Comparative detection of Karenia mikimotoi by exponential rolling circle amplification (E-RCA) and double-ligation E-RCA

Karenia mikimotoi is a globally distributed, toxic, bloom-forming dinoflagellate. The development of rapid, precise and sensitive detection methods is essential for the field monitoring of this harmful alga. In this study, exponential rolling circle amplification (E-RCA) and double-ligation E-RCA (dlE-RCA) were established for the detection of K. mikimotoi. The partial large subunit rDNA (D1-D2) of K. mikimotoi was PCR amplified, cloned and then sequenced. The resultant sequence was used to perform alignment analysis for species-specific regions and consequently design padlock probes and primers for E-RCA and dlE-RCA. Both E-RCA and dlE-RCA detection protocols were established and their parameters were optimized. dlE-RCA can avoid self-cyclization of PLP compared with E-RCA. The optimized parameters were as follows: ligation temperature, 61 degrees C; ligation time, 60min (E-RCA)/30min (dlE-RCA); amplification temperature, 61 degrees C (E-RCA)/64 degrees C (dlE-RCA); and amplification time, 30min (E-RCA)/40min (dlE-RCA). Specificity tests showed that both E-RCA and dlE-RCA were specific for K. mikimotoi. Sensitivity comparison indicated that E-RCA was 10-fold more sensitive than PCR and the sensitivity of dlE-RCA was comparable with that of PCR. Tests with simulated field samples suggested that the developed E-RCA and dlE-RCA obtained detection limits of 1 and 10 cells, respectively. Positive E-RCA and dlE-RCA could be confirmed by visual observation of coloration reaction with the addition of fluorescent SYBR Green I dye to the reaction tube. The developed E-RCA and dlE-RCA were also efficient for field samples with target cell densities ranging from 1 cell mL(-1) to 1000 cells mL(-1). These results suggest that the established E-RCA and dlE-RCA detection protocols show promising applications in the field monitoring of K. mikimotoi.