CYP2C19显色型基因芯片的研究

Study of colorful gene chip for CYP2C19 genotyping

目的提供一种基于显色原理的CYP2C19基因芯片诊断方法。方法采用碱性磷酸酶促显色原理设计制备固定有*2、*3检测探针的CYP2C19基因芯片,通过EDTA抗凝外周血样本提取基因组DNA,PCR扩增CYP2C19*2、*3突变片段并与芯片在43℃杂交,经洗涤、显色后扫描获取图像,经软件分析输出基因型并对基因芯片性能进行系统评价。结果 CYP2C19显色型基因芯片检出限为20 ng基因组DNA;与基因组DNA同等浓度鱼精DNA检测信号阴性;用*2/*2、*1/*3基因型样本重复检测10次,结果一致;常见浓度的甘油三酯、总胆固醇、胆红素和完全溶血对测定没有影响。临床三家医院临检实验室对1000例临床样本的检测与双向测序方法比对验证,结果一致。结论为高通量基因诊断产品的产业化提供了一种实用的技术选择。

Objective Gene microarray as a new kind of genetic diagnosis technology has been applied in clinical examination. Currently, probe-target hybridization is usually detected and quantified by detection of fluorophore with several shortages, such as raw materials, expensive testing equipment and unstable detection signal. Here, we try to build a method detected by coloration for quickly detecting the genotypes of CYP2C19. Methods Genomic DNA is extracted from peripheral blood samples anticoagulated with EDTA. The DNA fractions were amplified by PCR and hybridized with the probes of CYP2C19＊2 and CYP2C19＊3 mutant alleles on the CYP2C19 gene chip on 43℃. After washing and developing the gene chip, the hybridization results were observed through gene chip scanning machine with Arraydoctor2.0 software. Results The detection limit of CYP2C19 colorful gene chip is 20 ng genomic DNA; Negative control groups which is hybridized with equal concentration of Salmon sperm DNA show no bands; The measurement of samples of＊2/＊2,＊1/＊3 genotype alleles are repeated 10 times and the results are also consistent. Triglyceride, cholesterol, bilirubin and hemolysis which are contained in blood samples have no influence to the results. 1000 clinical samples were detected by clinical laboratory from three hospitals. The detection results from gene chip were same as sequencing results. Conclusion Our study provides a technological meaning for clinical application using high-throughput gene detection related to personalized medicine.