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水样环境DNA分析包括水样采集、DNA提取和分析等流程,已成为监测濒危水生生物种群分布调查的重要手段.为减少在监测目标物种尤其濒危物种中的不确定性,对水环境DNA分析流程的优化至关重要.本研究以川陕哲罗鲑为目标物种,采用滤膜法采集养殖池中的水样,设计了250 mL、500 mL、1 L和2 L等4种水样采集量,分别采用PoweWater DNA Isolation kit和DNeasy Tissue and Blood DNA extraction kit提取水样环境DNA(e DNA),使用物种mtDNAD_loop区特异性引物进行PCR扩增,通过研究滤膜法、水样采集量和水样DNA提取方法对水样e DNA中目标基因检出率的影响,探索适宜的e DNA分析操作方案.结果表明:使用DNeasy Tissue and Blood DNA extraction kit提取的水样DNA中目的基因的检出率为100%,效果明显优于PoweWater DNA Isolation kit(目标基因的检出率为0);目标基因扩增条带的亮度随水样采样量的增加而增加,其中2 L水样目标基因的扩增效果较理想;序列比对结果显示,本试验从水样DNA中成功扩增得到了川陕哲罗鲑mtD NA Dloop区部分序列.表明DNA提取方法和水样采集量对目标物种的检出率有显著的影响,滤膜法、2 L水样采集量、DNeasy Tissue and Blood DNA extraction kit更适宜进行水样的DNA分析,mtD NA D-loop区可作为川陕哲罗鲑识别的特异性分子标记.
Water sample DNA analysis including water sampling, DNA extraction and analysis has become an important means of monitoring the distribution of endangered aquatic species. To reduce the uncertainty in monitoring target species, especially endangered species, DNA analysis of the aquatic environment The optimization of the process is very important.In this study, the Salmonella chuanmii was taken as the target species, and the water samples in the aquaculture pond were collected by the membrane filtration method. Four water samples of 250 mL, 500 mL, 1 L and 2 L were collected (EDNA) were extracted by using PoweWater DNA Isolation Kit and DNeasy Tissue and Blood DNA extraction kit respectively. The mtDNAD_loop region-specific primers were used for PCR amplification. The effects of membrane filtration, water sample collection and water Like DNA extraction method on the detection rate of target gene in water sample e DNA, and explore the appropriate eDNA analysis protocol.The results showed that the detection rate of the target gene in water sample DNA extracted by DNeasy Tissue and Blood DNA extraction kit Was 100%, the effect was significantly better than the PoweWater DNA Isolation kit (target gene detection rate of 0); target gene amplification bands with the sample size increased water samples increased, of which 2 L water samples The results of sequence alignment showed that the sequence of mDCDNADloop region was successfully amplified from the water sample DNA in this experiment.The results showed that DNA extraction method and the amount of water samples collected on the target species , The DNeasy Tissue and Blood DNA extraction kit was more suitable for the DNA analysis of water samples. The mtD NA D-loop region could be used as the identification marker Of specific molecular markers.