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ARS Home » Southeast Area » Athens, Georgia » U.S. National Poultry Research Center » Egg Safety & Quality Research » Research » Publications at this Location » Publication #306557

Research Project: Genetic Analysis of Poultry-Associated Salmonella enterica to Identify and Characterize Properties and Markers Associated with Egg-Borne Transmission of Illness

Location: Egg Safety & Quality Research

Title: A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples

Author
item Rothrock, Michael
item Hiett, Kelli
item Gamble, John - Oregon State University
item Caudill, Andrew - University Of Georgia
item Cicconi-hogan, Kellie - Former Ars Employee
item Caproaso, Gregory - Northern Arizona University

Submitted to: Journal of Visualized Experiments
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/10/2014
Publication Date: 3/31/2014
Citation: Rothrock Jr, M.J., Hiett, K.L., Gamble, J., Caudill, A., Cicconi-Hogan, K., Caproaso, G.J. 2014. A hybrid DNA extraction method for the qualitative and quantitative assessment of bacterial communities from poultry production samples. Journal of Visualized Experiments.94:e52161, doi: 10.3791/52161.

Interpretive Summary: 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.

Technical 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.