EMA-Real-Time PCR as a Reliable Method for Detection of Viable Salmonella in Chicken and Eggs

• Published in: Journal of food science 2010-04, Vol.75 (3), p.M134-M139
• Main Authors: Wang, Luxin, Mustapha, Azlin
• Format: Article
• Language: English
• Subjects: accuracy; Animals; Azides - chemistry; bacterial contamination; Biological and medical sciences; Cells; chicken; chicken meat; Chickens - microbiology; Comparative analysis; Deoxyribonucleic acid; detection; DNA; DNA, Bacterial - isolation & purification; DNA, Bacterial - metabolism; dye binding; dyes; eggs; Eggs - microbiology; ethidium bromide monoazide; False Positive Reactions; food analysis; food composition; food contamination; Food industries; Food Industry - methods; Food Microbiology; food pathogens; Fundamental and applied biological sciences. Psychology; General aspects; Limit of Detection; Meat - microbiology; Methods of analysis, processing and quality control, regulation, standards; Microbial Viability; Polymerase chain reaction; Polymerase Chain Reaction - methods; Poultry; rapid methods; real-time PCR; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA, Bacterial - isolation & purification; RNA, Bacterial - metabolism; Salmonella; Salmonella - genetics; Salmonella - isolation & purification; Salmonella enteritidis - genetics; Salmonella enteritidis - isolation & purification; Salmonella Food Poisoning - prevention & control; Salmonella typhimurium - genetics; Salmonella typhimurium - isolation & purification; Studies; Time Factors; viability; viable Salmonella
• ISSN: 0022-1147; 1750-3841
• DOI: 10.1111/j.1750-3841.2010.01525.x

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 10³ to 10⁹ CFU/mL for pure cultures and 10⁵ to 10⁹ 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.