菌落PCR技术快速鉴别丝状真菌的实验研究  

Rapid identification of filamentous fungi by colony PCR

作　　者：张晓利[2] 吕雪莲 沈永年[4] 吕桂霞[4] 王淼淼[4] 葛一平[1] 刘维达[1] 

机构地区：[1]中国医学科学院北京协和医学院皮肤病研究所真菌科,南京210042 [2]暨南大学临床医学博士后流动站,广州510632 [3]大连皮肤病医院 [4]苏州大学第一附属医院

出　　处：《中华皮肤科杂志》2011年第8期556-559,共4页Chinese Journal of Dermatology

基　　金：科技部重大专项（2008zxlo004-002）;临床重点学科建设项目[2007-2009年度卫生部部属（管）医院临床学科重点项目（第7号）];卫生部公益性行业科研专项经费项目（200802032）

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。Objective 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 con- ventional 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 con- ventional 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.

关 键 词：有丝分裂孢子真菌 菌落PCR 限制件内切醢图谱浃 序列比对 

分 类 号：R440[医药卫生—诊断学]