Rapid Extraction and Analysis of Salmonella enterica from Fish and Kitchen Surfaces

Introduction: The demand for seafood is increasing globally, and currently in the United States, approximately 1/10 of imported seafood is rejected due to Salmonella enterica contamination.  Salmonella is among the leading causes of foodborne illness caused by seafood.  Currently slowed by plating or the combination of long extraction and analysis times, the need for rapid, on-site testing for this pathogen is essential to prevent the spread of disease. 

Purpose: This test demonstrates successful Salmonella extraction from inoculated fish and kitchen surface swab using an Evogen ONE-UP extraction protocol and rapid LAMP amplification on the Optigene Genie II. The objective is to provide a combined rapid extraction and detection system.

Methods: Fresh fish samples were inoculated with 1ml S. enterica at 4,000 CFU/ml, or kitchen surfaces which had been in contact with the fresh fish were inoculated and dried for 30 minutes, then swabbed, and both swabs (in 1ml media) and fish samples (in 50ml media) were incubated at 37 °C for 24 hours. DNA from 200μl of each sample was extracted and purified by the Evogen ONE-UP extraction protocol, which included lysis with Evogen ONE, a bead-binding step, a wash step, and an elution step.  Finally, eluted DNA was amplified via LAMP on an Optigene Genie II using primers targeting the S. enterica ttrRSBCA locus.

Results: All fish cultures showed amplification within 10 to 16 minutes on the Genie II, with the no template culture not showing any amplification.  Similarly, fish surface swabs showed amplification within 10 to 13 minutes.

Significance:  The combined system rapidly produced high quality DNA in that provided excellent amplification signals via LAMP.  This combination of instruments demonstrates a simple and effective test method for detecting Salmonella that may easily be performed at any food testing or manufacturing facility.