猪瘟病毒实时定量PCR检测方法的建立及初步应用

Establishment and Preliminary Application of Real-time PCR Assay for Quantitative Detection of Classical Swine Fever Virus

为建立一种快速检测猪瘟病毒的基因检测方法,根据GenBank中猪瘟病毒(CSFV)基因组NS2基因序列设计并合成引物和Taq Man探针,通过各项条件优化并以10倍稀释度的质粒为标准品进行实时定量PCR扩增制作标准曲线,建立检测CSFV的实时定量PCR方法。该方法的检测灵敏度可达每微升10个拷贝,比常规PCR方法的灵敏度高出1个数量级;对标准品质粒检测的线性范围是1.0×109～1.0×101μL-1。对1.0×107、1.0×105、1.0×103μL-13种稀释度的标准品质粒进行重复试验,批内变异系数分别是0.30%、0.85%、0.43%,批间变异系数分别是0.81%、1.36%、0.63%,具有良好的重复性。应用该方法对45份临床样品进行检测,检出26份阳性样品,而常规PCR只检测出19份阳性样品,说明该实时定量PCR方法的敏感性高于常规PCR。可见,该方法具有敏感性高、特异性强、重复性好的特点,可用于检测病料中的猪瘟病毒。

Real-time PCR assay of Classical swine fever virus（CSFV） was established using a pair of primers and an internal Taq Man fluorogenic probe derived from the NS2 region of CSFV genome.The variety of conditions was optimized and the standard curve was achieved by using quantitative concentration of serial 10 fold dilutions of recombined plasmid DNA.The sensitivity of the assay was 10 μL-1,which was 10 times higher than that of the conventional PCR.The linear range was from 1.0×109 to 1.0×101 μL-1.Data of the repetitive tests showed good reproducibilities and the coefficients variations of 1.0×107,1.0×105,1.0×103 μL-1 within and between batches were 0.30%,0.85%,0.43% and 0.81%,1.36%,0.63%,respectively.The detection of 45 clinical sample showed that 26 of them were CSFV positive by the real-time PCR assay,in comparison only 19 of them were detected positive in routine PCR,indicating that the real-time PCR assay is more sensitive than routine PCR.In conclusion,the method has high sensibility,specificity and good reproducibilities,and could be applied to detect concentrations of CSFV.