A Versatile Colorimetric Diagnostic Platform Based on Primer Exchange Reaction Cascades Driven Loop-Mediated Isothermal Amplification
Xiaoya Liu, Hai Peng, Xiaoping Ye, Xiang Zhang, Ge Xu, Xianxian Zhao
Abstract
The primer exchange reaction (PER), as a well-established and sophisticated isothermal nucleic acid amplification strategy, has been extensively employed to amplify various genetic biomarkers. However, the amplification efficiency of conventional PER is generally unsatisfactory due to its linear signal-amplifying mechanism. In this study, we developed a v ersatile c olorimetric platform by rationally integrating P rimer e xchange reaction with efficient L oop-mediated isothermal a mplification (LAMP), termed vcPeLa, for the rapid and sensitive detection of cancer-related biomarkers, such as microRNAs (miRNAs) and carcinoembryonic antigen (CEA). The PER cascade in the vcPeLa can be directly initiated by the target, resulting in the production of long single-stranded DNA products with repeated functional sections. These repeats in the product can serve as the template to produce a significant number of double stem-loop DNAs with the cooperation of DNA polymerase and hairpin structure primer probes. These DNAs are the primary starting materials for subsequent LAMP. A substantial quantity of pyrophosphate can be recognized by Cu 2+ -chelated pp Probe (pyrophosphate sensing probe). The Cu 2+ is removed from the Cu 2+ -chelated pp Probe as a result of the formation of a complex between Cu 2+ and pyrophosphate, which results in color changes. Consequently, the vcPeLa platform is feasible to detect 0.31 fM miRNA and 0.043 ng/mL CEA with high selectivity and stability without the requirement of introducing any additional reaction steps or sample transfer operations in comparison to conventional assays. Therefore, this facile and ultrasensitive vcPeLa platform provides a new promising tool for cancer diagnosis and biomarker screening.