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Detection and quantification of the toxic microalgae Karenia brevis using lab on a chip mRNA sequence-based amplification

Detection and quantification of the toxic microalgae Karenia brevis using lab on a chip mRNA sequence-based amplification
Detection and quantification of the toxic microalgae Karenia brevis using lab on a chip mRNA sequence-based amplification
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.
0167-7012
189-195
Loukas, Christos-Moritz
cda31664-e0d8-46d9-b9d2-162484c18baf
McQuillan, Jonathan S.
697cdf72-f353-4779-b64a-45494f29772f
Laouenan, Florian
37796527-85f1-42fd-a45d-c119bafe702d
Tsaloglou, Maria-Nefeli
99ab30ba-15da-4d25-86ba-608d127f8369
Ruano-lopez, Jesus M.
d4ff6945-0de2-42b7-a225-4fb68385a018
Mowlem, Matthew C.
6f633ca2-298f-48ee-a025-ce52dd62124f
Loukas, Christos-Moritz
cda31664-e0d8-46d9-b9d2-162484c18baf
McQuillan, Jonathan S.
697cdf72-f353-4779-b64a-45494f29772f
Laouenan, Florian
37796527-85f1-42fd-a45d-c119bafe702d
Tsaloglou, Maria-Nefeli
99ab30ba-15da-4d25-86ba-608d127f8369
Ruano-lopez, Jesus M.
d4ff6945-0de2-42b7-a225-4fb68385a018
Mowlem, Matthew C.
6f633ca2-298f-48ee-a025-ce52dd62124f

Loukas, Christos-Moritz, McQuillan, Jonathan S., Laouenan, Florian, Tsaloglou, Maria-Nefeli, Ruano-lopez, Jesus M. and Mowlem, Matthew C. (2017) Detection and quantification of the toxic microalgae Karenia brevis using lab on a chip mRNA sequence-based amplification. Journal of Microbiological Methods, 139, 189-195. (doi:10.1016/j.mimet.2017.06.008).

Record type: Article

Abstract

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.

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Loukas et al 2017_REVISED - Accepted Manuscript
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Accepted/In Press date: 7 June 2017
Published date: 1 August 2017

Identifiers

Local EPrints ID: 412691
URI: http://eprints.soton.ac.uk/id/eprint/412691
ISSN: 0167-7012
PURE UUID: d4cb1b78-fd64-49c0-a27c-6efd8e1fd53a

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Date deposited: 26 Jul 2017 16:30
Last modified: 07 Oct 2020 05:18

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Contributors

Author: Christos-Moritz Loukas
Author: Jonathan S. McQuillan
Author: Florian Laouenan
Author: Maria-Nefeli Tsaloglou
Author: Jesus M. Ruano-lopez
Author: Matthew C. Mowlem

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