one-step real-time reverse transcription-polymerase chain reaction detection of classical swine fever virus using a minor groove binding probe

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.