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

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反应体系与程序条件下,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。

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.