Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)
Quantification of copepod gut content by differential length amplification quantitative PCR (dla-qPCR)
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DOI：
10.1007/s00227-008-1079-8
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发表时间：
2009-02
期刊：
影响因子：
2.4
通讯作者：
C. Troedsson;Paolo Simonelli;Verena Naegele;J. Nejstgaard;M. Frischer
中科院分区：
文献类型：
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作者：
C. Troedsson;Paolo Simonelli;Verena Naegele;J. Nejstgaard;M. Frischer
浮游动物的摄食率和选择性的量化对于理解海洋生态系统的构造机制至关重要。然而，方法学的局限性使得许多这些研究变得困难。最近，基于分子的方法已经证明，来自猎物物种的 DNA 可用于识别浮游动物的肠道内容物，此外，通过针对 18S rRNA 基因的定量 PCR (qPCR) 测定进行的定量肠道内容物估计已用于估计阑尾动物和桡足类动物的摄食率。然而，虽然基于标准单引物的 qPCR 测定对滤食性阑尾动物 Oikopleura dioica 进行了定量，但桡足类 Calanus finmarchicus 的摄食率始终被低估。在这项研究中，我们测试了猎物 DNA 在被桡足类摄入后迅速消化的假设，并描述了一种基于 qPCR 的检测方法，即差异长度扩增 qPCR (dla-qPCR)，以解释 DNA 消化。该测定利用多个引物组来扩增猎物 18S rRNA 基因的不同大小片段，并根据这些片段的差异扩增来估计和校正消化程度。将此方法应用到C.与标准 qPCR 相比，finmarchicusfedRhodomonas marina 显着改善了定量饲喂估计。 dla-qPCR 的发展代表了在不涉及基于培养的操作的情况下评估原位桡足类摄食率的定量方法的重大进步。
Quantification of feeding rates and selectivity of zooplankton is vital for understanding the mechanisms structuring marine ecosystems. However, methodological limitations have made many of these studies difficult. Recently, molecular based methods have demonstrated that DNA from prey species can be used to identify zooplankton gut contents, and further, quantitative gut content estimates by quantitative PCR (qPCR) assays targeted to the 18S rRNA gene have been used to estimate feeding rates in appendicularians and copepods. However, while standard single primer based qPCR assays were quantitative for the filter feeding appendicularianOikopleura dioica, feeding rates were consistently underestimated in the copepodCalanus finmarchicus. In this study, we test the hypothesis that prey DNA is rapidly digested after ingestion by copepods and describe a qPCR-based assay, differential length amplification qPCR (dla-qPCR), to account for DNA digestion. The assay utilizes multiple primer sets that amplify different sized fragments of the prey 18S rRNA gene and, based on the differential amplification of these fragments, the degree of digestion is estimated and corrected for. Application of this approach toC. finmarchicusfedRhodomonas marinasignificantly improved quantitative feeding estimates compared to standard qPCR. The development of dla-qPCR represents a significant advancement towards a quantitative method for assessing in situ copepod feeding rates without involving cultivation-based manipulation.