Detection of food-borne pathogens bacteria Staphylococcus aureus in contaminated meat and milk product by real-time PCR

Abstract

The development of rapid, sensitive, and specific detection methods has increasingly become the attention in handling food poisoning cases. In this study, the method of Real-Time PCR (RT-PCR) with nuc gene target was developed for the detection of Staphylococcus aureus in food samples of meat and milk. The results showed that the primer nuc gene with Gradient PCR could detect Staphylococcus aureus bacteria from the pure culture at 57-61 oC by producing amplicons measuring 135 base pairs. Amplification by RT-PCR in the same sample at an annealing temperature of 58oC and a concentration of 50 ng gave a Ct value of 18.88-19.56. The primer specificity test of that gene shows that the primer can recognize the target bacteria with a strong fluorescent signal at the Melting Curve of 79.02 oC, whereas non-target bacteria give low signals and different Tm values. The primer sensitivity of the nuc gene has a detection limit (LOD) of 98.4 pg/µL or 8.8 x 102 CFU/mL. Based on these results, the RT PCR method with the nuc gene has the potential to be a method of detecting specific, sensitive, and rapid for Staphylococcus aureus bacteria in a food sample.