Detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time RT-PCR assay

Abstract

Background: As a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples.Methods: A pair of primers specific for highly conserved regions of the BVDV-1 5′-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples. Results: The results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them. Conclusions:Taken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.

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

“…The result of qPCR, as the gold standard technique for the detection of BVDV, validated the BVDV infection of the prepared samples with 7.2×107–5.5×10 11 virus copies/ml ( S2 Fig ). According to the reported standard curve for BVDV in literature, the least concentration of the virus which can be amplified by the qPCR technique is less than 10 copies/ml [ 53 ].…”

Diagnosis of bovine viral diarrhea virus: an overview of currently available methods

“…Compared to conventional RT-PCR, the assay exhibited a significantly higher positive detection rate (17.2%, 29/169) when used to test a total of 169 aerosol samples collected from six dairy herds. Furthermore, a concordance rate of 100% was achieved between the assay and the BVDV RPA-LFD assay in detecting the positive samples (Hou et al, 2020). An interesting study reported that the SYBR Green I real-time RT-PCR targeting 5'-UTR and nested RT-PCR are superior to antigen-capture ELISA (Ag ELISA) for BVDV detection in aborted fetus samples over a 22-year period (Spetter et al, 2020).…”

Induction of the Unfolded Protein Response during Bovine Alphaherpesvirus 1 Infection

“…Total cellular RNA was purified using Trizol reagent (Thermo Fisher Scientific, Hampton, NH, USA) according to the manufacturer's instructions. RT-PCR was performed by using PrimeScript™ One Step RT-PCR Kit Ver.2 (TaKaRa Bio Inc., Otsu, Japan) [39][40][41]. The XBP1 gene was amplified by PCR with XBP1-F: 5 -CTGAAAAACAGAGTAGCAGCTCAGA-3 and XBP1-R: 5 -TCAGTTCATTAATGGCTTCCAGC-3 .…”