发卡型万能传导在基于核酸分子线路的基因诊断中的应用和性能优化

Adaption and Optimization of Universal Hairpin Transduction in Gene Diagnostics Based on Nucleic Acid Circuits

核酸等温扩增技术作为核酸体外扩增技术,其反应过程始终维持在恒定温度下。与聚合酶链反应相比,核酸等温扩增反应具有优异的便携型,因而被视为最有望实现基因快检甚至即时快检的体外基因扩增方法。然而,由于反应过程中假阳性扩增频发、反应后对产物的检测方法缺乏特异性和灵敏度等缺点,限制了其在实际分析检测中的应用。通过构建发卡型结构万能中转探针,成功地将恒温扩增产物转到一套性能良好的已知核酸分子线路上;借助核酸分子线路的百倍放大性能和序列特异性,实现对上游基因序列信息的精准识别和放大信号输出。针对不同的待测序列,仅需改变发卡型中转探针的序列,即可实现对不同序列目标物的检测。基于中转探针的重要性,本研究对中转探针的设计原理和方法进行了重点阐述,提出并验证了一套行之有效的普适性设计规律,确保中转探针良好的中转效率(信噪比)。利用这一规律获得的中转探针,与核酸分子线路偶联,可成功为低至近单分子(20个拷贝)的模型基因提供显著荧光和电化学信号输出。

Isothermal nucleic acid amplifications,as powerful as polymerase chain reaction but functioning at a constant temperature, are considered to be very promising technique in achieving point-of-care gene diagnostics. However,until now,their practical applications are still seriously lagged by the bad reliability resulting from the problems such as false positive amplification and low signal amplitude. In this work,a universal transduction method in which any sequence（ including loop-mediated isothermal amplification products） could be transduced via a hairpin transducer into a catalyst of a well-engineered circuit（ catalytic hairpin assembly,CHA） was established. Because CHA circuit could amplify tens to hundreds fold with especially high sequence specificity,it could provide both accuracy and high amplitude for sequence detection.And for a new targeting sequence,the only sequence needed to be changed was the hairpin transducer. Due to the importance of the transducer,we provided and verified a universal designing rule-set to guarantee the transducing efficiency（ signal to background ratio） of the transducer. Transducers designed following this rule set were then proved to be very efficient in detecting pathogen gene targets. As less as near single molecule（ 20 copies） of pathogen genes could be detected with significant fluorescent and electrochemical signals.