摘要目的 探讨菌落PCR在检测病原性丝状真菌方面的应用价值.方法 初步建立用于丝状真菌的菌落PCR检测技术,用19种丝状真菌标准株进行验证,所有菌落PCR扩增产物进行测序,并选取8种菌株的菌落PCR产物和酶切结果与常规PCR进行比较,检测其准确性和可靠性.结果 19株菌中有16株(84.2%)菌落PCR成功扩增内转录间隔(ITS)区,ITS区基因序列分析鉴定菌种正确,与NCBI数据库中相同菌种的相似度为96%～100%;与常规PCR进行比较的8株菌中,除构巢曲霉菌落PCR扩增结果为阴性外,其他菌种菌落PCR产物及酶切条带与常规PCR基本一致.结论 与常规PCR相比,菌落PCR检测丝状真菌操作简单,省时省力,鉴定菌种具有较高的准确性和可靠性,可以用于丝状真菌的快速鉴定.

abstractsObjective To estimate the application value of colony PCR in the detection of pathogenic filamentous fungi. Methods Colony PCR was established and performed to amplify the internal transcribed spacer (ITS) region of 19 species (strains) of filamentous fungus followed by sequencing analysis. At the same time, DNA extracts from 8 of the 19 species of filamentous fungus were subjected to conventional PCR. Hha I and Hinf I endonucleases were used for restriction fragment length polymorphism (RFLP) analysis of the conventional and colony PCR products. Comparison analysis was carried out between the colony and conventional PCR. Results Of the 19 strains, 16(84.2%) yielded positive results by colony PCR; sequence analysis of the PCR products of ITS region revealed a 96% - 100% similarity with the reference sequence (NCBI database)of corresponding fungi. The amplification product length and RFLP profile of these products from the 8 species of filamentous fungus, except for those from Aspergillus nidulans, were consistent between the colony and conventional PCR. Conclusions Compared with conventional PCR, colony PCR-based detection of filamentous fungi is easy to operate, time and labor-saving, with high accuracy and reliability, and can be applied to the rapid identification of filamentous fungi.