Comparison of loop-mediated isothermal amplification with hyperbranched rolling circle amplification as a simple detection method for Heterosigma akashiwo

Abstract

The fish-killing alga Heterosigma akashiwo is a globally distributed, toxic, and bloom-forming raphidophyte that has caused great losses to the fishing industry in many coastal countries. Therefore, rapid and sensitive detection methods should be developed to present timely warning of harmful algal blooms. In this study, hyperbranched rolling circle amplification (HRCA) was established for the detection of H. akashiwo and compared with loop-mediated isothermal amplification (LAMP) in terms of specificity and sensitivity. The partial D1–D2 sequence of the large subunit (LSU) of rDNA of H. akashiwo was used to design a specific padlock probe for HRCA and two pairs of specific primers for LAMP. The parameters for HRCA were optimized. Cross-reactivity tests showed that the specificity of the developed HRCA for H. akashiwo was greater than that of LAMP in this study. The sensitivities of HRCA and LAMP were comparable and were 10-fold higher than that of regular PCR. These methods also yielded a detection limit of 20 fg/μL for the recombinant plasmid containing the target LSU D1–D2 and 1 cell for target species. The test with the simulated field samples indicated that the developed HRCA obtained a detection limit of 5 cells mL−1, which was lower than the warning cell density (100 cells mL−1) of H. akashiwo. The visual detection of positive HRCA could be achieved via coloration reaction with the addition of fluorescent SYBR Green I dye to the amplification products. The developed HRCA was also efficient for field samples with target cell densities ranging from 10 cells mL−1 to 1000 cells mL−1. Therefore, the proposed HRCA detection protocols are possibly applicable to the field monitoring of H. akashiwo.