Fondazione IRCCS Cà Granda Ospedale Maggiore Policlinico, Pediatric Highly Intensive Care Unit, 20100 Milan, Italy.
Department of Clinical Sciences and Community Health, University of Milan, 20100 Milan, Italy.
Int J Mol Sci. 2018 Oct 20;19(10):3256. doi: 10.3390/ijms19103256.
Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for . Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples.
不同的步骤和条件已在文献中报道用于分析痰中的微生物群的 DNA 提取。我们旨在测试二硫苏糖醇（DTT）和痰液样本的酶处理，并确定最适合用于分析痰液微生物群的 DNA 提取技术。比较了 DTT 处理和无 DTT 处理在 DNA 提取产量和实时 PCR 16S rRNA 基因的重复性之间的中位值和变异系数。比较了有无溶菌酶和溶葡萄球菌酶的处理在实时 PCR 中的中位值。比较了两种基于酶的和三种基于珠粒的 DNA 提取技术的 DNA 提取产量、实时 PCR 16S rRNA 基因和微生物组分析。DTT 处理降低了 DNA 提取产量和实时 PCR 重复性之间的变异系数。溶葡萄球菌酶（0.18 或 0.36mg/ml）和溶菌酶处理增加了检测。一种基于酶的试剂盒提供了最高的 DNA 产量和 16S rRNA 基因实时 PCR，在 alpha 多样性指数方面没有显著差异。使用 DTT 和溶葡萄球菌酶/溶菌酶处理以及酶试剂盒的条件似乎更适合分析痰液样本的微生物组。
