摘要
目的通过分离纯化、培养特性分析和表型特征鉴定来确定头癣患者病原体犬小孢子菌,检测与分析该菌株胞外酶活性和致病功能,通过基因扩增和序列测定,建立了基于ITS序列PCR快速诊断技术。方法采用PDA培养基划线培养,根据真菌学检查确诊犬小孢子菌感染,采用API-ZYM酶活检测系统对该犬小孢子菌临床株分别进行19种胞外酶活性检测;设计真菌5.8S rDNA和28S rDNA基因间隔序列特异性引物扩增并测序后,运用GenBank的BLAST软件搜索比对,从分子水平确诊犬小孢子菌感染。结果头部皮肤真菌镜检阳性,经培养鉴定为犬小孢子菌;19种胞外酶检测结果显示,12种胞外酶具有活性(其中脂酶2种、肽酶3种、水解酶2种、糖发酵酶5种);该菌株扩增的ITS序列基因片段大小为737 bp,比对结果显示为犬小孢子菌特异性序列。结论犬小孢子菌可导致头癣病,其临床症状及致病机制可能与其所分泌的12种胞外酶活性有关,结合培养特性和临床表现,通过PCR快速诊断技术,能够快速准确地鉴别出犬小孢子菌感染。
Objective To detect Microsporum canis in tinea capitis patients by purification, separation, culture characteristics analysis and phenotypic characterization identification. The extracellular enzyme activity and pathogenic function of the strain were detected and analyzed. A rapid PCR method based on ITS sequence was established by gene amplification and sequence analysis. Methods PDA medium was used for streak culture, and the Microsporum canis infection was confirmed according to the mycological examination. 19 kinds of extracellular enzyme activities were detected by API-ZYM enzyme activity detection system. Specific primers of 5.8 S rDNA and 28 S rDNA spacer sequence were designed and used for amplifying and sequencing. GenBank BLAST software was used to search and compare, and the infection of Microsporum canis was confirmed at molecular level. Results Mycological examination of patient’s head skin was positive, and it was identified as Microsporum canis by culture. 19 kinds of extracellular enzymes detection results showed that 12 kinds of extracellular enzymes were active(include2 kinds of lipases, 3 kinds of peptidases, 2 kinds of hydrolases and 5 kinds of sugar fermentases). The amplicon length of ITS regions in this isolate was 737 bp, and comparison result showed that it was a specific sequence of Microsporum canis. Conclusion Microsporum canis can cause tinea capitis, and its clinical symptoms and pathogenesis may be related to the activities of 12 kinds of extracellular enzymes. Combination of culture and clinical manifestations, as well as rapid diagnosis technology by PCR, can identify Microsporum canis infection rapidly and accurately.
作者
寿晓岚
朱永泽
胡庆丰
吕火烊
周永列
葛玉梅
SHOU Xiao-lan;ZHU Yong-ze;HU Qing-feng;LV Huo-yang;ZHOU Yong-lie;GE Yu-mei(Clinical Laboratory,Deqing People's Hospital,Deqing ,Zhejiang 313299,China)
出处
《中国卫生检验杂志》
CAS
2019年第3期269-271,274,共4页
Chinese Journal of Health Laboratory Technology
基金
浙江省医药卫生科技项目(2017KY004)
国家卫生和计划生育委员会科研基金(WKJ-ZJ-1604)
湖州市科技计划项目(2017GY55)
关键词
犬小孢子菌
胞外酶
ITS序列
快速诊断
Microsporum canis
Extracellular enzymes
ITS sequence
Rapid diagnosis
