Exploring Rapid and Efficient Protocol for Isolation of Fungal DNA
Exploring Rapid and Efficient Protocol for Isolation of Fungal DNA
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探索快速有效的真菌 DNA 分离方案
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发表时间：
2017
期刊：
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通讯作者：
K. Raj
中科院分区：
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作者：
S. Tripathy;M. Maharana;D. Ithape;D. Lenka;D. Mishra;A. Prusti;D. Swain;M. R. Mohanty;K. Raj
快速、可靠地检测病原微生物是诊断食源性疾病的基本要求。通过 PCR 检测临床和植物样品中的真菌病原体需要有效的 DNA 提取方法。有多种提取方案可用于分离 DNA。似乎没有一种提取方法对于所有生物体都是最佳的（Bolano 等人，2001）。根据所使用的菌株，真菌可能会在培养基上形成菌丝生长（丝状）或孢子。在分离之前将真菌冷冻干燥或冻干。刚性细胞壁中的真菌核酸酶和高多糖含量（甘露聚糖、β-葡聚糖和几丁质）（Chaffin 等，1998）增加了从丝状真菌中分离 DNA 的难度（Muller 等，1998；Fredricks 等，2005）。因此，需要额外的裂解步骤，例如机械破坏或超声处理、酶消化或使用有毒化学品（Alaey 等人，2005）。 CTAB提取法最初是由Doyle和Doyle于1987年开发的用于植物组织提取的方法。它被认为在去除碳水化合物方面效果良好，但它既耗时又费力，并且不能从包括真菌菌丝体和孢子在内的所有生物样品中获得令人满意的 DNA 产量。现在有多种改进的 CTAB 方法（Saghai-Maroof 等，1984）可用于使用有机溶剂提取和纯化真菌 DNA（Blin 和 Stafford，1976）。此外，几本DNA International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 3 (2017) pp. 951-960 期刊主页：http://www.ijcmas.com
Rapid and reliable detection of pathogenic organisms is a basic requirement for diagnosis of food-borne illness and diseases. The detection of fungal pathogens in clinical and plant samples by PCR requires an efficient method of DNA extraction. A variety of extraction protocol is available for isolation of DNA. No single extraction method seems to be optimal for all organisms (Bolano et al., 2001). Fungus may form mycellial growth (filamentous) or spores on the media depending upon the strain used. The fungus is freeze dried or lyophilized before isolation. Fungal nucleases and high polysaccharide content (mannan, β-glucans and chitin) in rigid cell wall (Chaffin et al., 1998) add to the difficulties in isolating DNA from filamentous fungi (Muller et al., 1998; Fredricks et al., 2005). Therefore, additional lysis steps, such as mechanical disruption or sonication, enzymatic digestion or use of toxic chemicals are required (Alaey et al., 2005). The CTAB extraction method was originally developed by Doyle and Doyle in 1987 for plant tissue extraction. It was considered superior for removing carbohydrates but it is timeconsuming and labor-intensive and does not result DNA yields satisfactorily from all biological samples including fungal mycelia and spores. A variety of modified CTAB methods are now available (Saghai-Maroof et al., 1984) for extraction and purification of fungal DNA using organic solvents (Blin and Stafford, 1976). Besides, several DNA International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 3 (2017) pp. 951-960 Journal homepage: http://www.ijcmas.com