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A novel split DNA aptamer (SDA) probe enables isothermal detection of single nucleotide variations (SNVs) without expensive equipment. This mix-and-read format offers a cost-effective alternative for genetic analysis and pathogen detection.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Hybridization probes are essential for detecting single nucleotide variations (SNVs) in DNA and RNA.
  • Conventional Taqman probes require expensive quantitative polymerase chain reaction (qPCR) instruments.
  • Isothermal amplification methods need probes that can selectively detect SNVs at a constant temperature.

Purpose of the Study:

  • To design and validate a novel split DNA aptamer (SDA) hybridization probe for isothermal SNV detection.
  • To develop a cost-effective and accessible alternative to qPCR-based detection methods.
  • To demonstrate the probe's utility in detecting clinically relevant targets.

Main Methods:

  • Design of a split DNA aptamer (SDA) probe utilizing a light-up fluorophore system.
  • Testing the probe's ability to bind a dapoxyl dye and generate fluorescence upon analyte hybridization.
  • Application of SDA for SNV detection in the inhA gene of Mycobacterium tuberculosis.
  • Integration of SDA with isothermal nucleic acid sequence based amplification (NASBA) for Zika virus detection.

Main Results:

  • The SDA probe exhibits a 120-fold increase in fluorescence upon forming a dye-binding site with the target analyte.
  • SDA successfully differentiated SNVs in the Mycobacterium tuberculosis inhA gene at ambient temperatures.
  • The probe detected a conserved region of the Zika virus after isothermal NASBA amplification.

Conclusions:

  • Split DNA aptamer (SDA) probes offer a sensitive and selective method for isothermal SNV detection.
  • This approach provides a viable, cost-effective alternative to qPCR for mix-and-read formats.
  • SDA technology has broad applications in genetic analysis and infectious disease diagnostics.