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

The overall goal of the following experiment is to develop a novel DNA extraction technique that combines mechanical DNA homogenization with PCR inhibitor removal and the gold standard and somatic extraction method to environmental samples along the poultry production continuum. This is accomplished by first dividing out an environmental sample into micro centrifuge tubes containing a mixture of beads to effectively homogenize the complex environmental samples. The second step is to place the tubes into a high powered homogenizer to efficiently release nucleic acids from the bacterial cells and the environmental matrix.

Next, the homogenate is used as the starting material for the gold standard enzymatic DNA extraction method through the PCR inhibition binding phase. The final step is to place the samples into A DNA extraction robotic workstation to provide the user with extracted DNA samples amenable to a host of downstream applications, ultimately quantitative PCR and microbiome analysis using the aluminum MiSeq platform, were used to show that this novel hybrid method provides the best combination of qualitative and quantitative assessments of total bacterial communities compared to processing samples with the mechanical or enzymatic extraction method alone. This method provides insight into the analysis of microbial communities along the poultry production continuum, but it can also be applied to other agricultural systems such as swine cattle or crop production that contains similarly complex environmental samples.

Demonstrating this procedure today will be Kaitlyn Griffin, a student in my laboratory. To begin the experiment, set a water bath to 95 degrees Celsius. Next, weigh out three replicates of 0.33 grams to analyze a total of one gram of room temperature, soil, or fecal material into a two milliliter lysing matrix ET-tube change gloves between samples.

Add 825 microliters of sodium phosphate buffer and 275 microliters of pre lysis solution or PLS to a sample tube. Then vortex the samples for 15 seconds after mixing centrifuge the tubes at 14, 000 Gs for five minutes. Decant the supernatant and add 700 microliters of buffer A SL.Then vortex the tube for five seconds.

Open one tube at a time to prevent contamination. Next, ensure that there is a headspace of 10%of the total volume in the conical tube before placing the samples into the fast prep 24 instrument. Homogenize the samples at a speed of six meters per second for 40 seconds.

Note the difference between fast prep 24 and vortexing after homogenization. Centrifuge the sample at 14, 000 Gs for five minutes. Transfer the supernatant to a sterile two milliliter micro centrifuge tube to the remaining sample in the lysing matrix ET-tube at an additional 700 microliters of a SL buffer re homogenize and centrifuge.

Using the previous settings, place plastic locking clips on the sample tubes to ensure they will not pop open and incubate the supernatants in a 95 degree Celsius water bath for five minutes. Next, open each micro centrifuge tube to release the pressure, recap the tubes and vortex them for 15 seconds. Centrifuge the samples at 14, 000 GS for one minute.

After centrifugation transfer 1.2 milliliters of the supernatant into a sterile two milliliter micro centrifuge tube. Then place the blister pack containing the tab directly over the open micro centrifuge tube and gently push the tab out of the blister pack into the tube. To avoid contact between the inhibit X tab and the tube, add one inhibit X tab to each sample.

Next, vortex the tubes until the samples turn to a uniformly off-white liquid. Incubate the samples for one minute at room temperature and centrifuge them at 14, 000 cheese for five minutes while avoiding any remaining particles at the bottom of the tubes. Transfer all liquid to a sterile 1.5 milliliter micro centrifuge tube.

Then centrifuge the tubes at full speed for five minutes. Before starting automated DNA purification, make sure the workstation contains the appropriate amount of filter tips and buffers. Next, add ellucian tubes and filter tubes to the rotor adapters and add 400 microliters of each sample to the middle slot of the rotor adapter.

Place the rotor adapters in the workstation centrifuge and ensure all of the micro centrifuge tube lids are properly secured within the rotor adapter. Add proteinase K solution to a sterile 1.5 milliliter micro centrifuge tube and place it into slot A on the workstation. Add two milliliter safe lock micro centrifuge tubes to the shaker section of the workstation, and ensure the lids of the sample tubes are securely placed.

Using the touch screen on the workstation, select the DNA stool human stool pathogen detection protocol and read through the subsequent screens to make sure the workstation was loaded correctly. Once all check screens are passed, select start to run the protocol. Then remove the samples from the rotor adapters.

Combine the three replicate purifications for an individual sample using a centrifugation evaporation based system. Re elute the sample to a final volume of 100 microliters with tris EDTA buffer. Finally, cap the samples and place them at minus 20 degrees Celsius until they are needed for downstream analysis.

This graph demonstrates that the mechanical method consistently provided the overall highest total bacterial normalized gene abundance estimates for fecal and litter samples. The enzymatic method had the lowest estimates in all cases. The novel hybrid extraction method produced the best combination of qualitative and quantitative total bacterial community estimates.

Of the three extraction methods tested. You now should have a better understanding of how to perform this novel, semi-automated hybrid DNA extraction method and see its utility in the qualitative and quantitative assessment of microbial communities from complex environmental samples.