【摘要】 抗病毒RNA干扰(RNA interference,RNAi)机制在哺乳动物中保守存在,宿主通过病毒源siRNA(virus-derived small interfering RNA,vsiRNA)引导Ago2蛋白剪切病毒RNA,从而发挥抗病毒作用.为了建立快速检测宿主抗流感病毒(PR8ΔNS1)和野田村病毒(NoVΔB2)特异性siRNA分子的方法,根据病毒特异性siRNAs序列设计茎环引物和扩增引物,筛选出具有高特异性和灵敏度的茎环引物和PCR扩增引物(16768,3280和24～45),成功建立了茎环法RT-qPCR检测哺乳动物病毒源siRNA表达水平的系统.该方法快速简便,准确性高,兼具较高的特异性与灵敏度,可作为小RNA测序替代方案检测siRNA或在测序前对宿主产生的病毒源siRNA进行有效监测与评价.更多还原

【Abstract】 Antiviral RNA interference(RNAi)is conserved in mammals.Diverse hosts produce virus-derived small interfering RNAs(vsiRNAs)to guide Ago2 to cleave virus RNA genome,directing antiviral immunity by RNAi.To establish a real-time RT-PCR assay for detection of specific siRNAs derived from influenza A virus(PR8ΔNS1)and nodamura virus(NoVΔB2),a real-time RT-PCR method was developed with a stem-loop primer and a pair of PCR primers targeting the viral siRNAs sequence in PR8ΔNS1 and NoVΔB2.Three groups stem-loop primer and PCR primers(16768,3280 and 24—45)was screened out as indicators after evaluating ΔCt and vsiRNA expression level between mock and treatment groups,a system for detection mammalian antiviral vsiRNAs by stem-loop primer was built.The method was capable of fast-quantitative detection viral siRNAs,which showed high specificity and sensitivity simultaneously.Hence,the system may be used as the alternative solution of small sequence or monitor host antiviral siRNAs before sequencing.更多还原