A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Mechanical Homogenization of Environmental Poultry Production Samples

1. Prior to extraction, set a water bath to 95 °C and allow the water bath time to reach that temperature.
2. Weigh out 0.33 g of soil or fecal material into a 2 ml Lysing Matrix E tube.
   1. Do not exceed 0.33 g of sample in the tube, since this will cause the following solutions to exceed the capacity of the tube.
   2. Thaw frozen samples to RT prior to weighing.
   3. In order to analyze a total of 1 g of soil/feces, weigh out 3 replicate 0.33 g samples for each individual environmental sample.
   4. Store samples at -20 &#....

Results

For this study, fresh fecal droppings and litter samples were recovered from a commercial broiler house (~25,000 birds) in the southeastern US. The broilers (Gallus gallus) were Cobb-500 crosses, and they were 59 days old at the time of sampling. Fresh fecal and litter samples were recovered from four distinct areas within the house (near cooling pad, near the waterer/feeder lines, in between the waterer/feeder lines, and near exhaust fans), and samples from each of these areas contained five pooled samples from.......

Discussion

The DNA extraction method used effected the quantitative and qualitative total bacterial community estimates for both the fecal and litter samples, supporting the sample analyses dependent nature of DNA extraction methods seen previously^1,3,6. For both the fecal and litter samples, the order of performance of the DNA extraction methods was different for the quantitative (Mechanical > Hybrid > Enzymatic) and the qualitative (Enzymatic > Hybrid > Mechanical) total bacterial community estimates. W.......

Materials

List of materials used in this article

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