Sequence-Specific Probe-Mediated Isothermal Amplification for the Single-Copy Sensitive Detection of Nucleic Acid

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. < 0.1), and anti-interference ability in detecting nucleic acid samples. A clinical trial demonstrated the perfect effectiveness of this method in the diagnosis of rotavirus infection and consistency with the gold standard method used in the clinic (p > 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.