5-carboxytetramethylrhodamine-succinimidyl-ester and Opportunistic-Infections

Despite the great importance of Aureobasidium pullulans in biotechnology, the fungus had emerged as an opportunistic human pathogen, especially among immunocompromised patients. Clinical detection of this rare human fungal pathogen presently relies on morphology diagnosis which may be misleading. Thus, a sensitive and accurate quantitative molecular assay for A. pullulans remains lacking. In this study, we presented the microscopy observations of A. pullulans that reveals the phenotypic plasticity of the fungus. A. pullulans-specific primers and molecular beacon probes were designed based on the fungal 18S ribosomal RNA (rRNA) gene. Comparison of two probes with varied quencher chemistry, namely BHQ-1 and Tamra, revealed high amplification efficiency of 104% and 108%, respectively. The optimized quantitative real-time PCR (qPCR) assays could detect and quantify up to 1 pg concentration of A. pullulans DNA. Both assays displayed satisfactory performance parameters at fast thermal cycling mode. The molecular assay has great potential as a molecular diagnosis tool for early detection of fungal infection caused by A. pullulans, which merits future study in clinical diagnosis.

Topics: Ascomycota; Base Sequence; Biological Assay; DNA Probes; DNA, Fungal; Early Diagnosis; Humans; Immunocompromised Host; Microscopy; Molecular Sequence Data; Mycoses; Opportunistic Infections; Phenotype; Phylogeny; Real-Time Polymerase Chain Reaction; Rhodamines; RNA, Ribosomal, 18S