Development and Validation of a Colorimetric Reverse Transcriptase Loop-Mediated Isothermal Amplification Assay for...

Area of Science:

• Virology
• Molecular Biology
• Public Health Entomology

Background:

• Eastern equine encephalitis virus (EEEV) poses a significant threat, causing outbreaks in the eastern USA.
• Current surveillance and control of EEEV and other mosquito-borne diseases face challenges, particularly in environmental sampling of mosquito populations.
• Existing detection methods like RT-PCR can be complex and require specialized equipment.

Purpose of the Study:

• To develop and validate a novel, user-friendly assay for detecting EEEV in mosquito vectors.
• To provide a cost-effective and accessible alternative for vector surveillance programs.
• To identify biomarkers and establish a colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for EEEV.

Main Methods:

• Development and validation of a colorimetric RT-LAMP assay for EEEV detection.
• Utilized viral culture, experimentally spiked mosquito pools, and previously tested mosquito pools for validation.
• Assessed assay sensitivity by detecting low viral titers and tested its efficiency in pooled mosquito samples.

Main Results:

• The RT-LAMP assay successfully detected EEEV with a visual color change (pink to yellow).
• The assay demonstrated high sensitivity, detecting viral titers as low as approximately 10% of that in a single infectious mosquito.
• Efficiently tested up to 250 mosquitoes per reaction in pooled samples with no false positives from known or potential EEEV vectors.

Conclusions:

• The developed colorimetric RT-LAMP assay is a highly accurate and simple method for EEEV detection.
• This assay offers a cost-effective alternative to RT-PCR for vector surveillance, requiring minimal equipment.
• The assay's ease of use and visual output can significantly aid mosquito abatement programs in controlling EEEV.