Establishment of a real-time fluorescence quantitative PCR method for the detection of clinical Exophiala dermatitidis

Abstract

Abstract: Objective To establish a novel fluorescent quantitative PCR (qPCR) method for the detection of Exophiala dermatitidis based on TaqMan probe technology.Methods Specific primers and fluorescent labeling probes were designed, according to the sequence of internal transcribed spacer (ITS) regions of E.dermatitis genomic sequence (GenBank:JN675373.1). And the condition of qPCR was optimized.E.dermatitis isolated from clinical specimens were tested as positive control, and other species of fungi and bacteria were used as negative control, to evaluate the detective effect of specificity, sensitivity and repeatability.Results In this study, primers and probes were designed to amplify Exophiala dermatitidis specific sequence, and Exophiala dermatitidis isolated from clinical specimens obtained obvious amplification curve in the reaction, whereas the 20 negative control strains, such as Exophiala spinous,Aspergillus fumigatus,Candida albicans,Cryptococcus neoformans,Penicillium marneffei,et al were not amplified in the CT value≤38 range. The qPCR exhibited high sensitivity (10 Cp/μL) and a good linearity from 10³ to 10⁷ copies (R²=1.000).Conclusion We successfully established a quantitative PCR method with high specificity, sensitivity, good repeatability for detection of E.dermatitis.The method could contribute to the early diagnosis of E.dermatitis infection and targeted therapy.

Key words: Exophiala dermatitis, ribosomal genes, fluorescence quantitative polymerase chain reaction

CLC Number:

R379.9

WANG Fang, MU Chao, ZHAO Jing-ya, GAO Li, HU Xiao-hua, HAN Li, TIAN Shu-guang. Establishment of a real-time fluorescence quantitative PCR method for the detection of clinical Exophiala dermatitidis[J]. Chinese Journal of Mycology, 2013, 8(6): 321-324.

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