茎环法RT-qPCR定量检测细胞抗猪瘟病毒siRNA的表达

Quantitative detection of anti-classical swine fever virus siRNA expression in cells by stem-loop RT-qPCR

摘要:

RNAi (RNA interference) 已成为特异抗病毒治疗研究的热点，但siRNA (small interfering RNA) 的定量检测仍是评价RNAi抗病毒效果的瓶颈。为了检测抗CSFV特异siRNA分子 (siN1和siN2) 在细胞中的表达水平，设计并以交叉组合方法筛选了具有较高特异性和灵敏度的siRNA特异茎环引物 (SLP-N1-6和SLP-N2-8)，成功地建立了最优的siN1和siN2的茎环法RT-qPCR检测方法。该方法表现出良好的特异性和较高的灵敏度，能检测出102至108个拷贝的siRNA，至少可达7个数量级的检测范围，平行性好 (Rsq=0.999)，扩增效率高 (Eff. =98.2％)。茎环法RT-qPCR能准确地定量检测抗CSFV的PK-15细胞克隆的siN1/siN2表达水平，可结合常规的检测病毒水平的间接免疫荧光和TCID50等技术定量评价RNAi抗CSFV的有效性，为未来抗猪瘟转基因猪的抗病毒效果评价提供了先进的检测技术。

关键词:抗猪瘟病毒小干扰RNA，茎环引物，MGB探针，实时定量PC

Abstract:

RNA interference (RNAi) is a promising technology in development of specific antiviral therapy, but the quantitative detection of small interfering RNA (siRNA) expressed in vivo is the main challenge to assess its antiviral effect. In order to detect the siRNA molecules (siN1 and SiN2) particularly expressed in cells to inhibit the replication of classical swine fever virus (CSFV), serial specific stem-loop primers were designed and synthesized. Two of them (SLP-N1-6 and SLP-N2-8) were selected by screening in cross combination and successfully used in establishment of an optimal stem-loop RT-qPCR, which showed high specificity and sensitivity in detection of anti-CSFV siRNA expressed in PK-15 cells. The method was capable of detecting 102 to 108 copies of siRNA molecule with good parallel relationship (Rsq=0.999) and high amplification efficiency (Eff. = 98.2%). Therefore, the established stem-loop RT-qPCR can be used as an ideal tool in quantitative assessment of the anti-CSFV effects of RNAi in combination with detection of viral antigens using indirect immunofluorescent assay and TCID50, providing a novel technique for evaluating the antiviral effects of the siRNA expressed in anti-CSFV transgenic pigs to be established in future.

Key words:anti-CSFV siRNA, stem-loop primer (SLP), MGB probe, real-time PCR