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

Abstract

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'-untranslated 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 10 3 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.

Diagnosis of Ruminant Viral Diseases with Loop-Mediated Isothermal Amplification

“…P25 primers were able to detect both strains with higher sensitivity compared to primers of previous study by Aebischer et al However, authors reported that P25 primer set weakly cross-reacts with pestivirus D and H. In clinical samples, BVDV RT-LAMP test had comparable results with RT-qPCR. An additional species-specific primer set (P26) designed to differentiate BVDV-1 from BVDV-2 as well [ 60 ].…”

A label-free G-quadruplex aptamer/gold nanoparticle-based colorimetric biosensor for rapid detection of bovine viral diarrhea virus genotype 1

“…Bovine viral diarrhea virus (BVDV), the cause of bovine viral diarrhea (BVD) disease, belongs to the family Flaviviridae genus Pestivirus [ 1 ]. Among 11 different species of Pestivirus (A-K), species A and B, known as BVDV-1 and BVDV-2 respectively, are the most important [ 2 ]. The BVDV contains a positive-sense single-stranded RNA (12.3–16.5 kb) encoding four structural and eight non-structural proteins [ 3 – 5 ].…”

“…Various techniques are implemented for the detection of acute or persistent BVDV infections. These techniques include virus isolation, PCR-based methods, ELISAs and more recent techniques such as loop-mediated isothermal amplification (LAMP), dot-blot assay [ 2 , 16 – 18 ]. Since these methods are laborious, time and cost-consuming, developing new accurate and rapid detection techniques such as biosensors, as a new generation of sensing technology, is essential.…”