上海海洋大学学报2025，Vol.34Issue(5)：968-977,10.DOI:10.12024/jsou.20250804913
基于环境DNA多重PCR技术的大薸入侵监测引物开发与验证
Development and validation of primers for monitoring the invasion of water lettuce(Pistia stratiotes L.)using environmental DNA based multiplex PCR technology
摘要
Abstract
Pistia stratiotes L.(water lettuce),as a highly detrimental invasive plant species in China's freshwater ecosystems,has posed significant threats to aquatic ecological security through its rapid proliferation.To enable early monitoring and control of this species,this study developed a highly sensitive and specific detection system based on environmental DNA(eDNA)technology.By integrating k-mer indexing strategies with chloroplast genome alignment analysis,we designed four sets of species-specific multiplex PCR primers(PS_7,PS_8,PS_9,PS_10).Experimental validation demonstrated that:(1)All individual primers and their combinations effectively amplified target DNA from tissue samples,aquaculture water,and natural water samples;(2)The detection limits reached 0.69× 10-2 ng/μL for individual primers PS_7 and PS_10,0.34×10-1 ng/μL for PS_8,and 0.17 ng/μL for PS 9.The multiplex PCR system achieved a detection limit of 1.1 ×10-5 ng/μL,representing approximately a 625-fold improvement in sensitivity compared to the best single primer(P＜0.01);(3)Specificity tests confirmed no cross-reactivity with closely related species such as Spirodela polyrhiza.The entire detection process can be completed within 3 hours.This established detection system provides reliable technical support for early warning and monitoring of Pistia stratiotes invasions,while serving as a practical model for developing rapid eDNA-based detection methods for aquatic invasive plants.关键词
大薸/入侵物种/环境DNA/多重PCR/引物设计Key words
Pistia stratiotes/invasive species/environmental DNA(eDNA)/multiplex PCR/primer design分类
生物科学引用本文复制引用
陈雨萱,陆亮,方敏瑶,李晨虹..基于环境DNA多重PCR技术的大薸入侵监测引物开发与验证[J].上海海洋大学学报,2025,34(5):968-977,10.基金项目
国家重点研发计划(2022YFC2601301) （2022YFC2601301）
临港新片区海洋生物医药科技创新型平台(RWS-2025-004) （RWS-2025-004）
