Development Of A Multiplex Polymerase Chain Reaction Assay For The Parallel Detection Of Harmful Algal Bloom-forming Species Distributed Along The Chinese Coast

In recent years,harmful algal blooms?HAB?have adverse effects on the marine ecological environment,public health,and marine economy.Thus,methods for the accurate and rapid identification of harmful algal species are urgently needed for the effective monitoring of the occurrence of HAB.However,the accurate identification of harmful algal species through microscopy observation is complicated by the small sizes,complex structures,and highly similar morphological features of different algal species.Morphological examination based on optical or electronic microscopy is time consuming and laborious.Moreover,many kinds of microalgae can cause HAB in the natural ocean environment.Thus,the development of an analytical method for the parallel detection of several harmful algae is necessary.This study explored the application of multiplex PCR?m PCR?in the detection of six harmful algae species that are commonly distributed in Chinese coastal areas.Microalgal species distributed along the Chinese coast and with highest HAB-forming frequencies reported in recent years were used as m PCR targets.These species included Karlodinium veneficum?Kv?,Chattonella marina?Cm?,Skeletonema spp.,Scrippsiella trochoidea?St?,Karenia mikimotoi?Km?,and Prorocentrum donghaiense?Pd?.Molecular identification of six target algae species was first performed by cloning and sequencing.The genomic DNA of the target species was extracted,and the internal transcribed spacer?ITS?genes or the D1-D2 region of the large subunit?LSU?rDNA genes of the target species were PCR-amplified.PCR products were purified.Purified PCR products were inserted into the p MD?18-T Vector and transformed into competent Escherichia coli.Positive clones were identified through colony PCR and then sequenced.Sequencing results were analyzed with the online BLASTn search tool on the NCBI website to determine the correctness of the target species.According to the sequencing results of each target algae species,the algal species with similar blood relationship or the sequences with higher similarity were downloaded from Genebank.Related sequences retrieved from Gen Bank and sequences obtained in this study were aligned using Clustal W implemented with the program Bioedit.Species-specific and genus-specific regions were visually identified for primer design and adjusted using the software Primer Premier 5.0.To confirm the specificity of the developed m PCR system,each m PCR primer was subjected to a cross-reactivity test with 25 representative microalgae that are widely distributed along the Chinese coast and all target algal species.Specific m PCR primers were designed from the internal transcribed spacer rDNA or large subunit rDNA gene of the target algal species.Expected amplification fragments with sizes of 437 bp,343 bp,254 bp,198 bp,137 bp,and 83 bp were obtained for Kv,Cm,Skeletonema spp.,St,Km,and Pd,respectively.The m PCR system was initially established and the reaction conditions were optimized.The optimized m PCR conditions were as follows: Kv,Cm,Skeletonema spp.,St,Km,and Pd primer concentrations of 0.10 ?M,0.15 ?M,0.10 ?M,015 ?M,0.20 ?M,and 0.20 ?M,respectively;Mg2+ concentration of 3.5 m M;d NTP concentration of 0.6 m M;Taq DNA polymerase concentration of 0.10 U ?L^-1;and annealing temperature of 52 °C.subsequent m PCR tests were performed on the optimized m PCR system.The m PCR system was further confirmed to be specific by the combination of DNA templates of different target algae species added to the m PCR amplification system.The stability of the developed m PCR system was tested by mixing target genomic DNA with nontarget genomic DNA at different concentration ratios.The results indicated that background DNA did not affect the performance of the established m PCR system.The results of the sensitivity test showed that the detection limit of the proposed m PCR system for Kv,Cm,Km,and Pd was 0.6 ng ?L^-1 and that for Skeletonema spp.and St was 0.06 ng ?L^-1.Additional m PCR analysis with spiked field samples revealed that the detection limit of the m PCR system for Km,Pd,and Kv was 60 cells m L^-1,whereas that for Cm,Skeletonema spp.,and St was 6 cells m L^-1.The convenience and accuracy of the established m PCR assay were further validated through tests with field samples from the East China Sea.The proposed m PCR assay is characterized by parallel analysis,strong specificity,and stability and can be used to supplement morphology-based detection methods for algal species.