A highly sensitive and versatile fluorescent biosensor for pathogen nucleic acid detection based on toehold-mediated strand displacement initiated primer exchange reaction.
Analytica chimica acta(2022)
摘要
Existing detection methods for pathogen nucleic acid detection, such as polymerase chain reaction (PCR), are complicated and expensive to perform. 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 the target, the blocked hairpin substrate is released by toehold-mediated strand displacement, which triggers the primer exchange reaction amplification. Then, multiple long tandem-repeat single-strands generated by PER open the molecular beacon to recover the fluorescence signal. The t-PER protocol also successfully directly detected human papilloma virus from clinical cervical swab samples, with consistent results compared to real time-polymerase chain reaction (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 enabled specific and sensitive detection of pathogen nucleic acid in a format that might hold great potential for point-of-care infection diagnosis.
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