红掌毁灭炭疽菌的分子检测

Molecular detection of Colletotrichum destructivum in Anthurium andraeanum

根据GenBank中炭疽属Colletotrichum不同种的ITS序列差异,设计了毁灭炭疽菌Colletotrichumdestructivum的特异性引物F1/ITS4,由此建立的PCR检测体系可以从80个毁灭炭疽菌菌株中扩增得到1条486bp的特异性条带,而扩增其他近似或相关种的菌株时没有相应的特异性条带。该检测体系对毁灭炭疽菌基因组DNA的扩增灵敏度达到10pg。将引物F1/ITS4与ITS区通用引物进行套式PCR扩增后,检测灵敏度至少提高10 000倍,每克土中含有200个毁灭炭疽菌分生孢子时即可检测出。进一步利用此检测体系对携带病原菌的灌溉水、发病组织进行检测,均能快速准确地检测出病原菌。

Colletotrichum destructivum is the pathogen of anthracnose in Anthurium andraeanum.Based on internal transcribed space（ITS） sequences of Colletotrichum genus,a pair of specific primers（F1 and ITS4） to detect C.destructivum was synthesized.The primer sets amplified a single PCR band of 486 bp with DNA extracted from C.destructivum isolated from A.andraeanum,while other relative strains within different species had no corresponding band.The detection sensitivity was 10 pg of genomic DNA.When using ITS1/ITS4 as the first round primes and F1/ITS4 as the second round primes,the detection sensitivity increased 10 000-fold to 10 fg.The detection sensitivity for the soil pathogens was 200-conidia per gram soil.The PCR-based method developed here could stably and quickly detect the pathogen from water samples and diseased plants.