277/284).                                                                                               

Key words: bovine viral diarrhea virus (BVDV); recombinase polymerase amplification (RPA); lateral flow dipstick; bulk tank milk