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Abstract This work aimed to develop a SYBR Green Ι Real-time fluorescent quantitative PCR assay for detection of bovine toll-like receptors mRNA. According to the gene sequences of bovine's Tol1-like receptors (BoTLR-2, 3, 4, 5, 7, 8 and 10) available in GenBank, eight pairs of primers based on their conserved regions were designed with bovine glyceraldehyde-3-phosphate dehydrogenase (BoGAPDH) as an interna control to construct Real-time fluorescent quantitative PCR assay. The results showed a good linear relationships(r2>0.991) between the Ct value and the concentration of positive plasmid for each gene on the condition that the concentration of positive plasmids were limited from 1×102 to 1×109 copies /μL. The melting curve analysis showed the product was specific to a single peak, with high sensitivity and specificity. Reproducibility showed that the minimum detectable concentration of positive plasmids of TLRs and GAPDH gene were 100 copies /μL and 10 copies /μL. And the intra-assay and inter-assay coefficient of variation values were maintained at less than 3.5%. The clinical sample test showed that TLR3 and TLR8 mRNA levels in the induction of early high peak at 4h, while the level of TLR4 and TLR7 contrast, higher late in the induction, peaked at 24 h.These data showed that the detection method in this study successfully can be use for the detection of clinical samples and provids a technical platform in the quantitative analysis of BoTLRs on the mRNA levels.
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Received: 21 May 2010
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