多重不对称PCR探针熔解曲线法在一管中检测多个肥胖易感基因方法的建立

Establishment of an assay for simultaneous detection of multiple obesity-susceptibility genes based on multiplex PCR probe-based fluorescence melting curve analysis in one tube

目的针对中国人群4个常见的肥胖易感基因位点(FTO基因rs1421085位点、APOA5基因rs662799位点、FABP2基因rs1799883位点、ADRB3基因rs4994位点),建立一种方便、精准、低成本的检测方法。方法结合前期研究中发现的与中国人群肥胖密切相关的4个基因位点,设计引物对、荧光探针以及对应等位基因的靶序列,通过熔解曲线法,验证荧光探针与对应等位基因的靶序列的工作效果,确认不同基因型的熔解温度。采用PCR-Sanger测序法为金标准,对收集的人唾液样本进行检测,选取12个基因型的样本,开发多重不对称PCR荧光探针熔解曲线法进行检测。随后,使用多重不对称PCR探针熔解曲线法与PCR-Sanger测序法分别对200例志愿者的唾液样本进行基因分型检测,比较两种方法的结果与优缺点。结果多重不对称PCR探针熔解曲线法可以在一管中同时对4个常见的肥胖易感基因位点进行检测,准确度与PCR-Sanger测序法相同,且与PCR-Sanger测序法需有扩增、电泳、纯化、s测序的复杂步骤相比,可在一管中一次完成检测,操作更为便捷,且成本更加节省。结论本研究建立了一种针对4个常见肥胖易感基因位点的方便、精准、低成本的基因分型方法,为人群肥胖易感基因的批量筛查提供了一种有效的检测手段。

Objective To establish a simple, accurate, and low-cost gene detection assay for 4 common obesity-susceptibility single nucleotide polymorphisms(SNPs),including rs1421085 in FTO gene, rs662799 in APOA5 gene, rs1799883 in FABP2 gene and rs4994 in ADRB3 gene. Methods Based on 4 obesity-susceptibility SNPs identified by previous study in Chinese population, primer, fluorescent probes and target sequences were designed, the corresponding target oligonucleotide sequences were confirmed.The fluorescence melting curve analysis(FMCA) was used to verify the working effect of fluorescent probes and target sequences of corresponding alleles and determine the melting temperatures.The Sanger sequencing method was used as golden standard method, the human salivary samples of 12 genotypes were used to establish multiplex PCR probe-based FMCA assay.Two hundred samples collected from volunteers were detected by multiplex PCR probe-based FMCA method and Sanger sequencing method to compare results, advantages and disadvantages of the two methods. Results The multiplex PCR probe-based FMCA method can achieve simultaneous detection of 4 common obesity-susceptibility SNPs in one tube.Compared with the Sanger sequencing method, the established method showed the same accuracy and did not require complicated procedures such as amplification, electrophoresis, purification and sequencing.All steps could be finished in 1 tube which made it more convenient and cheap. Conclusion A convenient, accurate and low-cost genotyping method for 4 common obesity susceptibility SNPs was established, which provides an effective large scale population screening method for obesity-susceptibility genes.