학술논문
Real-time isothermal RNA amplification of toxic marine microalgae using preserved reagents on an integrated microfluidic platform.
Document Type
Article
Author
Source
Subject
*NUCLEIC acid amplification techniques
*MICROALGAE
*CHEMICAL reagents
*MICROFLUIDIC analytical techniques
*NUCLEIC acid isolation methods
*AMPLIFICATION reactions
*ANNEALING of metals
*
*
*
*
*
*
Language
ISSN
0003-2654
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. [ABSTRACT FROM AUTHOR]