【摘要】 目的制备金(gold,Au)纳米颗粒HBV DNA基因探针,通过透射电镜直接检测乙肝患者血清中的HBV DNA,实现不经过PCR扩增检测DNA。方法制备Au纳米颗粒和Au纳米颗粒HBV DNA基因探针,用荧光标记法检测纳米颗粒探针表面寡核苷酸的覆盖数和杂交效率。在检测体系中加入乙肝患者血清中提取的HBV DNA,透射电镜下观察。结果制备的Au纳米颗粒粒径为(10±5)nm,水相分散良好,在520nm有Au特征性的最大吸收峰,经寡核苷酸修饰后,其最大吸收峰改变为524nm。Au纳米颗粒基因探针表面寡核苷酸的最大覆盖数目为(132±10)条,最大杂交效率为(22±3)%。在液相中与HBV DNA反应后,透射电镜下可观察到Au纳米颗粒凝聚成大的聚集体。结论成功制备了Au纳米颗粒HBV DNA基因探针,可直接用于HBV DNA的检测。更多还原

【Abstract】 Objective To prepare gold (Au) nanoparticle-supported HBV DNA gene probes for direct detection of HBV DNA extracted from positive serum of hepatitis B patients with transmission electron microscopy (TEM).Methods Alkanethiol-modified oligonucleotide was bound with self-made gold nanoparticles to prepare HBV DNA gene probes through covalent binding of gold-sulfur.By fluorescence-labeled method the number of thiol-derivatized single-stranded oligonucleotides and the efficiency of hybridization with complementary oligonucleotides in solution were determined.Compositive target DNA or HBV DNA extracted from positive serum of hepatitis B patients was added to the detection system containing nanoparticle HBV DNA gene probes.Results TEM showed the prepared gold nanoparticles were well-dispersed with (10±5) nm of diameter.Compared with bare Au nanoparticles, the absorbance peak of oligonucleotide-modified Au nanoparticles shifted from 520nm to 524nm.The maximal number of hexaethiol 30-mer oligonucleotide covered on the surface of gene probe was (132±10) per Au nanoparticle,and the maximal percentage of hybridization on nanoparticles was (22±3)%.When compositive target DNA or HBV DNA extracted from serum of hepatitis B patients was added to the detection system,TEM showed self-assembled nanoparticles formed large network aggregates.Conclusion Au nanoparticle-supported gene probes prepared in this study could be successfully used for direct detection of HBV DNA.更多还原