论文部分内容阅读
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,AxonGenePix4000B芯片扫描仪阅读芯片,GenePixPro4·0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 establish a fast and accurate chip technology for detecting common susceptibility gene loci in diabetic mitochondrial DNA. Methods The wild-type and amino acid residues of 16 common susceptible loci (1888,3200,3206,3243,3290,3316,3394,3593,3426,12026,12258,14577,14693,14709,16189,16223) Mutant probes were spotted onto aldehyde-modified substrates by a Packard chip spotter and fixed overnight at room temperature with moisturization. The gene chip was used to detect type 2 diabetes mellitus in 52 known and 52 unknown sites. Each sample was tested in parallel for 3 times, and the results were analyzed by AxonGenePix4000B chip reader and GenePixPro4.0 chip image analysis software. Results The distribution of chip samples was uniform, clear, good regularity, no leakage and even point; the coefficient of variation of fluorescence intensity between parallel points was 0.62% ~ 9.64%, the same between the chips was The background signal was low (the background fluorescence intensities of the mutant and wild-type probes were 43.25 ± 2.10 and 44.56 ± 2.63, respectively); the background signal was subtracted (T = 8.6, P = 0.000), which is easy to judge the results. The mutant cut-off values of screening and diagnostic experiments were initially set. According to this standard, the fluorescence intensity of positive probe was significantly higher than that of negative probe As a result, the false positive rate of the screening experiment was 11.11% and the false negative rate was 0.53%. The false positive rate of the diagnostic test was 2.22% and the false negative rate was 6.41%. In addition to the site of 16189, Other known mutations were verified and the A12026G mutation and C16223T polymorphism were detected in unknown mutant samples. The results were consistent with the sequencing results. Conclusion The chip can be used for rapid screening and test diagnosis of 15 common susceptibility loci in mitochondrial DNA of diabetes mellitus.