P3-57 Streamlined Food Pathogen DNA Extraction Using a Small Benchtop Automated Instrument

Tuesday, July 28, 2015
Hall B (Oregon Convention Center)
Brad Hook , Promega Corporation , Madison , WI
Chris Moreland , Promega Corporation , Madison , WI
Trista Schagat , Promega Corporation , Madison , WI
Introduction: Every year, pathogenic microbe contamination in foods causes serious and sometimes fatal infections worldwide.  It is, therefore, important to monitor food sources carefully.  Application of molecular techniques is allowing fast and selective foodborne pathogen testing. 

Purpose: Here we examine the utility of a small benchtop automated instrument, the Maxwell® 16, and novel cellulose-based magnetic particles for purification of amplifiable bacterial DNA from enriched food cultures. 

Methods: Using enumerated aliquots of viable bacteria, we created 25 g food samples (lettuce, milk or ground beef) of specific pathogen contamination (1, 10, or 100 CFU).  Bacteria tested were Salmonella enterica, Listeria monocytogenes, Staphylococcus aureus (milk and ground beef only) and Shiga Toxin-producing Escherichia coli O157:H7 (lettuce and ground beef only).  After a standard overnight incubation to enrich for live microbes, we evaluated both a direct extraction (fast) protocol and a lysozyme treatment (maximum sensitivity) protocol for DNA extraction using a custom Maxwell® 16 kit.  Following extraction, probe-based qPCR was used to detect purified microbial DNA.  Extractions were performed in triplicate for each microbe, food type, and microbe concentration. 

Results: We found that following both the fast and maximum sensitivity protocols, we were able to detect down to at least 10 viable microbes in each food tested, and in some cases the single microbe cultures were also positive.  The difference in positive microbe detection between the fast and maximum sensitivity protocols was ≥ 5 Ct.  The method was compared to an AFNOR and AOAC-certified manual purification protocol and found to give comparable results. 

Significance: This study supports the use of Maxwell® 16 for automated purification of microbial DNA from enriched cultures for food safety testing.