A novel G-quadruplex aptamer with high affinity for the specific detection of equine herpesvirus type 1: Comprehensive biophysical and analytical characterization.

Overview

• This research developed a new G-quadruplex aptamer that can specifically detect equine herpesvirus type 1 (EHV-1) with very high sensitivity and affinity, offering a rapid and cost-effective diagnostic method.

Background

• Equine herpesvirus type 1 (EHV-1) causes serious diseases in horses, including respiratory, neurological, and reproductive problems.
• Early and accurate detection of EHV-1 is crucial for controlling outbreaks and managing the disease effectively.
• Current diagnostic methods like PCR require complex procedures including DNA extraction and gel electrophoresis, which limits their use in field settings.

Aptamer Development

• The study designed the first G-quadruplex forming aptamer specifically targeting EHV-1.
• An in silico (computer-based) approach was used to design the aptamer sequence predicted to form a stable G-quadruplex structure.
• This G-quadruplex conformation was experimentally confirmed using:
  • Circular dichroism (CD) spectroscopy, which identifies characteristic signals of G-quadruplex folding.
  • Crystal violet fluorescence assay, which binds specifically to G-quadruplex DNA and shows increased fluorescence.

Binding Affinity and Specificity Evaluation

• The aptamer’s ability to bind to EHV-1 was tested with multiple analytical methods:
  • Colorimetric assays that produce visible color changes upon aptamer-virus binding.
  • Enzyme-linked apta-sorbent assay (ELASA), similar to ELISA but using aptamers instead of antibodies to detect the virus.
  • Surface plasmon resonance (SPR) to measure real-time binding kinetics and affinity.
  • Fluorescence-based analysis to further confirm binding interaction and specificity.
• SPR experiments showed the aptamer binds EHV-1 with very high affinity in the picomolar range, indicating strong and specific interaction.

Sensitivity and Diagnostic Performance

• The aptamer allowed detection of as few as 10 viral particles per milliliter:
  • This sensitivity is 100 times better than conventional PCR, which detects around 1000 viral particles per milliliter.
• ELASA results demonstrated excellent diagnostic accuracy:
  • Area under the curve (AUC) was 0.96, indicating high reliability of the aptamer-based test.
• The method does not require:
  • DNA extraction steps.
  • Use of primers or gel electrophoresis.

Implications and Conclusions

• The aptamer offers a rapid, cost-effective, and easy-to-use diagnostic tool suitable for point-of-care testing.
• Its high sensitivity and specificity make it valuable for early detection of EHV-1, potentially improving disease outbreak management.
• By eliminating complex sample preparation, the test is particularly advantageous in low-resource or field environments where traditional PCR is impractical.
• The study sets the groundwork for further development and potential commercialization of aptamer-based diagnostics for veterinary pathogens.