Adaption and Optimization of Universal Hairpin Transduction in Gene Diagnostics Based on Nucleic Acid Circuits / 分析化学

ABSTRACT

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.