Cross-priming amplification for detection of bovine viral diarrhoea virus species 1 and 2
PBN-AR
Instytucja
Państwowy Instytut Weterynaryjny - Państwowy Instytut Badawczy
Informacje podstawowe
Główny język publikacji
en
Czasopismo
JOURNAL OF APPLIED MICROBIOLOGY
ISSN
1364-5072
EISSN
1365-2672
Wydawca
WILEY-BLACKWELL
DOI
URL
Rok publikacji
2015
Numer zeszytu
3
Strony od-do
632-639
Numer tomu
119
Identyfikator DOI
Liczba arkuszy
Słowa kluczowe
en
BVDV-1 and BVDV-2
bovine viral diarrhoea virus
cross-priming 3 amplification
detection
isothermal amplification
Streszczenia
Język
en
Treść
Aims The 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 Results Three and five specific primers, respectively, for the detection of BVDV-1 and BVDV-2, were designed on the basis of the sequences of the 5′UTR region. Incubation temperature and reaction time were determined. The optimal incubation conditions using water bath were 63°C for 75 min. 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. Conclusions CPA is a rapid method for the detection of BVDV-1 and BVDV-2 in field samples from PI animals. Significance and Impact of Study This is the first report on the application of the CPA method for the detection of BVDV
Cechy publikacji
ORIGINAL_ARTICLE
Inne
System-identifier
633017
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