Flow-LAMP: Label-Free Digital LAMP Using Scatter-Based Flow Cytometry on Vortex-Generated Polydisperse Gel Beads

Area of Science:

• Biotechnology
• Molecular Diagnostics
• Analytical Chemistry

Background:

• Accurate nucleic acid quantification is crucial for clinical diagnostics.
• Digital PCR adoption is hindered by fluorescence detection and microfluidics.

Purpose of the Study:

• To develop a label-free, cost-effective digital nucleic acid detection assay.
• To integrate loop-mediated isothermal amplification (LAMP) with flow cytometry for quantification.

Main Methods:

• Developed Flow-LAMP assay using vortex emulsification for agarose gel beads.
• Utilized flow cytometry (FSC and SSC) to analyze bead volume and amplification.
• Magnesium pyrophosphate precipitate indicates positive amplification within gel beads.

Main Results:

• Side scatter (SSC) correlated with amplification products; forward scatter (FSC) reflected bead volume.
• Achieved a limit of detection of 38.15 copies/μL for Epstein-Barr virus plasmid.
• Flow-LAMP results showed strong correlation with qPCR and digital PCR in clinical samples.

Conclusions:

• Flow-LAMP provides accurate, accessible digital nucleic acid detection.
• Eliminates need for fluorescent labels and specialized microfluidics.
• Offers a cost-effective alternative using standard laboratory equipment.