应用反向线点杂交技术鉴定临床常见曲霉属和毛霉目真菌

中国真菌学杂志　 2011, Vol. 6 　Issue (5): 261-266.

应用反向线点杂交技术鉴定临床常见曲霉属和毛霉目真菌

赵作涛, 李丽丽, 王晓阳, 万喆, 陈伟, 李若瑜

北京大学第一医院皮肤性病科, 北京大学真菌和真菌病研究中心, 北京100034

收稿日期:2011-01-28 出版日期:2011-10-28 发布日期:2011-10-28
通讯作者: 李若瑜,E-mail:lrymm@medmail.com.cn E-mail:lrymm@medmail.com.cn
作者简介:赵作涛,男(汉族),博士,助理研究员.E-mail:zhaozuotao@yahoo.com
基金资助:
卫生部公益基金(200802026);北京大学第一医院青年基金

Simultaneous identification of Aspergillus and Mucorales species by reverse line blot(RLB) hybridization assay

ZHAO Zuo-tao, LI Li-li, WANG Xiao-yang, WAN Zhe, CHEN Wei, LI Ruo-yu

Research Center of Medical Mycology and Mycoses, Department of Dermatology and Venerology, First Hospital, Peking University, Beijing 100034, China

Received:2011-01-28 Online:2011-10-28 Published:2011-10-28

摘要/Abstract

摘要： 目的应用反向线点杂交技术(reverse line blot hybridization,RLB)快速鉴定临床常见的曲霉属和毛霉目真菌。方法收集我院真菌和真菌病研究中心保存的5种曲霉菌(烟曲霉、黄曲霉、黑曲霉、土曲霉、构巢曲霉)和7种毛霉目真菌(冻土毛霉菌、总状毛霉菌、卷枝毛霉菌、少根根霉、小孢根霉、微小根毛霉、伞状犁头霉),共计98株菌株。利用真菌通用引物ITS1和ITS4对菌株进行PCR扩增,用12个真菌种特异性探针与扩增后产物进行反向线点杂交。将RLB结果与真菌传统形态学鉴定结果、ITS区DNA测序结果进行比较。结果 RLB可以正确鉴定98株实验菌株,与形态学方法和ITS区测序方法鉴定结果100%一致,种特异性探针之间未见交叉杂交,显示出该方法的高度敏感性和特异性。8株阴性对照菌株(白念珠菌、茄病镰刀菌、尖端赛多孢、马尔尼菲青霉、疣状瓶霉、棒曲霉、日本曲霉以及雅致小克银汉霉),使用RLB方法无法鉴定。通过烟曲霉基因组DNA浓度10倍倍比稀释法验证RLB的敏感性为1.8×10^-3 ng/μL。结论 RLB技术为实验室早期快速诊断、鉴定临床常见的曲霉属和毛霉目真菌提供参考。

关键词: 曲霉, 毛霉, PCR, 反向线点杂交

Abstract: Objective To Rapidly differentiate Aspergillus and Mucorales species by reverse line blot hybridization(RLB).Methods Totally 98 isolates including five Aspergillus strains(Aspergillus fumigatus,Aspergillus flavus,Aspergillus niger,Aspergillus terreus and Aspergillus nidulans) and seven Mucorales species(Mucor heimalis,Mucor racemosus,Mucor cercinelloidea,Rhizopus arrhizus,Rhizopus microsporus,Rhizomucor pusillus and Absidia corymbifera) were obtained from Research Center for Medical Mycology and Mycoses,Peking University.ITS1 and ITS4 fungal universal primers were chosen for PCR amplification,and the amplified products were used for reverse line blot hybridization with 12 fungal specie-specific probes.RLB data were compared with those by traditional fungal morphology and ITS sequencing methods.Results RLB showed high sensitivity and specificity,with 100% correct identification percentage of all the isolates and no cross hybridization between the species-specific probes.Eight negative control stains(Candida albicans,Fusarium solani,Scedosporium apiospermum,Penicillium marneffei,Exophiala verrucosa,Aspergillus clavatus,Aspergillus japonicus and Cunninghamella elegans) also showed negative by RLB.The analytical sensitivity of RLB was 1.8×10^-3 ng/μL by 10-fold serial dilution of Aspergillus fumigatus genomic DNA.Conclusions The RLB assay provides a rapid and reliable option for laboratory diagnosis and identification of Aspergillus and Mucorales species.

Key words: Aspergillus, Mucorales, PCR, Reverse line blot(RLB) hybridization

中图分类号:

赵作涛, 李丽丽, 王晓阳, 万喆, 陈伟, 李若瑜. 应用反向线点杂交技术鉴定临床常见曲霉属和毛霉目真菌[J]. 中国真菌学杂志, 2011, 6(5): 261-266.

ZHAO Zuo-tao, LI Li-li, WANG Xiao-yang, WAN Zhe, CHEN Wei, LI Ruo-yu. Simultaneous identification of Aspergillus and Mucorales species by reverse line blot(RLB) hybridization assay[J]. Chinese Journal of Mycology, 2011, 6(5): 261-266.

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