基于Taqman探针和染料相结合的液滴数字PCR用于PIK3CA基因E545K三种基因型的检测

发布时间：2018-05-24 15:04

本文选题：液滴数字PCR + TaqMan探针　；参考：《大连医科大学》2016年硕士论文

【摘要】：目的:针对目前实时定量PCR(real-time quantitative PCR,qPCR)和液滴数字PCR(droplet digital PCR,ddPCR)只能检测已知突变位点的不足,尝试建立一种基于Taqman探针和染料相结合的ddPCR方法用于检测未知突变序列,并应用该方法检测PIK3CA基因E545K突变位点的三种基因型(野生型、杂合突变型和纯合突变型)。方法:本研究属于方法学研究,即先建立方法,然后与已有方法进行统计学分析比较,确定方法的可靠性。1.选择包含PIK3CA基因的野生型(MCF-7细胞)和杂合突变型(H460细胞)细胞系以及合成包含PIK3CA基因的纯合突变型双链DNA,针对PIK3CA基因E545K突变位点设计合成特异性引物及一对野生型(FAM标记)和突变型(VIC标记)探针。2.提取各细胞系的核酸,PCR扩增后测序扩增产物,确定各细胞系包含的PIK3CA基因E545K位点的基因型。3.实时定量PCR检测引物和探针的特异性,初步建立基于Taqman探针和DNA结合染料(EvaGreen)联合使用的qPCR反应体系,并优化反应体系,使三种基因型纯样本之间的qPCR终荧光信号值差别最大,从而便于对三种基因型纯样本进行鉴别区分。4.理论上qPCR反应体系可以直接应用于ddPCR,但在实际实验中需要对体系的某些条件进行微调,然后以qPCR反应体系为基础,应用ddPCR建立和优化双探针法(野生型+突变型)与野生型探针联合EvaGreen染料的方法,用于PIK3CA基因E545K突变位点三种基因型的检测,确保能够从ddPCR结果图中清晰的区分不同基因型的液滴团。5.应用上述两种ddPCR方法对同时含有三种基因型的混合样本进行定量检测,分别得到三种基因型的拷贝数,对三组数据分别做配对样本均数的t检验,通过P值判断两种检测方法所得到的结果的差异是否具有统计学差异,验证方法的可靠性。结果:1.由染料法qPCR优化体系及PCR循环条件,得到最佳退火温度为62℃,此时体系扩增效率(amplification efficiency,E)E=0.918l。良好的扩增效率可降低ddPCR中各液滴之间因扩增效率不同导致的荧光信号差异和结果的不准确。2.采用qPCR双探针体系分别检测三种基因型纯样本,由扩增曲线可知探针特异性良好。3.初步建立了联合使用野生型探针和EvaGreen染料的qPCR体系,分别对等拷贝数的三种基因型纯样本进行扩增检测,优化后各样本间野生型探针(VIC)信号差值最大。4.基于ddPCR建立的双探针法体系,经优化可用于检测同时含有三种基因型的混合样本,包含不同基因型的液滴团可清晰区分。5.基于ddPCR建立的联合使用野生型探针和EvaGreen染料的体系,优化后也可检测同时包含三种基因型的混合样本,各基因型对应的液滴团清晰可分。6.应用ddPCR的双探针法和联合使用野生型探针和EvaGreen染料两种方法分别对同一组含有三种基因型的混合样本进行检测,分别得到三种基因型的拷贝数,分别对三组样本数据进行配对样本均数的t检验。野生型组:P=0.9540.05;杂合突变型组:P=0.4510.05;纯合突变型组:P=0.3080.05,说明两种检测方法差异无统计学意义。结论:成功建立了基于ddPCR联合使用TaqMan探针和染料检测PIK3CA基因E545K突变位点三种基因型的方法。该方法只使用野生型探针,可检测与此野生型探针序列不匹配的任何SNP或其他类型的突变,打破了普通双探针法只能对突变型探针所设计的已知突变位点进行检测的局限,有望降低多突变位点检测的成本,并用于突变的筛查。
[Abstract]:Objective: in order to detect the deficiency of the known mutation site in real-time quantitative PCR (real-time quantitative PCR, qPCR) and droplet digital PCR (droplet digital PCR, ddPCR), a ddPCR method based on the combination of Taqman probe and dye is tried to detect the unknown mutation sequence, and the method is used to detect the mutation of the mutation. Three genotypes of the loci (wild, heterozygous and homozygous mutant). Methods: This study belongs to methodological study, first to establish a method, and then to compare with the existing methods for statistical analysis, and to determine the reliability of the method.1. selection of the wild type (MCF-7 cell) and heterozygous mutant (H460 cell) cell lines containing the PIK3CA gene and the cell lines, as well as the cell lines of the heterozygous mutant (H460 cell). The homozygous mutant double stranded DNA containing the PIK3CA gene was synthesized and the specific primers were designed for the E545K mutation site of the PIK3CA gene, and a pair of wild type (FAM markers) and the mutant (VIC marker) probe.2. were used to extract nucleic acid from each cell line. PCR amplified the product after amplification and determined the genotype.3. of the PIK3CA gene E545K loci of each cell line. The specificity of the primers and probes was detected by real-time quantitative PCR. The qPCR reaction system based on the combination of Taqman probe and DNA binding dye (EvaGreen) was initially established, and the reaction system was optimized to make the maximum difference between the qPCR terminal fluorescence signal values between the three genotypes of pure samples. Thus, the.4. theory was differentiated from the pure samples of the three genotypes. The