A ratiometric fluorescent biosensor for rapid detection of Burkholderia pseudomallei by dual CRISPR/Cas12a trans-cleavage assisted signal enhancement

Melioidosis caused by Burkholderia pseudomallei (Bp) infection has high mortality in tropical and subtropical regions. A rapid and sensitive method for its detection is urgently needed. Herein, a ratiometric fluorescent sensor with dual recognition sites of target was designed for sensitive detection of Bp DNA. The sensor was constructed with a TAMRA-labeled DNA as probe, which could adsorb on polymer dots (Pdots) at acidic pH to produce TAMRA fluorescence at the excitation wavelength of Pdots via fluorescence resonance energy transfer (FRET). In the presence of target DNA, the trans-cleavage activity of CRISPR/Cas12a was activated with the dual recognition sites, which digested the probe DNA to weaken the FRET. With the fluorescence ratio of Pdots to TAMRA as the detection signal, a ratiometric fluorescent biosensing was thus achieved. This method with dual CRISPR/Cas12a trans-cleavage assisted signal enhancement showed a detectable range of target DNA from 1.0 pM to 10 nM, and could be successfully used to detect Bp DNA extract with a detection time of 40 min. The excellent performance such as high sensitivity, good specificity, acceptable accuracy and short analytical time along with convenient operation demonstrated its potential in clinic diagnosis of melioidosis.