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一种改进的DNA提取方法,适用于基于PCR从黄麻和其他黏液植物中检测双生病毒。

An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants.

作者信息

Ghosh Raju, Paul Sujay, Ghosh Subrata Kumar, Roy Anirban

机构信息

Plant Virus Laboratory and Biotechnology Unit, Division of Crop Protection, Central Research Institute for Jute and Allied Fibres, Barrackpore, Kolkata 700120, West Bengal, India.

出版信息

J Virol Methods. 2009 Jul;159(1):34-9. doi: 10.1016/j.jviromet.2009.02.020. Epub 2009 Mar 3.

Abstract

A relatively quick and inexpensive modified cetyl trimethylammonium bromide method for extraction of DNA from leaf materials containing large quantities of mucilage is described. The modification including use of more volume of extraction buffer and dissolving crude nucleic acid pellet in 1 M NaCl, reduced markedly the viscosity of the mucilage and thus in the final purification step yielded a larger quantity of mucilage-free DNA suitable for subsequent PCR-based detection of begomoviruses. The method was standardized with jute samples with yellow mosaic disease and validated with different other mucilaginous-hosts with low titre of begomoviruses. DNA isolated using this method showed consistency in yield and compatibility with PCR for detection of begomoviruses from different mucilaginous plant species. The method was compared for efficacy with other reported methods and it was found to be superior over the existing methods described for isolation of DNA from mucilaginous hosts. Thus the method described could be used on a wider scale for reliable and consistent detection of begomoviruses from mucilaginous hosts for characterization and variability study.

摘要

本文描述了一种相对快速且廉价的改良十六烷基三甲基溴化铵法,用于从含有大量黏液的叶片材料中提取DNA。该改良方法包括使用更多体积的提取缓冲液,并将粗核酸沉淀溶解在1 M NaCl中,显著降低了黏液的粘度,因此在最终纯化步骤中获得了大量不含黏液的DNA,适用于随后基于PCR的双生病毒检测。该方法在患有黄花叶病的黄麻样品上进行了标准化,并在其他低滴度双生病毒的黏液宿主上进行了验证。使用该方法分离的DNA在产量上具有一致性,并且与用于检测来自不同黏液植物物种的双生病毒的PCR兼容。将该方法与其他报道的方法进行了效果比较,发现它优于现有的用于从黏液宿主中分离DNA的方法。因此,所描述的方法可更广泛地用于从黏液宿主中可靠且一致地检测双生病毒,以进行特征分析和变异性研究。

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