为建立定量检测猪Ⅰ型干扰素信号传导因子mRNA的TaqMan荧光定量PCR检测方法,本研究针对猪Ⅰ型干扰素信号传导因子视黄酸诱导基因-1 (RIG-1)、Toll样受体-9(TLR-9)、干扰素调节因子-3(IRF-3)和干扰素调节因子-7 (IRF-7)的基因序列设计了特异性的引物和探针,分别构建了各自的阳性标准重组质粒,同时选择β -actin作为管家基因,建立了检测猪RIG-1、TLR-9、IRF-3、IRF-7的Taq Man荧光定量PCR方法.结果表明:所建立的检测方法荧光定量PCR扩增均无非特异性产物产生；检测下限均达1.0×101 copies/μL；批内及批间变异系数均小于3％.利用该方法对猪细小病毒(PPV)感染48 h的ST细胞中RIG-1、TLR-9、IRF-3和IRF-7mRNA的表达水平进行了检测.结果表明,RIG-1、IRF-3和IRF-7转录水平呈现上调趋势,而TLR-9转录水平呈现下调趋势.本研究建立的TaqMan荧光定量PCR方法特异性强、敏感性高、重复性好,适于猪Ⅰ型干扰素信号传导因子的定量检测分析.%In this study, a real-time PCR assays based on TaqMan probes for detection of retinoic acid-inducible gene 1 (R1G-1), toll-like receptor 9 (TLR-9), interferon regulate factor 3 (IRF-3), interferon regulate factor 7 (IRF-7) and β-actin were established with specific primers and probes of each gene. The results indicated that the assays were specific and highly sensitive with a detection limit of 1.0 × 10 copies/Ml, and the coefficient of variation were less than 3% for both inter- and intra-assay. The established assays were used to detect Mrna transcription levels of RIG-1, TLR-9, IRF-3 and IRF-7 controled with β -actin in ST cells infected with porcine parvovirus. The results showed the transcription levels of TLR-9mRNA was down-regulated, and others showed up-regulared. The assays with highly specific, sensitive and reproducibility could be used to detect and quantify the signal transduction factors in porcine cells.
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