upper qPCR reaction system can be directly applied to ddPCR, but in actual experiments, some conditions of the system need to be adjusted. Then, based on the qPCR reaction system, the method of combining the double probe (wild type + mutant) and wild type probe combined with EvaGreen dye is established and optimized by ddPCR, which is used for the three genes of the E545K mutation site of the PIK3CA gene. To ensure that the two ddPCR methods were used to detect the mixed samples containing three genotypes at the same time, and the number of copies of the three genotypes was obtained, and the three sets of data were tested by the t test of the average number of paired samples, and two kinds of P values were used to judge two kinds of ddPCR. Whether the difference between the results obtained by the detection method has statistical difference, and the reliability of the method is verified. 1. the optimum annealing temperature is 62 C by the dye method qPCR optimization system and the PCR cycle condition. The good amplification efficiency of the system amplification efficiency (amplification efficiency, E) E= 0.918l. can reduce the cause of each droplet in ddPCR. The difference of the fluorescence signal and the inaccuracy of the results were detected by the different amplification efficiency. The qPCR dual probe system was used to detect three pure samples of the genotypes. The amplification curve showed that the probe specificity was good.3., and the qPCR system of the combined use of wild type probe and EvaGreen dye was preliminarily established, and the pure samples of the three genotypes of the peer-to-peer number were identified respectively. The double probe system based on ddPCR based on the maximum.4. signal difference of the wild type probe (VIC) between the samples was optimized. The optimization could be used to detect the mixed samples containing three genotypes. The droplets containing different genotypes could clearly distinguish.5. based on ddPCR and EvaGreen dyeing. The system of material can also be optimized to detect the mixed samples containing three genotypes at the same time. The corresponding droplets of each genotype can be clearly divided into two kinds of mixed samples containing three genotypes, including three kinds of genes, respectively, using the double probe method of.6. using ddPCR and the combined use of wild type probe and EvaGreen dye. The copy number of the type is t test for the average number of paired samples of three groups of samples. The wild type group: P=0.9540.05; the heterozygous mutant group: P=0.4510.05; the homozygous mutant group: P=0.3080.05, indicating that there is no statistical difference between the two detection methods. Conclusion: a successful establishment of a ddPCR combined with a TaqMan probe and a dye for the detection of the PIK3CA gene E. The method of three genotypes of the 545K mutation site. This method only uses a wild type probe to detect any SNP or other types of mutations that do not match the sequence of the wild type probe. It breaks the limitation that the common double probe can only detect the known mutation sites designed by the mutant probe, and it is expected to reduce the detection of multiple mutation sites. Ben, and for mutation screening.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R440

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