Rapid Detection Of Haemful Marine Microalgae By Recombinaae Polymerase Amplification

Algal blooms involving toxic or harmful phytoplankton are a global problem that has occurred in many coastal and shelf ecosystems and has negatively affected human activities for several years,moreover,serious damage to marine ecosystems and aquaculture.Therefore,establishing a rapid detection method for causative species is necessary to reduce or prevent detrimental effects of HABs?Harmful blooms,HABs?.At the same time,molecular biology technology provides a new idea for the identification and quantitative detection of red tide algae.The rapid development of this technology has become the main position in the field of detection.In this study,the performance of recombinase poly amplification?RPA?was used to couple with later flow dipstick?LFD?assay for the detection of Karlodinium veneficum,Prorocentrum minimum and P.donghaiense.The RPA-LFD assays establish herein is sensitive,specific,rapid,and convenient for the detection and it may be promising for application.In this paper,three species of harmful microalgae were selected as target and their internal transcribe spacers?ITS?sequences were obtain by cloning and sequencing.Subsequently,the obtained sequences were processed and analyzed by bioinformatics software,and their specific regions were visually detected.Primer Premier 5.0 was used to design specific primers and probes.RPA-LFD systems of three target microalgae are established and their amplification conditions were optimized to verify the specificity,stability by simulating natural water samples and collection eight natural water samples from the East China Sea.In this study,a method?RPA-LFD?was developed for the rapid detection of common harmful K.veneficum.The primers for RPA and the detection probe for LFD were designed to specially target the internal transcribed spacer of K.veneficum based RPA primer principles.The results show RPA-LFD could highspecify detection K.veneficum at optimal conditions of 35 ?,30 min.The RPA products can be visually detected by the naked eyes through an LFD after an automatic chromatography for 5 min at room temperature.In addition,the lowest detection limit of RPA-LFD was 10 ng ?L^-1 of genomic DNA,10 copies ?L^-1 and0.1 cell m L^-1,which was 100-fold sensitive than conventional PCR.The developed RPA-LFD was established for the rapid detection harmful microalgae of P.minimum.Specific primers and probes were designed after monoclonal sequencing of ITS sequences of P.minimum.The specificity,sensitivity,practicability and stability of proposed RPA-LFD was well validated.In addition,various RPA conditions including amplification time,amplication temperature,magnesium ion concentration the amplification reaction were optimized.The optimal RPA-LFD can recognize the target algal species from water samples with a cell density of 10 cells and 8.1 × 10^-2ng ?L^-1,2.5 × 10 copies ?L^-1.The RPA-LFD was established for the practical detection of P.donghaiense.The partial large subunit r DNA?TW81 and AB28?of P.donghaiense was PCR amplified,cloned and then sequenced.The relevant representative sequences were downloaded from Gen Bank and aligned analysis for species-specific regions and consequently design specifity primers and probes for RPA-LFD.Specificity tests showed that RPA-LFD was specific for P.donghaiense and cross-reactions with other tested algae.Sensitivity comparison indicated that RPA-LFD was 6.79 × 10^-2ng ?L^-1,6.01 × 10¹ copies ?L^-1and 10-fold more sensitive than PCR.Tests with simulated field samples suggested that the developed RPA-LFD obtained detection limits 10 cells,respectively.Positive RPA-LFD could be detection three brands in eight natural water samples and more sensitive the conventional PCR.