Journal
HARMFUL ALGAE
Volume 9, Issue 1, Pages 116-122Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.hal.2009.08.010
Keywords
IC-NASBA; Internal control RNA; Karenia brevis; Quantitative NASBA
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Funding
- National Oceanic and Atmospheric Administration Coastal Ocean Program [NA06NOS4780230]
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Nucleic acid sequence-based amplification (NASBA) is an isothermal method used to amplify RNA and has been used for clinical, environmental, and food testing applications. Quantification of RNA by real-time NASBA occurs by comparing time to positive (TTP) fluorescence values, similar to threshold cycle (Ct) values in PCR, of unknown samples to a standard curve of known RNA titers. Incorporation of an internal control RNA molecule (IC-RNA) has been used to increase precision and accuracy of real-time NASBA and also serves as an indicator of NASBA inhibition. A real-time IC-NASBA assail was developed targeting the ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) large-subunit gene (rbcL) of the harmful algal bloom (HAB) causing dinoflagellate Karenia mikimotoi. This assay is sensitive to one K. mikimotoi cell and 1 x 10(3) copies of in vitro transcript with a high degree of specificity against closely related organisms. Enumeration of K. mikimotoi from environmental samples by IC-NASBA was not significantly different from microscopic cell counts (P = 0.156, alpha = 0.05) performed by the Fish and Wildlife Research Institute (FWRI. a division of the Fish and Wildlife Conservation Commission, St. Petersburg, FL), the agency responsible for monitoring red tide status throughout the state. In addition, the IC-NASBA enumeration had a good linear relationship (r(2) = 0.887) with FWRI microscopic cell counts. IC-NASBA is an alternative method for the rapid and reliable detection and quantification of K. mikimotoi from marine waters. (C) 2009 Elsevier B.V. All rights reserved.
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