| With the global climate change,eutrophication of seawater and the intensification of human activities,the spread of harmful algae in the world is accelerated and more widely distributed.In particular,some toxic microalgae can form harmful algal blooms through explosive proliferation,produce biotoxins,destroy the balance of marine ecosystem,affect the survival of aquatic animals and the sustainable development of fishery economy,and threaten the health and safety of aquatic food.Therefore,real-time monitoring of harmful aquatic microalgae in the marine environment has become an urgent problem.However,at present,there is still a lack of accurate,efficient and rapid detection method for harmful microalgae,especially the field detection technology that can be directly applied to marine aquaculture base or fishery management.Therefore,this paper focus on typical aquatic harmful algae species in Chinese coast,including Karenia mikimotoi,Alexandrium tamarense,Chattonella marina Prorocentrum minimum,and Heterosigma akashiwo,based on specific primers and taxonomic probes,using special primer tag design strategies,taking gel electrophoresis,membrane based taxonomic chip and lateral flow chromatographic strip as detection platforms,multiplex PCR（MPCR）,MPCR-membrane based reverse dot blot hybridization（MPCR-RDBH）,and MPCR based nucleic acid chromatography test strip（MPCR-NACST）technologies were successively established.The main research results of this paper are as follows:（1）Molecular identification of typical aquatic harmful microalgae.Genomic DNA was extracted from 5 test algae strains（K.mikimotoi,A.tamarense,C.marina,P.minimum,and H.akashiwo）that were stored in the laboratory or purchased.Then,ribosomal large subunit unit（LSU r DNA D1-D2）and the internal transcribed spacer（ITS）were used as molecular markers for algae identification.Combined with morphological classification,double identification were performed for test algae strains.PCR amplification,molecular cloning and sequencing and bioinformatics analysis showed that the LSU r DNA and ITS sequences of all tested algae strains were 99%or 100%similar to the corresponding algae species in NCBI database,and the E value was 0,indicating that the tested algae strains were the target algae species of this study.It lays the foundation for the establishment of the next specific multiple nucleic acid detection method.（2）Design of specific primers and classification probes.The LSU r DNA or ITS gene sequences of targeted algae species were identified by BLASTn homology retrieval and multiple comparison analysis,and the specific primers were designed,screened and verified.The length of 5 pairs of specific primers was 20 bp,GC content difference was less than 8%,Tm value difference was less than 5℃,and the length difference of each amplified product was more than 50 bp.The results of single PCR and cross amplification experiment showed that all the five pairs of specific primers could amplify the target bands in accordance with the expectation,and none of the primers could be cross-amplified with non-target algae,indicating that the designed primers had good specificity.In addition,using the actin gene of Litopenaeus vannamei as template,five classification probes were designed based on Tm value,GC content,sequence similarity and other parameters.（3）Establishment of multiple detection technology-MPCR for typical aquatic harmful algae species.Based on the above specific primers,the MPCR amplification system was preliminarily established and systematically optimized to obtain the optimal MPCR system.The detection specificity,sensitivity,stability and practicability of MPCR were evaluated experimentally.The results showed that,the single or multiple target bands were amplified in accordance with genomic DNA of single or multiple target species which were added randomly into the MPCR system.No cross-reaction occurred between the primers,and non-specific amplification products appeared,indicating that the good specificity of MPCR system.When different concentrations of interfering algae were added to the system for MPCR,5 bands could be amplified and no target bands appeared,indicating that the system was not affected by interfering algae.The results of simulated natural water showed that the limit of MPCR system for simultaneous detection of 5 target algae cells was 4 cells m L-1,and the limit of detection of target algae genome DNA was 1 ngμL-1.Finally,aquaculture water samples were collected for MPCR practical testing,and it was found that the analysis results of target algal species in water samples by this method were basically consistent with the results of traditional microscopic examination.（4）Establishment of MPCR-membrane-based RDBH technology for typical aquatic harmful algae species.Based on the established MPCR system,MPCR amplification and Biotin labeling were performed by designing and adding a label sequence at the 5’end of the reverse primer.The reverse complementary sequence of the label sequence was used as the detection probe,and the probe was tail-added and fixed on the nylon membrane by UV crosslinking.After preparing the membrane based classification chip,the MPCR products was hybridized with the membrane chip.Then,the MPCR-RDBH system was preliminarily established,and optimization was carried out.Furthermore,the specificity,sensitivity and anti-interference performance of MPCR-RDBH were evaluated.The results showed that the specificity of MPCR primers and probes was excellent,they neither hybridized with PCR products of non-target algae,nor cross-reacted with MPCR products of other target algae.In the presence of interfering algal species,the hybridization system can complete the detection of target algal species,indicating that the established MPCR-RDBH technology is s Tab..The detection limit of MPCR-RBDH for target algae DNA was 100 pgμL-1,and the simulated natural water samples showed that the method could simultaneously detect 5 target algal cells with a limit of 4×10-1cells m L-1.Finally,the practicability of MPCR-membrane based RDBH was evaluated by collecting aquaculture water samples,and it was found that the method could accurately and sensitively detect the presence of target algae in the aquaculture samples,which was consistent with microscopic examination results,indicating that the established MPCR-RDBH has certain practicability.（5）Preliminary establishment of MPCR-NACST technology for typical aquatic harmful algae species.Firstly,the forward and reverse primers with label sequences were used to establish MPCR amplification system,and MPCR products containing double label sequences were prepared.With an ingenious joint primer design and colloidal gold as tracer molecule,using hybridizedation of joint primer with colloidal gold probes,detection probes and control probes,we developed a hand–prepared NACST,which can be used for multiple analysis of MPCR products.Then,MPCR was directly applied to the analysis of NACST,and the nucleic acid analysis was completed through colored spots,and the MPCR-NACST method was successfully established.Finally,the main parameters of MPCR for NACST analysis,including primer concentration and annealing temperature were optimized,and the specificity of the MPCR-NACST was evaluated by using 21 species of common microalgae along the coast of China.The results showed that NACST did not cross-react with PCR products of non-target algae,indicating that MPCR-NACST has excellent specificity.In conclusion,this study focuses on the typical harmful microalgae in coastal aquatic products in China.Based on the MPCR technology of multiple detection,the visual MPCR membrane based rdbh and convenient mpcr-nacst detection technology are established with membrane based classification chip and nucleic acid chromatography test strip as the analysis platform respectively,in order to simplify the nucleic acid analysis method step by step,shorten the detection time and get rid of the dependence on special instruments and equipment,In order to meet the actual needs of mariculture or fishery management for the detection of harmful microalgae in aquatic products,and provide a sharp tool for the monitoring of harmful algae species for marine aquaculture and fishery practice. |
