一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

摘要

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

摘要

的DNA提取协议的功效可以是高度依赖于样品被调查的的类型和执行的下游分析的类型。考虑到采用新的细菌群落分析技术（ 例如，microbiomics，宏基因组学）越来越流行在这些学科范围内的农业和环境科学等诸多环境样品可能是生理化学和微生物学的独特（ 如粪便和垫料/寝具样本家禽生产谱），适当和有效的DNA提取方法需要慎重选择。因此，一种新型的半自动混合DNA提取方法与环境家禽生产样品使用专门开发的。该方法是DNA提取的两种主要类型的组合:机械和酶。两步强烈的机械同质化的步骤（使用珠跳动专门针对环境下制定TAL样本）加入到"金标准"的酶的DNA提取方法的开始的粪便样品来增强去除细菌和DNA从样品基质和改善革兰氏阳性细菌的社区成员的恢复。一旦混合方法的酶法提取部分开始，剩下的净化过程是使用机器人工作站，以增加样品通量，降低样加工误差的自动化。在比较严格的机械和酶的DNA提取方法，这种新的混合方法加工的家禽粪便和垃圾样品时，考虑提供定量时（使用16S rRNA基因定量PCR）和定性（使用microbiomics）的总细菌群落的估计最佳的整体性能相结合。

引言

When analyzing complex clinical or environmental samples (e.g., feces, soils), there are two main methodologies used for the extraction of DNA. The first is a mechanical disruption of the matrix using an intense bead-beating step, while the second is an enzymatic disruption of the matrix to chemically release bacterial cells and inhibit PCR inhibitors from the matrix simultaneously. Given the different means by which these two types of extraction methods work, it is not surprising that previous studies demonstrated that the appropriate DNA extraction method is both highly sample and analysis dependent. Comparative DNA extraction studies previously showed that some methods are more appropriate for improved DNA quality and quantity from environmental samples^1-3, while others demonstrated improvements for community-level analyses such as denaturing gradient gel electrophoresis (DGGE)^4-6, terminal restriction fragment length polymorphism (T-RFLP)⁷, automated ribosomal intergenic spacer analysis (ARISA)⁸, and phylogenetic microarrays⁹. Therefore, appropriate DNA extraction methods need to be used, or developed, according to the types of environmental samples and the types of analyses being performed on those samples, especially given the recent advancements in bacterial community analyses.

Next generation sequencing, in conjunction with more quantitative community assessments (e.g., quantitative PCR (qPCR)), is becoming more prevalent in the environmental and clinical sciences, however, very little research has been performed to determine the effect of DNA extraction methods on these data sets. Most DNA extraction comparison studies dealt with microbiomic community estimates from human or human model samples^10,11, not agricultural animal samples. The few poultry-focused next generation sequencing studies dealt with specific metagenomic^12,13 or microbiomic¹⁴ questions; they did not discuss the effect of DNA extraction method on the resulting microbiomic analyses. Considering the complex nature of environmental samples related to poultry production (e.g., feces, litter/bedding, pasture soil), DNA extraction methods need to be carefully selected. Poultry-related environmental samples are known to contain large numbers of PCR inhibitors and up to 500-fold DNA extract dilutions have been required for PCR and subsequent downstream analysis^15-17. Therefore it is essential that DNA extraction methods be optimized for these types of samples in order to not only physically disrupt the matrix, but also to be able to reduce/eliminate the large number of inhibitors that are present.

The QIAamp DNA Stool Mini Kit, an enzymatic extraction method, has been considered the “gold standard” when extracting DNA from difficult gut/fecal samples^1,18,19 and has been applied successfully to poultry environmental samples^8,14. The enzymatic removal of PCR inhibitors through the use of a proprietary matrix is one of the greatest advantages of using this method for these types of environmental samples, as is the ability to significantly improve throughput (and reduce sample processing error) using automated workstations. One major disadvantage is the lack of a mechanical homogenization step to physically disassociate bacterial cells from the environmental matrix. When testing gut and fecal samples of non-poultry origin, the addition of a bead-beating or mechanical disruption step within a DNA extraction protocol significantly increased extraction efficiency⁹, DNA yield/quality^1,4,5 and significantly improved downstream community analyses in terms of richness, diversity, and coverage^5,6,11. These studies compared not only mechanical bead-beating methods to the “gold standard” enzymatic method, but some also added the mechanical bead-beating step to the enzymatic protocol to improve results^6,9,11.

