Direct colony PCR for rapid identification of varied microalgae from freshwater environment
Direct colony PCR for rapid identification of varied microalgae from freshwater environment
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DOI：
10.1007/s10811-012-9895-0
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发表时间：
2013-04-01
影响因子：
3.3
通讯作者：
Ramya, Mohandass
中科院分区：
文献类型：
--
作者：
Radha, Sudhakar;Fathima, Anwar Aliya;Ramya, Mohandass
微藻物种的分子鉴定至关重要，涉及基因组中存在的特定标记的测序，这些标记是属所特有的。PCR是鉴定微藻的重要手段，传统方法制备PCR模板DNA需要大量藻细胞，耗时长。减少这些并发症的一种简单方法是直接使用微藻菌落从基因组扩增所需的DNA片段。在这项研究中，一个简单的细胞破碎的方法，使用高压灭菌的玻璃粉，已被用于微藻的直接菌落PCR。从淡水样品中选择了4种形态不同的微藻株，用3种不同的分子标记（rbcL、internal transcribed spacer 2和18 S rDNA）对PCR扩增反应进行了评价。使用较少数量的细胞（0.04 x 10（5））、最少量的玻璃粉（0.5 mg）和Milli-Q水（用于内部转录间隔区标记）优化PCR扩增。根据序列相似性，将分离到的菌株鉴定为Desmodesmus sp. JQ 782747、Coelastrum proboscideum JQ 898144、Chlorella sorokiniana JQ 898145和Scenedesmus sp. JQ 782746。这种直接的微藻菌落PCR被证明是一种简单、快速的检测各种微藻的方法。
Molecular identification of microalgal species is vital and involves sequencing of specific markers present in the genome, which are unique to a genus. PCR is a vital tool for identification of microalgae; the preparation of template DNA for PCR by traditional methods requires large amount of algal cells and a time-consuming process. One simple way to reduce these complications is to use the microalgal colonies directly for amplification of required DNA fragments from the genome. In this study, a simple cell-disrupting method, using autoclaved glass powder, has been used for direct colony PCR of microalgae. Four morphologically different microalgal strains were chosen from freshwater samples, and the PCR amplification reaction was evaluated with three different molecular markers (rbcL, internal transcribed spacer 2, and 18S rDNA). PCR amplification was optimized with less number of cells (0.04 x 10(5)), minimal quantity of glass powder (0.5 mg), and in the presence of Milli-Q water for internal transcribed spacer marker. The isolated strains were identified as Desmodesmus sp. JQ782747, Coelastrum proboscideum JQ898144, Chlorella sorokiniana JQ898145, and Scenedesmus sp. JQ782746 based on sequence similarity. This direct microalgal colony PCR proves to be a simple and rapid method for detection of varied microalgal species.