猪繁殖与呼吸综合征病毒细胞受体Sn和CD163 TaqMan荧光定量RT-PCR方法的建立  

Establishment of TaqMan fluorescence quantitative RT-PCR assay for PRRSV Sn and CD163 receptor detection

作　　者：韩梓峰[1,2] 刘永刚[2] 石文达[2] 王刚[2] 何玉利[2] 王淑杰[2] 刘鹤[2] 董建国[2] 武嘉男[2] 蔡雪辉[1,2] 

机构地区：[1]东北农业大学,黑龙江哈尔滨150001 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/基础诊断研究室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2012年第4期297-300,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家863计划(2011AA10A213);哈尔滨市科技攻关项目(2010AA6AN083)

摘　　要：为建立检测猪繁殖与呼吸综合征病毒(PRRSV)允许细胞表面的Sn和CD163受体的方法,本研究设计针对Sn和CD163基因的特异性引物和荧光探针,建立了检测PRRSV Sn和CD163受体的荧光定量RT-PCR方法。结果显示,该方法在检测101copies/μL～108copies/μL模板范围内具有良好的线性关系。标准曲线的相关系数r值均大于0.996,扩增效率分别为100%和107%;该检测方法对Sn和CD163的检测下限均为10拷贝,敏感性高;批内重复试验和批间重复试验的变异系数均小于5%,具有良好的重复性。利用该方法对PRRSV感染肺泡巨噬细胞(PAM)72 h后Sn和CD163受体mRNA的转录水平进行了检测,结果表明Sn和CD163受体的转录水平显著上调。本研究为PRRSV病毒感染后两种主要受体变化趋势的研究提供了有效的检测方法。For detection receptor of porcine reproductive and respiratory syndrome virus（PRRSV） in permissive cells,a real time TaqMan RT-PCR was developed using specific primers and fluorogenetic probe based on the gene of Sn and CD163 receptor of PRRSV.The established method had a linear dynamic range from 101 copies/μL to 108 copies/μL.The result demonstrated that the real-time RT-PCR assay was highly specific and sensitive with a detection limit of 10 copies for both Sn and CD163.The coefficient of variation for both intra-and inter-assay was less than 5%.The assay was used to detect Sn and CD163 mRNA transcription in porcine alveolar macrophage（PAM） cells at 72 hours post infected with PRRSV.The result showed the transcription levels of Sn and CD163 mRNA were both up-regulated significantly.The assay demonstrated that the real-time RT-PCR assay was highly specific and sensitive.

关 键 词：猪繁殖与呼吸综合征病毒 Sn受体 CD163受体 荧光定量RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]