| 丁鹏,卢占军.柑橘黄龙病菌LAMP检测制样方法的比较[J].中国南方果树,2018,47(1): |
| 柑橘黄龙病菌LAMP检测制样方法的比较 |
| Comparative Test for DNA Extraction Methods of Cirtrus Huanglongbing Suitable for LAMP Assay |
| 投稿时间：2017-11-27 修订日期：2017-11-27 |
| DOI： |
| 中文关键词: 柑橘黄龙病 环介导等温扩增 制样方法 核酸 |
| 英文关键词:Cirtrus Huanglongbing LAMP extraction methods DNA |
| 基金项目:国家自然科学基金(编号31560602)；江西省科技计划重大项目(编号20152ACF60003)；江西省杰出青年人才资助计划项目(编号20171BCB23074)；江西省2016年度研究生创新专项资金项目(编号YC2016-S408)；江西省科技计划重大项目(编号20161ACF60016)；江西现代农业科研协同创新专项经费(编号JXXTCX2015002-003)；江西省科技计划重大项目(编号20143ACF60012)。 |
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| 中文摘要: |
| 分析比较适合大规模柑橘样品黄龙病LAMP检测的制样方法,为柑橘黄龙病的防治提供技术支持。分别采用CTAB-trion法、磁珠法、裂解法、浸提法四种制样方法提取病原菌核酸,利用LAMP和常规PCR进行扩增,并从制备步骤、简易程度、核酸质量、成本、耗时及环保等方面比较,确定最佳制样方法。CTAB-trion法和磁珠法提取的核酸质量最好,其OD260/OD280值在1.8-2.0之间,满足LAMP扩增要求,但操作步骤繁琐、困难,成本、耗时较高且CTAB-trion法需使用苯酚和氯仿试剂；裂解法提取的核酸质量虽不高,但可满足LAMP扩增,同时其操作步骤极少且易,成本低、耗时少,相对环保；浸提法提取的核酸量少、质量差,无法满足LAMP扩增要求。裂解法是柑橘黄龙病LAMP检测的最佳制样方法,与LAMP联用可建立成本小、操作简便、高效快速、环境友好、结果可视化、无需昂贵特殊仪器、适合大规模柑橘样品的黄龙病检测。 |
| 英文摘要: |
| Several DNA extraction methods apply to a great amount of citrus samples for Huanglongbing LAMP assay were compared. Provide technical support for the prevention and control of Cirtrus Huanglongbing.DNA genome of citrus leaves with suspicious Huanglongbing symptoms was prepared, using CTAB-trion method, magnetic bead method, cracking method, soaking extracting method, and amplified by LAMP and PCR respectively,. Optimal method will evaluate based on preparing procedure, operation program, the quality of DNA, prepare time, spend costs and environment-friendly.The results showed the DNA prepared by CTAB-trion method and magnetic bead method was the best,the range of OD260/OD280 was 1.8-2.0, and the DNA can be detected by LAMP, but preparing procedure is tedious, need long preparing time and spend more costs, what’s more, CTAB-trion method need phenol and chloroformSreagents. The DNA prepared by cracking method was useful to detection although DNA quality was not as good as CTAB-trion and magnetic bead method, furthermore preparing procedure easy, environment-friendly, short prepare time and less costs. the DNA extracted by soaking extracting method wasn’t suitable for LAMP assay because of low Purity and concentration . Cracking method was superior to other methods for LAMP assay. It would be an easy and simple to handle, eco-friendly, theSresultsScan beSvisualized, economical, time saving, needn’t expensive and special instrument and can apply to a great amount of samples for Cirtrus Huanglongbing detection with LAMP assay. |
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