基于Holliday交叉链迁移的新型多重突变检测技术的构建及其在卵巢癌基因检测中的应用研究

Multiplexed mutation detection can provide abundant and comprehensive genetic information, which is profound for cancer diagnosis and therapy. Current methods are not able to perform rapid, sensitive and low-cost multiplexed mutation detection, which limits their large-scale clinical applications. Nucleic acid probes, especially the probe/blocker composition system, hold the potential for achiving the goal due to their high sensitivity and rapidness. However, the sensitivity and specificity of the probe/blocker composition system are negatively correlated when changing with the concentration and length of the blocker strand, which lead to cumbersome optimization, diverse reaction conditions and thereby failure of multiplexed mutation detection. In this project, we aim to construct a novel probe/blocker composition sytem and its theoretical model based on Holliday junction branch migration. Through the system, we anticipate to break the aforementioned negative correlation and achieve rapid, sensitive and low-cost multiplexed mutation detection. Taking the 130 driver mutations of BRCA gene as our modelling targets, we plan to develop multiplexed mutation detection array for 100 blood DNA samples (peripheral blood leucocyte DNA, reflecting germline mutations), 50 plasma cell-free DNA samples and corresponding tumor tissue DNA samples (reflecting somatic mutations) of ovarian cancer patiens. Ultimately, we aim to construct a universal platform for multiplexed mutation detection, which offers a powerful tool for genetic testing and liquid biopsy of cancers.

多重突变检测能够提供丰富全面的基因突变信息，对癌症的诊疗具有重要意义。现有检测技术难以实现快速、高灵敏、低成本的多重突变检测（>10重），因而大规模临床推广受限。核酸探针技术尤其是探针/竞争链组合检测体系因其快速、高灵敏等特点，有望实现该目标。但组合体系的特异性和灵敏度随竞争链变化时存在负相关关系，导致优化过程繁琐，反应条件不统一，无法实现多重突变检测。本项目拟构建基于Holliday交叉链迁移的探针/竞争链组合检测体系及其理论模型，打破这一负相关关系，实现快速、高灵敏、低成本的多重突变分析。项目拟以BRCA基因130种致病突变为目标，对100例卵巢癌患者血液DNA（外周血白细胞DNA，反映胚系突变），50例卵巢癌患者血浆游离DNA、癌组织DNA（可反映体系突变）进行检测，建立卵巢癌BRCA基因130重突变检测技术和普适的多重突变分析平台，为癌症基因检测、液体活检提供强大的分析工具。

多重突变检测能够提供丰富全面的基因突变信息，对癌症的诊疗具有重要意义。现有检测技术难以实现快速、高灵敏、低成本的多重突变检测（>10重），因而大规模临床推广受限。核酸探针技术尤其是探针/竞争链组合检测体系因其快速、高灵敏等特点，有望实现该目标。但组合体系的特异性和灵敏度随竞争链变化时存在负相关关系，导致优化过程繁琐，反应条件不统一，无法实现多重突变检测。本项目针对实际致病基因构建基于Holliday交叉链迁移的探针/竞争链组合体系（命名为“SELECT”系统），打破灵敏度和区分度的反比关系，实现检测不同目标突变时，序列设计和反应条件“零优化”和“统一化”。设计中无需任何优化，区分度都在140以上。在此基础上，项目开发了核酸酶和链迁移探针相结合的策略，进一步提高了探针系统的灵敏度和特异性，检测限低至0.01%，并对20例临床标本进行了检测，与高通量测序结果完全一致。基于本项目的研究成果，申请人与国药集团基因科技有限公司开展了产学研合作，开发了试剂盒，正在申报医疗器械注册证。本项目进展顺利，取得了较为丰硕的成果，所发明的新技术、产生的试剂盒为肿瘤基因突变检测提供了强大的工具，具有重要的科学意义和应用价值。