A Ratiometric Fluorescent Biosensor Based on Exonuclease III Amplification Strategy for Pathogenic Bacterial Gene Detection

Abstract

Food-borne pathogenic bacteria seriously threaten public health.Based on the mechanism of fluorescence resonance energy transfer (FRET), a ratiometric fluorescence biosensor was constructed by integration of Exo Ⅲ-based signal amplification strategy.The Cy3 labeled R1-DNA firstly hybridized with Cy5 labeled R2-DNA to form duplex of R1/R2.Cy3 showed a low fluorescence response while Cy5 showed a high fluorescence response.The addition of target pathogenic bacterial gene (Lac Z gene) could de-hybridize the R1/R2, resulting in the fluorescence decreasing of Cy5 and the fluorescence recovering of Cy3.Under the assistance of Exo Ⅲ, the signal change was further amplified.The detection of limit reached as low as 5.29 pmol/L.The linear detection range was from 10 pmol/L to 2000 pmol/L.The developed ratiomtric detection strategy significantly reduced the possibility of false-positive and false-negative detection results.