咖啡叶锈病菌单管巢式PCR检测体系的建立与应用  

Establishment and Application of Single Tube Nested PCR Detection System for Hemileia vastatrix Causing Coffee Leaf Rust

作　　者：吴伟怀[1] 刘宝慧 汪全伟[3] 鹿鹏鹏 贺春萍[1] 梁艳琼[1] 黄兴[1] 易克贤[1,4] WU Weihuai;LIU Baohui;WANG Quanwei;LU Pengpeng;HE Chunping;LIANG Yanqiong;HUANG Xing;YI Kexian(Key Laboratory of Integrated Pest Management on Tropical Crops,Ministry of Agriculture and Rural Affairs,Key Laboratory for Monitoring and Control of Tropical Agricultural Pests,Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,China;College of Plant Protection,Nanjing Agricultural University,Nanjing 210095,China;Institute of Information and Technology,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,China;Sanya Research Institute,Chinese Academy of Tropical Agricultural Sciences,Sanya 572000,China)

机构地区：[1]中国热带农业科学院环境与植物保护研究所/农业农村部热带作物有害生物综合治理重点实验室,海南海口571101 [2]南京农业大学植物保护学院,江苏南京210095 [3]中国热带农业科学院科技信息研究所,海南海口571101 [4]中国热带农业科学院三亚研究院,海南三亚572000

出　　处：《特产研究》2023年第5期1-7,15,共8页Special Wild Economic Animal and Plant Research

基　　金：中国热带农业科学院基本科研业务费专项资金(1630042017021);FAO/IAEA合作研究项目(20380);中葡咖啡锈病联合研究和技术交流项目(12200031)。

摘　　要：由专性寄生菌咖啡驼孢锈菌(Hemileia vastatrix)引起的咖啡叶锈病是小粒种咖啡生产中的一种毁灭性病害,建立高效、准确的分子检测技术对于该病害的快速鉴定与监测具有重要意义。本研究基于咖啡叶锈病菌ITS序列保守区域设计该病原菌的单管巢式引物对,通过单因素试验筛选外引物与内引物退火温度后,进一步对单管巢式PCR检测反应体系的内、外引物浓度、dNTPs浓度、以及rTaq酶用量等4个关键因素进行正交优化试验,从而建立高效的单管巢式PCR检测反应体系。结果表明,咖啡叶锈病菌单管巢式PCR外引物HvF1/HvR1、内引物HvF2/HvR2的最佳退火温度分别为63℃与52℃。正交试验显示,在20μL的反应体系中各组分的最佳含量分别为:10×rTaq Buffer 2.5μL,2.5 mmol/L dNTPs 3.2μL,rTaq DNA Polymerase(5 U/μL)1.8μL,7μmol/L的HvF2/HvR2各1μL,1nmol/L的HvF1/HvR1各1μL,模板DNA1μL,ddH207.5μL。所建立的单管巢式PCR检测体系具有高度特异性,能从含有咖啡叶锈病菌DNA模板中检测出特异性条带,而其他供试的非咖啡叶锈病菌DNA模板中扩增不出任何条带。该检测技术体系的最低检测限为5 fg/μL,是普通PCR灵敏度的100倍。采集的22份早期疑似病样经检测鉴定均含有咖啡驼孢锈菌。本试验建立的单管巢式检测技术可用于咖啡叶锈病菌的快速鉴定,可用于病害监测预警。Coffee leaf rust,caused by the obligate parasite Hemileria vastatix,is a devastating disease in the production of Coffea Arabica.It is of great practical significance to establish an efficient and accurate molecular detection technique for rapid identification and monitoring of this disease.In the present study,a single-tube nested primer for H.vastatrix was designed based on ITS sequences that have conservative region,after screening the annealing temperature of the outer and inner primers by single factor test,four key factors,such as the concentration of inner and outer primers,the concentration of dNTPs and the dosage of rTaq enzyme,were optimized by the orthogonal design,thus,an efficient single-tube nested PCR system was established.Then the specificity and sensitivity of the single-tube nested PCR detection system were analyzed.The results showed that when the optimal annealing temperature of the single-tube nested PCR primer HvF1/R1 and the inner Primer HvF2/HvR2 were 63℃and 52℃respectively,and the optimum contents of each component in the 20μL reaction system were selected as follows:10×rTaq Buffer 2.5μL,2.5 m mol/L dNTPs 3.2μL,rTaq DNA polymerase(5 U/μL)1.8μL,7μmol/L HvF2/HvR2 primers each 1μL,1 nmol/L HvF1/HvR1 primers each 1μL,template DNA 1μL,dd H_2O 7.5μL.The single-tube nested PCR detection system can only detect the specific bands from the DNA template containing the H.vastatrix,while the other non-H.vastatrix DNA template cannot detect any bands,show a high degree of specificity.Based on this,the minimum detection limit of the single-tube nested PCR detection system is 5 fg/μL,which is 100 times higher than that of the conventional PCR system with sensitivity of 0.5 pg/μL.Twenty-two early suspected samples were tested and identified to all contain H.vastatix by single-tube nest PCR.The method established in this study can be used for rapid identification of H.vastatix,disease monitoring and early warning.

关 键 词：咖啡叶锈病 驼孢锈菌 单管巢式PCR 

分 类 号：S435.712[农业科学—农业昆虫与害虫防治]