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EMA-real-time PCR as a reliable method for detection of viable Salmonella in chicken and eggs
1Food Science Program, Div. of Food Systems and Bioengineering, 256 WCS Wing, Eckles Hall, Univ. of Missouri, Columbia, MO 65211, USA.
Abstract:
Culture-based Salmonella detection takes at least 4 d to complete. The use of TaqMan probes allows the real-time PCR technique to be a rapid and sensitive way to detect foodborne pathogens. However, unlike RNA-based PCR, DNA-based PCR techniques cannot differentiate between DNA from live and dead cells. Ethidium bromide monoazide (EMA) is a dye that can bind to DNA of dead cells and prevent its amplification by PCR. An EMA staining step prior to PCR allows for the effective inhibition of false positive results from DNA contamination by dead cells. The aim of this study was to design an accurate detection method that can detect only viable Salmonella cells from poultry products. The sensitivity of EMA staining coupled with real-time PCR was compared to that of an RNA-based reverse transcription (RT)-real-time PCR. To prevent false negative results, an internal amplification control was added to the same reaction mixture as the target Salmonella sequences. With an optimized EMA staining step, the detection range of a subsequent real-time PCR was determined to be 10(3) to 10(9) CFU/mL for pure cultures and 10(5) to 10(9) CFU/mL for food samples, which was a wider detection range than for RT-real-time PCR. After a 12-h enrichment step, EMA staining combined with real-time PCR could detect as low as 10 CFU/mL Salmonella from chicken rinses and egg broth. The use of EMA with a DNA-based real-time PCR can successfully prevent false positive results and represents a simple, yet accurate detection tool for enhancing the safety of food.
Insights
Ethidium bromide monoazide (EMA) combined with real-time PCR offers a rapid method to detect only viable Salmonella in food. This approach prevents false positives from dead cells, enhancing food safety detection.
Area of Science:
- Food microbiology
- Molecular diagnostics
- Pathogen detection
Background:
- Traditional Salmonella detection methods are time-consuming (≥4 days).
- Real-time PCR offers rapid and sensitive pathogen detection but cannot distinguish live from dead cells.
- Ethidium bromide monoazide (EMA) is a DNA-binding dye that inhibits PCR amplification of DNA from dead cells.
Purpose of the Study:
- To develop an accurate method for detecting only viable Salmonella in poultry products.
- To evaluate the sensitivity of EMA staining coupled with real-time PCR compared to RNA-based reverse transcription (RT)-real-time PCR.
- To prevent false positive results from dead cell DNA and false negatives using an internal amplification control.
Main Methods:
- Optimized EMA staining followed by DNA-based real-time PCR.
- Incorporated an internal amplification control to prevent false negatives.
- Compared EMA-real-time PCR with RNA-based RT-real-time PCR for sensitivity and accuracy.
Main Results:
- Optimized EMA staining coupled with real-time PCR achieved detection ranges of 10^3–10^9 CFU/mL (pure cultures) and 10^5–10^9 CFU/mL (food samples).
- This method detected as low as 10 CFU/mL Salmonella in chicken rinses and egg broth after a 12-h enrichment.
- EMA-real-time PCR demonstrated a wider detection range than RT-real-time PCR.
Conclusions:
- EMA staining combined with DNA-based real-time PCR effectively inhibits false positive results from dead cells.
- This method provides a simple, accurate, and rapid tool for detecting viable Salmonella in food products.
- Enhances overall food safety by ensuring reliable pathogen detection.
