[Journal of Microbiological Methods] Detection and quantification of the toxic microalgae Karenia br
Now and again, the rapid proliferation of certain species of phytoplankton can give rise to Harmful Algal Blooms, which pose a serious threat to marine life and human health. Current methods of monitoring phytoplankton are limited by poor specificity or by the requirement to return samples to a highly resourced, centralised lab. The Lab Card is a small, microfluidic cassette which, when used in tandem with a portable Lab Card Reader can be used to sensitively and specifically quantify harmful algae in the field, from nucleic acid extracts using RNA amplification; a sensitive and specific method for the enumeration of potentially any species based on their unique genetic signatures. This study reports the culmination of work to develop a Lab Card-based genetic assay to quantify the harmful algae Karenia brevis using mRNA amplification by the Nucleic Acid Sequence Based Amplification (NASBA) method. K. brevis cells were quantified by amplification of the rbcL gene transcript in nucleic acid extracts of K. brevis cell samples. A novel enzyme dehydration and preservation method was combined with a pre-existing reagent Gelification method to prepare fully preserved Lab Cards with a shelf-life of at least six weeks prior to use. Using an internal control (IC), the Lab Card-based rbcL NASBA was demonstrated for the quantification of K. brevis from cell extracts containing between 50 and 5000 cells. This is the first demonstration of quantitation of K. brevis using IC-NASBA on a Lab Card.
Christos-Moritz Loukas a, b, Jonathan S. McQuillan a, , , Florian Laouenan c, Maria-Nefeli Tsaloglou a, b, Jesus M. Ruano-Lopez c, Matthew C. Mowlem a a National Oceanography Centre, European Way, Southampton SO14 3ZH, United Kingdom b Department of Ocean and Earth Science, University of Southampton Waterfront Campus, European Way, Southampton SO14 3ZH, United Kingdom c IK4-IKERLAN, J. M. Arizmendiarrieta, 2, 20500 Arrasate-Mondragón, Guipuzcoa, Spain Received 22 May 2017, Revised 7 June 2017, Accepted 7 June 2017, Available online 8 June 2017
Show less https://doi.org/10.1016/j.mimet.2017.06.008