糖尿病线粒体DNA常见易感基因位点检测芯片的研制

Development of a chip for detecting the diabetes-associated common loci in mitochondrial genome

目的建立一种快速、准确的检测糖尿病线粒体DNA常见易感基因位点的芯片技术。方法将氨基修饰的16个常见易感基因位点（1888，3200，3206，3243，3290，3316，3394，3593，3426，12026，12258，14577,14693，14709，16189，16223）的野生型和突变型探针，通过Packard芯片点样仪点样于醛基修饰的基片上，室温保湿过夜固定。采用该基因芯片对52份已知和52份未知突变位点的2型糖尿病标本进行检测，每份样本平行测3次，Axon GenePix4000B芯片扫描仪阅读芯片，GenePix Pro4．0版芯片图像分析软件分析结果。结果芯片样点分布均匀、清晰、规整度好、无漏点和连点；片内平行点之间的荧光强度变异系数为0．62％～9．64％，芯片间相同位点之间为1．00％～13．46％；背景信号低（突变型探针和野生型探针的背景荧光强度分别为43．25±2．10，44．56±2．63）；减去背景信号的阳性探针荧光强度明显高于阴性探针（t=8．6，P=0．000），易于判断结果；初步设定筛选和诊断实验的突变型cut—off值；按此标准判断实验结果，筛选实验的假阳性率为11．11％，假阴性率为0．53％；诊断试验的假阳性率为2．22％，假阴性率为6．41％；除16189位点外，验证其他已知突变位点，并在未知突变样本中检出了A12026G突变和C16223T多态性，结果与测序结果一致。结论该芯片可用于糖尿病线粒体DNA15个常见易感基因位点的快速筛查和检验诊断。

Objective To develop a chip for rapid detecting the diabetes-associated common loci in mitochondrial genome. Methods The amino modified wild-type and mutant probes of 16 loci （1888, 3200, 3206, 3243, 3290, 3316, 3394, 3593, 3426, 12026, 12258, 14577, 14693, 14709, 16189, 16223） in mitochondrial genome were immobilized onto the activated slides with aldehyde surface using Biochip Arrayer, then they were incubated overnight in a humid chamber at room temperature. The chips were used to detect 16 loci in 52 known and 52 unknown mutant samples, each sample was repeatedly measured 3 times. After hybridization, Axon GenePix 4000B scanner and GenePix Pro 4. 0 software were used to scan and analyze the chips. Results The printed spots on the chip showed distinct and regular on the scanned image. The intra-chip coefficient of variation of parallel spots ranged from 0.62% to 9. 64% , and the interchip coefficient of variation of the same locus ranged from 1.0% to 13.46%. The background signals of mutant and wild-type probes were 43.25 ±2. 10 and 44. 56 ±2.63, respectively. The fluorescence intensity without background of positive probe was significantly higher than that of negative probe （ t = 8. 6, P =0. 000） . The cut-off values of mutant type for screen and diagnostic tests were set up, their false positive rates were 11.11% and 2.22%, respectively, and their false negative rates were 0.53% and 6.41%, respectively. Except for locus16189, all the known mutant loci were confirmed, and A12026G mutation and 16223 C/T polymorphism were detected in unknown mutant samples. The results were further verification by DNA sequencing. Conclusion The chip could be used for rapid detecting 15 common diabetes-associated loci in mitochondrial genome.