Kumar Ankit, Singh Jyoti, Panwar Deepak, Singh Anupma, Thapa Ravi Singh, Kumar Rakesh, Pratap Dharmendra
Plant Molecular Virology Laboratory, Department of Genetics and Plant Breeding, Chaudhary Charan Singh University, Meerut, Uttar Pradesh, 250004, India.
School of Agricultural Sciences, IIMT University, Meerut, UP, 250001, India.
Arch Microbiol. 2024 Nov 14;206(12):468. doi: 10.1007/s00203-024-04176-0.
The extraction of DNA from okra (Abelmoschus esculentus) is challenging due to its high mucilage and polysaccharide content, which can hinder both the yield and quality of DNA. In this study, an improved DNA isolation method is described incorporating a key modification being the use of solution I (1 M NaCl and 2% Sarcosyl) as a pre-treatment before applying the CTAB buffer, resulting in high-purity genomic DNA in just 1 h and 45 min., making it suitable for handling large sample sizes due to its rapid processing capabilities. This enhanced DNA extraction method was crucial for the accurate and rapid molecular detection of Okra enation leaf curl virus (OELCuV), a monopartite begomovirus that has spread across various regions of India. Transmitted by the whitefly (Bemisia tabaci), OELCuV causes leaf curling, enations, and stunted growth in okra, leading to significant yield losses. The surveys conducted during the 2020-21 and 2021-22 sowing seasons revealed disease incidence ranging from 14.03 to 67.57%. The extracted DNA via the improved DNA extraction method enhanced the speed of PCR based molecular identification of OELCuV, using virus-specific coat protein primers. The amplified CP genes were cloned and sequenced to study the CP gene based diversity among OELCuV isolates from different states of India. The CP gene nucleotide identity among the studied OELCuV isolates ranged from 95.57 to 99.27%, while comparison with previously reported Indian OELCuV CP sequences, the nucleotide identity ranged from 89.35 to 98.83%. The successful application of this optimized DNA extraction method sped up the detection process but also holds promise for broader use in the molecular study of okra and other mucilaginous crops, particularly in the rapid and reliable identification of begomoviruses. The optimized DNA extraction method significantly accelerated the detection of OELCuV, demonstrating its efficiency and reliability. This method shows strong potential for broader applications in the molecular study of okra and other mucilaginous crops, making it a valuable tool for future research and disease management.
从秋葵（黄秋葵）中提取DNA具有挑战性，因为其黏液和多糖含量高，这会影响DNA的产量和质量。在本研究中，描述了一种改进的DNA分离方法，其中一个关键改进是在应用CTAB缓冲液之前使用溶液I（1 M NaCl和2% 肌氨酸钠）进行预处理，从而在仅1小时45分钟内获得高纯度基因组DNA，由于其快速处理能力，使其适用于处理大量样本。这种改进的DNA提取方法对于秋葵曲叶病毒（OELCuV）的准确快速分子检测至关重要，OELCuV是一种单分体双生病毒，已在印度各地区传播。由烟粉虱传播，OELCuV会导致秋葵叶片卷曲、畸形和生长受阻，从而导致显著的产量损失。在2020 - 21年和2021 - 22年播种季节进行的调查显示，发病率在14.03%至67.57%之间。通过改进的DNA提取方法提取的DNA提高了基于PCR的OELCuV分子鉴定速度，使用病毒特异性外壳蛋白引物。对扩增的CP基因进行克隆和测序，以研究来自印度不同邦的OELCuV分离株基于CP基因的多样性。所研究的OELCuV分离株之间的CP基因核苷酸同一性在95.57%至99.27%之间，而与先前报道的印度OELCuV CP序列相比，核苷酸同一性在89.35%至98.83%之间。这种优化的DNA提取方法的成功应用加快了检测过程，也有望在秋葵和其他黏液性作物的分子研究中得到更广泛应用，特别是在双生病毒的快速可靠鉴定方面。优化的DNA提取方法显著加快了OELCuV的检测，证明了其效率和可靠性。该方法在秋葵和其他黏液性作物的分子研究中具有广阔的应用潜力，使其成为未来研究和病害管理的宝贵工具。
