Tool for quantification of staphylococcal enterotoxin gene expression in cheese

Area of Science:

• Food Microbiology
• Molecular Biology
• Food Safety

Background:

• Cheese harbors diverse microbes, some beneficial for flavor, others causing spoilage or health risks.
• Staphylococcus aureus is a significant foodborne pathogen producing enterotoxins in food.
• Understanding enterotoxin gene expression during cheese production is crucial for food safety.

Purpose of the Study:

• To develop an efficient method for total RNA recovery from cheese for gene expression studies.
• To establish a robust strategy using reverse transcription-quantitative PCR (RT-qPCR) to analyze enterotoxin gene expression.
• To identify reliable reference genes for normalizing RT-qPCR data in cheese matrices.

Main Methods:

• Developed an efficient RNA extraction protocol from cheese.
• Utilized RT-qPCR to quantify gene expression.
• Investigated expression of 10 reference genes to find stable internal controls.
• Monitored expression of staphylococcal enterotoxin genes sea and sed.

Main Results:

• Successfully recovered pure, undegraded RNA from cheese suitable for RT-qPCR.
• Identified ftsZ, pta, and gyrB as the most stable reference genes during cheese manufacture.
• Quantified expression of sea and sed genes during the initial 72 hours of cheese production.
• Correlated mRNA expression data with actual enterotoxin production.

Conclusions:

• The developed RNA extraction and RT-qPCR method is effective for studying S. aureus enterotoxin gene expression in cheese.
• Stable reference genes (ftsZ, pta, gyrB) are essential for accurate gene expression analysis in cheese.
• This study provides insights into the dynamics of enterotoxin gene expression during cheese production, aiding in food safety assessments.