Evaluation of a Commercial Real Time PCR Assay Performance with Various Spices
Erica Miller 1, Daniel R. DeMarco 1, J. David Legan 2, Joelle Mosso 3, and Chris Crowe1
Eurofins Microbiology Laboratories, Inc.: 1 Louisville, KY; 2 Madison, WI, 3 Fresno, CA
ABSTRACT
Introduction
Contamination of spices and seasoning blends with Salmonella remains a public health concern. Many spices contain antimicrobial components and/or assay inhibitors, which challenge pathogen detection methods, especially those using PCR for detection. Hence, test kit manufacturers often omit spices when validating methods and there is a paucity of spice validation data in comparison with other commonly tested food matrices.
Purpose
To evaluate the performance of a commercial real-time PCR Salmonella detection method with spice matrices having antimicrobial and PCR inhibitory properties and to determine if any method modifications are required to obtain acceptable results.
Methods
Ground black pepper, roasted garlic powder, dried oregano, dried clove, and basil were weighed and prepared following FDA BAM protocols. Duplicate samples of each were inoculated with ~10 CFU of Salmonella Typhimurium and tested using Gold Standard Diagnostics’ BACGene Salmonella method following the manufacturer’s protocol, both unmodified and with a BHI regrow step prior to lysis.
Results
All samples except dried basil yielded presumptive results when tested either with or without a BHI regrow. Post enrichment, target Cq values for all matrices were similar and quite low (20.7 avg. Cq, 0.4 SD) suggesting growth was robust. Dried basil showed significant PCR inhibition as evidenced by no amplification of the internal positive control. A 3h BHI regrow step post primary enrichment, eliminated the inhibition and presumptive results were obtained with low Cq values similar to what were observed with the non PCR inhibitory matrices.
Significance
The method reliably detected Salmonella in most spices tested without modification of the standard protocol, and in dried basil after a short BHI regrow step. Importantly, the total incubation time for the enrichments was not changed and all results were obtained in 24 hours or less.
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