高灵敏纳米金比色芯片检测乙型肝炎病毒DNA及其变异

A Visual DNA Microarray for High Sensitivity Detection of Hepatitis B Virus DNA and Single Nucleotide Polymorphisms

构建了新型纳米金比色芯片,利用Taq DNA连接酶的连接特异性,将其与乙型肝炎病毒DNA(HBV-DNA)靶序列完全互补杂交的捕获探针(固定在芯片上)和纳米金修饰的探针连接成一条链,从而将纳米金颗粒固定到芯片点阵上,再通过银染反应放大,形成裸眼可见的显色信息。通过点阵的位置及灰度,即可判断HBV-DNA靶序列的单碱基突变,并得出相对定量信息。本实验对不同浓度的HBV-DNA靶序列进行了检测。结果显示:此技术对单碱基突变有很强的特异性识别能力,并且具有较高的灵敏度(约10pmol/L),在10～100pmol/L浓度范围内表现出较好的线性关系。该技术检测时间短(<1h)、操作简单、不需要特殊的检测设备,具有很好的临床应用前景。

Hepatitis B is a major disease which causes serious public health problems worldwide.Herein,a visual DNA microarray was developed for sensitive and specific detection of hepatitis B virus DNA（HBV-DNA） and single nucleotide polymorphisms.5′ end amino-modified oligonucleotide,which was immobilized on microarray surface acted as capture probe,and another part of gold nanoparticle-labeled oligonucleotide were designed to bind specifically to complementary targets HBV-DNAt,and then linked as a single-strand DNA via Taq DNA ligase.The black image of microarray spots,resulting from silver stain on gold nanoparticles,was used to detect visually target HBV-DNA and single nucleotide polymorphisms.Capture probe with single base mismatch was prepared to detect specificity and sensitivity of hybridization on microarray.Target HBV-DNA with different concentration had been tested,the result indicated that this method had good specificity in the discrimination of single nucleotide polymorphisms and showed high sensitivity（about 10 pmol/L）.There is also a linear relationship between the signal intensity of microarray spots and the concentration of target DNA at the concentration range of 10-100 pmol/L.For its high sensitivity and good specificity,this visual detecting technique has potential applications in clinical fields.