Rapid and Highly Sensitive Detection of Leishmania by Combining Recombinase Polymerase Amplification and...

Nucleic acid detection is widely used to identify infectious diseases and ensure food safety. However, conventional PCR-based techniques are time consuming. Thus, this study aims to combine recombinase polymerase amplification (RPA), which enables the rapid amplification of even trace amounts of nucleic acid fragments within 10-40 min at 37-42 °C, and solution-processed oxide thin-film transistor (TFT) technology, which exhibits high detection sensitivity, to detect Leishmania. A single-stranded anti-probe was incorporated into the RPA primer to facilitate effective hybridization between the RPA product and the immobilized probe on the solution-processed oxide TFT. The RPA-amplified product carrying an anti-probe enabled specific binding to the chip surface. Changes in current were monitored before and after sample incubation to identify the target nucleic acids in the samples accurately. The proposed method achieved a remarkable limit of detection of 10¹ copies/μL of the Leishmania HSP70 fragment within 30 min. The design of the probes on the solution-processed oxide TFT surface and the anti-probe simplified the detection of other target nucleic acids, eliminating the need to denature DNA double-strands for specific binding during nucleic acid detection. Thus, the novel method offers the advantage of requiring minimal reagent resources and eliminates the need for complex procedures.