Abstract:

This study aimed to develop a real-time reverse-transcription polymerase chain reaction (RT-PCR) method based on SYBR Green Ι fluorescent for detection of horse Toll-like receptor mRNA. According to the gene sequence in conservative region of horse’s Toll-like receptors available in GenBank, the specific primers were designed with β-actin as an internal control to construct Real-time RT-PCR assay. The standard curves showed good linear relationships with the correlation (r²>0.990) and efficiency about 1.0 in the range of 1×10² to 1×10⁸ copies·μL^-1. The melting curve analysis showed that the product was specific to a single peak, and no primer-dimers, with high specificity and sensitivity. The good reproducibility was obtained and the coefficient of variation were less than 3.5% for the intra-assay and inter-assay. The clinical sample test indicated that the TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8 and TLR9 mRNA were transcripted in medulla, and TLR4 mRNA level was highest, but TLR9 mRNA level was lowest. This assay could be successfully used for the detection of clinical samples and provide a technical platform to research horse TLRs at the mRNA levels in the quantitative analysis.