Detection Of Toxigenic Marine Microalgae By Double Probes Rolling Circle Amplification

The amount of nutrients in the marine environment has increased by human activities.It causes marine microalgae to multiply and form harmful algae blooms（HABs）.Harmful algae that cause HABs destroy the ecosystem in water area,severely affect the economic development of coastal areas,and threaten human health.Therefore,the establishment of efficient and accurate detection method for harmful algae plays an important role in preventing the occurrence of HABs.In this study,a new isothermal amplification technology,double probes rolling circle amplification（dpRCA）,was established and combined with lateral flow dipstick（LFD）.The results showed dpRCA-LFD not only improves the specificity of the detection,but also makes the observation of the reaction results more rapid and intuitive.This technology can be used for field analysis of harmful algae in the future.In this study,the internal transcribed spacers（ITS）of Chattonella marina,Prorocentrum minimum,and Amphidinium carterae were selected as the target sequence respectively.Their complete sequence information obtains by PCR amplification,cloning and sequencing.The obtained sequences were verified by BLAST.The sequences were downloaded with a similarity higher than 80%.The above sequences were analyzed by Clustal W Multiple Alignmentmultiple using the software BioEdit.The specific regions of the ITS sequences were found by visual inspection.According to the design principles of probes and primers of dpRCA,specific primers and probes were designed by Primer Premier 5.0 combining the specific region with the gene sequence of a distant relative species,respectively.The dpRCA reaction conditions of C.marina which can produce toxins were optimized to establish dpRCA-LFD for C.marina,and the specificity,sensitivity,spiked and natural samples were tested under the optimal conditions.The results showed that the optimal circular ligation temperature and cycle number of circular ligation were 58℃ and 12,respectively.The optimal nucleic acid amplification temperature was 60℃,and the optimal amplification time was 60 min.The specific results showed that only C.marina could be amplified by dpRCA,while there was no amplification by other algae.The detection limits（DL）for genomic DNA,recombinant plasmids and spiked samples were 8.3×10^–3 ngμL^-1,7.8 copiesμL^–1,and 10 cells mL^-1,respectively.The verification of 8 natural samples showed that the established dpRCA-LFD could detect C.marina specifically.dpRCA-LFD was established to providing a new method for accurately identifying P.minimum.The optimization results of each reaction condition showed that the optimal condition for the circular ligation was 56℃ for 9 cycles,and the optimal condition for the amplification reaction was 60℃ for 45 min.Under these conditions,the sensitivity of the genomic DNA of P.minimum and the recombinant plasmid DNA containing the ITS sequence was tested.The DL of dpRCA-LFD was 6.8×10^–4 ngμL^-1 and 37 copiesμL^–1,respectively.The results of spiked samples showed that the lowest detectable cell concentration of dpRCA-LFD was 1 cell mL^-1.The results of specificity and natural samples showed that the established dpRCA-LFD could only detect P.minimum,which had high specificity and practicability.A specific detection method,dpRCA-LFD,was established with the object of A.carterae.The cycle number of circular ligation,the circular ligation temperature,the nucleic acid amplification time and the amplification temperature were optimized.The results showed that the optimal reaction conditions for dpRCA were as follows:cycle number of ligation reaction,10;ligation temperature,61°C;amplification time,50 min;and amplification temperature,59℃.The specificity and sensitivity of this method were tested by combining the dpRCA products with LFD.The established dpRCA-LFD did not cross-react with non-target algae,indicating that this method has good specificity.The sensitivity test showed that the DL of dpRCA-LFD for genomic DNA,recombinant plasmid and cell concentration were 4.5×10^–4 ngμL^-1,1.3 copiesμL^–1 and 1 cell mL^-1,respectively.The results of natural samples showed that dpRCA-LFD could accurately detect A.carterae in complex environments and it is expected to become a common method for field test.