According to the results from the above studies, bacterial community analyses (both qualitative and quantitative) could be improved from poultry-related environmental samples through the addition of a mechanical homogenization step to the enzymatic method. Therefore, the goal of this study was twofold: (1) to develop a novel DNA extraction technique that utilizes the most desirable aspects of both the mechanical (powerful homogenization step) and enzymatic (PCR inhibitor removal and automation) extraction methods and (2) compare the quantitative (via qPCR) and qualitative (via microbiomics) bacterial community assessments of this novel method to representative mechanical and enzymatic methods.

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研究方案

环境家禽生产样品1.机械同质化

1. 在提取之前，设定一水浴中以95℃，并让水浴时间达到该温度。
2. 称出0.33克土壤或粪便物质的到2ml裂解矩阵E管。
   1. 不超过0.33克在管样品的，因为这会导致以下的解决方案，以超过所述管的容量。
   2. 解冻冷冻样品RT之前称重。
   3. 为了共1克土壤/粪便进行分析，称出3复制0.33克样本为每个单独的环境样本。
   4. 商店样品在萃取前-20℃圆锥形基质管内，如果需要的话。
3. 添加825微升磷酸钠缓冲液和275微升的PLS溶液到样品管中。混合使用涡旋为〜15秒，然后离心样品在14000×g离心5分钟。
4. 倒出superna坦并加入700μlBuffer ASL的。混合使用涡旋5秒。
   1. 确保有顶部空间（〜10％总体积）在锥形管可用在这一点上。如果没有顶部空间，该管将有一种倾向在接下来的均化步骤可能导致交叉污染和/或样品损失泄漏。
5. 将样品成FastPrep 24仪器，并且使样品均质化，在6.0米/秒，40秒的速度。
6. 离心匀浆样品在14000×g离心5分钟。将上清液转移到一个无菌的2ml微量管中。
7. 为了最大限度地提高来自样品的DNA回收率，重复步骤1.4至1.6，将上清液合并到同一无菌，加入2ml微量离心管中。

从样品匀浆抑制剂2.酶抑制

注:此协议使用的QIAamp DNA凳子套件。

1. 孵育上清液在95℃水浴中5分钟，以最大化从上清液内的任何剩余的细胞中的DNA的恢复。
   1. 孵育在70℃下对含有大部分革兰氏阴性生物体的样本。然而，如果革兰氏阳性生物体存在（这是与家禽粪便样品的情况下），孵育在95℃。
   2. 使用在离心管塑料锁定夹以确保该管不会"弹出"开放式和可能丢失的样品体积作为压力可以在这些密封的离心管中建立起来的。
2. 打开每个微量离心管中以释放压力，再盖的微量离心管，并混合使用涡旋15秒。
3. 离心样品以14000×g离心1分钟，取出1.2上清液毫升，并将其放置到一个新的无菌的2ml微量管中。
4. 加1 InhibitEx标签到每个样品中，并混合使用涡旋直到样品变得均匀白色/灰白色液体。
   1. 避免吨ouching所述InhibitEx标签而将其放入装有样品的离心管中。要做到这一点，将直接包含的标签在开放的离心管的泡罩包装，轻轻推离卡的泡罩包装，并移入离心管中。
5. 孵育在室温（〜25℃）的样品1分钟，离心机在14000×g离心5分钟。
6. 所有的液体转移到一个无菌的1.5 ml离心管和离心机在14000×g离心5分钟。
   1. 避免传输液体时可能已沉淀在离心管的底部，在步骤2.5的末端任何剩余的颗粒。

