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

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、...

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

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.