A one-step real-time reverse transcription-polymerase chain reaction detection of classical swine fever virus using a minor groove binding probe
文献类型： 外文期刊
作者： Wen, Guoyuan 1 ; Yang, Jun 1 ; Luo, Qingping 1 ; Hu, Zhibin 3 ; Song, Nianhua 3 ; Zhang, Rongrong 1 ; Wang, Hongling 1 ;
作者机构： 1.Hubei Acad Agr Sci, Inst Anim Husb & Vet Sci, Wuhan 430070, Peoples R China
2.Hubei Key Lab Anim Embryo & Mol Breeding, Wuhan 430070, Peoples R China
3.Hubei Prov Anim Dis Control Ctr, Wuhan 430064, Peoples R China
关键词： viral genome;genome copy number;end-point dilution comparison
期刊名称：VETERINARY RESEARCH COMMUNICATIONS （ 影响因子：2.459； 五年影响因子：2.352 ）
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收录情况： SCI
摘要： 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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