Biosensor for PCR amplicons by combining split trehalase and DNA-binding proteins: A proof of concept study

The use of polymerase chain reaction (PCR) technology in low-cost settings has gained significant attention due to its ability to amplify and detect specific bacterial pathogen genes, aiding in the diagnosis of infectious diseases. PCR amplicons can be visualized by conventional endpoint agarose gel electrophoresis and fluorochrome-enabled real-time PCR. However, this is not practical in on-field tests due to cumbersome instrumentation, labor-intensive reaction preparation, and long time-to-results. Many studies have combined microfluidic devices or electrochemical dyes with PCR technology to enhance in-field operability. However, the high cost of manufacturing high-precision microfluidic chips and the dependence on non-portable readout equipment limit their further development. In this paper, we present a proof-of-principle study of a novel method combining split enzyme technology and DNA-binding proteins for the convenient and efficient detection of amplified genetic material from bacterial pathogens. The amplicon binding split trehalase assay (ABSTA) relies on incorporating specific recognition sequences of DNA-binding protein SpoIIID in tandem within one of the PCR primers. Applied by a Gram-type specific PCR assay, ABSTA was capable of discriminating Staphylococcus devriesei and Escherichia coli in <90 min after colony PCR amplicons bound split trehalase fragments-fused SpoIIID and triggered split enzyme complementation. The salt concentration, protein reagents versus DNA substrate ratio, direction and linker length of tandem recognition sites required for the complementation were optimized. The glucose produced by restored enzymatic activity was detectable by glucometer. With limited requirements for reaction preparation and the compatibility of ABSTA with commercially available handheld glucometers, this test platform has substantial potential to be implemented into a future point-of-care (POC) diagnostic tool for detecting pathogen specific genes with further improvement.