基于颗粒表面桥式扩增的核酸多重检测技术研究

Multiple PCR, with the high sensitivity and specificity, paly important roles in the field of molecular diagnosis such as blood screening and inherited disease detection. However, traditional multiplex PCR method is difficult to make each target independently amplify without interfere to each other. So the number of targets can simultaneous amplified in one tube is limited. We employed the new technology which can make each target just amplifying on the surface of proper solid particles to avoid interference among different primers. And then, we combined this technology with convective PCR to resolve the problem of particle settling and with xMAP to detecting the multiplex PCR products. Finally, we will construct a new platform for multiple amplification and detection, which can used in different areas such as early cancer screening, infectious disease and inherited disease diagnosis, blood screening, and scientific research.

多重PCR因其高灵敏度与特异性的特点在血液筛查、遗传病检测等诸多核酸诊断领域具有十分重要的意义。然而，目前的多重PCR，仅靠引物设计与体系调整很难做到各靶标独立、互不干扰的扩增，也限制了其可同时扩增的靶标数目。本研究引入固相颗粒表面的核酸扩增技术，使得每个靶标的扩增集中于以特定固相颗粒为载体的表面，从根本上解决多重PCR引物间相互干扰问题；并结合管式对流PCR技术与xMAP技术，解决固相颗粒沉降与超多重扩增产物检测的难点，最终建立一种新型的多重PCR扩增与检测平台，可广泛应用于癌症检测、传染病检测、遗传病鉴定、血液筛查、科学研究等诸多领域，为核酸多靶标检测带来革命性的突破。

多重PCR因其既具备高灵敏度高特异性，又具备可一次检测多个靶标的特点，在血液筛查、病原体鉴定、伴药诊断、遗传病检测等众多方面具有十分广泛的应用。然而目前在临床中应用的主流多重PCR方法很难做到各靶标独立、互不干扰的扩增；此外，PCR仪荧光通道的数量也大大限制了可在一个反应中同时检测的重数。针对上述问题，本研究建立了一种在荧光编码微球上标记引物，在对流PCR运动流场中完成微球表面桥式扩增，并应用xMAP技术对微球进行解码和产物分析的多重核酸检测方法。阐明了引物标记密度与扩增效率之间的关系、固相颗粒表面、引物、酶、杂交探针之间相互位阻与扩增效率的之间的关系。建立了既能保障稳定流场又能提供固相扩增最适温度场的对流PCR反应参数；并在上述基础之上，建立了通用的固相颗粒表面多重扩增方法，该方法比xMAP方法灵敏度高约100倍。