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There is currently the lack of a method for precisely monitoring the progress of isothermal amplification reactions by means of sequence-specific fluorescent probes like the TaqMan probe used in the PCR system. Here, we created a circular fluorescent probe-mediated isothermal amplification (CFPA) method. This novel method uses two circular fluorescent probes and Bst DNA polymerase to construct an overlapping structure that can be cut off by flap structure-specific endonuclease 1, separating the fluorescence and quenching groups on the probes. The results showed single-copy sensitivity, ultrahigh specificity, stability (C.V. 0.05). In summary, we present a new, reliable, and precise isothermal amplification approach for applications in biomedical research and the clinical accurate diagnosis of pathogen infections.
Ye et al. (Fri,) studied this question.