A fluorogenic-probe hydrolysis (TaqMan) real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay based on amplification of a 93 bp fragment from 5’un-translated region (5’UTR) of classical swine fever virus was used for detection and absolute quantification of the virus in clinical and tissue samples. For determining analytical sensitivity, an in vitro transcript RNA containing 5’UTR of classical swine fever virus (CSFV) strain Alfort187 from plasmid pCRXLV324-6 was used as a positive control and a standard for quantification of CSFV genomic RNA copies. The real-time quantitative RT-PCR (qRT-PCR) assay was used to assess the CSFV shedding from naturally infected pigs in whole blood, nasal swab and also in tissue samples. Used qRT-PCR was specific and sensitive as it could detect as low as 16.3 copies of CSFV genomic RNA. The assay was also reproducible as shown by satisfactory low intra-assay (0.80 % to 1.87 %) and inter-assay (1.00% to 3.80%) coefficient of variation with an efficiency of 102.3% and R² of 0.993. Thus, the real-time qRT-PCR assay described here allows rapid, specific and sensitive laboratory detection and quantification of CSFV genomic RNA copies.

中文翻译：

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通过一步式TaqMan实时RT-PCR分析定量经典猪瘟病毒载量

基于经典猪瘟病毒5'非翻译区（5'UTR）的93 bp片段的扩增，采用荧光探针水解（TaqMan）实时逆转录酶聚合酶链反应（RT-PCR）分析检测和绝对定量临床和组织样本中的病毒。为了确定分析灵敏度，在体外包含来自质粒pCRXLV324-6的经典猪瘟病毒（CSFV）菌株Alfort187的5'UTR的转录本RNA用作阳性对照和定量CSFV基因组RNA拷贝的标准。实时定量RT-PCR（qRT-PCR）分析用于评估全血，鼻拭子以及组织样品中自然感染猪的CSFV脱落。使用的qRT-PCR具有特异性和敏感性，因为它可以检测低至16.3个拷贝的CSFV基因组RNA。如令人满意的低批内（0.80％至1.87％）和批间（1.00％至3.80％）的变异系数所示，该方法的重现性也很高，效率分别为102.3％和R ²为0.993。因此，此处描述的实时qRT-PCR分析可快速，特异性和灵敏地对CSFV基因组RNA拷贝进行实验室检测和定量。