Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis
Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis
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DOI：
10.1128/aem.00931-15
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发表时间：
2015-08-01
影响因子：
4.4
通讯作者：
Gin, Karina Yew-Hoong
中科院分区：
文献类型：
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作者：
Te, Shu Harn;Chen, Enid Yingru;Gin, Karina Yew-Hoong
近几十年来，有害蓝藻水华的发生日益增多，通常与水质恶化和不良公共健康影响有关，已成为全世界关注的问题。实时定量 PCR (qPCR) 等分子技术的使用在有害蓝藻物种的检测和监测中已变得越来越流行。先前已经建立了多重 qPCR 测定法，可量化几种产毒蓝藻物种；然而，还没有分子检测方法可以同时检测多种水华形成物种。微囊藻属和圆柱藻属是最常见的两个属，已知能够产生微囊藻毒素和圆柱藻毒素肝毒素。在本研究中，我们设计了引物和探针，能够基于圆柱藻属物种的 RNA 聚合酶 C1 基因和微囊藻属物种的 c-藻蓝蛋白 β 亚基样基因对这些属进行定量。双重检测是针对两种分子技术——qPCR 和液滴数字 PCR (ddPCR) 开发的。优化后，qPCR 和 ddPCR 检测均与标准品具有较高的线性度和定量相关性。两种技术的比较表明，qPCR具有更高的灵敏度、更宽的线性动态范围、更短的分析时间，并且更具成本效益，使其成为初步筛选的合适方法。然而，ddPCR 方法具有较低的变异性，并且能够处理双链检测中发现的 PCR 抑制和竞争效应，从而为水华样品提供更精确和准确的分析。
The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.