基于扩增阻碍突变系统实时荧光PCR的IL28B基因SNP位点联合检测方法的建立

Establishment of combined detection of IL28B SNP sites based on the amplification refractory mutation system and real-time PCR

目的 建立基于扩增阻碍突变系统和实时荧光定量PCR相结合的IL28B基因多SNP位点联合检测方法,为进一步研究丙型肝炎患者抗病毒治疗效果提供技术平台.方法 分别构建包含IL28B基因的rs12979860和rs8099917两个SNP位点的野生型和突变型质粒标准品;采用ARMS技术,结合Taqman探针技术,建立能够同时检测上述两个SNP位点的实时荧光ARMS-PCR方法.然后采用本方法检测60例丙型肝炎住院患者血清标本并以基因测序结果为金标准进行方法学评价.结果 实时荧光ARMS-PCR能够有效的鉴别rs 12979860位点的CC、CT、TT基因型和rs8099917位点的TT、TG和GG基因型,通过和测序结果进行比对,二者符合率为100％ （P ＜0.05）.结论 本研究建立的双重实时荧光ARMS-PCR能够快速可靠的对IL28B的rs12979860和rs8099917位点进行基因型多态性的联合检测,为慢性丙型肝炎患者实现个体化治疗奠定基础.

Objective Establish combined detection method of IL28B SNP sites based on the amplification refractory mutation system and real-time PCR to provide a technical platform for further study of the effect of antiretroviral therapy in the patients with hepatitis C.Methods Constructed wild-type and mutant plasmid standard of rs12979860 and rs8099917 in IL28B respectively.Using ARMS technology,combined with Taqman probe technology,to establish the real-time fluorescent ARMS-PCR method which could simultaneously detect the two SNP sites.Then 60 serum samples were analyzed by ARMS-PCR,and gene seguencing was used for verification.Results The genotype CC,CT or TT of rs12979860 and the genotype TT,TG or GG of rs8099917 could be well identified by ARMS-PCR and the coincidence rate was 100% （P 〈0.05）,compared to gene sequencing,both of the sensitivity and spceficity of ARMS-PCR were 100%.Conclusion The dual real-time fluorescence ARMS-PCR can quickly,reliable combined detect rs12979860 and rs8099917 in IL28B,Lay the foundation for individual treatment of hepatitis C patients with chronic hepatitis.