环境DNA方法对进口亲虾携带水中宿主物种的鉴定

Use of Enviromental DNA Method to Identify Host Species in Water Carried by Imported Parent Shrimp

为深入了解进口亲虾携带水中物种情况,本试验设计凡纳滨对虾物种特异性引物,采用环境DNA(eDNA)方法对境外进口亲虾携带水进行凡纳滨对虾物种鉴定,分析不同过滤膜、不同样本体积对于获取eDNA浓度的影响;对比不同来源水样、不同过滤膜和样品取样时间等因素对水样中凡纳滨对虾物种鉴定的影响;用宏基因组测序方法对水过滤膜eDNA进行测序分析。结果显示,本试验所设计的凡纳滨对虾物种特异性引物特异性好、灵敏度高,可以有效地从虾体和水中进行凡纳滨对虾物种鉴定;硝酸纤维素膜、玻璃纤维膜均有不同的过滤优势,水样品体积增加,得到的eDNA浓度也随之增加;虾体生存时间、样品取样时间对于宿主物种鉴定均会产生重要影响;宏基因组测序所得的对虾属序列中,凡纳滨对虾的物种序列占40%,物种丰度占比最高。结果表明,eDNA检测方法具有较好的灵敏性和特异性,可以作为监测水生动物携带水外来物种入侵的一个有效手段。

In order to deeply analyze the host species in the water carried by imported parent shrimp,this study utilized Penaeus vannamei(syn:Litopenaeus vannamei)species specific primers and the environmental DNA(eDNA)method to identify P.vannamei in the water carried by imported parent shrimp.The influence of different filter film and sample volume on the concentration of eDNA was examined;the effects of water samples from different sources,different filtration membranes and sampling time on the identification of P.vannamei were compared.The metagenome of water filtration membrane eDNA was sequenced and analyzed.The results showed that the designed P.vannamei species specific primers have good specificity and high sensitivity,and effectively identified P.vannamei from shrimp body and water;nitrocellulose membrane and glass fiber membrane had different filtration advantages,and the concentration of eDNA increased with the increase of sample volume;the survival time of shrimp and the sampling time of samples had obvious effects on the identification of host species.Among the sequences of Penaeus obtained by metagenome sequencing,the species sequences of P.vannamei accounted for 40%,representing the highest proportion of all sequenced sequences.Thus,the eDNA method provides a sensitive and effective technique for monitoring aquatic animals carried in water.