果树学报2012，Vol.29Issue(6)：1121-1126,I0003,7.
柑橘黄龙病LAMP快速检测方法的建立及应用
Establishment and application of loop-mediated isothermal amplification assay for the detection of Citrus Huanglongbing
摘要
Abstract
【Objective】The objective of the study is to develop a loop-mediated isothermal amplification assay for rapid detection of Candidatus Liberibacter asiaticus.【Method】The detection method is based on the loop-mediated isothermal amplification(LAMP) reaction.Outer membrane protein gene of HLB pathogen was amplified by a set of four special primers that recognize six distinct sequences of the target.The reaction conditions of the method were optimized.Amplification products were visualized by naked eyes either as turbidity or in the form of a color change when SYBR Green I,a fluorescent dsDNA intercalating dye,was employed.The specificity and sensitivity of the method was also evaluated.【Result】The amplification can be finished in 70 minutes by incubating the mixed solution of all reagents in a single tube with Bst DNA polymerase at 63 ℃.The electrophoresis results showed ladder-like bands from LAMP products of HLB-positive samples.The lowest detection limit of the LAMP and Real-time PCR method was 3.9×101 copies,100 fold higher than conventional PCR.The HLB detection rate of LAMP for 105 field samples was 52.4%,and that of the conventional PCR method was 37.1%.The coincidence rate of the two methods was 84.7%.【Conclusion】The studies suggested that LAMP method was rapid,simple,specific and sensitive for the detection of HLB.The LAMP can be a new and alternative technique for rapid detection of citrus huanglongbing.关键词
柑橘黄龙病菌亚洲种/外膜蛋白基因/环介导等温扩增技术Key words
Candidatus Liberibacter asiaticus/Outer membrane protein gene/LAMP分类
农业科技引用本文复制引用
黄丽,苏华楠,唐科志,黄爱军,周常勇,李中安..柑橘黄龙病LAMP快速检测方法的建立及应用[J].果树学报,2012,29(6):1121-1126,I0003,7.基金项目
公益性行业专项 （）
教育部创新团队 （）
