Southern California Coastal Water Research Project, Costa Mesa, CA, USA.
PLoS One. 2013;8(1):e51273. doi: 10.1371/journal.pone.0051273. Epub 2013 Jan 4.
Molecular methods, such as DNA barcoding, have the potential to enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biomonitoring using benthic macroinvertebrates. Using higher volumes or concentrations of ethanol, requirements for shorter holding times, or the need to include additional filtering may increase cost and logistical constraints to existing biomonitoring programs. To address this issue we evaluated the efficacy of various ethanol-based sample preservation methods at maintaining DNA integrity. We evaluated a series of methods that were minimally modified from typical field protocols in order to identify an approach that can be readily incorporated into existing monitoring programs. Benthic macroinvertebrates were collected from a minimally disturbed stream in southern California, USA and subjected to one of six preservation treatments. Ten individuals from five taxa were selected from each treatment and processed to produce DNA barcodes from the mitochondrial gene cytochrome c oxidase I (COI). On average, we obtained successful COI sequences (i.e. either full or partial barcodes) for between 93-99% of all specimens across all six treatments. As long as samples were initially preserved in 95% ethanol, successful sequencing of COI barcodes was not affected by a low dilution ratio of 2∶1, transfer to 70% ethanol, presence of abundant organic matter, or holding times of up to six months. Barcoding success varied by taxa, with Leptohyphidae (Ephemeroptera) producing the lowest barcode success rate, most likely due to poor PCR primer efficiency. Differential barcoding success rates have the potential to introduce spurious results. However, routine preservation methods can largely be used without adverse effects on DNA integrity.
分子方法，如 DNA 条形码技术，具有增强全球生物监测计划的潜力。改变常规使用的样本保存方法以保护 DNA 免受降解，可能会对 DNA 条形码和宏基因组学在使用底栖大型无脊椎动物进行生物监测中的应用构成潜在障碍。使用更高的乙醇体积或浓度、缩短保存时间的要求，或者需要包括额外的过滤，可能会增加成本和对现有生物监测计划的后勤限制。为了解决这个问题，我们评估了各种基于乙醇的样本保存方法在保持 DNA 完整性方面的效果。我们评估了一系列从典型现场方案中最小程度修改的方法，以确定一种可以很容易地纳入现有监测方案的方法。从美国加利福尼亚州南部一条受干扰最小的溪流中采集底栖大型无脊椎动物，并对其进行六种保存处理之一。从每种处理中选择五个分类单元的十个个体，并进行处理以从线粒体基因细胞色素 c 氧化酶 I (COI) 生成 DNA 条形码。平均而言，我们在六种处理中的所有标本中获得了成功的 COI 序列（即完整或部分条形码）的比例在 93-99%之间。只要样品最初保存在 95%乙醇中，2∶1 的低稀释比、转移到 70%乙醇、丰富的有机物存在或长达六个月的保存时间都不会影响 COI 条形码的成功测序。条形码成功率因分类单元而异，Leptohyphidae（蜉蝣目）的条形码成功率最低，这很可能是由于 PCR 引物效率差所致。不同的条形码成功率有可能引入虚假结果。然而，常规保存方法可以在很大程度上使用，而不会对 DNA 完整性产生不利影响。
