中国果菜2024，Vol.44Issue(12)：20-24,30,6.DOI:10.19590/j.cnki.1008-1038.2024.12.004
柑橘黄龙病PCR检测技术优化
Optimization of PCR Detection Technology for Citrus Huanglongbing
摘要
Abstract
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 solution β-Mercaptoethanol β-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 0.4-1.0 μmol/L,and primer dimers were still formed at primer concentrations of 0.1 μmol/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 0.8 μmol/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Key words
Citrus Huanglongbing/DNA extraction/PCR/detection system分类
农业科技引用本文复制引用
杨亚,申超峰,石卓艺,周闰,彭丁文,朱宝德,王丽艳,孙倩楠,唐志敏,何艳晴..柑橘黄龙病PCR检测技术优化[J].中国果菜,2024,44(12):20-24,30,6.基金项目
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 （木虱）
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) （2024YFD2300802）
