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首页> 外文期刊>Canadian Journal of Plant Pathology: Revue Canadienne de phytopathologie >Comparison of RNA extraction methods for RT-PCR detection of Coconut cadang-cadang viroid variant in orange spotting oil palm leaves
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Comparison of RNA extraction methods for RT-PCR detection of Coconut cadang-cadang viroid variant in orange spotting oil palm leaves

机译：RT-PCR检测橙色斑点油棕叶椰子山豆-山豆类病毒变体的RNA提取方法比较

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摘要

Coconut cadang-cadang viroid (CCCVd) is a lethal disease that has devastated the coconut industry in the Philippines. Recently, a CCCVd variant associated with orange spotting of foliage, which is a phenomenon reported in the Malaysian oil palm industry, occurring as an isolated palm with no similar symptoms on the adjacent neighbouring palms. The presence of this viroid in oil palms is difficult to detect as it is found in very low concentrations. Thus, an efficient RNA extraction method for isolating the CCCVd variant and to detect it through reverse transcriptase polymerase chain reaction (RT-PCR) using specific primers for CCCVd was required. Leaf samples from symptomatic and asymptomatic oil palms were collected from various locations in Malaysia, namely, Negeri Sembilan, Selangor and Perak. The samples were subjected to three RNA extraction methods -natrium chloride EDTA TrisHCl mercaptoethanol extraction (NETME), polyethylene glycol extraction (PEG) and cetyltrimethylammonium bromide extraction (CTAB). The quantification of the RNA, based on optical density (OD) and concentration, showed that the CTAB extraction was the best, followed by the NETME extraction. The RNA extraction using PEG resulted in poor yields and had the lowest purity. The viroid was detected in all of the samples extracted via CTAB and NETME by RT-PCR with reduced starting material compared with PEG extracted RNA. A BLAST analysis indicated that the viroid sequences are highly conserved and shared a 93% sequence identity with the CCCVd246 variant from oil palm. Phylogenetic analysis showed that the variant belonged to an outgroup from other cocadviroids found in monocots.
机译：椰子cadang-cadang类病毒（CCCVd）是一种致命疾病，已经摧毁了菲律宾的椰子产业。最近，CCCVd变种与叶子的橙色斑点相关，这是马来西亚油棕行业中报道的一种现象，它是一种孤立的棕榈，在相邻的相邻棕榈上没有类似的症状。由于在很低的浓度下发现了类病毒，因此很难检测到它在油棕中的存在。因此，需要一种有效的RNA提取方法，用于分离CCCVd变体并使用CCCVd的特异性引物通过逆转录酶聚合酶链反应（RT-PCR）进行检测。从有症状的和无症状的油棕的叶子样品采集自马来西亚的各个地方，即森美兰州，雪兰莪州和霹雳州。样品经过三种RNA提取方法-氯化钠EDTA TrisHCl巯基乙醇提取（NETME），聚乙二醇提取（PEG）和十六烷基三甲基溴化铵提取（CTAB）。根据光密度（OD）和浓度对RNA进行定量，结果表明CTAB提取效果最好，其次是NETME提取。使用PEG提取RNA导致收率低且纯度最低。与PEG提取的RNA相比，在通过CTAB和NETME通过RT-PCR提取的所有样品中检测到类病毒，起始原料减少。 BLAST分析表明类病毒序列是高度保守的，并且与来自油棕的CCCVd246变体具有93％的序列同一性。系统发育分析表明，该变体属于单子叶植物中发现的其他类球虫的外群。

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