Nanoporous Materials for Biological Application Research Group (NAMBAR), Sustainability Research Alliance, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia.
Folia Microbiol (Praha). 2011 Sep;56(5):459-67. doi: 10.1007/s12223-011-0070-9. Epub 2011 Sep 10.
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.
尽管出芽短梗霉在生物技术中具有重要意义，但它已成为一种机会性人类病原体，尤其是在免疫功能低下的患者中。目前，这种罕见的人类真菌病原体的临床检测依赖于形态学诊断，这可能会产生误导。因此，仍然缺乏针对出芽短梗霉的敏感和准确的定量分子检测方法。在本研究中，我们介绍了出芽短梗霉的显微镜观察结果，揭示了该真菌的表型可塑性。基于真菌的 18S 核糖体 RNA(rRNA)基因，设计了出芽短梗霉特异性引物和分子信标探针。比较了两种具有不同猝灭化学物质的探针，即 BHQ-1 和 Tamra，发现它们的扩增效率分别高达 104%和 108%。优化的定量实时 PCR(qPCR)检测方法可以检测和定量高达 1 pg 浓度的出芽短梗霉 DNA。两种检测方法在快速热循环模式下均表现出良好的性能参数。该分子检测方法具有作为出芽短梗霉引起的真菌感染早期检测的分子诊断工具的巨大潜力，值得在临床诊断中进一步研究。
