基于功能纳米颗粒的流感病毒快速分子诊断新方法研究

Influenza virus poses a serious threat to human health and life, because of its high infectivity and high morbidity. Currently, antigen test and PCR-based molecular diagnosis are used to detect influenza virus in the clinical lab. However, antigen test is lowly sensitive, and is not used to distinguish the subtypes of influenza A virus. Although the sensitivity of PCR is high, it is still too tough to be widely applied in most hospitals because of its high level of operation technique and cost. In China, the infection of influenza virus is usually predicted by clinical sighs and symptoms. But it is highly uncertain. Therefore, it is urgent to develop a rapid, simple, sensitive and accurate assay for the diagnosis of influenza virus. .In this program, we propose to develop novel approaches for diagnosis and subtype of influenza virus by integration of magnetic separation, recombinase polymerase amplification, multicolor CdSe/ZnS QDs coding and microplate chemiluminescence. Firstly, viral nucleic acid is isolated using functional magnetic particles based on the study of interface interaction between nucleic acid and particles. Secondly, viral nucleic acid are amplified using recombinase and polymerase. Finally, the product is detected by nucleic acid fluorescent probe, chemiluminescence and multicolor CdSe/ZnS QDs, respectively. This novel assay can be used to test and subtype influenza virus in clinical lab as well as on site, and it also provides a new way for the development of molecular diagnosis.

流感病毒因其传染性强、致病率高而严重威胁着人类的健康和生命。流感病毒检测主要是抗原检测和基于PCR的分子诊断技术。抗原检测的灵敏度较低，且不能鉴定亚型。虽然PCR检测灵敏度高，但技术要求和成本较高，很难广泛推广应用。在我国，流感病毒感染主要依靠临床体征和症状诊断。然而，临床体征和症状预测具有极大不确定性。因此，急需发展快速、简便、灵敏、准确的流感病毒分子诊断新方法。本项目整合磁分离、重组酶聚合酶扩增、多色CdSe/ZnS量子点编码和微孔板化学发光，建立流感病毒检测和分型的新方法。基于功能性纳米颗粒与核酸的界面作用研究，建立核酸磁分离方法；利用重组酶聚合酶扩增靶基因，而后借助核酸荧光探针、化学发光和多色量子点标记技术分析RPA产物。新型分子诊断技术，既能用于流感病毒的实验室检测和分型，指导病人及时精准用药，又能用于流感病毒的现场调查和即时检测，同时还为分子诊断发展提供了新的思路。

流感病毒因其传染性强、致病率高而严重威胁着人类的健康和生命。本项目整合重组酶聚合酶扩增和核酸免疫侧流层析检测技术建立流感病毒检测和分型的新方法。利用重组酶聚合酶扩增靶基因，而后借助核酸免疫侧流层析技术分析RPA产物。.研究结果表明，利用重组酶聚合酶扩增和核酸免疫侧流层析技术同时实现了甲型流感病毒与乙型流感的检测，同时也建立用于甲型流感病毒H1和H3分型检测的方法，并且最低可检测500拷贝和50拷贝的甲型与乙型流感病毒。临床标本的应用检测表明，与实时荧光RT-PCR技术相比，该方法在敏感性、特异性、阳性预测值和阴性预测值上均有表现优异。.因此，新型分子诊断技术，既能用于流感病毒的实验室检测和分型，指导病人及时精准用药，又能用于流感病毒的现场调查和即时检测，同时还为分子诊断发展提供了新的思路。