热带作物学报2017，Vol.38Issue(12)：2334-2339,6.DOI:10.3969/j.issn.1000-2561.2017.12.021
甘蔗褐锈病菌巢式PCR分子检测方法的建立
Establishment of a Nested PCR Detection System of Puccinia melanocephala Sydow Causing Sugarcane Brown Rust Disease
摘要
Abstract
The objective of this study is to develop a nested-PCR method to rapidly and accurately detect sugarcane brown rust.In the present study,based on the differences in the sequences of internal transcribed spacer (ITS) ITS1-ITS4 sequence ribosomal DNA of P.melanocephala in sugarcane and the other closely related species at the NCBI web,two pairs of primers PM2F/R and PM3F/R were designed and a PCR assay was developed.Among the same host and rust fungi of different genera including brown rust was implemented to determine the primer specificity that two pairs of primers were amplified,only two PCR bands of 474 bp and 363 bp were amplified with DNA extracted from all isolates of P.melanocephala in sugarcane,while other tested isolates had no corresponding bands.The detection sensitivity increased 100-fold to 0.001 ng/μL genomic DNA by developing a nested PCR procedure with bacterial universal primer pair PM2F/R as the first round primers and PM3F/R as the second round primers.Therefore,this study set up a fast,sensitive and accurate nested PCR detection system of sugarcane brown rust pathogen.It has important significance in early diagnosis,and rapid detection and research on epidemiology of sugarcane brown rust.关键词
甘蔗褐锈病/黑顶柄锈菌/巢式PCR分子检测Key words
Sugarcane brown rust disease/Puccinia melanocephala Sydow/nested PCR detection system分类
农业科技引用本文复制引用
汪涵,易克贤,吴伟怀,杨旭光,杨先锋,李锐,郑金龙,黄兴,梁艳琼,贺春萍..甘蔗褐锈病菌巢式PCR分子检测方法的建立[J].热带作物学报,2017,38(12):2334-2339,6.基金项目
中国热带农业科学院橡胶研究所省部重点实验室/科学观测实验站开放课题(No.RRI-KLOF201506). （No.RRI-KLOF201506）