使用QIAcube工作站机器人工作站3.自动化DNA纯化

注:塑料消耗品的数量，在离心机内的样本转子适配器的结构中，和缓冲器/溶液的所需体积依赖于NUMBER是正在运行的样品。

1. 加入洗脱管和过滤管的转子适配器中的适当插槽。对于每个样品，加入400微升至转子适配器的中间槽。根据被纯化的样本数将转子适配器在工作站离心机在正确安排。
   1. 确保所有的离心管盖子被正确地固定在转子适配器中因为不这样做可能会导致在的离心步骤的纯化方案之一剪切。
2. 加入1000微升和200微升过滤提示所需数量的工作站，并填写所提供的缓冲瓶缓冲所需音量。
   注意:需要这种纯化方案（AL，AW1，AW2，和AE）的缓冲器都包含所述的QIAamp DNA粪便Mini试剂盒之内。用户需要提供所需要的AW BU的100％的乙醇ffers并且如在纯化过程中所用的溶液。
3. 添加提供的蛋白酶K的解决方案所需要的量进入无菌1.5 ml离心管，并将其放置到工作站上插槽A。此外，添加所需数量（等于样本的数量被净化）2毫升安全锁的离心样品管的RB到工作站的摇床截面。
   1. 保证样品管的盖子被安全地放置在工作站上的相应插槽中，因为不这样做将导致一个错误，当机器开始扫描工作站，以确保所有需要的塑料和液体可用于要求运行。
4. 使用触摸屏的工作站上，选择DNA凳 - 人类凳 - 病原体检测协议，并通过后续屏幕阅读，以确保工作站被正确加载。一旦所有的检查屏幕被传递，选择开始运行这个协议。
   1. 如果从超过12个样品中提取DNA，开始均质化处理（步骤1）为下一个组样品中，由于12个样本的运行花费〜72分钟，以完成在工作站上。
5. 从转子适配器在-20℃，直到需要用于后续下游分析取出样品，帽他们，和地点。
   1. 在这一点上，结合了3个重复纯化用于使用离心分离/蒸发为基础的系统的个体的样品（总分析量= 1克）。结合的重复和重新洗脱至100微升的Tris-ETDA缓冲器的最终体积。

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结果

在这项研究中，新鲜的粪便排泄物和垃圾样品从商业肉鸡舍（〜25000鸟）在美国东南部恢复。肉鸡（ 鸡内金 ）是柯布-500十字架，他们分别为59日龄，在采样的时间。新鲜的粪便和垃圾样本来自四个不同的区域恢复的房子内（靠近散热垫，附近的饮水器/馈线，在饮水器/馈线之间，靠近排风扇），并从这些地区的样本中含有5汇集样品从该区域内。从房子的四个方面的样本中提取定量/定性分?...

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讨论

所使用的DNA提取方法影响的定量和定性的总细菌群落估计为粪便和垃圾样本，配套样品分析的DNA提取方法依赖性质^1,3,6以前见过。对于这两种粪便和垃圾样本的DNA提取方法的性能订的是定量（机械>混合>酶）和质（酶>混合>机械）总细菌群落的估计不同。而混合方法没有产生最高定量或定性的估计，在两种情况下，混合方法产生的统计学相似性能最高的提取方法（ 图1，表2）...

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材料

Name	Company	Catalog Number	Comments
Lysing Matrix E tube	MPBio	6914-050	Different sizes available and the last 3 numbers of the cat. No. indicate size (-050 = 50 tubes, -200 = 200 tubes, -1000 = 1,000 tubes)
Sodium Phosphate Solution	MPBio	6570-205	Can be purchased individually, or also contained within the FastDNA Spin Kit for feces (Cat. No. 116570200)
PLS Buffer	MPBio	6570-201
Buffer ASL (560 ml)	Qiagen	19082
FastPrep 24 homogenizer	MPBio	116004500	48 x 2 ml HiPrep adapter (Cat. No. 116002527) available to double throughput of mechanical homogenization step
QIAamp DNA Stool Mini Kit	Qiagen	51504
QIAcube24 (110V)	Qiagen	9001292	Preliminary results show that QIAcube HT (Cat. No. 9001793) can be used to improve throughput, but different consumables are required of this machine and more comparative work needs to be done.
Filter-Tips, 1,000 ml (1024)	Qiagen	990352
Filter-Tips, 200 ml (1024)	Qiagen	990332
QIAcube Rotor Adapters (10 x 24)	Qiagen	990394	For 1.5 ml microcentrifuge tubes included with in the rotor adapter kit there is an alternative.  It is Sarstedt Micro tube 1.5 ml Safety Cap, Cat. No. 72.690
Sample Tubes RB (2 ml)	Qiagen	990381	Alternative: Eppendorf Safe-Lok micro test tube, Cat. No. 022363352