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P3-59 Comparative Analysis of Genomic DNA Extraction Strategies from Gouda Cheese

Wednesday, August 3, 2016
America's Center - St. Louis
Sartaj S. Narula, Illinois Institute of Technology, Bedford Park, IL
Christina K. Carstens, U. S. Food and Drug Administration, Bedford Park, IL
Joelle K. Salazar, U. S. Food and Drug Administration, Bedford Park, IL
Vriddi M. Bathija, Illinois Institute of Technology, Bedford Park, IL
Kristin M. Schill, U. S. Food and Drug Administration, Bedford Park, IL
Mary Lou Tortorello, U. S. Food and Drug Administration, Bedford Park, IL
Introduction: The safety and quality of cheeses can be influenced by their native microbial populations. Procedures used to determine native microbiota include PCR coupled to next-generation sequencing technology. Specific quality and quantities of microbial DNA are necessary to ensure the effectiveness of these procedures.

Purpose: The purpose of this study was to compare five DNA extraction strategies from Gouda cheese to identify a high-yield method for use in downstream procedures.

Methods: Methods for DNA extraction included (1) PowerFood® Microbial DNA Isolation Kit (MO BIO Laboratories), (2) PowerSoil® DNA Isolation Kit (MO BIO Laboratories), (3) DNeasy Blood & Tissue Kit (Qiagen), (4) DNeasy Blood & Tissue Kit with pre-processing protocol A, and (5) DNeasy Blood & Tissue Kit with pre-processing protocol B. For protocol A, cheese was homogenized with 2% sodium citrate and centrifuged. For protocol B, cheese was homogenized with a solution of 0.5% sodium chloride, 1% casitone, and 2% sodium citrate, incubated for 1 h at 37°C, homogenized, and centrifuged. DNA samples were quantitated using Qubit® technology. 

Results: Method (1) produced the highest DNA yield of all five techniques at 2053±158.7 ng/g of cheese, P<0.05. Results of methods (2) and (3) were below the level of detection (2 ng). Methods (4) and (5) produced 618±0.8 and 186±16.4 ng/g of cheese, respectively. Therefore, pre-processing of Gouda cheese using protocol A coupled with a commercial kit produced approximately 400 ng/g of cheese more DNA than when using protocol B.

Significance: Results from this study will help to identify high-yield DNA extraction methods from Gouda cheese to be used with downstream molecular procedures.

Back to: Poster Session 3 – Antimicrobials, Laboratory and Detection Methods, Microbial Food Spoilage, Modeling and Risk Assessment, Retail and Food Service Safety and Sanitation
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