Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA
Detecting an elusive invasive species: a diagnostic PCR to detect Burmese python in Florida waters and an assessment of persistence of environmental DNA
复制标题
DOI：
10.1111/1755-0998.12180
复制
发表时间：
2014-03-01
影响因子：
7.7
通讯作者：
Avery, Michael L.
中科院分区：
文献类型：
--
作者：
Piaggio, Antoinette J.;Engeman, Richard M.;Avery, Michael L.
最近的研究表明，从水体中的水生脊椎动物中检测环境DNA（eDNA）是可能的。缅甸蟒蛇（Python bivittatus）是佛罗里达的一种半水生入侵物种，其难以捉摸的性质和神秘的颜色使其难以被发现。我们的目标是开发一种诊断PCR方法来检测水生生物中的双条拟步行虫，这可以帮助管理人员监测这一入侵物种。首先，我们使用捕获的P.bivittatus来确定是否可以从水样中分离和扩增爬行动物DNA。我们还评估了两种DNA提取方法和两种DNA提取试剂盒在eDNA制备中常用的效果。结果表明，用醋酸钠沉淀法和QIAamp DNA微试剂盒分离的所有水样中均检测到双纹裸鲤线粒体细胞色素B基因片段。接下来，我们设计了双纹裸腹鱼特异性引物，并评估了eDNA在水中的降解率。我们的引物没有扩增出近缘物种的DNA，我们发现双带拟青霉DNA在96 h内一直可检测到。最后，我们从南佛罗里达的六个现场取样。来自五个地点的样本，其中P.bivittatus已被观察到，检测呈阳性的eDNA。最后的网站是阴性的，没有事先记录的证据P.bivittatus。本研究表明，双带拟步行虫的cDNA可以从水样中提取，因此，这种方法是一种新的和有前途的技术，用于管理入侵爬行动物。
Recent studies have demonstrated that detection of environmental DNA (eDNA) from aquatic vertebrates in water bodies is possible. The Burmese python, Python bivittatus, is a semi-aquatic, invasive species in Florida where its elusive nature and cryptic coloration make its detection difficult. Our goal was to develop a diagnostic PCR to detect P.bivittatus from water-borne eDNA, which could assist managers in monitoring this invasive species. First, we used captive P.bivittatus to determine whether reptilian DNA could be isolated and amplified from water samples. We also evaluated the efficacy of two DNA isolation methods and two DNA extraction kits commonly used in eDNA preparation. A fragment of the mitochondrial cytochrome b gene from P.bivittatus was detected in all water samples isolated with the sodium acetate precipitate and the QIAamp DNA Micro Kit. Next, we designed P.bivittatus-specific primers and assessed the degradation rate of eDNA in water. Our primers did not amplify DNA from closely related species, and we found that P.bivittatus DNA was consistently detectable up to 96h. Finally, we sampled water from six field sites in south Florida. Samples from five sites, where P.bivittatus has been observed, tested positive for eDNA. The final site was negative and had no prior documented evidence of P.bivittatus. This study shows P.bivittatus eDNA can be isolated from water samples; thus, this method is a new and promising technique for the management of invasive reptiles.