基于数字PCR的红耳龟环境DNA动态监测及其影响因素研究

The red-eared slider is listed in the top 100 of the world’s worst invasive species，which seriously threatens the ecological security of the invaded areas and has been spreading in thenatural habitats in China. Early monitoring of invasive alien species is key to effective prevention and control, and environmental DNA (eDNA) technology is a powerful tool for early surveillance. At present, real-time PCR method which has insufficient detection sensitivity and is easily interfered by PCR inhibitors, have been used for quantitative detection of eDNA from invasive species. The concentration of eDNA in the environment is dynamic change, and the biological and abiotic factors may affect eDNA shedding and decay rates, thereby affecting the accuracy of eDNA detection. In this study, the dynamic changes of the eDNA concentration of red-eared slider under different population densities and different environmental conditions were quantitatively monitored by using the emerging digital PCR in laboratory microcosm experiments. The effects of population density and environmental factors on the red-eared slider eDNA shedding and decay rates were analyzed. In the field monitoring experiments, the relationship between population density and eDNA concentration of red-eared slider and the influence of environmental factors on it were analyzed.This study will provide technical support for the effective application of eDNA technology in the monitoring of red-eared slider populations.

红耳龟是全球100个最具威胁性的入侵物种之一，严重威胁入侵地区的生态安全，已在我国野外呈不断蔓延态势。外来入侵物种的早期监测是对其进行有效防控的关键，环境DNA（eDNA）技术是早期监测的有力工具。目前，对入侵物种eDNA的定量分析通常采用荧光定量PCR技术，其检测灵敏度不够而且容易受PCR抑制剂干扰。环境介质中eDNA浓度是动态变化的，生物和非生物因素可能会影响其产生和降解速率，进而影响eDNA检测的准确性。本项目以红耳龟为研究对象，采用新兴的数字PCR技术，通过室内微宇宙实验，对不同种群密度和不同环境条件下红耳龟eDNA浓度的动态变化进行定量监测，掌握其动态变化规律，分析种群密度和环境因子对红耳龟eDNA产生和降解速率的影响，并结合野外种群监测实验，分析种群密度与红耳龟eDNA浓度的关联性，以及环境因素对其产生的影响。本研究将为eDNA技术在红耳龟野外种群监测中的有效应用提供技术支撑。

外来物种入侵是造成生物多样性丧失的最重要因素之一，早期监测是外来入侵物种有效防控的关键。基于环境DNA(environmental DNA, eDNA)的调查方法以其快速、灵敏、高效等独特优势，为外来入侵物种的调查监测提供了新的工具。红耳龟（Trachemys scripta elegants）是全球100个最具威胁性的入侵物种之一，严重威胁入侵地区的生态安全。本项目以红耳龟为研究对象，1）设计、筛选并验证红耳龟特异性检测引物和探针，建立微滴式数字PCR（ddPCR）定量检测限，ddPCR对红耳龟的定量限为1.47±0.23copies/μL；2）通过室内微宇宙实验解析不同种群密度和盐度条件下红耳龟环境DNA（eDNA）脱落和降解的速率，结果显示 eDNA产生速率与种群密度呈正相关，通过指数衰减曲线拟合不同种群密度下红耳龟环境DNA的降解系数为0.012～0.016/h。盐度对红耳龟eDNA降解速率有显著影响，高盐度下eDNA降解更慢；3）通过半自然条件下的控制实验研究红耳龟eDNA在不同时间和空间尺度上散布、脱落和降解的动态变化规律，结果显示红耳龟eDNA在水平方向上的扩散距离有限，主要集中于6m范围内，eDNA检出率和浓度随距离增加急剧下降，eDNA在底泥中的持留时间相对水样中更长。研究成果为红耳龟及其他龟类野外种群的调查监测提供了参考依据和技术支撑。