2株临床分离烟曲霉菌株病原学检测及分子鉴定方法比较与分析  

Comparison and analysis of two strains of Aspergillus fumigatus from clinical isolates between pathogenic biology and molecular identification

作　　者：杜伟勤 王淑峰[2] 薛婷 DU Weiqin;WANG Shufeng;XUE Ting(Department of Clinical Laboratory,the People′s Hospital of Lvliang,Lvliang,Shanxi 033000,China;Department of Clinical Laboratory,the First Hospital of Shanxi Medical University,Taiyuan,Shanxi 030001,China;Department of Respiratory and Critical Care Medicine,the First Hospital of Shanxi Medical University,Taiyuan,Shanxi 030001,China)

机构地区：[1]吕梁市人民医院医学检验科,山西吕梁033000 [2]山西医科大学第一医院医学检验科,山西太原030001 [3]山西医科大学第一医院呼吸与危重症医学科,山西太原030001

出　　处：《检验医学与临床》2021年第20期2937-2941,共5页Laboratory Medicine and Clinic

基　　金：山西省高等学校科技创新项目(2020L0201、2020L0223);山西医科大学省级博士启动基金项目(SD1905);山西医科大学校级博士启动基金项目(XD1905)。

摘　　要：目的对2株临床分离的烟曲霉菌株进行病原学检测和分子鉴定,并对不同检测方法进行比较分析。方法收集2例侵袭性肺曲霉菌病(IPA)患者痰液标本,采用直接压片、亚甲蓝染色和改良GMS染色,以及分离、培养等病原学检测,应用rodA、anxc4、β-tub、ITS、cyp51A和cyp51B基因进行PCR、环介导等温扩增(LAMP)、分子克隆和Sanger测序。结果痰涂片镜检可见典型的烟曲霉鹿角样菌丝、分生孢子头和分生孢子;真菌显色培养基上菌落颜色由白色转为蓝绿色;rodA、anxc4、β-tub、ITS、cyp51A和cyp51B与Genbank数据库中烟曲霉相应序列的同源性均在99.7%以上;LAMP扩增产物肉眼可见白色沉淀,琼脂糖凝胶电泳呈典型梯度条带,HNB可视化检测结果呈淡蓝色,real-time LAMP呈“S”型扩增曲线。结论临床疑似烟曲霉感染时,可通过LAMP进行快速分子鉴定,辅助临床提升诊断效率,为临床治疗提供依据。Objective To detect two strains of Aspergillus fumigatus from clinical isolates using pathogenic biology and molecular identification,and to compare and analyze the different methods.Methods Sputum samples from patients with invasive pulmonary aspergillosis(IPA)were collected.Sputum smears were prepared and detected using direct compression,methylene blue staining method and modified GMS method to observe the morphological characteristics.The fungal isolation and culture techniques of pathogenic biology as well as the fungal chromogenic medium were performed.Meanwhile,conservative genes including rodA,anxc4,β-tub,ITS,cyp51A and cyp51B were examined using polymerase chain reaction(PCR),loop-mediated isothermal amplification(LAMP),molecular-cloning and Sanger sequencing.Results Typical morphological features of Aspergillus fumigatus including hyphae shaped like antlers,conidial heads and conidia.The colony colors of Aspergillus fumigatus changed from white to aquamarine blue on the fungal chromogenic medium.Compared with previously published nucleotide sequences of Aspergillus fumigatus related genes such as rodA,anxc4,β-tub,ITS,cyp51A and cyp51B in the Genbank database,the sequencing results in this study showed that the homology was more than 99.7%.The amplification products of LAMP could be tested by white precipitate visible to the naked eye,agarose gel electrophoresis showed typical gradient bands,and HNB visual detection results showed light blue,real-time LAMP showed an"S"type amplification curve.Conclusion The rapid and accurate molecular identification could be performed by LAMP from those with suspected Aspergillus fumigatus infection in order to improve the efficiency of the auxiliary clinical diagnosis and provide reference for clinical therapy.

关 键 词：烟曲霉 病原学检测 分子鉴定 环介导等温扩增 

分 类 号：R446.5[医药卫生—诊断学] R372[医药卫生—临床医学]