实时荧光定量PCR法用于人类SLC01B1和ApoE基因多态性检测的方法学评价

目的：探讨在ISO15189实验室认可体系下，实时荧光定量 PCR法检测人类SLC01B1和ApoE基因多态性的方法学评价。方法本文收集经Sanger测序确定基因型的20份DNA样本。采用实时荧光定量PCR法对其中2份样本的SLCO1B1*1b、SLCO1B1*5、ApoE2和ApoE4共4个多态性位点分别批内重复扩增检测10次，以评价方法的精密度；再使用该方法扩增所有样本的4个多态性位点，每个位点检测一次，并与Sanger测序法结果比对，以评价方法的准确性；使用实时荧光定量PCR法对其中1份经梯度稀释的DNA样本进行扩增，以评价方法的最低检测限。结果2份DNA样本扩增所得Ct值变异系数均小于2．5％（≤1％）；与Sanger测序法结果对比，实时荧光定PCR法结果准确性达100％（20／20）；DNA浓度在0．964 ng／μl时，该方法仍可正确检出其基因型。结论借助经典实时荧光定量PCR法，我们能准确、快速地检测临床样本SLC01B1和ApoE基因多态性，用于指导正确合理使用他汀类药物。

Evaluation of real-time fluorescence quantitative PCR assay for human SLC01B1 and ApoE polymorphisms

Objective In the ISO15189 laboratory accreditation system ,we evalutate real-time fluorescence quantitative PCR as-say for human SLC01B1 and ApoE polymorphisms .Methods We collected twenty DNA samples of which the genotypes had been confirmed by Sanger sequencing .Four polymorphism loci ,SLCO1B1*1b ,SLCO1B1*5 ,ApoE2 and ApoE4 ,of two samples was tested respectively 10 times in one run to evaluate the precision of real-time fluorescence quantitative PCR assay .Further ,we applied the assay to amplify four polymorphism loci of all samples ,one test for each locus ,and we aligned the PCR results to the Sanger se-quencing outputs to access its accuracy .We diluted one random DNA sample in gradient and tested via real-time fluorescence quanti-tative PCR to evaluate the minimum detectable concentration .Results All Ct values ,due to PCR amplification reaction ,of two DNA samples showed less than 2 .5% of co-efficiency variations (≤1% ) .Compared with Sanger sequencing data ,this assay showed 100% accuracy .The genotype of DNA concentrating at 0 .964ng/μl could still be correctly detected by this experimental system . Conclusion With the help of classic quantitative PCR assay ,we can quickly and precisely determine SLC01B1 and ApoE polymor-phisms of clinical samples and guide the proper and rational use of statins .