A highly sensitive and versatile fluorescent biosensor for pathogen nucleic acid detection based on toehold-mediated strand displacement initiated primer exchange reaction

Abstract Existing methods for pathogen nucleic acid detection, such as PCR, require complicated operation and expensive facility. Here, we report a simple and versatile strategy for highly sensitive detection of pathogen nucleic acid based on toehold-mediated strand displacement initiated primer exchange amplification (t-PER). In the presence of target, the blocked hairpin substrate would be released by toehold-mediated strand displacement, which triggered the primer exchange reaction amplification. Then, multiple long tandem-repeat single-strands generated by PER could open the molecular beacon to recover the fluorescence signal. Based on the target selection and conditions optimization, the developed platform could detect down to 18 fM of target in 1 hour of process time with high discrimination factor. It is also successful applied for direct detection of HPV from clinical cervical swab samples, the results were consistent with those obtained from RT-PCR. Moreover, the versatility and clinical feasibility of this method was further confirmed by measuring Epstein-Barr virus, Hepatitis B virus, and Ureaplasma Urealyticum from different clinical samples (serum samples and urine samples). This simple platform enables specific and sensitive detection of pathogen nucleic acid in a format that might hold great potential for point-of-care infection diagnosis.