利用寡核苷酸芯片检测乙型肝炎病毒基因突变

目的　利用寡核苷酸芯片平行分析乙肝病毒表面抗原区S、前核心抗原pre C区、X区、聚合酶P区 12个已知的突变位点。方法　针对S、pre C、X、P区的 12个突变位点 ,设计位于乙肝病毒反义链的 2 4条寡核苷酸探针和两对PCR引物 ,探针长度为 14～ 18bp ,其 5′端连有氨基己烷和T15间隔子 ,合成后经点样仪点到醛基化玻片上。两对PCR引物分别用于扩增S与P区内的 5个突变位点和X、前C区内的 7个突变位点 ,其中上游引物含有荧光标记。不对称PCR扩增所得到的荧光标记的单链DNA与寡核苷酸阵列杂交、清洗后经扫描分析实验结果。结果　在对 12个乙肝阳性样品前核心抗原C和X区的突变检测中 ,存在 176 2A→T ,176 4G→A联合突变的有 2例、1896G→A突变的 3例、同时存在 176 2A→T、176 4G→A联合突变和 1896G→A突变的 1例、未存在任何突变的样品 6例。在 12个乙肝阳性样品的表面抗原S的突变检测中 ,未发现有突变出现。部分样品的随机测序结果与寡核苷酸阵列杂交结果一致。结论　寡核苷酸芯片适于快速、平行、大量检测突变

Mutation of hepatitis B virus detected by hybridization in oligonucleotide array

Objective To detect 12 mutation sites located in S, pre-C, X and P region of hepatitis B virus genome. Methods 12 pairs of oligonucleotide probes were designed in the antisense strand with amino linker and poly T15 spacer at their 5 terminal, the length of which was 14-18bp. Synthesized probes were immobilized on aldehyde modified glass slides. One pair of PCR primers was used for amplification of the part of S, P region which contain 5 mutation sites and the other pair of primers for fragment of X and pre-C region which contain 7 mutation sites. Both of upper primers were fluorescence labeled at their 5 terminal. Single-strand fluorescence marked DNA acquired by asymmetric PCR was hybridized to oligonucleotide array and signal intensities were collected after scanning. Results Among 12 positive serum samples, no mutation was detected in surface antigen. While in pre-core and core region, T1762-A1764 mutant was observed in 2 specimens and A1896 mutant found in 3 specimens, 1 sample was tested to hold T1762-A1764 and A1896 simultaneously and no mutant was identified in other 6 samples. Random DNA sequencing result was identical to the results of oligonucleotide array. Conclusion Oligonucleotide array is a fast method to detect mutations in parallel.