肝炎与艾滋病的高特异高灵敏高通量快速并行分子检测新方法研究

Hepatitis and AIDS are the major infectious diseases that endanger human health and being the need for early detection of cases treatment so as to limit the spread of the diseases. Protein detection methods are still generally the widely used methods but their disadvantages containing low sensitivity and complex operation and missed detection or wrong detection make them difficult to detect many diseases simultaneously and promptly. Although real-time fluorescence quantitative PCR and other detection methods have been developed as hepatitis and HIV diagnostic reagents but high cost detection, complex sample handling and detection procedures difficult to achieve large-scale and widespread applications. Therefore there is very urgent need to come up with an accurate, rapid, highly sensitive, high-throughput and suitable for clinical parallel multiple diseases nucleic acid detection methods for hepatitis and HIV. In this program, combined with nano-magnetic separation technology, comparative genomics, multiple loop-mediated amplification technology and microplate analysis techniques, a variety of specific detection sites are in situ cycling amplified on the surface of silicon-coated and iron core-shell nano-magnetic particles to hybridize with different types of probes and applied chemiluminescence detection of parallel detection of hepatitis and AIDS. Thus both parallell detection of hepatitis and AIDS, and significantly enlargement for nucleic acid information are realized and enabled high specificity and highly sensitive and high-throughput analysis of low copy number nucleic acid information of the diseases. Meanwhile the program would provide methodological supports for developing the clinical and large-scale molecular detection system with independent intellectual property rights in the near future.

肝炎和艾滋病是危害人类健康的重大传染性疾病，需要及早发现病例治疗，从而限制疾病的传播。目前蛋白检测仍是普遍广泛使用的方法，但灵敏度很低，操作复杂，经常会出现漏检或检测不及时，且很难实现多种疾病的同时检测。虽然用实时荧光定量PCR和其他检测方法已研制出肝炎和艾滋病诊断试剂，但因检测成本较高，样品处理和检测技术也较复杂，难以实现大规模的应用推广。因此亟需研究出准确、快速、高灵敏、高通量且适用于临床的多重疾病核酸并行的检测方法。本课题综合应用纳米磁分离技术、比较基因组学、多重环介导扩增技术以及微孔板分析技术，通过在硅包铁核壳纳米磁性颗粒表面对多种特异检测位点进行在位循环扩增，与不同类型的修饰探针进行杂交化学发光检测，既能实现肝炎和艾滋病并行检测，而且核酸信息显著放大，从而实现低拷贝数核酸信息的高特异性、高灵敏和高通量分析，同时可为研究具有自主知识产权的临床大规模分子检测系统提供方法学支撑。

本研究综合应用纳米磁分离技术、化学发光技术、多重环介导扩增技术以及微孔板分析技术，通过在硅包铁核壳纳米磁性颗粒表面对肝炎的核酸分子进行多种特异性的在位循环扩增，并与不同类型的修饰探针进行杂交化学发光检测，实现了肝炎（主要为乙肝和丙肝两种类型）的等温、快速并行分子检测的目的，分别建立了基于多重PCR扩增和化学发光乙肝和丙肝病毒的高灵敏度检测新技术以及基于环介导等温扩增、化学发光的肝炎病毒检测新方法，并获得基于磁性纳米颗粒的核酸（DNA和RNA）提取试剂盒，本研究成果不仅为研究出准确、快速、高灵敏、高通量且适用于临床的多重疾病核酸并行的检测方法进行有效的探索，也为开发具有自主知识产权的临床大规模分子检测系统提供方法学支撑。与此同时，本研究还对基于聚苯乙烯微球和化学发光技术进行乙肝的蛋白质分子检测技术和电化学检测技术进行了探索性研究，并获得积极有效的数据资料，这些都为肝炎及艾滋病的早期检测技术和高通量的血液筛查奠定了坚实的基础。