Detection and identification of opportunistic Exophiala species using the rolling circle amplification of ribosomal internal transcribed spacers

作者：Najafzadeh, M. J.; Dolatabadi, S.; Keisari, M. Saradeghi; Naseri, A.; Feng, P.^*; de Hoog, G. S.

来源：Journal of Microbiological Methods, 2013, 94(3): 338-342.

DOI：10.1016/j.mimet.2013.06.026

• Exophiala
• Black yeasts
• Identification
• Rolling circle amplification

Deep infections by melanized fungi deserve special attention because of a potentially fatal, cerebral or disseminated course of disease in otherwise healthy patients. Timely diagnostics are a major problem with these infections. Rolling circle amplification (RCA) is a sensitive, specific and reproducible isothermal DNA amplification technique for rapid molecular identification of microorganisms. RCA-based diagnostics are characterized by good reproducibility, with few amplification errors compared to PCR. The method is applied here to species of Exophiala known to cause systemic infections in humans. The ITS rDNA region of five Exophiala species (E. dermatitidis, E. oligosperma, E. spinifera, E. xenobiotica, and E. jeanselmei) was sequenced and aligned in view of designing specific padlock probes to be used for the detection of single nucleotide polymorphisms (SNPs) of the Exophiala species concerned. The assay proved to successfully amplify DNA of the target fungi at the level of species; while no cross-reactivity was observed. Amplification products were visualized on 1% agarose gels to verify the specificity of probe-template binding. Amounts of reagents were minimized to avoid the generation of false positive results. The sensitivity of RCA may help to improve early diagnostics of these difficult to diagnose infections.

• 出版日期2013-9
• 单位河北医科大学; 中国人民解放军第二军医大学; 北京大学; 天津市中医药研究院附属医院; 中山大学