Establishment Of A LAMP-LFD Detection Technique For Several Important Blooming Algae In The East China Sea

In recent years,the degree of eutrophication in the offshore waters has become more and more serious and led to the increasing number of global red tides.Therefore,development of efficient and sensitive methods for the detection of red tide microalgae is necessary.In this paper,we established a novel method to detection three red tide algae thats common in the East China Sea,namely,Amphidinium carterae,Alexandrium catenella,and Karenia mikimotoi by loop–mediated isothermal amplification?LAMP?combined with chromatographic lateral flow dipstick?LFD?.In this paper,the internal transcribed spacer or large subunit was used as target for detection.First,the target sequences were obtained by genomic DNA extraction and cloning sequence,the specific LAMP primers and detection probes were design,synthesis,and screening according to this target sequences.Secondly,the LAMP–LFD detection system was establishment for the target algae,and the LAMP detection were optimized.Finally,the specificity,sensitivity and practicality of LAMP–LFD were verified and evaluated.The ITS sequence of the A.carterae was cloned and used to design a sets of LAMP primers and a detection probe.After optimizing the LAMP reaction system,the specificity of LAMP–LFD was confirmed by cross reactivity tests with the common microalgae on the coast of the East China Sea,the results showed that LAMP–LFD has specificity for A.carterae.Both the genomic DNA and recombinant plasmid containing the ITS region gene of A.carterae were used to compare the sensitivity of the LAMP–LFD,The results showed that the detection limits of LAMP–LFD were 8.34×10^-44 ng·?L^-11 and 1.86×10⁴ copies·?L^-1,respectively,the sensitivity were all 100 times more than conventional PCR.The simulated samples results showed the detection limit of the LAMP–LFD with the simulated samples was approximately 10 cells·mL^–1.the sensitive was 10 times more than conventional PCR.The ITS sequence of A.catenella was cloned,and three sets of LAMP primers?AcLF1,AcLF2,and AcLF3?were successfully designed with the target sequence,the primers were screened and determine the optimal primer was AcLF2,and a detection probes was designed.Using the optimal primer to establish the detection system and optimized LAMP system.Cross reactivity tests results showed LAMP–LFD has specificity for A.catenella.LAMP–LFD detection limit for A.catenella genomic DNA and ITS cloning plasmids were 4.63×10^-44 ng·?L^-11 and1.26×10⁴ copies·?L^-1,both showing the sensitivity is 10 times higher than the SYBR Green I dye method and 100 times more than conventional PCR.The simulated samples results showed the detection limit of the LAMP–LFD with the simulated samples was approximately 10 cells·mL^–1,the sensitive was 100 times more than conventional PCR.The ITS sequence of K.mikimotoi was cloned and used to design two sets of LAMP primers?KmLF1 and KmLF2?.Two sets of primers?KmLF3 and KmLF4?were further designed with the LSU sequence obtained earlier in the laboratory.The primers were screened and determine the optimal primer was KmLF3,and a detection probe was designed based on the LSU sequence.The LAMP reaction system has been optimized.Cross reactivity tests results showed LAMP–LFD has specificity for K.mikimotoi,LAMP–LFD detection limit for K.mikimotoi genomic DNA were 1.70×10^-44 ng·?L^-11 and the sensitivity was 100 times than conventional PCR.LAMP–LFD detection limit for K.mikimotoi LSU cloning plasmids was 6.21×10³copies·?L^-1,The sensitivity is 10 times than LAMP and SYBR Green I dyes,1000 times than conventional PCR.The simulated samples results showed the detection limit of the LAMP–LFD with the simulated samples was approximately10^-11 cells·mL^-1,the sensitive was 1000 times more than conventional PCR.All in all,LAMP–LFD was a rapid and accurate molecule detection method of harmful algae.