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首页> 外文期刊>Journal of Residuals Science & Technology >Optimization of a Rapid DNA Extraction and Purification Protocol for Wastewater Biosolids
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Optimization of a Rapid DNA Extraction and Purification Protocol for Wastewater Biosolids

机译：废水生物固体中快速DNA提取和纯化方案的优化

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The aim is to optimize a rapid DNA extraction protocol for biosolids that provides high recovery and purity, and to evaluate potential biases on both quantitative and qualitative microbial analyses. A protocol with 3-step sequential DNA extractions using a modified lysis buffer and purification by a commercial DNA kit was developed and compared to a previously known high recovery solvent-based extraction method, a commercial soil DNA kit with single extraction, and the same commercial kit protocol except extracted with the modified lysis buffer. The developed protocol showed more than 7 times DNA recovery compared to the commercial kit, and comparable E. coli concentrations to the solvent-based method. Direct adoption of the commercial soil kit showed significantly lower recovery and underestimation of E. coli compared to the high recovery protocols. A simple switch of the lysis buffer also improved the DNA recovery by 5 times. Species diversity indexes from pyrosequencing analysis on the other hand showed scatter results from all protocols and only as much as 76% similarity was observed among any paired protocols. Phylogenetic analysis showed a shift of dominance as DNA recovery increases. Overall, this work shows that the developed protocol allows extraction of high quality and quantity DNA within 3 hr. However, qualitative comparisons of species structure may still vary.
机译：目的是为生物固体优化快速DNA提取方案，以提供高回收率和纯度，并评估定量和定性微生物分析的潜在偏差。制定了使用改良的裂解缓冲液进行三步连续DNA提取并通过商业DNA试剂盒纯化的方案，并将其与以前已知的基于高回收率溶剂的提取方法，单次提取的商业土壤DNA试剂盒以及相同的商业方法进行了比较试剂盒方案，不同之处在于用改良的裂解液提取。与市售试剂盒相比，开发的方案显示出超过7倍的DNA回收率，并且大肠杆菌浓度与基于溶剂的方法相当。与高回收率方案相比，直接采用商业化土壤试剂盒显示出大大降低了大肠杆菌的回收率和低估了大肠杆菌。裂解缓冲液的简单切换也使DNA的回收率提高了5倍。另一方面，焦磷酸测序分析的物种多样性指数显示了所有方案的分散结果，在任何成对方案之间仅观察到高达76％的相似性。系统发育分析显示，随着DNA回收率的提高，优势地位发生了变化。总的来说，这项工作表明，开发的方案可以在3小时内提取高质量和高数量的DNA。但是，物种结构的定性比较仍可能有所不同。

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