猪NLRP3基因Real-time PCR检测方法的建立及应用

Establishment and Application of Real-time PCR for Detection of Porcine NLRP3 Gene

根据NCBI NLRP3 cds(NM-001256770)序列设计1对特异性引物,构建标准品质粒pMD18T-NLRP3-189。通过反应条件优化、稳定性、敏感性等试验,成功建立了NLRP3的SYBR GreenⅠreal-time PCR检测方法,用该方法定量检测猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)感染后的猪肺组织中NLRP3表达水平。最佳引物浓度为0.15μmol/L,建立的标准曲线方程为y=-3.5196x+36.968,E值为0.96,最低检测量为84个拷贝,熔解曲线有单一峰,批内变异系数CV为0.067%~0.184%,批间变异系数为0.221%~0.594%,重复性好。该方法检测仔猪在感染Mhp后肺组织中的NLRP3 mRNA表达水平,感染组与健康组相比差异显著(P<0.01),NLRP3表达水平明显增高,在感染后14 d可达到高峰,平均mRNA拷贝数达18000左右,可持续42 d。表明该检测方法稳定性良好,精确度高,特异性强,为进一步研究猪炎症小体NLRP3的激活及其相关炎症反应机制提供了技术支持。

The objective of the current study was to establish and apply a quantitative method to detect the level of expression of the porcine NLRP3 gene.A pair of specific primers were designed based on the sequence of NCBI NLRP3 cds(NM-001256770)and a standard plasmid containing the gene,pMD18T-NLRP3-189,was constructed in this study.The SYBR GreenⅠreal-time PCR detection method for the NLRP3 gene was successfully established through the optimization of reaction conditions,stability and sensitivity tests,and the method was used to quantitatively detect the expression of the NLRP3 gene in porcine lung tissue at different stages after Mycoplasma hyopneumoniae(Mhp)infection.The results showed that the optimal concentration of primers in the 20μL system of this detection method was 0.15μmol/L,the established standard curve equation was y=-3.5196x+36.968,the E value was 0.96,the minimum detection amount was 84 copies.The melting curve had a single peak,and the intra-assay coefficient of variation,was in a range of 0.067%to 0.184%.The inter-assay coefficient of variation ranged from 0.221%to 0.594%,with good repeatability.This method was used to detect the expression of NLRP3 gene in porcine lung tissue after Mhp infection.The results showed that the infected group was significantly different from the uninfected control group(P<0.01).The expression of NLRP3 gene in the lung tissue of piglets after Mhp infection.Reached a peak at 14 days after infection,with a copy number of about 18000,which was detectable for 42 days post-infection.The results showed that the detection method had good stability,high accuracy and strong specificity,which provided technical support for further research on the activation of porcine inflammasome NLRP3 and its related inflammatory response mechanism.