剑麻紫色卷叶病相关植原体单管巢式PCR检测技术建立与优化  

Establishment and Optimization of Single-tube Nested PCR Detection Technique for Phytoplasma Related to Sisal Purple Leafroll Disease

作　　者：鹿鹏鹏 吴伟怀[2] 郑金龙[2] 王桂花[2] 贺春萍[2] 林培群[2] 黄兴[2] 梁艳琼[2] 易克贤[2] LU Peng-Peng;WU Wei-Huai;ZHENG Jin-Long;WANG Gui-Hua;HE Chun-Ping;LIN Pei-Qun;HUANG Xing;LIANG Yan-Qiong;YI Ke-Xian(College of Plant Protection,Hainan University,Haikou 570228,China;Hainan Key Laboratory for Monitoring and Control of Tropical Agricultural Pests/Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,China)

机构地区：[1]海南大学植物保护学院,海口570228 [2]中国热带农业科学院环境与植物保护研究所/海南省热带农业有害生物监测与控制重点实验室,海口571101

出　　处：《农业生物技术学报》2021年第7期1426-1434,共9页Journal of Agricultural Biotechnology

基　　金：2019年海南省基础与应用基础研究计划(自然科学领域)高层次人才项目(2019RC282);国家重点研发项目(2018YFD0201100);财政部和农业农村部:国家麻类产业技术体系剑麻生理与栽培岗位(CARS-16-E16);中央级公益性科研院所基本科研业务费专项(1630042019028;1630042020015)。

摘　　要：剑麻紫色卷叶病是近年来剑麻(Agave sisalana)上发生的一种毁灭性病害。本课题组前期研究表明,该病与植原体高度相关。建立一种高效的分子检测技术对于植原体的深入研究及病害监测与检测非常必要。本研究拟建立剑麻紫色卷叶病相关植原体的高效单管巢式PCR检测技术。首先对单管巢式PCR反应体系的内外引物退火温度进行单因素试验;确定退火温度后,利用正交设计的方法对单管巢式PCR反应体系的内外引物浓度、dNTPs浓度和Ex Taq酶用量等关键因素进行优化。结果显示,当外引物Sis-F1/R1退火温度为64℃、内引物Sis-F2/R2退火温度为54℃,通过正交设计试验最终筛选出的最佳反应体系(25μL)为:10×Ex Taq Buffer 2.5μL,2.5 mmol/L dNTPs 5μL,Ex Taq DNA Polymerase1μL,15μmol/L的内引物Sis-F2/R2各1μL,0.02μmol/L的外引物Sis-F1/R1各1μL,模板DNA 1μL,ddH2O 11.5μL。上述反应体系只对紫色卷叶病植株DNA模板扩增出条带,具有高度特异性;最低检测限为植原体质粒浓度≥1 fg/μL。本研究所建立的剑麻紫色卷叶病相关植原体单管巢式PCR检测体系为后续病害监测、相关性调查、致病性以及功能验证等方面的研究提供技术支持。Sisal purple leafroll disease(SPLD) is a devastating disease that has occurred on sisal(Agave sisalana) in recent years. Previous studies by this research group have shown that the disease is highly associated with phytoplasmas. It is necessary to establish an efficient molecular detection technology for indepth research on phytoplasma or disease monitoring and detection. To this end, this study intends to establish an efficient single-tube nested PCR detection technique for phytoplasma related to sisal purple leafroll disease. Firstly, the annealing temperature of inner and outer primers in the single tube nested PCR reaction system was studied by single-factor test. After the annealing temperature was determined, an orthogonal design was used to optimize key factors such as inner and outer primer concentration, dNTPs concentration,and Ex Taq enzyme dosage of single-tube nest PCR detection system. The results showed that when the annealing temperature of outer primer Sis-F1/R1 was determined to be 64 °C, and inner primer Sis-F2/R2 was 54 °C, the best reaction system(25 μL) was finally selected by orthogonal design test as follows: 10×Ex Taq Buffer 2.5 μL, 2.5 mmol/L dNTPs 5 μL, Ex Taq DNA Polymerase 1 μL, 15 μmol/L inner primers SisF2/R2 each 1 μL, 0.02 μmol/L outer primers Sis-F1/R1 each 1 μL, template DNA 1 μL, ddH2 O 11.5 μL. The above reaction system only amplified bands from the DNA template of the plants with purple leafroll disease, which had a high degree of specificity. The lowest detection limit of the detection system was a phytoplasma concentration of ≥ 1 fg/μL. The single tube nested PCR reaction system for the detection of phytoplasma related to sisal purple leafroll disease established in this research could provide technical support for follow-up researches on disease monitoring, correlation investigation, pathogenicity and functional verification.

关 键 词：剑麻 植原体 单管巢式PCR 正交试验 

分 类 号：S41-30[农业科学—植物保护]