Zika virus lateral flow assays using reverse transcription-loop-mediated isothermal amplification

Area of Science:

• Molecular Biology
• Virology
• Biotechnology

Background:

• The global spread of Zika virus (ZIKV) necessitates urgent development of field-ready diagnostic tools.
• Existing diagnostic methods may lack the speed and accessibility required for rapid outbreak response.

Purpose of the Study:

• To develop and optimize a rapid, field-deployable diagnostic method for Zika virus (ZIKV) detection.
• To evaluate the efficacy of combining reverse transcription loop-mediated isothermal amplification (RT-LAMP) with a lateral flow assay (LFA) for ZIKV diagnosis.

Main Methods:

• Optimization of ZIKV-specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) for biological samples (PBS, urine, plasma).
• Specificity confirmation using BtsI restriction enzyme digestion and end-point detection across different sample matrices.
• Development and optimization of a lateral flow assay (LFA) using gold nanoparticles (AuNPs) and a ZIKV-specific probe for rapid detection of RT-LAMP products.

Main Results:

• ZIKV RT-LAMP successfully detected ZIKV RNA in 40 minutes at 63°C with no off-target amplification.
• The lateral flow assay (LFA) enabled rapid (<5 minutes) and simple detection of RT-LAMP products without sample preparation.
• The combined RT-LAMP and LFA method demonstrated high specificity and feasibility across various sample types.

Conclusions:

• The integrated ZIKV RT-LAMP and LFA assay provides a rapid, accurate, and point-of-care diagnostic solution.
• This method is suitable for field deployment, aiding in the prevention and control of Zika virus outbreaks.
• The assay's independence from complex laboratory equipment enhances its utility in resource-limited settings.