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 10(2) to 10(8) copies of siRNA molecule with good parallel relationship (R(sq) = 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.
Animals ; Cell Line ; Classical swine fever virus ; genetics ; isolation & purification ; metabolism ; RNA Interference ; RNA, Small Interfering ; analysis ; genetics ; metabolism ; RNA, Viral ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; Swine ; Transfection ; Viral Nonstructural Proteins ; genetics ; metabolism ; Virus Replication