Automated Loop-mediated Amplification for Rapid Microalgae Monitoring – ALARMM

Harmful algal blooms (HABs) are sporadic events presenting a serious health risk with economic impact on shellfish and tourism industries. Thus, the detection of toxic microalgae has become a critical challenge, making phytoplankton monitoring with efficient on-field devices essential for the early detection of HABs.
PCR and LAMP emerged as central detection methods for targeted nucleic acids (NA) due to high sensitivity and selectivity. While PCR requires successive temperature cycles, LAMP method benefits from an isothermal protocol with an easier device integration for on-field analysis. Both methods still suffer from limitations in sensitivity and relative quantification. Digital techniques emerged as a new paradigm for the quantification of NA. The sample is partitioned into several thousand droplets containing none or several NA sequences. The end-point fluorescence of each droplet is measured after amplification with only the ones presenting sequences illuminating (positive droplets). The fraction of positive droplets allows for an absolute quantification of the NA concentration. While PCR is adapted for end-point fluorescence measurement, it suffers from interfering agents causing false negative. The LAMP method is less sensitive to interference but self-priming may lead to amplification and then false positive occurs.
Our aim is to by-pass the self-priming drawback of LAMP by measuring in real-time the fluorescence of each droplet and discriminate between real amplification and false positives. For this, we expect to design primers for the multiplexed detection of algae, to develop microfluidic cartridges for the sample preparation and droplets production and to build a portable device coupling pressure actuation and optics for the droplets generation and their fluorescence monitoring. Finally, we intend to validate our prototype by assessing the improved detection limit and by testing the detection of microalgae of interest in real sea water samples.