Rothrock Michael J, Hiett Kelli L, Gamble John, Caudill Andrew C, Cicconi-Hogan Kellie M, Caporaso J Gregory
Egg Safety and Quality Research Unit, USDA-Agricultural Research Service;
Poultry Microbiological Safety and Processing Research Unit, USDA-Agricultural Research Service.
J Vis Exp. 2014 Dec 10(94):52161. doi: 10.3791/52161.
The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the "gold standard" enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.
DNA提取方案的效果可能高度依赖于所研究的样本类型以及后续进行的分析类型。鉴于新的细菌群落分析技术（如微生物组学、宏基因组学）在农业和环境科学中的应用越来越普遍，并且这些学科中的许多环境样本在物理化学和微生物学方面可能具有独特性（例如家禽生产领域的粪便和垫料样本），因此需要谨慎选择合适且有效的DNA提取方法。因此，专门开发了一种新型半自动混合DNA提取方法，用于处理环境家禽生产样本。该方法是两种主要DNA提取类型的结合：机械提取和酶提取。在用于粪便样本的“金标准”酶法DNA提取方法开始时，添加了两步强烈机械匀浆步骤（使用专门为环境样本配制的珠磨法），以增强从样本基质中去除细菌和DNA的能力，并提高革兰氏阳性细菌群落成员的回收率。一旦启动混合方法的酶提取部分，剩余的纯化过程就使用机器人工作站自动进行，以提高样本通量并减少样本处理误差。与严格的机械和酶法DNA提取方法相比，在处理家禽粪便和垫料样本时，考虑总细菌群落的定量（使用16S rRNA qPCR）和定性（使用微生物组学）估计，这种新型混合方法提供了最佳的综合整体性能。
