基因扩增联合测序法对痰标本分离的60株真菌病原学鉴定

Pathogenic identification of 60 fungal strains isolated from sputum specimens by gene sequencing

目的:应用PCR扩增真菌ITS2区基因,结合基因测序法对痰标本中分离的60株真菌进行病原学鉴别。方法:收集痰标本分离的真菌菌落60份及2种真菌标准菌株(白色念珠菌,烟曲霉菌)培养菌落,分别提取基因组DNA,半巢式PCR扩增ITS2区,扩增产物经测序后,结果与NCBI基因库中的核酸序列进行比对,确定致病菌的种属,并对其分布特征进行分析。结果 :2种真菌标准菌株的ITS2区基因序列比对结果与种属一致。痰标本中60株真菌测序鉴定结果为:曲霉菌属24株(其中烟曲霉菌17例、黑曲霉菌3例、黄曲霉菌2例、土曲霉菌1例、变色曲霉1例),青霉菌属3株(小刺青霉菌1株、娄地青霉菌1株、产黄青霉菌1株),念珠菌属29株(白色念珠菌20株、热带念珠菌3株、近平滑念珠菌2株、光滑念珠菌2株、鲁西坦念珠菌1株、季也蒙毕赤酵母菌1株),其它丝状菌还有暗孢节菱孢菌2株,淡紫拟青霉菌1株和串珠状赤霉菌1株。结论:基因测序法能快速、简便、准确地鉴别痰培养的真菌种属,为本地区呼吸道真菌性疾病病原菌流行病学调查及个性化治疗奠定基础。

Objective：60 fungal strains from sputum specimens was identified by using PCR to amplify fungal ITS2 region .Methods ：The 60 filamentous fungi isolated from sputum specimens were collected .The genomic DNA of the 60 fungal strains and 2 standard fungal strains（Candida albicans ,Aspergillus fumigatus）were extracted respectively ,and then ,the ITS2 region of them were amplified by semi-nested PCR ,the amplified products were sequenced ,the results were compared with the DNA sequences in GenBank of NCBI to determine the pathogenic fun‐gus species .The distribution characteristics was analysed .Results ：The genus and species identified by the sequences of the ITS2 regions of the 3 standard fungal strains were accord with the sequences in NCBI .The identified results of the 60 strains from the sputum specimens were as follows ：24 strains of Aspergillus（including 17 cases of Aspergil‐lus fumigatus ,3 cases of Aspergillus niger ,2 cases of Aspergillus flavus ,1 case of Aspergillus terreus and 1 case of Aspergillus variecolor ）;3 strains of Penicillium （including 1 case of Penicillium spinulosum ,1 case of Penicillium roqueforti ,1 case of Penicillium Chrysogenum ）;29 cases of Candida （20 cases of Candida albicans ,3 cases of Candi‐da tropicalis ,2 cases of Candida parapsilosis ,2 cases of Candida glabrata ,1 case of Candida lusitaniae and 1 case of Pichia guilliermondii）;other filamentous fungi including 2 cases of Arthriniu mphaeospermum ,1 case of Paecilomyces lilacinus and 1 case of Gibberella moniliformis ） .Conclusion：Gene sequencing is a rapid ,simple and accurate method to identify the genus and species of fungi cultured from sputum specimen .It layed the foundation for the epidemiology of local pathogenic fungi in respiratory tract disease and the personalized treatment .