芋疫霉菌的巢式PCR检测

Nested-PCR Detection of Taro Leaf Blight Pathogen Phytophthora colocasiae

【目的】建立芋疫霉菌快速、准确的PCR检测技术,为芋疫病流行规律监测和综合防控提供科学依据。【方法】根据芋疫霉菌与其他疫霉菌种类Ypt1基因序列差异,设计了1对芋疫霉菌PCR检测特异引物PCOF/PCOR,并对该引物的特异性、灵敏性和应用性进行了验证。【结果】在优化的反应体系与扩增条件下,PCOF/PCOR引物能特异性地从芋疫霉菌基因组DNA中扩增出1条172 bp的条带,而其他供试病原菌均无扩增条带。在25μL PCR反应体系中,PCOF/PCOR引物对芋疫霉菌基因组DNA的检测灵敏度为100 pg,而以疫霉菌Ypt1基因通用引物ph1F/Yph2R为第一轮引物,PCOF/PCOR为第二轮引物,进行巢式PCR扩增,能检测到10 fg芋疫霉菌基因组DNA,检测灵敏度提高了10 000倍。采用巢式PCR,可从芋疫病发病的叶片和未显症叶片组织中检测到芋疫霉菌,检出率分别为100%和57.5%。【结论】所建立的巢式PCR可应用于芋疫霉菌的快速、特异和高灵敏度检测。

【Objective】To develop a PCR assay for rapid and accurate detection,epidemiology information,and integrated disease management on Phytophthora colocasiae,the pathogen of taro phytophthora blight.【Method】A pair of species-specific primers,PCOF/PCOR,for P.colocasiae was designed based on the differences in Ras-related protein(Ypt1)gene sequence between P.colocasiae and other species in the same genus.The specificity,sensitivity and applicability of the primers were evaluated.【Result】With the optimized reaction conditions and amplification,PCOF/PCOR amplified only a single band of 172 bp with genomic DNA extracted from all P.colocasiae strains,while the other tested pathogens had no corresponding band.The sensitivity of conventional PCR method using PCOF/PCOR as primers was 100 pg of genomic DNA in a 25μL reaction solution.Whereas,the newly developed nested-PCR performed using Ypt1 gene universal primers ph1F/Yph2R for the first-round and PCOF/PCOR for the second-round increased 10 000-fold on the sensitivity to 10 fg.The nested-PCR methodology could positively detected P.colocasiae 100%in diseased leaves or 57.5%in symptom-free infected tissues.【Conclusion】The newly established nested-PCR assay could be used for rapid,specific and sensitive detection of P.colocasiae.