Development of a digital loop-mediated isothermal amplification using a digital polymerase chain reaction device.

Area of Science:

• Molecular Biology
• Biotechnology
• Environmental Science

Background:

• Traditional isothermal amplification methods face limitations in quantification accuracy and partition counts.
• Low partition numbers in conventional assays can lead to variability and molecular dropout.
• There is a need for high-throughput, stable quantification of low-copy targets.

Purpose of the Study:

• To establish a robust digital loop-mediated isothermal amplification (dLAMP) workflow.
• To leverage the QIAcuity nanoplate-based digital PCR (dPCR) system for enhanced isothermal reactions.
• To achieve high-throughput absolute quantification without standard curves.

Main Methods:

• Optimized dLAMP reactions targeting the mitochondrial CO1 region of Limnoperna fortunei.
• Utilized diluted EvaGreen Master Mix on the QIAcuity dPCR system.
• Compared dLAMP performance against conventional turbidimetric LAMP.

Main Results:

• dLAMP achieved stable and clear detection down to 1.52 × 10¹ copies.
• The digital assay demonstrated enhanced quantitative stability compared to bulk assays.
• High partition density (up to 26,000 partitions) reduced molecular dropout risk.

Conclusions:

• The QIAcuity-based dLAMP workflow offers a user-friendly and precise tool for absolute quantification.
• This method is suitable for diagnostics, food safety, and environmental DNA (eDNA) monitoring.
• The platform overcomes limitations of traditional isothermal reactions for low-copy target detection.