柑橘黄龙病PCR检测技术优化  

Optimization of PCR Detection Technology for Citrus Huanglongbing

作　　者：杨亚 石卓艺 周闰 彭丁文 朱宝德 王丽艳 孙倩楠 唐志敏 何艳晴 申超峰 YANG Ya;SHI Zhuoyi;ZHOU Run;PENG Dingwen;ZHU Baode;WANG Liyan;SUN Qiannan;TANG Zhimin;HE Yanqing;SHEN Chaofeng(Chenzhou Institute of Agricultural Sciences,Chenzhou 423000,China;Chenzhou Citrus Huanglongbing Inspection and Testing Center,Chenzhou 423000,China;Chenzhou Citrus Detoxification Technology Research and Development Center,Chenzhou 423000,China;Chenzhou Agricultural Molecular Technology Application Research Center,Chenzhou 423000,China;Chenzhou Citrus Huanglongbing(Citrus psyllid)Monitoring and Control Technology Research and Development Center,Chenzhou 423000,China;Xiangnan University,Chenzhou 423000,China;Chenzhou Agriculture and Rural Bureau,Chenzhou 423000,China)

机构地区：[1]郴州市农业科学研究所,湖南郴州423000 [2]郴州市柑橘黄龙病检验检测中心,湖南郴州423000 [3]郴州市柑橘脱毒技术研发中心,湖南郴州423000 [4]郴州市农作物分子技术应用研发中心,湖南郴州423000 [5]郴州市柑橘黄龙病(木虱)监测与防控技术研发中心,湖南郴州423000 [6]湘南学院,湖南郴州423000 [7]郴州市农业农村局,湖南郴州423000

出　　处：《中国果菜》2024年第12期20-24,30,共6页China Fruit & Vegetable

基　　金：郴州市技术研发中心项目:郴州市柑橘脱毒技术研发中心、郴州市柑橘黄龙病(木虱)监测与防控技术研发中心;国家重点研发计划课题早中熟柑橘大面积丰产增效轻简化栽培技术集成示范(2024YFD2300802)。

摘　　要：为提高Jagoueix等建立的柑橘黄龙病PCR检测体系的检测效率和检出率,本研究对基因组DNA提取、PCR体系和PCR程序进行优化,分析了不同萃取次数、裂解温度和抗氧剂组合对总DNA质量的影响,并设置不同引物浓度梯度和退火温度梯度,研究其对检测效果的影响。结果表明,萃取次数对DNA质量基本无影响;水浴65℃裂解比常温裂解提取的DNA质量更优,但总体差别不大;DNA提取液中添加β-巯基乙醇、β-巯基乙醇和PVP或者二者均不加入,对最终的基因组DNA的质量和纯度影响不大;引物浓度为0.4~1.0μmol/L出现稳定特征条带,引物浓度为0.1μmol/L时仍会产生引物二聚体;在66.5℃退火温度下的检测结果最优。因此,采取一次萃取、常温裂解、不添加抗氧化剂可简化DNA提取步骤;将引物浓度调整为0.8μmol/L效果最佳;退火温度66.5℃可提高检出率;优化后检测体系操作简便、稳定性好、检测率高,适于柑橘黄龙病的批量检测。In order to improve the efficiency and detection rate of citrus Huanglongbing detection system,the author combined the amplification system established by Jagoueix et al.to optimize DNA extraction,PCR system,and PCR program.This study used different extraction times,different cracking temperatures,and different combinations of antioxidants to study the impact on DNA quality,as well as by setting primer concentration gradients and annealing temperature gradients to study their impact on detection performance.The results showed that the extraction frequency had little effect on DNA quality.The quality of DNA extracted by water bath lysis at 65℃was better than that by room temperature lysis,but the overall difference was not significant.Adding to DNA extraction solutionβ-Mercaptoethanolβ-Mercaptoethanol and PVP,both of which were not added,had little effect on the quality and purity of the final total DNA.Stable characteristic bands appeared at primer concentrations of 0.4-1.0μmol/L,and primer dimers were still formed at primer concentrations of 0.1μmol/L The detection results were optimal at an annealing temperature of 66.5℃.Therefore,adopting a single extraction,room temperature lysis,and no addition of antioxidants can simplify the DNA extraction process.Adjust the primer concentration to 0.8μmol/L had the best effect.Annealing temperature of 66.5℃could improve the detection rate.The optimized detection system was easy to operate,stable,and had a high detection rate,making it suitable for batch detection of Huanglongbing.

关 键 词：柑橘黄龙病 DNA提取 PCR 检测体系 

分 类 号：S436[农业科学—农业昆虫与害虫防治]