Detecting of viable Staphylococcus aureus by loop-mediated isothermal amplification coupling with propidium monoazide in dairy products

In recent years, the increasing number of Staphylococcus aureus outbreaks linked to food product highlighted the need to develop technique with rapid, simple and accurate. The conventional technologies for rapid and sensitive detection of S. aureus viable cells in produce had several limitations. The signal from viable versus dead cells could not be distinguished due to the persistence of DNA after the cell death, resulting in overestimate the number of cells. In the present study, some problems had been solved by developing a new concept with DNA-intercalating dye propidium membrane (PMA) combining with loop-mediated isothermal amplification (LAMP) . The test was used to analyse S. aureus and routine dairy products. a series of primers targeted six distinct sequences of nuc gene were designed, which was characteristic of S. aureus. Also, the LAMP assay and the performance of PMA-LAMP for detecting viable S. aureus were optimized. Moreover, they improved the method for extraction DNA from dairy samples. In pure culture, the detection limit of PMA-LAMP was 3. 2CFU/mL, up to 100-fold more sensitive than PCR. In dariy products, PMA-LAMP assay could detect as less as 5×101CFU/mL compared to that of PCR-PMA was 5×103CFU/mL. The complete LAMP-PMA assay for took about 6h, demonstrating the method was rapid and convenient. In conclusion, PMA-LAMP offers a novel DNA-based detection method for distinction between viable and dead cells with wide application in food products.