Gaget V, Keulen A, Lau M, Monis P, Brookes J D
Ecology and Environmental Sciences, School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia.
Australian Water Quality Centre, South Australia Water Corporation, Adelaide, SA, Australia.
J Appl Microbiol. 2017 Jan;122(1):294-304. doi: 10.1111/jam.13332. Epub 2016 Dec 6.
Benthic Cyanobacteria produce toxic and odorous compounds similar to their planktonic counterparts, challenging the quality of drinking water supplies. The biofilm that benthic algae and other micro-organisms produce is a complex and protective matrix. Monitoring to determine the abundance and identification of Cyanobacteria, therefore, relies on molecular techniques, with the choice of DNA isolation technique critical. This study investigated which DNA extraction method is optimal for DNA recovery in order to guarantee the best DNA yield for PCR-based analysis of benthic Cyanobacteria.
The conventional phenol-chloroform extraction method was compared with five commercial kits, with the addition of chemical and physical cell-lysis steps also trialled. The efficacy of the various methods was evaluated by measuring the quantity and quality of DNA by UV spectrophotometry and by quantitative PCR (qPCR) using Cyanobacteria-specific primers. The yield and quality of DNA retrieved with the commercial kits was significantly higher than that of DNA obtained with the phenol-chloroform protocol.
Kits including a physical cell-lysis step, such as the MO BIO Power Soil and Biofilm kits, were the most efficient for DNA isolation from benthic Cyanobacteria.
These commercial kits allow greater recovery and the elimination of dangerous chemicals for DNA extraction, making them the method of choice for the isolation of DNA from benthic mats. They also facilitate the extraction of DNA from benthic Cyanobacteria, which can help to improve the characterization of Cyanobacteria in environmental studies using qPCRs or population composition analysis using next-generation sequencing.
底栖蓝藻产生的有毒和有气味的化合物与其浮游同类相似，这对饮用水供应质量构成挑战。底栖藻类和其他微生物产生的生物膜是一种复杂的保护基质。因此，监测以确定蓝藻的丰度和鉴定依赖于分子技术，而DNA分离技术的选择至关重要。本研究调查了哪种DNA提取方法最适合DNA回收，以确保基于PCR的底栖蓝藻分析获得最佳DNA产量。
将传统的酚 - 氯仿提取方法与五种商业试剂盒进行比较，还尝试了添加化学和物理细胞裂解步骤。通过紫外分光光度法测量DNA的数量和质量，并使用蓝藻特异性引物通过定量PCR（qPCR）评估各种方法的效果。商业试剂盒回收的DNA产量和质量明显高于酚 - 氯仿方法获得的DNA。
包括物理细胞裂解步骤的试剂盒，如MO BIO Power Soil和Biofilm试剂盒，是从底栖蓝藻中分离DNA最有效的方法。
这些商业试剂盒可实现更高的回收率，并消除DNA提取中的危险化学品，使其成为从底栖垫中分离DNA的首选方法。它们还便于从底栖蓝藻中提取DNA，这有助于在使用qPCR的环境研究中改善蓝藻的表征，或在使用下一代测序的种群组成分析中发挥作用。
