Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Application of Engineered Zinc Finger Proteins Immobilized on Paramagnetic Beads for Multiplexed Detection of Pathogenic DNA

  • Shim, Jiyoung (Department of Chemistry, Western Kentucky University) ;
  • Williams, Langley (Department of Chemistry, Western Kentucky University) ;
  • Kim, Dohyun (Department of Mechanical Engineering, Myongji University) ;
  • Ko, Kisung (Department of Medicine, College of Medicine, Chung-Ang University) ;
  • Kim, Moon-Soo (Department of Chemistry, Western Kentucky University)
  • Received : 2021.06.20
  • Accepted : 2021.07.07
  • Published : 2021.09.28
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Abstract

Micro-scale magnetic beads are widely used for isolation of proteins, DNA, and cells, leading to the development of in vitro diagnostics. Efficient isolation of target biomolecules is one of the keys to developing a simple and rapid point-of-care diagnostic. A zinc finger protein (ZFP) is a double-stranded (ds) DNA-binding domain, providing a useful scaffold for direct reading of the sequence information. Here, we utilized two engineered ZFPs (Stx2-268 and SEB-435) to detect the Shiga toxin (stx2) gene and the staphylococcal enterotoxin B (seb) gene present in foodborne pathogens, Escherichia coli O157 and Staphylococcus aureus, respectively. Engineered ZFPs are immobilized on a paramagnetic bead as a detection platform to efficiently isolate the target dsDNA-ZFP bound complex. The small paramagnetic beads provide a high surface area to volume ratio, allowing more ZFPs to be immobilized on the beads, which leads to increased target DNA detection. The fluorescence signal was measured upon ZFP binding to fluorophore-labeled target dsDNA. In this study, our system provided a detection limit of ≤ 60 fmol and demonstrated high specificity with multiplexing capability, suggesting a potential for development into a simple and reliable diagnostic for detecting multiple pathogens without target amplification.

Keywords

Acknowledgement

This research was supported by the Kentucky Biomedical Research Infrastructure Network (KBRIN)-Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number 2P20GM10343614, and National Science Foundation (NSF)-International Research Experiences for Students (IRES) Award under grant number IIA-1358222.

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