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

This study established a robust digital loop-mediated isothermal amplification (dLAMP) workflow using the QIAcuity nanoplate-based dPCR system to overcome the limitations of traditional isothermal reactions and low partition counts. By optimizing reactions targeting the mitochondrial CO1 region of Limnoperna fortunei with diluted EvaGreen Master Mix, the method achieved high-throughput absolute quantification across a wide dynamic range without requiring standard curves. Results demonstrated that while conventional turbidimetric LAMP showed moderate variability accompanied by occasional delayed amplification at low concentrations, dLAMP maintained stable and clear detection at 1.52 × 10¹ copies. Despite minor saturation effects at high concentrations and stochasticity at extremely low levels, the QIAcuity-based dLAMP demonstrated enhanced quantitative stability compared to the conventional bulk assay. This user-friendly platform potentially reduces the risk of molecular dropout through high partition density (up to 26,000 partitions), offering a practical and precise tool for diagnostics, food safety, and environmental DNA (eDNA) monitoring, especially for low-copy target detection.