Abstract
The aim of this study was to develop a one-step real-time reverse transcription-polymerase chain reaction assay using the minor groove binding probe (MGB rRT-PCR) for rapid and quantitative detection of classical swine fever virus (CSFV). The method, which targets the 5′-nontranslated region (5′NTR) of the viral genome, detected all CSFV isolate tested, but not heterologous pathogens. Using an in vitro transcript of the 5′NTR as a quantitative standard for the CSFV genome copy number, the assay had a detection limit of 10 copies/reaction, and the standard curve had a linear range from 10 to 107 copies/reaction, with good reproducibility. As determined by an end-point dilution comparison, in most case, the sensitivity of the MGB rRT-PCR was approximately 10-fold higher than that of virus isolation and the rRT-PCR using the standard Taqman probe (standard rRT-PCR). The agreement between the MGB rRT-PCR and standard rRT-PCR, or virus isolation was 93.3% and 76.7%, respectively, when detecting 261 field samples. Due to its rapidity, high specificity and sensitivity, the MGB rRT-PCR assay provides a valuable tool for diagnosis and molecular studies of CSFV biology.
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Acknowledgements
The author would like to thank Dr. Zishu Pan (State Key Laboratory of Virology, Wuhan University) for kindly providing virus antibody and constructive criticisms of the manuscript. This work was supported by grant from Hubei key laboratory of Animal Embryo and Molecular Breeding (2008ZD08).
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Wen, G., Yang, J., Luo, Q. et al. A one-step real-time reverse transcription-polymerase chain reaction detection of classical swine fever virus using a minor groove binding probe. Vet Res Commun 34, 359–369 (2010). https://doi.org/10.1007/s11259-010-9363-8
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DOI: https://doi.org/10.1007/s11259-010-9363-8