Department of Nursing, Tokai University, Bohseidai, Isehara-shi, Kanagawa, Japan.
J Biosci Bioeng. 2010 Jan;109(1):83-8. doi: 10.1016/j.jbiosc.2009.06.015. Epub 2009 Jul 14.
The present study aimed to optimize a real-time PCR assay to quantitate airborne fungi collected on a gelatin filter. In particular, the study optimized conditions for the DNA extraction and real-time PCR amplification to accurately measure airborne fungal concentrations. First, time of fine bead homogenization to extract the DNA from fungal cells was optimized to maximize the DNA yield and prepare the DNA suitable for sensitive and precise quantification by a subsequent real-time PCR analysis. Second, a condition for the real-time PCR amplification was optimized to successfully amplify and quantitate the extracted fungal DNA. In particular, a dilution ratio of the DNA extracts to be introduced to PCR was optimized to achieve an appropriate balance between mitigating PCR inhibition and securing detection sensitivity. Since concentrations of airborne fungi generally observed in indoor and outdoor environments (i.e., 10(1)-10(4) CFU m(-3)) were found to be near the limit of quantification by the generally-used molecular-based detection technique in conjunction with use of gelatin filters, optimizations of these conditions were found to be crucial. Our preliminary result showed that a culture-based method underestimated concentrations of airborne environmental fungi by 1 to 2 orders of magnitude compared to those characterized by the real-time PCR assay.
本研究旨在优化实时 PCR 分析方法,以定量采集于明胶滤膜上的空气中真菌。特别是,本研究优化了 DNA 提取和实时 PCR 扩增的条件,以准确测量空气中真菌的浓度。首先,优化了用于从真菌细胞中提取 DNA 的精细珠匀浆时间,以最大限度地提高 DNA 产量,并为后续的实时 PCR 分析做好准备,确保获得敏感和精确的定量结果。其次,优化了实时 PCR 扩增的条件,以成功扩增和定量提取的真菌 DNA。特别是,优化了要引入 PCR 的 DNA 提取物的稀释比例,以在减轻 PCR 抑制和确保检测灵敏度之间取得适当的平衡。由于室内和室外环境中通常观察到的空气中真菌浓度(即 10(1)-10(4) CFU m(-3))接近通常与明胶滤膜一起使用的基于分子的检测技术的定量极限,因此这些条件的优化至关重要。我们的初步结果表明,与实时 PCR 分析相比,基于培养的方法低估了空气中环境真菌的浓度,相差 1 到 2 个数量级。