猪FoxP3、IL-10双重实时荧光定量PCR方法建立及应用OA北大核心

This study was to establish a dual RT-qPCR method for quantitative detection of FoxP3 and IL-10 transcription.FoxP3 and IL-10 amplification primers and fluorescent probes were designed and synthesized.Primer concentration and annealing temperature were opti-mized.RT-qPCR method was established,and FoxP3 and IL-10 mRNA contents in peripheral Treg and immune organs infected with PCV2 were detected.Finally,Western blot was used to detect protein expression and verify the reliability of the method.The results were that the established RT qPCR method presented an"S"type amplification curve,with R2≥0.99.None of the TGF-β,IFN-γ,IL-2,IL-4,IL-6,and IL-17 nucleic acid templates or the negative controls had any amplification curves,with a minimum detection concentration of 101 copies/μL.The coefficients of variation within and between the batches were 0.87％and 1.84％,respectively.The peripheral blood Treg FoxP3 and IL-10 mRNA levels were both significantly increased in the PCV2 challenge group,with FoxP3 mRNA reaching its peak on 14 days of the experiment and IL-10 mRNA reaching its peak on 10 days.PCV2 infection resulted in an increase in FoxP3 and IL-10 transcrip-tion in the thymus,lymph nodes,and spleen.The Western blot analysis showed that FoxP3 was significantly higher than that in the control group on days 14 and 28,and IL-10 was significantly higher on days 10 and 14;which were consistent with the RT qPCR results.In this study,pig FoxP3 and IL-10 dual RT-qPCR had specificity,sensitivity,and stable and reliable detection points,which was of great signifi-cance for evaluating the immune response level and immune homeostasis in pigs.