Development of real-time RT-PCR assays based on SYBR GreenI for detection of goat Toll-like receptors

For detection of the three goat Toll-like receptors TLR2,TLR4 and TLR9 and the housekeeping gene β-actin,a series of primers were designed according to the sequences.The recombinant plasmids containing the target genes were used to optimize assay condition of developing a SYBR Green Ⅰ real-time RT-PCR.The results showed that the Ctof TLR2,TLR4,TLR9 and β-actin cytokines had a good linear relationship(R2>0.984).These assays were single specific melting peak for every cytokine.The coefficients of variation were lower than 2 percent for both intra-and inter-assay.The established assays were successfully used to detect TLR2 and TLR9 mRNA expression levels in peripheral blood mononuclear cells(PBMCs) in goats experimentally infected with recombinant goatpox virus(△ N1 L strain),indicating△N1 L strain could significantly increase the level of innate immune response in vivo.The established real-time RT-PCR could be applied to the development of a genetic vaccines for goatpox.