遗传性耳聋基因芯片检测方法的建立

发布时间：2018-01-03 17:10

本文关键词：遗传性耳聋基因芯片检测方法的建立　出处：《中国优生与遗传杂志》2016年11期 　论文类型：期刊论文

【摘要】：目的以膜芯片技术为平台,建立一种遗传性耳聋基因芯片检测方法(PCR-反向点杂交法)。方法根据近期在我国国内开展的大规模耳聋分子流行病学调查结果及Gene Bank上报道的基因信息,设计并合成20条探针及5对引物检测与遗传性耳聋相关的四个热点基因(GJB2、GJB3、SLC26A4和12S r RNA)中的10个突变位点,10条探针点至尼龙膜上制成反向杂交膜芯片,应用五重PCR技术一管完成所有位点的扩增,建立了一种一次实验就可以检测非综合征遗传性耳聋四个热点基因上10个位点突变的方法。结果遗传性耳聋基因芯片检测方法对10种基因型检测的灵敏度为10ng/μL;除肝素抗凝剂外,EDTA和枸橼酸钠抗凝剂都不是本检测方法的干扰物质,溶血、脂血和黄疸样本都不影响本方法的检测结果;应用这种方法检测临床样本,检测结果与测序结果比较,阳性符合率为100%,阴性符合率为100%,总符合率为100%,经χ2检验分析,遗传性耳聋基因检测方法与测序法没有明显差别;重复性测试结果显示在不同的实验条件下检测结果完全一致,该方法能稳定重复检出。结论成功的建立了一种灵敏度高、特异性好、检测结果稳定、可靠、通量较高的遗传性耳聋基因芯片检测方法。
[Abstract]:Objective to take the membrane chip technology as the platform. Establishment of a method for genetic Deafness Gene Chip Detection by PCR- reverse Dot Hybridization (PCR- reverse Dot Hybridization). Methods according to the results of molecular epidemiological investigation on deafness and the gene information reported on Gene Bank in China recently. 20 probes and 5 pairs of primers were designed and synthesized to detect four hot genes associated with hereditary deafness: GJB2 and GJB3. Ten mutation sites of SLC26A4 and 12s r RNAs were used to produce reverse hybridization membrane chip on nylon membrane. All the sites were amplified by using a single tube of quintuple PCR technique. A method was established to detect 10 locus mutations in four hot spots of non-syndromic hereditary deafness. Results the sensitivity of the microarray method for detecting 10 genotypes of hereditary deafness was as follows: 1. 10ng / 渭 L; Besides heparin anticoagulant EDTA and sodium citrate were not interfering substances in this method. Hemolysis lipid blood and jaundice samples did not affect the results of this method. Compared with the results of sequencing, the positive coincidence rate was 100, the negative coincidence rate was 100, and the total coincidence rate was 100. The results were analyzed by 蠂 2 test. There was no significant difference between the methods of gene detection and sequencing of hereditary deafness. The results of repeatability test showed that the detection results were completely consistent under different experimental conditions, and the method could be detected stably and repeatedly. Conclusion A new method with high sensitivity, good specificity, stable and reliable detection results has been established successfully. A high throughput genetic microarray method for the detection of hereditary deafness.
【作者单位】：深圳市南山区妇幼保健院检验科;
【基金】：深圳市南山区技术研发和创意设计项目(卫生类)(南科研卫2014020)
【分类号】：R764.43;R440
【正文快照】： 耳聋是人类最常见的致残原因之一,严重影响着人类的生活质量[1]。全球共有3.6亿人存在不同程度的听力障碍,我国现有听力残疾者2004万人,7岁以下的聋儿达80万人,每1000个新生儿中就有1~3名聋儿,我国每年新生聋儿达3万人,每年新发迟发性耳聋患儿超过8万人[2,3]。耳聋已经成为我

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