原创《甘蔗褐锈病菌巢式PCR分子检测方法建立》：本文是一篇关于甘蔗论文范文，可作为相关选题参考,和写作参考文献。
摘 要 为建立甘蔗褐锈病菌巢式PCR分子检测方法,本研究利用真菌DNA内源转录间隔区通用引物ITS1/ITS4扩增甘蔗黑顶柄锈菌DNA,并对其扩增产物进行克隆测序,获得序列于NCBI网站进行比对分析,并于该序列多态性丰富区域设计了2对引物PM2F/R、PM3F/R.通过对同寄主不同病原菌、以及不同属的锈菌DNA进行PCR检测以验证引物的特异性.结果表明,在优化的单一PCR反应体系和程序条件下,2对引物均仅能从甘蔗黑顶柄锈菌中扩增出约474 bp和363 bp的特异条带,而从其它真菌DNA中均扩增不出任何条带.进一步将引物PM2F/R作为第一轮扩增引物、PM3F/R作为第二轮扩增引物进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达0.001 ng/μL,较常规PCR提高100倍.由此表明,依据本研究所设计的2对引物而建立的快速、灵敏、准确的甘蔗黑顶柄锈菌的检测技术,对病原菌的早期诊断、快速检测及病害流行学研究具有重要意义.
关键词 甘蔗褐锈病；黑顶柄锈菌；巢式PCR分子检测
中图分类号 S435.661 文献标识码 A
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.
Key words Sugarcane brown rust disease； Puccinia melanocephala Sydow； nested PCR detection system
doi 10.3969/j.issn.1000-2561.2017.12.021
由黑頂柄锈菌（Puccinia melanocephala Sydow）引起的甘蔗褐锈病是我国甘蔗种植区普遍发生的一种病害.该病依赖夏孢子传播,借风力附着在叶片表面,经气孔侵入植物体内.主要为害叶片,老叶较幼叶易感病.病斑分布较分散,多发自叶顶端.初期病斑长形,淡绿色至 ；后期病斑扩大,长2～10 mm,有时可达30 mm,宽1～3 mm.夏孢子堆呈红褐色,主要着生在叶背,严重时,叶面也可见.受害后的甘蔗分蘖减少,蔗茎变细[1-4].甘蔗褐锈病一旦发生,很容易引起流行,发病田地一般减产15%～30%,严重时减幅可达40%以上[5-6],已成为世界上普遍发生,危害最严重的甘蔗病害之一.自1977年该病在我国首次发生以来,现已遍及云南、福建、四川、江西、海南和广东等蔗区,造成甘蔗产量及其含糖量的极大损失[7-9],严重影响了蔗糖产业的持续稳定发展[10-11].
目前甘蔗褐锈病的防治仍以加强检疫、推广脱毒健康苗和选用抗病品种为主,以栽培管理和药剂防治为辅.因此,建立一种快速、高效、实用的甘蔗褐锈病早期检测技术,对病害防治具有重大意义.迄今为止,针对甘蔗黑顶柄锈菌的检测技术已有传统田间诊断、免疫学诊断、分子检测诊断等[12],其中传统田间诊断一直以来都是甘蔗锈病的主要检测方法,该方法主要通过外部形态观察发病症状,显微观察孢子形态确认是否感病,但耗时长,灵敏度较低,结果的准确性和可信度也在很大程度上取决于检测人员的技术水平和经验,很容易错过病害防治的最佳时期；免疫学诊断也存在耗时长,易出现假阳性等缺点[13]；比较而言,以PCR为基础的分子检测技术具有快速、灵敏度较高的优点.
甘蔗论文参考资料：
结论：甘蔗褐锈病菌巢式PCR分子检测方法建立为适合甘蔗论文写作的大学硕士及相关本科毕业论文，相关甘蔗糖蜜开题报告范文和学术职称论文参考文献下载。
