Broad detection and quick differentiation of bovine viral diarrhea viruses 1 and 2 by a reverse transcription loop-mediated isothermal amplification test

抄録

For broad detection of pestivirus A (bovine viral diarrhea virus 1: BVDV1) and pestivirus B (BVDV2) by a reverse transcription loop-mediated isothermal amplification (RT-LAMP) test, the P25 primer set was designed using nucleotide sequences of 5’-UTR region of 1454 BVDVs. The base coverage of each primer against diverse BVDVs were more than 99% in each base position. The one step LAMP test with the P25 primer set could detect both BVDV1 (TK) and BVDV2 (KZ), but did not amplify 5 other bovine viruses. Detection limit of the LAMP test was 10³ copies of synthesized DNAs, and 10⁻³ and 10⁻⁴ dilutions of viral RNAs of TK and KZ strains, respectively, whereas that with current Aebischer’s primer set was 10⁻² dilution and negative of these RNAs, respectively. All of the 63 viral RNA samples of persistently infected (PI) cattle, consisting of the 1a (12), 1b (31), 1c (11), and 2a (9) subgenotypes, were broadly detected with the P25, while only 65% of them were positive with Aebischer’s primer set. The validation study showed that the RT-LAMP test with the P25 had 100% sensitivity and 100% specificity against that with updated Vilcek’s PCR primers. Also, by using the P26 primer set which contained 3 species-specific primers, all 63 RNA samples were clearly distinguished from BVDV1 or BVDV2 by the typing RT-LAMP test. These results indicate that the one step RT-LAMP test using P25 or P26 primer sets would be useful for broad detection and rapid differentiation of BVDV1 and BVDV2.