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首页> 外文期刊>Communications in Agricultural and Applied Biological Sciences >AN OPTIMIZED METHOD FOR EXTRACTION AND DETECTION OF COCONUT CADANG-CADANG VIROID (CCCVD) FROM OIL PALM
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AN OPTIMIZED METHOD FOR EXTRACTION AND DETECTION OF COCONUT CADANG-CADANG VIROID (CCCVD) FROM OIL PALM

机译：从油棕中提取和检测椰子卡丹-卡丹病毒（CCCVD）的优化方法

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摘要

Coconut cadans-cadang viroid (CCCVd) causes the lethal cadang-cadang disease of coconut palms in the Philippines and it is recently reported to be associated with the orange spotting disease on oil palm in Malaysia. The low concentration of the viroidRNA in oil palm as well as the high content of polyphenols and polysaccharides in this plant which interfere with the purification steps makes it difficult to extract and detect this viroid from oil palm. A previously described method was modified and optimized for extraction and detection of CCCVd from infected oil palms. Briefly, 7 g of leaf material was homogenized in a mortar or a blender using liquid nitrogen. 10 ml of extraction buffer (100 mM Tris-HCl pH 7.5,100 mM NaCl, 10 mM EDTA) along with 100 mM 2-mercaptoethanol and 10 ml water saturated phenol was added to the frozen powder. After centrifuging at 4deg C, 4000 g for 30 min, the aqueous phase was extracted once more with phenol then once with chloroform-isoamyl alcohol (24:1). After addingsodium acetate, pH 5.6 to 200 muM, the mixture was precipitated with 2.5 vol ethanol overnight in -20 freezer and then the pellet was washed with 70% ethanol and air-dried. One milliliter of 8 M LiCl was added to the dried pellet and after shaking overnight at 4deg C and another centrif ugation step the supernatant was collected and precipitated again with ethanol and then the resulting pellet was washed and air-dried. To carry out northern blotting, samples equivalent to 40 g of plant tissue were mixed with formamide buffer and loaded onto a 12% polyacrylamide gel containing 7 M urea and after separation by electrophoresis, were electroblotted onto membrane and fixed by UV cross-linking. Pre-hybridization and hybridization using hybridization buffer(50% formamide, 25%SSPE, 0.1% Ficol and PVP, 0.1 % SDS, 0.02 % DNA (5mg/ml)) was carried out at 45 deg C for 90 min and 16 h, respectively followed by two low stringency washes (0.5X SSC, 0.1% SDS, at room temperature for 5 min) and one high stringency wash (0.1X SSC, 0.1% SDS at 60deg C for 1 hour). In vitro synthesized DIG-labeled full-length CCCVd(-) RNA probe was used in hybridization step. DIG Nucleic Acid Detection Kit (Roche) instructions were followed for detection procedure and as a result theblue bands corresponding to the position of the viroid were appeared on the membrane. The result of this study showed the ability of DIG labeled probe in detection of the viroid and also provided a suitable extraction and hybridization method for the detection of CCCVd from oil palm.
机译：在菲律宾，椰子树豆-豆荚类病毒（CCCVd）引起致命性的椰子树豆树-豆荚类疾病，据报道，这与马来西亚油棕上的橙色斑点病有关。油棕中viroidRNA的浓度低，以及该植物中高含量的多酚和多糖会干扰纯化步骤，因此很难从油棕中提取和检测该类病毒。修改并优化了先前描述的方法，用于从受感染的油棕中提取和检测CCCVd。简要地，使用液氮在研钵或搅拌机中将7g叶材料均质化。将10 ml提取缓冲液（100 mM Tris-HCl pH 7.5、100 mM NaCl，10 mM EDTA）以及100 mM 2-巯基乙醇和10 ml水饱和苯酚添加到冷冻的粉末中。在4℃，4000g下离心30分钟后，用苯酚再萃取一次水相，然后用氯仿-异戊醇（24∶1）萃取一次。在将pH 5.6的乙酸钠添加至200μM之后，将混合物用2.5体积的乙醇在-20冰箱中沉淀过夜，然后将沉淀用70％乙醇洗涤并风干。将一毫升的8 M LiCl加入干燥的沉淀中，并在4℃振荡过夜后，再进行一次离心分离步骤，收集上清液，并再次用乙醇沉淀，然后将所得沉淀洗涤并风干。为了进行Northern印迹，将相当于40g植物组织的样品与甲酰胺缓冲液混合，并加载到含有7M尿素的12％聚丙烯酰胺凝胶上，并通过电泳分离，然后电印迹到膜上并通过UV交联固定。使用杂交缓冲液（50％甲酰胺，25％SSPE，0.1％Ficol和PVP，0.1％SDS，0.02％DNA（5mg / ml））进行预杂交和杂交，在45°C下进行90分钟和16小时，分别进行两次低严格洗涤（0.5X SSC，0.1％SDS，在室温下5分钟）和一次高严格洗涤（0.1X SSC，0.1％SDS在60°C下1小时）。体外合成的DIG标记的全长CCCVd（-）RNA探针用于杂交步骤。遵循DIG核酸检测试剂盒（Roche）的说明进行检测，结果在膜上出现了与类病毒位置相对应的蓝带。这项研究的结果表明，DIG标记探针具有检测类病毒的能力，也为从油棕中检测CCCVd提供了一种合适的提取和杂交方法。

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