乙型肝炎病毒核心启动子及前C区突变检测基因芯片的制备及临床应用

目的 建立检测乙型肝炎病毒核心启动子及前C区突变基因芯片并探讨其临床应用的价值.方法 设计并合成针对核心启动子1762/1764、1814及前C区1896位点突变的特异性探针,制备寡核苷酸芯片.采用不对称PCR对该区域进行扩增,扩增产物与芯片杂交后分析结果,并评价该方法的特异性、灵敏度.采用该方法检测138例HBV DNA阳性血清标本.结果 该方法能够特异地检测乙型肝炎病毒核心启动子1762/1764、1814及前C区1896位点,灵敏度达1×101拷贝/μl.138例HBV DNA阳性血清标本中,T1762/A1764突变40例（28.99%）,C1814突变11例（7.97%）,A1896突变16例（11.59%）.前C区A1896突变在高拷贝组中明显高于低拷贝组（P＜0.01）.结论 本研究建立的基因芯片能够准确同时检测HBVV核心启动子区突变和前C区突变,前C区A1896突变可能与HBV复制状态有关.

Establishment of microarray for detecting mutation in HBV pre-core/core and basic core promoter regions

Objective To develop a sensitive and specific microarray for detecting mutations of HBV pre-core/core and basic core promoter regions in the clinic. Methods Site-specific oligonucleotide probes were designed and immobilized to microarray slides and hybridized to HBV gene fragments amplified with specific biotin-labeled primer using asymmetrical PCR. The specificity and sensitivity of the method were estimated. And the microarray was applied to detect 138 clinical serum samples with HBV-DNA.Results The mutations of HBV pre-core/core and basic core promoter regions can be specifically detected using the microarray, and the sensitivity was 1 × 101 copies/μl. Among 138 samples,40 samples had T1762/A1764 mutation, 1 lsamples had C1814 mutation, and 16 samples had A1896 mutation. The A1896 mutation rate in high HBV-DNA load group was significantly higher than that in low HBV-DNA load group (P ＜ 0. 01 ). Conclusion An DNA microarray assay was successfully established to detect the mutations in HBV pre-core/core and basic core promoter regions. The A1896 mutation in Pre-core/core region maybe involve in duplication of HBV.