Abstract
The aim of the present study was to establish a method of EvaGreen real time fluorescence quantitative polymerase chain reaction (qPCR) for detecting quickly bovine rotavirus (BRV) in fecal samples from calves with diarrhea. The specific primers were designed and synthesized according to BRV VP7 gene in Genbank. VP7 gene was cloned into the pMD18-T vector. The bovine viral diarrhea virus, bovine coronavirus, porcine epidemic diarrhea virus, bovine mycobacterium tuberculosis and negative control were detected with qPCR. Plasmids in five 10-fold gradients were detected using qPCR. The results show that a EvaGreen qPCR was established in the present study. Only BRV displayed amplification curve; cross-reactivity between BRV and the other viruses or bacteria was not observed. The amplification curve of pMD18-T vector plasmid (diluted in 10-1 to 103 gradient) was typical S shape. The detection limit of qPCR was 8.0 copies/μL, namely 100 plasmid concentrations. The coefficients of variation in both intra-assay and inter-assay was less than 2%. The established EvaGreen qPCR had a high specificity, sensitivity and reproducibility. It can be applied to clinical diagnosis and epidemiological surveys of BRV. Key words: Rotavirus, VP7 gene, evagreen, real-time quantitative PCR, bovine.
Highlights
Neonatal calf diarrhea is a common disease affecting the newborn calf worldwide, threatening the cattle production along with significant morbidity and mortality and inducing severe economic losses (Wei, 2011)
The aim of the present study was to establish a method of EvaGreen real time fluorescence quantitative polymerase chain reaction for detecting quickly bovine rotavirus (BRV) in fecal samples from calves with diarrhea
The bovine viral diarrhea virus, bovine coronavirus, porcine epidemic diarrhea virus, bovine mycobacterium tuberculosis and negative control were detected with Quantitative PCR (qPCR)
Summary
Neonatal calf diarrhea is a common disease affecting the newborn calf worldwide, threatening the cattle production along with significant morbidity and mortality and inducing severe economic losses (Wei, 2011). Group A rotaviruses (RVA) are known to be important viral diarrheal agents in infants and young animals, including calves. The numbers of the rotavirus-associated mortality are estimated to be 453,000 in 2008 (Tate, 2012; 2013). The numbers of deaths is high in developing countries (Abe et al, 2009). Rotavirus remains an important cause of morbidity. Rotaviruses possess 11 segments of double-stranded ribonucleic acid (dsRNA) and two outer capsid proteins: VP4 (encoded by gene segment 4) and VP7 (encoded by gene segment 7, 8 or 9 depending on the strain), both of which are independently responsible for virus neutralization (Estes, 2001). RVA strains are antigenically heterogeneous, and are classified in multiple G and P types de-
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