Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a dinoflagellate)
Development of a real-time PCR assay for rapid detection and quantification of Alexandrium minutum (a dinoflagellate)
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DOI：
10.1128/aem.70.2.1199-1206.2004
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发表时间：
2004-02-01
影响因子：
4.4
通讯作者：
Magnani, M
中科院分区：
文献类型：
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作者：
Galluzzi, L;Penna, A;Magnani, M
海洋甲藻属Alexandrium包括许多产生麻痹性贝类中毒（PSP）的神经毒素的物种，这在人类中可能导致肌肉麻痹，神经系统症状，在极端情况下甚至死亡。沙蚤是西地中海盆地分布最广的有毒沙蚤种类。对沿海水域有害藻类存在的监测通常还包括对浮游植物种群的显微镜检查。这些程序耗时且需要大量的分类学经验，从而限制了可以分析的标本数量。由于不同属和种的遗传多样性，分子工具也可以帮助检测海洋野外样品中目标微生物的存在。在这项研究中，我们开发了一种基于实时pcr的检测方法，用于快速检测固定保存的环境样品和培养物中亚历山大菌属的所有有毒物种。此外，我们还建立了一种实时定量PCR方法来定量海水样品中的微囊藻细胞。在5.8S rDNA区域设计亚历山大属特异性引物。引物特异性通过BLAST和其他鞭毛藻和硅藻的代表性样本的DNA扩增得到证实。利用含有it1.5.8 s - its2 a . minutum序列的质粒与培养的a . minutum细胞构建标准曲线，测定每个细胞5.8S rDNA拷贝的绝对数量。因此，在含有A. minutum细胞的样品中定量5.8S rDNA拷贝后，我们也能够估计细胞的数量。采用该方法分析了来自Arenys Harbor （catalalan Coast, Spain）的几种固定褐花海样，并将定量结果与标准显微镜计数方法进行了比较。这两种方法的结果相当，证实了实时PCR是一种有效的、快速的替代方法，可用于沿海水域监测中目标浮游植物物种的检测和定量。
The marine dinoflagellate genus Alexandrium includes a number of species which produce neurotoxins responsible for paralytic shellfish poisoning (PSP), which in humans may cause muscular paralysis, neurological symptoms, and, in extreme cases, death. A. minutum is the most widespread toxic PSP species in the western Mediterranean basin. The monitoring of coastal waters for the presence of harmful algae also normally involves microscopic examinations of phytoplankton populations. These procedures are time consuming and require a great deal of taxonomic experience, thus limiting the number of specimens that can be analyzed. Because of the genetic diversity of different genera and species, molecular tools may also help to detect the presence of target microorganisms in marine field samples. In this study, we developed a real-time PCR-based assay for rapid detection of all toxic species of the Alexandrium genus in both fixative-preserved environmental samples and cultures. Moreover, we developed a real-time quantitative PCR assay for the quantification of A. minutum cells in seawater samples. Alexandrium genus-specific primers were designed on the 5.8S rDNA region. Primer specificity was confirmed by using BLAST and by amplification of a representative sample of the DNA of other dinoflagellates and diatoms. Using a standard curve constructed with a plasmid containing the ITS1.5.8S-ITS2 A. minutum sequence and cultured A. minutum cells, we determined the absolute number of 5.8S rDNA copies per cell. Consequently, after quantification of 5.8S rDNA copies in samples containing A. minutum cells, we were also able to estimate the number of cells. Several fixed A. minutum bloom sea samples from Arenys Harbor (Catalan Coast, Spain) were analyzed using this method, and quantification results were compared with standard microscopy counting methods. The two methods gave comparable results, confirming that real-time PCR could be a valid, fast alternative procedure for the detection and quantification of target phytoplankton species during coastal water monitoring.