Development of a multiplex polymerase chain reaction assay for the parallel detection of harmful algal bloom-forming species distributed along the Chinese coast
Development of a multiplex polymerase chain reaction assay for the parallel detection of harmful algal bloom-forming species distributed along the Chinese coast
复制标题
开发多重聚合酶链式反应检测方法，用于平行检测中国沿海分布的有害藻类水华形成物种
DOI：
10.1016/j.hal.2019.02.008
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发表时间：
2019-04-01
期刊：
影响因子：
6.6
通讯作者：
Sun, Rui
中科院分区：
文献类型：
--
作者：
Sun, Yan-Jie;Chen, Guo-Fu;Sun, Rui
有害藻华对海洋生态环境、公众健康和海洋经济产生了严重影响。因此，迫切需要准确、快速识别有害藻种的方法，以有效监测有害藻种的发生。由于海洋环境中存在多种形成有害赤潮的藻类，因此必须建立一种有害藻类的平行检测方法。本研究建立了一种多重PCR（mPCR）方法，可同时检测沿着中国沿海常见的六种赤潮微藻：Karlodiniwn veneficum（Kv），Chattonella marina（Cm），锥状斯氏藻（St）、米氏凯伦藻（Km）和东海原甲藻（Pd）。根据目的藻的rDNA内转录间隔区或大亚基rDNA基因设计特异性引物。优化了mPCR反应条件。每种mPCR引物与其他微藻进行交叉反应性测试，以确认所开发的mPCR系统的特异性。系统稳定性试验结果表明，供试DNA的背景浓度不影响已建立的mPCR系统的性能。灵敏度试验结果表明，拟定的mPCR系统对Kv、Cm、Km和Pd的检测限为0.6 ng mu L-1，对螺旋藻属的检测限为0.6 ng mu L-1。St为0.06 ng μ L~（-1）。用加标的现场样品进行的额外mPCR分析显示，对于Km、Pd和Kv，mPCR系统的检测限为60个细胞，而对于Cm、螺旋藻属，St为6个细胞。通过对现场样品的检测，进一步验证了所建立的mPCR方法的简便性和准确性。该方法具有平行分析、特异性强、稳定性好等特点，可作为藻类形态学检测方法的补充。
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: Karlodiniwn 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 mu L-1 and that for Skeletonema spp. and St was 0.06 ng mu L-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.