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目的建立以Taq Man探针实时荧光PCR技术为基础的核苷酸结合寡聚化结构域样受体3(Nucleotide-binding oligomerization domain,Leucine rich Repeat and Pyrin domain containing,NLRP3)基因单核苷酸多态性(Single nucleotide polymorphism,SNP)检测方法。方法以20份健康人血液样本为对象,选择NLRP3基因的两个SNP位点(rs3806265和rs35829419),针对每个位点分别设计1对PCR引物和2条Taq Man探针,进行实时荧光PCR扩增,对SNP位点进行分型。用常规PCR和基因测序的方法对Taq Man探针实时荧光PCR分型的结果进行验证。结果用建立的Taq Man探针实时荧光PCR分型法对20份样本进行检测,其中rs3806265位点发现TT基因型3份,TC基因型8份,CC基因型9份;rs35829419位点发现CC基因型20份,无AC和AA基因型。分型结果与常规PCR及基因测序分型方法完全一致。结论基于Taq Man探针技术的实时荧光PCR方法简单、快速,可实现对NLRP3基因位点的分型检测。
OBJECTIVE: To establish a single nucleotide of Nucleotide-binding oligomerization domain (Leucine rich repeat and Pyrin domain containing, NLRP3) gene based on Taq Man probe real-time fluorescence PCR Single nucleotide polymorphism (SNP) detection method. Methods Two SNP loci (rs3806265 and rs35829419) of NLRP3 gene were selected from 20 healthy human blood samples. One pair of PCR primers and two TaqMan probes were designed for each locus, and real-time PCR Increased, the SNP site type. The results of real-time TaqMan probe real-time PCR were validated by conventional PCR and gene sequencing. Results 20 samples were detected by Taq Man probe real-time PCR. Among them, 3 TT genotypes, 8 TC genotypes and 9 CC genotypes were found in rs3806265 locus. CC gene was found in rs35829419 locus 20 copies, no AC and AA genotypes. Typing results are in good agreement with conventional PCR and genotyping methods. Conclusion The real-time PCR method based on Taq Man probe technology is simple and rapid and can detect the genotype of NLRP3 locus.