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首页> 外文期刊>Marine biology >Real-time PCR assay for detection and relative quantification of Liocarcinus depurator larvae from plankton samples
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Real-time PCR assay for detection and relative quantification of Liocarcinus depurator larvae from plankton samples

机译：实时荧光定量PCR检测浮游生物样品中的拟生化石Li幼虫并进行相对定量

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摘要

Accurate species identification of decapod crustacean larvae is required to understand their population distributions, life cycle dynamics and interactions with their habitats. Analysis of plankton samples using morphological taxonomic methods and microscopy is time-consuming, requires highly skilled and trained operatives and may often be inaccurate. As complementary tools to classical identification methods, recent work has focused on the development of molecular approaches and shows their feasibility for species-specific identification. This study has developed real-time PCR assays utilising species-specific Taqman~(R) probes designed in the cytochrome oxidase I (COI) gene of Liocarcinus depurator, Necora puber, Carcinus maenas and Cancer pagurus. Our study then employed the probe and primers designed for L. depurator to obtain accurate identification and relative abundance estimates of L. depurator larvae in plankton samples collected between March 2005 and October 2006. Ranges of larval abundances were derived from a standard curve created from plankton samples spiked with a known number of larvae reared in the laboratory. Inhibition of the PCR reaction was shown to be an important factor and our results suggested that 0.1 ng of DNA as template provided accurate identification and avoided inhibition. Real-time PCR was shown to provide accurate species identification on unsorted plankton samples and could be suitable for the estimation of larval abundances in the plankton, although more work must be done to improve the accuracy of those estimations.
机译：需要准确鉴定十足纲甲壳类幼虫的种类，以了解其种群分布，生命周期动态以及与生境的相互作用。使用形态学分类学方法和显微镜对浮游生物样品进行分析非常耗时，需要熟练且训练有素的操作人员，并且可能经常不准确。作为经典识别方法的补充工具，最近的工作集中在分子方法的发展上，并显示了它们在物种特异性识别中的可行性。这项研究利用在Liocarcinus depurator，Necora puber，Carcinus maenas和Cancer pagurus的细胞色素氧化酶I（COI）基因中设计的物种特异性Taqman®探针，开发了实时PCR检测方法。然后，我们的研究使用了专为L. depurator设计的探针和引物，以获得2005年3月至2006年10月收集的浮游生物样品中L. depurator幼虫的准确鉴定和相对丰度估计。样品中掺入了已知数量的在实验室饲养的幼虫。已显示抑制PCR反应是一个重要因素，我们的结果表明，以0.1 ng DNA为模板可提供准确的鉴定并避免抑制。研究表明，实时PCR可以在未分类的浮游生物样品上提供准确的物种识别，并且可能适用于浮游生物中幼虫丰度的估计，尽管必须做更多的工作来提高这些估计的准确性。

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