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首页> 外文期刊>Australasian Plant Pathology >Optimization of total nucleic acid extraction method for detecting Coconut cadang-cadang viroid variants in oil palm
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Optimization of total nucleic acid extraction method for detecting Coconut cadang-cadang viroid variants in oil palm

机译：用于检测油棕中椰子Cadang-Cadang Viroiders的总核酸提取方法的优化

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摘要

Variants of Coconut cadang-cadang viroid (CCCVd) with over 90% sequence similarity to CCCVd in coconut have recently been associated with orange spotting disease in oil palm, especially in low concentrations in infected hosts. Thus, there is a need to extract high-quality nucleic acid for molecular detection of the viroid. Total nucleic acid (TNA) was extracted from oil palm leaf samples with orange spotting symptoms collected from different states in Malaysia using a modified and optimized version of the conventional natrium chloride EDTA Tris-HCL mercaptoethanol extraction method. The modifications involved additional ethanol and lithium chloride precipitation stages, thereby eliminating the need for further purification through non-denaturing polyacrylamide gel electrophoresis (PAGE). The modified procedure yielded a mean volume of 60 mu g RNA per 10 g of fresh tissue which is approximately four times the volume produced by the conventional extraction method. Furthermore, the modified method resulted in higher purity than the conventional method according to the ratios of absorbance at wavelengths of 260/280 nm and 260/230 nm, which were 1.838 and 1.883 with the modified method and 1.085 and 0.765 with the conventional method. The modified method enabled extraction of high-quality RNA from all samples investigated. The extracted RNA was suitable for cDNA synthesis and PCR amplification and showed consistent detection of CCCVd-like RNA at approximately 250 amplicons. Using the conventional method, detection of CCCVd-like RNA was inconsistent and only feasible after the PAGE purification step.
机译：椰子卡康 - 卡康病人（CCCVD）的变异性与椰子中CCCVD超过90％的序列相似性最近与油棕中的橙色斑点疾病有关，特别是在感染宿主中的低浓度。因此，需要提取高质量的核酸以进行病原体检测。从油棕榈叶样品中萃取总核酸（TNA），使用常规的氯化钠EDTA Tris-HCL巯基乙醇萃取方法的改性和优化版本从马来西亚的不同状态收集的橙色斑点症状。修饰涉及额外的乙醇和氯化锂沉淀阶段，从而消除了通过非变性聚丙烯酰胺凝胶电泳（页面）进一步纯化的需要。改性过程产生每10克新鲜组织的60μgRNA的平均体积，其大约四倍由常规提取方法产生的体积。此外，根据260/280nm和260/230nm的波长的吸光度比率，改性方法比常规方法更高，其为1.838和1.883，用改性方法和1.085和0.765，具有常规方法。改性方法使能来自研究的所有样品的高质量RNA。提取的RNA适用于cDNA合成和PCR扩增，并在约250个扩增子下显示出一致的CCCVD样RNA检测。使用常规方法，检测CCCVD样RNA的检测不一致，并且在页面纯化步骤之后仅可行。

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