title: Real-time isothermal RNA amplification of toxic marine microalgae using preserved reagents on an integrated microfluidic platform.
abstract: Quantitation of specific RNA sequences is a useful technique in marine biology that can elucidate cell abundance, speciation and viability, especially for early detection of harmful algal blooms. We are thus developing an integrated microfluidic system for cell concentration and lysis, RNA extraction/purification and quantitative RNA detection for environmental applications. The portable system is based on a microfluidic cartridge, or "lab-card", using a low-cost injection moulded device, with a laminated lid. Here we present real-time isothermal RNA amplification using reagent master-mixes preserved on-chip in a gel at 4 °C for up to eight months. We demonstrate quantitation by reference to an internal control in a competitive assay with 500 cell equivalents of the toxic microalga Karenia brevis. Annealing of primers, amplification at 41 °C and real-time fluorescence detection of the internal control and target using sequence-specific molecular beacons were all performed on-chip.
authors.affiliation: Sensors Development, Ocean Technology and Engineering Group, National Oceanography Centre, Southampton, SO14 3ZH, UK. M.Tsaloglou@soton.ac.uk
authors.forename: Maria-Nefeli
authors.initials: MN
authors.lastname: Tsaloglou
authors.forename: Florian
authors.initials: F
authors.lastname: Laouenan
authors.forename: Christos-Moritz
authors.initials: CM
authors.lastname: Loukas
authors.forename: Lisandro Gabriel
authors.initials: LG
authors.lastname: Monsalve
authors.forename: Christine
authors.initials: C
authors.lastname: Thanner
authors.forename: Hywel
authors.initials: H
authors.lastname: Morgan
authors.forename: Jesus M
authors.initials: JM
authors.lastname: Ruano-López
authors.forename: Matthew C
authors.initials: MC
authors.lastname: Mowlem
mesh_terms: D002478:Cells, Cultured; D057097:Harmful Algal Bloom; D058086:Microalgae / Q000235:genetics*; D046210:Microfluidic Analytical Techniques; D044085:Microfluidics; D021141:Nucleic Acid Amplification Techniques; D012313:RNA / Q000032:analysis; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics*; D021161:Self-Sustained Sequence Replication
chemical_list: D012313:RNA; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase