Detection and quantification of Karenia mikimotoi using real-time nucleic acid sequence-based amplification with internal control RNA (IC-NASBA)

Abstract

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 assay 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 × 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, α = 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.