A multiplex qPCR targeting hepato- and neurotoxigenic cyanobacteria of global significance
A multiplex qPCR targeting hepato- and neurotoxigenic cyanobacteria of global significance
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DOI：
10.1016/j.hal.2011.11.001
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发表时间：
2012-03-01
期刊：
影响因子：
6.6
通讯作者：
Neilan, Brett A.
中科院分区：
文献类型：
--
作者：
Al-Tebrineh, Jamal;Pearson, Leanne A.;Neilan, Brett A.
有毒的水华蓝藻是一种全球性的健康危害。这些光合微生物产生一系列次级代谢物毒素，包括微囊藻毒素、节球藻毒素和柱精子蛋白等肝毒素以及蛤蚌毒素等神经毒素。这些毒素可能威胁饮用水供应的安全，而石房蛤毒素可能在贝类中积累到危险水平，影响海产品行业。已经描述了几种分子方法用于检测和定量产毒蓝藻，然而，迄今为止，还没有用于同时检测和定量肝毒素和神经毒素产生属的方法。本文介绍了一种四重定量PCR（qPCR）检测和定量毒素基因的微囊藻毒素，节球藻毒素，柱孢菌素和石房蛤毒素生物合成途径的发展和验证。本试验中使用的引物和探针是从大多数代表性蓝藻属的毒素生物合成基因内的保守区域设计的。优化qPCR测定以在单个反应中可靠地确定蓝藻毒素生物合成基因的拷贝数以及内部蓝藻16S rDNA对照。在蓝藻毒素基因之间的扩增效率和再现性是相似的，而毒素基因的反应灵敏度范围为每个反应10（2）至10（6）个基因拷贝。这种多重qPCR检测是检测和定量实验室和现场样品中潜在有毒蓝藻的有力工具。这种技术将使水质和食品安全当局能够更好地预测、评估和减少未来有害藻华事件的影响。皇冠版权所有（C）2011由Elsevier B.V.出版。保留所有权利。
Toxic bloom-forming cyanobacteria are a global health hazard. These photosynthetic microorganisms produce a suite of secondary metabolite toxins including hepatotoxins such as microcystin, nodularin and cylindrospermopsin and neurotoxins such as saxitoxin. These toxins can threaten the safety of drinking water supplies and in the case of saxitoxin, can accumulate to dangerous levels in shellfish, affecting the seafood industry. Several molecular methods have been described for the detection and quantification of toxigenic cyanobacteria, however, to date there is no method for the simultaneous detection and quantification of hepatotoxin and neurotoxin producing genera. This paper describes the development and validation of a quadruplex quantitative-PCR (qPCR) assay capable of detecting and quantifying toxin genes from the microcystin, nodularin, cylindrospermopsin and saxitoxin biosynthesis pathways. The primers and probes employed in this assay were designed from conserved regions within toxin biosynthesis genes from most of the representative cyanobacterial genera. The qPCR assay was optimized to reliably determine the copy number of cyanotoxin biosynthesis genes, as well as an internal cyanobacteria 16S rDNA control, in a single reaction. Amplification efficiency and reproducibility were similar among the cyanotoxin genes, while the sensitivity of the reaction for the toxin genes ranged from 10(2) to 10(6) gene copies per reaction. This multiplex qPCR assay is a powerful tool for detecting and quantifying potentially toxic cyanobacteria in laboratory and field samples. Such technology will enable water quality and food safety authorities to better forecast, evaluate and reduce the impact of future harmful algal bloom events. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.