林业科学2016，Vol.52Issue(10)：161-166,6.DOI:10.11707/j.1001-7488.20161020
等温扩增技术快速检测棕榈疫霉
A Method for Rapidly Identifying Phytophthora palmivora Using the LAMP Technique
摘要
Abstract
Objective]The oomycetes pathogen Phytophthora palmivora is one of the most devastating pathogens in agricultural and forestry production. It is important to establish suitable molecular methods for detecting the pathogen with a rapid,cost - effective and efficient assay in predicting the occurrence of P. palmivora,controlling the spread of P. palmivora and reducing the economic losses. [Method]In this study,the loop - mediated isothermal amplification ( LAMP) assay was used to establish a method for visual detection of P. palmivora with targeting the IGS gene. Multiple sets of LAMP primers were designed according to the IGS target using the LAMP technique. The LAMP reaction system and conditions were optimized to select a definitive set of primers. The set of primers were also verified by the specificity, sensitivity and application of the LAMP assay. A visualization indicator,namely,hydroxynaphthol blue ( HNB ) was brought into the PpIGS-LAMP assays prior to amplification. [Result]The result showed that the PpIGS-LAMP assay efficiently amplified the target element in 80 min at 64 ° C. All isolates of Phytophthora species,Pythium species,and fungi were used to determine the speci? city of the LAMP assay. A positive colour ( sky blue) was only observed in the presence of P. palmivora by addition of hydroxynaphthol blue prior to amplification,whereas none of other isolates showed a colour change. The detection limit of the PpIGS-specific LAMP assay for P. palmivora was 10 fg·μL -1 of genomic DNA per reaction. Furthermore,the naturally P. palmivora-infected soil samples collected from diseased cassava root and inoculated cassava root were evaluated for detection P. palmivora. [Conclusion]In conclusion,this is the first report of the application of the LAMP assay technique for the rapid and speci? c detection of P. palmivora. These results suggest that this IGS-specific LAMP provides a rapid,high sensitivity,high specificity,visualization for detecting P. palmivora in plants and in production fields. The PpIGS-LAMP assay developed in this research efficiently amplified the target element in 80 min at 64 ℃,significantly shortened the testing time and reduced the cost of the government. Establishment of PpIGS-LAMP method provides a new alternative means for the rapid detection of P. palmivora.关键词
棕榈疫霉/环介导等温扩增/PpIGS/羟基萘酚蓝Key words
Phytophthora palmivora/loop-mediated isothermal amplification ( LAMP)/PpIGS/hydroxynaphthol blue ( HNB)分类
农业科技引用本文复制引用
戴婷婷,吴小芹..等温扩增技术快速检测棕榈疫霉[J].林业科学,2016,52(10):161-166,6.基金项目
国家自然科学基金项目(31500526) （31500526）
中国博士后科学基金项目(2014M561657) （2014M561657）
江苏省博士后科学基金项目(1402159C) （1402159C）
江苏省高校优势学科建设工程项目(PAPD) （PAPD）
南京林业大学高层次人才启动基金项目(G2014003)。 （G2014003）
