摘要
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 （RIG-1）, toll-like receptor 9 （TLR-9）, interferon regulate factor 3 （IRF-3）, interferon regulate factor 7 （IRF-7） and 13-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.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2011年第12期953-956,共4页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(31001069
31172349
31172341)
关键词
Ⅰ型干扰素
信号传导因子
荧光定量PCR
检测
type I interferon
signal transduction factors
fluorescent quantitative PCR
detection
