Establishment and application of hyperbranched rolling circle amplification coupled with lateral flow dipstick for the sensitive detection of Karenia mikimotoi
2019
Zhang, Chunyun | Chen, Guofu | Wang, Yuanyuan | Zhou, Jin | Li, Chenghua
The dinoflagellate Karenia mikimotoi is a noxious and harmful algal bloom (HAB)-forming microalga. Establishing a rapid, accurate, and sensitive method of detecting this harmful alga is necessary to provide warnings of imminent HABs through field monitoring. Here, an isothermal amplification technique combined with a rapid analytical method for nucleic acid-based amplified products, i.e., hyperbranched rolling circle amplification (HRCA) coupled with lateral flow dipstick (LFD), hereafter denoted as HRCA-LFD, was established to detect K. mikimotoi. The HRCA-LFD assay relied on a padlock probe (PLP) targeting DNA template and an LFD probe targeting PLP. The sequenced internal transcribed spacer of K. mikimotoi through molecular cloning was used as the target of PLP. The optimized HRCA conditions was determined to be as follows: PLP concentration, 20 pM; ligation temperature, 65 °C; ligation time, 10 min; amplification temperature, 61 °C; and amplification time, 30 min. The developed HRCA-LFD assay was specific for K. mikimotoi, displaying no cross-reactivity with other common microalgae. Sensitivity-comparison tests indicated that HRCA-LFD assay was 100-fold more sensitive than PCR, with a detection limit of 0.1 cell mL−1 when used to analyze spiked field samples. The analysis with field samples also indicated that HRCA-LFD assay was suitable for samples with a target cell density range of 1–1000 cells mL−1. All of these results suggested that HRCA-LFD assay is an alternative method for the sensitive and reliable detection of K. mikimotoi from marine water samples.
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