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

Abstract

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 °C; ligation time, 60 min (E-RCA)/30 min (dlE-RCA); amplification temperature, 61 °C (E-RCA)/64 °C (dlE-RCA); and amplification time, 30 min (E-RCA)/40 min (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.