Development of a multiplex polymerase chain reaction assay for the parallel detection of harmful algal bloom-forming species distributed along the Chinese coast

Yan-Jie Sun; Guo-Fu Chen; Chun-Yun Zhang; Chang-Lu Guo; Yuan-Yuan Wang; Rui Sun

doi:10.1016/j.hal.2019.02.008

Development of a multiplex polymerase chain reaction assay for the parallel detection of harmful algal bloom-forming species distributed along the Chinese coast

Harmful Algae. 2019 Apr:84:36-45. doi: 10.1016/j.hal.2019.02.008. Epub 2019 Mar 16.

Authors

Yan-Jie Sun¹, Guo-Fu Chen², Chun-Yun Zhang³, Chang-Lu Guo⁴, Yuan-Yuan Wang¹, Rui Sun¹

Affiliations

¹ School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Weihai, 264209, PR China.
² School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Weihai, 264209, PR China. Electronic address: chenguofu@hitwh.edu.cn.
³ School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Weihai, 264209, PR China. Electronic address: zhangchunyun@hitwh.edu.cn.
⁴ School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Weihai, 264209, PR China. Electronic address: guochanglu@hitwh.edu.cn.

PMID: 31128811
DOI: 10.1016/j.hal.2019.02.008

Abstract

Harmful algal blooms (HABs) 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 HABS. A method for the parallel detection of harmful algal species must be established because various HAB-forming algal species coexist in the marine environment. This work developed a multiplex PCR (mPCR) method that can simultaneously detect six common HAB-forming microalgal species distributed along the coast of China: Karlodinium veneficum (Kv), Chattonella marina (Cm), Skeletonema spp., Scrippsiella trochoidea (St), Karenia mikimotoi (Km), and Prorocentrum donghaiense (Pd). Specific mPCR primers were designed from the internal transcribed spacer rDNA or large subunit rDNA gene of the target algal species. The mPCR conditions were optimized. Each mPCR primer was subjected to a cross-reactivity test with other microalgae to confirm the specificity of the developed mPCR system. The results of the system stability test indicated that the background concentration of DNA tested did not affect the performance of the established mPCR system. The results of the sensitivity test showed that the detection limit of the proposed mPCR system for Kv, Cm, Km, and Pd was 0.6 ng μL^-1 and that for Skeletonema spp. and St was 0.06 ng μL^-1. Additional mPCR analysis with spiked field samples revealed that the detection limit of the mPCR system for Km, Pd, and Kv was 60 cells, whereas that for Cm, Skeletonema spp., and St was 6 cells. The convenience and accuracy of the established mPCR assay were further validated through tests with field samples. The proposed mPCR assay is characterized by parallel analysis, strong specificity, and stability and can be used to supplement morphology-based detection methods for algal species.

Keywords: Detection; Harmful algal blooms; Harmful microalgae; Multiplex PCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

China
DNA, Ribosomal
Dinoflagellida*
Harmful Algal Bloom*
Multiplex Polymerase Chain Reaction

Substances

DNA, Ribosomal