利用真菌通用引物ITS1/ITS4,EF1-728F/EF1-986R对4种杨树溃疡病病原菌株和环境样本的核糖体DNA内部转录间隔区ITS序列和转录延伸因子EF-1α基因序列分别进行普通和多重PCR检测,再进行测序比对分析。结果表明:退火温度为55.6 ℃,各引物终浓度为0.2 μmol·L-1,可以成功地扩增出菌株和环境样本的目的条带,并通过测序比对鉴定出多种来自培养和环境病害组织中的溃疡病病菌,多重PCR能够检测到1 ng的基因组DNA。研究结果将为建立溃疡病病原多重PCR鉴定技术和以环境溃疡病病害样本为直接检测鉴定对象的高通量的基因芯片检测方法奠定基础。
Fungal pathogens causing poplar canker diseases are cosmopolitan in species, and have a wide range of hosts. These pathogens have diverse anamorphs and their morphology overlaps with each other; Their teleomorphs are hardly discovered in nature. Furthermore, the identification of these pathogens is usually limited by the geography, host and taxonomic knowledge. Therefore, a culture-independent method used to identify determine pathogens is expected to be developed for field diagnostics. Through amplifying, sequencing and analyzing multiplex nucleic acid templates and genetic marked target sequences, a multiplex PCR technique has been established and used to directly detect various environmental samples. In this study, four pathogen strains and environmental samples were amplified using fungal universal primers ITS1/ITS4 and EF1-728F/EF1-986R by general PCR and multiplex PCR. The amplicons were sequenced, and then aligned in GenBank. Our result showed that the multiplex PCR was able to successfully amplify the target gene and identify the pathogens from environmental samples in the condition of an annealing temperature of 55.6 ℃ and primers final concentration of 0.2 μmol·L-1. The multiplex PCR could amplify the target at the concentration level of approximately 1ng of genomic DNA. This method would provide a useful tool for the identification of canker pathogens by the multiplex PCR and the high-throughput DNA microarray detection of environmental samples.