摘要
本文首次报告用3’—末端核苷酸转移酶将生物素—11—dUTP标记到人工合成的21寡核苷酸上,该d(NMP)_(21)与HBV DNA长链缺口区附近的序列互补,并含HBV DNA的直接重复序列(Direct Repeat)。本文对其标记条件、检出方法进行观察比较,找出合适的条件,制成的生物素化d(NMP)_(21)探针可与标准的HBVDNA进行杂交,检测敏感度为25pg。用这种探针可以从乙型肝炎病人血清中检出HBV DNA。
A new method is described for labelling of oligonucleotide piobe of HBV. The oligonucleotide is a 21-nucletide sequence 5' -(CTTCGCTTCACCTCTGCACGT). It is complementary to a region near the end of single-stranded gap of HBV and contains the direct repeat (DR) sequence (underlined). The oligonucleotide was labelled with Biotin-11-dUTP by TdT.The results revealed that after 2 hours incubation the labelling efficiency is the highest. The polymerization in the presence of 1 mM Cobalt ion is better than the polymerization in the presence of 10 mM Cobalt ion or 10 mM Magnesium ion. The polymerization in the presence of dNTP is better than the polymerization in the absence of dNTP. When Biotin-11-dUTP is replaced by Biotin-7-dATP, the efficiency of labelling decreases.For colour development, we use two methods. The sensitivity of detection by preformed Streptavidin-Al kaline phosphatase complex is higher than the sensitivity of detection by adding the Streptavidin and Biotinated alkaline phospatase in series.Using this Biotinated oligonucleotide probe, we can detect as little as 25 Pg of standard HBV DNA. HBV DNA can also be detected in sera of Hepatitis B patients by dot-blot hybridization.
关键词
乙肝病毒
寡核苷酸探针
末端标记法
3'-Terminal Deoxynucleotidyl Transferase Biotin-11-dUTP Oligonuleotide Probe Hepatitis B Virus
