The development of a new technology for the detection of doublestranded (ds) DNA enables multiple biomedical applications including identifying multiple pathogens simultaneously. We previously employed colorimetric SEquence-Enabled Reassembly with TEM-1 β-lacatamase (SEER-LAC) to detect specific bacterial DNA sequence. SEER-Lac consists of the two inactive β-lactamase fragments which of each attached to a zinc finger protein (ZFP) would reassemble into an active full-length enzyme upon ZFPs binding to its target DNA. Here, we engineered two pairs of ZFPs which of each recognizes shiga toxin in E. coli O157 and staphylococcal enterotoxin B in Staphylococus Aureus, respectively. Biotin was simply conjugated to the detection probe ZFP, which allows for generating chemiluminescent signal in streptavidin-HRP (Horseradish peroxidase) assay upon ZFPs binding to their target DNA. Our assay generates DNA-dependent signal and allows for a detection limit of 0.5 nM without DNA amplification or DNA labeling. Our system can be developed into a simple multiplexed detection diagnostic for multiplexed detection of dsDNA.

Although DNA microarray can detect multiple DNA samples simultaneously, current detection techniques involve PCR and other traditional procedures. In this study, a sensitive, specific and rapid detection method, which eliminates PCR and other lengthy processes, for pathogenic DNA is presented. This technology is based on the hybridization of target DNA to the immobilized probe, extension of probe DNAs using the target-DNA as a template and signal generation by streptavidin-horseradish peroxidase and substrate. This method is highly specific and sensitive, allowing single-nucleotide-base mismatches discrimination and the detection at femtomole level. The experiments are designed to achieve short hybridization time. Therefore, satisfactory signal can be detected within minutes, allowing the rapid detection of multiple pathogenic DNA. Most importantly, the E. coli genomic DNA can be detected using this technology. In conclusion, this detection method is useful for applications including on-site pathogenic disease detection, crime scene investigation, and pathogen inspection in the environment.