Dataset Information

Development of a Real-Time Quantitative RT-PCR Assay for Detection of Bovine Rhinitis B Virus.

ABSTRACT: Bovine rhinitis B virus (BRBV) has been frequently identified in cattle diagnosed with bovine respiratory disease complex (BRDC) in recent years, suggesting its potential contribution to BRDC. The goal of this study was to develop a TaqMan-based real-time quantitative RT-PCR assay for efficient BRBV detection. A pair of primers and a probe were designed based on the 3D gene of the BRBV genome. The assay was specific for BRBV and able to exclude bovine rhinitis A virus, foot-and-mouth disease virus and Senecavirus A. The limit of detection of the assay was 4.46 copies per reaction. A standard curve was plotted, with a coefficient of determination of 0.999 in the concentration range of 10⁰-10⁸ copies/μl. The reproducibility of the assay was acceptable, with the standard deviations of cycle threshold values lower than 1.00 in both intra- and inter-assay. Of 200 samples collected from 150 head of cattle in recent years in China, 11% (22/200) of the samples tested positive in the assay, i.e., 4.6% (7/150) of the cattle were BRBV positive. This study provides an efficient diagnostic tool for the epidemiological investigations of BRBV.

SUBMITTER: Xie YL

PROVIDER: S-EPMC8212975 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Development of a Real-Time Quantitative RT-PCR Assay for Detection of Bovine Rhinitis B Virus.

Xie Yi-Lun YL Lv Dian-Hong DH Wen Xiao-Hui XH Zhai Qi Q Luo Man-Lin ML Wei Wen-Kang WK Chen Qin-Ling QL Zhai Shao-Lun SL

Frontiers in veterinary science 20210604

Bovine rhinitis B virus (BRBV) has been frequently identified in cattle diagnosed with bovine respiratory disease complex (BRDC) in recent years, suggesting its potential contribution to BRDC. The goal of this study was to develop a TaqMan-based real-time quantitative RT-PCR assay for efficient BRBV detection. A pair of primers and a probe were designed based on the 3D gene of the BRBV genome. The assay was specific for BRBV and able to exclude bovine rhinitis A virus, foot-and-mouth disease vir ...[more]

PMID: 34150897

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Project description: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.

Project description:BackgroundPseudorabies virus (PRV), a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo. Real-time RT-PCR is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of RNA molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection.ResultsIn this study, we have developed and applied a quantitative reverse transcriptase-based real-time PCR technique in order to profile transcription from the whole genome of PRV after lytic infection in porcine kidney cells. We calculated the relative expression ratios in a novel way, which allowed us to compare different PRV genes with respect to their expression dynamics, and to divide the PRV genes into distinct kinetic classes. This is the first publication on the whole-genome analysis of the gene expression of an alpha-herpesvirus by qRT2-PCR. We additionally established the kinetic properties of uncharacterized PRV genes and revised or confirmed data on PRV genes earlier examined by traditional methods such as Northern blot analysis. Our investigations revealed that genes with the same expression properties form clusters on the PRV genome: nested overlapping genes belong in the same kinetic class, while most convergent genes belong in different kinetic classes. Further, we detected inverse relationships as concerns the expressions of EP0 and IE180 mRNAs and their antisense partners.ConclusionMost (if not all) PRV genes begin to be expressed from the onset of viral expression. No sharp boundary was found between the groups of early and late genes classified on the basis of their requirement for viral DNA synthesis. The expressions of the PRV genes were analyzed, categorized and compared by qRT2-PCR assay, with the average of the minimum cycle threshold used as a control for the calculation of a particular R value. In principle, this new calculation technique is applicable for the analysis of gene expression in all temporally changing genetic systems.

Dataset Information

Development of a Real-Time Quantitative RT-PCR Assay for Detection of Bovine Rhinitis B Virus.

Publications

Development of a Real-Time Quantitative RT-PCR Assay for Detection of Bovine Rhinitis B Virus.

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