Cross-priming amplification for detection of bovine viral diarrhoea virus species 1 and 2

AimsThe aim of the study was the development of cross-priming amplification for ubiquitous detection of bovine viral diarrhoea virus (BVDV) species 1 and 2. Methods and ResultsThree and five specific primers, respectively, for the detection of BVDV-1 and BVDV-2, were designed on the basis of the sequences of the 5UTR region. Incubation temperature and reaction time were determined. The optimal incubation conditions using water bath were 63 degrees C for 75min. Reverse transcription step (RT) was not required. The results were visualized under UV-light as a bright yellow fluorescence in positive samples. Additional method for results interpretation was agarose gel electrophoresis. Positive samples showed the presence of ladder-like banding patterns, formed by harpin-like cross-priming amplification (CPA) products. Sensitivity of CPA was compared with conventional RT-PCR and real-time RT-PCR. The CPA detection limit was 3500 copies for BVDV-1 and 80000 copies for BVDV-2 per reaction. For RT-PCR it was 350 and 80 copies for BVDV-1 and BVDV-2, respectively, and for real-time RT-PCR it was 35 copies for BVDV-1 and 80 copies for BVDV-2. The sensitivity of the developed method is sufficient to detect persistently infected (PI) animals. Positive results were found in 24 of 25 BVDV isolates belonging to species 1 and 2. Additionally, one false-negative result for BVDV-2 was detected. There were no false-positive results in negative samples and in the negative control. Both sets of primers used for the detection of BVDV-1 and BVDV-2 were not able to detect atypical pestiviruses. CPA positive results were confirmed by RT-PCR and real-time RT-PCR. ConclusionsCPA is a rapid method for the detection of BVDV-1 and BVDV-2 in field samples from PI animals. Significance and Impact of StudyThis is the first report on the application of the CPA method for the detection of BVDV.