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比较液滴数字聚合酶链反应(PCR)、定量 PCR 和宏条形码在环境 DNA 中的物种特异性检测。

A comparison of droplet digital polymerase chain reaction (PCR), quantitative PCR and metabarcoding for species-specific detection in environmental DNA.

机构信息

Coastal and Freshwater Group, Cawthron Institute, Nelson, New Zealand.

Institute of Marine Science, University of Auckland, Auckland, New Zealand.

出版信息

Mol Ecol Resour. 2019 Nov;19(6):1407-1419. doi: 10.1111/1755-0998.13055. Epub 2019 Aug 7.

Abstract

Targeted species-specific and community-wide molecular diagnostics tools are being used with increasing frequency to detect invasive or rare species. Few studies have compared the sensitivity and specificity of these approaches. In the present study environmental DNA from 90 filtered seawater and 120 biofouling samples was analyzed with quantitative PCR (qPCR), droplet digital PCR (ddPCR) and metabarcoding targeting the cytochrome c oxidase I (COI) and 18S rRNA genes for the Mediterranean fanworm Sabella spallanzanii. The qPCR analyses detected S. spallanzanii in 53% of water and 85% of biofouling samples. Using ddPCR S. spallanzanii was detected in 61% of water of water and 95% of biofouling samples. There were strong relationships between COI copy numbers determined via qPCR and ddPCR (water R  = 0.81, p < .001, biofouling R  = 0.68, p < .001); however, qPCR copy numbers were on average 125-fold lower than those measured using ddPCR. Using metabarcoding there was higher detection in water samples when targeting the COI (40%) compared to 18S rRNA (5.4%). The difference was less pronounced in biofouling samples (25% COI, 29% 18S rRNA). Occupancy modelling showed that although the occupancy estimate was higher for biofouling samples (ψ = 1.0), higher probabilities of detection were derived for water samples. Detection probabilities of ddPCR (1.0) and qPCR (0.93) were nearly double metabarcoding (0.57 to 0.27 marker dependent). Studies that aim to detect specific invasive or rare species in environmental samples should consider using targeted approaches until a detailed understanding of how community and matrix complexity, and primer biases affect metabarcoding data.

摘要

目前,针对特定物种和群落范围的分子诊断工具正越来越频繁地用于检测入侵或稀有物种。然而,很少有研究比较过这些方法的敏感性和特异性。本研究采用定量 PCR(qPCR)、数字液滴 PCR(ddPCR)和靶向细胞色素 c 氧化酶 I(COI)和 18S rRNA 基因的宏条形码技术,对 90 个过滤海水和 120 个生物污损样本中的环境 DNA 进行了分析,以检测地中海扇虫 Sabella spallanzanii。qPCR 分析检测到 53%的水样和 85%的生物污损样本中存在 S. spallanzanii。使用 ddPCR 检测到 61%的水样和 95%的生物污损样本中存在 S. spallanzanii。qPCR 和 ddPCR 检测到的 COI 拷贝数之间存在很强的相关性(水 R  = 0.81,p  < 0.001,生物污损 R  = 0.68,p  < 0.001);然而,qPCR 的拷贝数平均比 ddPCR 低 125 倍。使用宏条形码技术,靶向 COI 时水中的检测率较高(40%),而靶向 18S rRNA 时的检测率较低(5.4%)。在生物污损样本中,这种差异不太明显(COI 为 25%,18S rRNA 为 29%)。占有率模型表明,尽管生物污损样本的占有率估计值较高(ψ  = 1.0),但水样的检测概率更高。ddPCR(1.0)和 qPCR(0.93)的检测概率几乎是宏条形码技术(0.57 至 0.27 与标记物相关)的两倍。在环境样本中检测特定入侵或稀有物种的研究,应考虑使用靶向方法,直到详细了解群落和基质复杂性以及引物偏倚如何影响宏条形码数据。

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