基于微滴式数字PCR方法的鱼类环境DNA样本处理与保存技术优化

OPTIMIZATION OF FISH ENVIRONMENTAL DNA SAMPLE PROCESSING AND PRESERVATION TECHNOLOGY BASED ON DROPLET DIGITAL PCR

以实验室内的鲫(Carassius auratus)为研究对象,利用微滴式数字PCR(Droplet Digital PCR,ddPCR)定量技术,优化了鱼类环境DNA(Environmental DNA,eDNA)样本的捕获、提取和保存方法,并对免DNA提取的PCR直扩技术进行了探索。研究结果如下:(1)在同一孔径、不同材质的6种滤膜中,捕获总量最高的是混合纤维素膜,最低的是聚碳酸酯膜,前者获得的ddPCR产物浓度可达后者的17倍;(2)提取DNA总量最高的是Qiagen DNeasy PowerWater kit,最低的是高盐法,前者获得的ddPCR产物浓度是后者的5.5倍;(3)不同滤膜和提取方法对最终的ddPCR产物总量有显著的交互作用(P<0.001);混合纤维素膜过滤和Qiagen DNeasy PowerWater kit提取的组合效果显著优于其他组合;(4)滤膜在-20℃冷冻保存和利用Longmire’s buffer保存液保存的效果最好;(5)免DNA提取PCR直扩实验结果表明,高保真酶可以直接对水样进行扩增。研究对eDNA操作流程中的关键步骤进行了详细比较,确定了鱼类eDNA样本处理与保存的最优方案,为建立eDNA标准化实验流程提供了参考依据。

In this study,the methods of fish environmental DNA(eDNA)capture,extraction and preservation were optimized by Droplet Digital PCR(ddPCR)quantitative technology using Carassius auratus in the laboratory,and the direct PCR technology without DNA extraction was preliminarily explored.The results showed that the mixed cellulose ester membrane produced the most ddPCR product,among the six kinds of filter membrane,and the polycarbonate membrane produced the lowest ddPCR product with only 1/17 of mixed cellulose ester membrane.The method which extracted the highest amount of DNA was the Qiagen DNeasy PowerWater kit,and the lowest was the high-salt method.The Qiagen DNeasy PowerWater kit yielded better results compared to the high-salt method,showing a 5.5-fold improvement in DNA yield.Different filter membranes and extraction methods have significant interactions on the total amount of final ddPCR product(P<0.001),filtration through the mixed cellulose ester membranes and Qiagen DNeasy PowerWater kit significantly outperformed other combinations of capture and extraction methods.Filter membranes stored at-20℃or stored in Longmire's buffer solution recovered the most ddPCR product.The results of direct PCR amplification without DNA extraction showed that the high-fidelity enzyme of Vazyme can directly amplify the water samples and obtain the target DNA product.This study,compared the key steps in the eDNA operation process in detail,and the optimal protocol of fish eDNA sample processing and preservation was determined,which provided a reference for the establishment of eDNA standardized experimental process.