生物素-亲和素系统用于DNA芯片显色分析乙型肝炎病毒基因多态性

To Analysis HBV Gene Polymorphism with the Techology of DNA Chip which Shown Colour under the Action of Biotin-amrexo System

目的利用生物素-亲和素系统使DNA芯片显色,用于乙型肝炎病毒基因(HBV DNA)多态性分析.方法将标记生物素的dUTP在双重聚合酶链反应(PCR)时掺入DNA样品目的断片,并将目的断片杂交于含HBV核苷酸序列(1896、1814、1762、1764、P区552)的野生型及突变型探针的DNA芯片上,用亲和素-碱性磷酸酶进行显色,并转印于尼龙膜上,再用光学扫描仪读取结果.结果 42例乙型肝炎患者HBV核苷酸序列1896、1814、1762、1764、P区552位点突变率分别为40%、31%、50%、50%、10%;该法检测敏感度为5.6×102病毒考贝数/ml;特异性高;强、弱阳性批内变异系数(CV)分别为15.1%和19.8%.结论生物素-亲和素系统用于HBV DNA多态性芯片显色,方法简便,不需特殊设备,易于推广应用.

Objective To make the DNA chip show colour under the action of biotin-amrexo system,then use it to analyse HBV DNA ploymorphism. Methods (1)Added the pattern purpose DNA pieces to the reactor when polymerase chain reac-tion(PCR) with dUTP labeltd biotin in processing, to hybridized the pattern purpose DNA pieces with the DNA chip which include the wild type and mutative type probe of HBV DNA sequence (1896,1814,1762,1764 and p-region 552) ;(2) Showed colours with amrexo-ALP;(3)Trans-print the colour to a nylon-film,then use a optical scanner to read it and gain results. Result In the 42 samples of HBV patients,the respective ratio of mutation about 1896,1814,1762,1764 and P-region 552 were 40% ,31 % ,50% ,50% and 10%;the sensitivity of the way was 5. 6 × 102copies/ml;the within-run CV was 15. 1% in high positive results and 19. 8% in low positive results. Conclusion The establishment of the way should make it capable that widely spread the techology of DNA chip.