基于环境DNA的岩原鲤检测及生物量评估  

Detection and biomass assessment of Procypris rabaudi based on environmental DNA

作　　者：闫卉果 董智玲 马婷婷 张连博 王晓艳 叶华[1] 姚维志[1] 何文平[1] YAN Huiguo;DONG Zhiling;MA Tingting;ZHANG Lianbo;WANG Xiaoyan;YE Hua;YAO Weizhi;HE Wenping(Key Laboratory of Freshwater Fish Reproduction and Development,Ministry of Education,College of Fisheries,Southwest University,Chongqing 400715,China;National Engineering Research Center of Marine Facilities Aquaculture,Zhejiang Ocean University,Zhoushan 316022,China)

机构地区：[1]西南大学水产学院,淡水鱼类资源与生殖发育教育部重点实验室,重庆400715 [2]浙江海洋大学,国家海洋设施养殖工程技术研究中心,浙江舟山316022

出　　处：《水产学报》2022年第6期1018-1026,共9页Journal of Fisheries of China

基　　金：国家自然科学基金(32071651);重庆市自然科学基金(cstc2020jcyj-msxmX0438);重庆市社会民生科技创新专项(cstc2016shmszx80087);中央高校基本科研业务费专项(XDJK2019C025)。

摘　　要：为建立岩原鲤基于环境DNA(eDNA)的实时荧光定量PCR(qPCR)检测方法,准确鉴别岩原鲤并探讨eDNA浓度与其生物量的定量关系,实验根据岩原鲤mtDNA中12S rRNA基因序列设计eDNA引物和TaqMan探针,利用PCR扩增出岩原鲤12S rRNA基因的序列,克隆入pMD19-T载体,构建重组质粒作为qPCR标准;使用梯度稀释质粒标准品作为模板,进行qPCR扩增,制作标准曲线,建立岩原鲤qPCR检测方法,并评价其特异性、灵敏性和应用效果。结果显示,引物和TaqMan探针对供试的岩原鲤样品出现荧光增长曲线,显示阳性扩增,而其他鱼类和空白对照均未得到扩增信号,表现为阴性;qPCR的阈值循环数(C_(t))与标准品拷贝数的线性关系好,且线性范围广,获得的标准曲线相关系数(R^(2))达到0.999,检测限位DNA浓度为5×10^(−6)ng/μL,扩增效率为94.7%;检测养殖不同数量岩原鲤的水体中eDNA浓度,目标DNA浓度和岩原鲤数量存在线性正相关性(R^(2)=0.957),得到岩原鲤DNA浓度与其个体数量的相关性曲线:y=131546x+77623;去除岩原鲤后,eDNA的拷贝数与时间负相关,其在水体环境中的存留时间为17 d。研究表明,该岩原鲤eDNA引物和TaqMan探针既能应用于水体中岩原鲤的定性检测,特异性高、时效性好;又能定量检测出环境中岩原鲤的密度,有利于反映岩原鲤在不同采样点的生物量及时空动态,从而为评价岩原鲤人工放流效果和管理保护资源提供参考。Species distribution and biomass are the basis for evaluating population dynamics and community structure in an ecosystem.Unfortunately,it is frequently full of challenges to capture the distribution of the rare species through traditional methods.Procypris rabaudi is a unique economic species in the upper reaches of the Yangtze River,the number of which has declined dramatically in recent years.Environmental DNA technology is a sensitive,low-cost and non-invasive new technology for species monitoring.It has great potential application in detecting endangered and invasive species.In order to establish a real-time quantitative PCR(qPCR)method for the detection of P.rabaudi and distinguish it from other fishes in the Yangtze River,this study designed eDNA primers and a TaqMan probe based on 12S rRNA gene sequence in mtDNA.The sequence of the 12S rRNA gene was amplified by PCR and cloned into the pMD19-T vector to construct the standard plasmid,and a qPCR method was developed for detection of P.rabaudi using serially diluted standard plasmid as templates.Subsequently the sensitivity,specificity and application effects of the method were evaluated.The results showed that the cycle threshold value(C_(t))of qPCR assay had a great linear relationship with the copy number of the standard plasmid.Amplification specificity analysis indicated that the method could specifically detect P.rabaudi.Then,eDNA was detected in the water samples in aquarium tanks with different numbers of P.rabaudi,The target DNA concentration and the number of P.rabaudi had a positive correlation of R^(2)was 0.957,and the correlation curve between DNA concentration and the individual number was obtained:y=131546x+77623.In addition,after the removal of P.rabaudi,the copies of eDNA were negatively correlated with time,and its retention time in the water environment was about 17 days.In this study,we found that the DNA primer and Taqman probe could be applied to the qualitative detection of P.rabaudi in water with high specificity,as well as estimate

关 键 词：岩原鲤 环境DNA 引物 探针 生物量 

分 类 号：S931[农业科学—渔业资源]