Development of PMA-LAMP for Rapid Detection of Staphylococcus aureus in Viable but Nonculturable State

Abstract

Staphylococcus aureus is a major foodborne pathogen. The viable but non-culturable (VBNC) state of S. aureus cannot be detected by traditional culturing methods while remain alive for long time and maintain the potential virulence. The objective of this study was to develop and optimize a method that combines propidium monoazide (PMA) treatment with loop-mediated isothermal amplification (LAMP) to detect VBNC cells of S. aureus. The cell suspension was treated with PMA in the dark for 10 min and was subsequently exposed to a 650 W halogen lamp for 5 min. Then the bacterial cells were harvested and DNA was extracted and amplified by LAMP. The primers targeted six distinct regions in the nuc gene of S. aureus was designed for the PMA-LAMP method. The results indicated that the treatment with 3 μg/mL PMA and a 5min light exposure was suitable for PMA-LAMP to distinguish VBNC cells from dead cells of S. aureus. The optimized assay was specific and sensitive; it could detect as low as 17 CFU/mL VBNC cells in pure culture, 17 CFU/g in spiked milk powder, and 170 CFU/g in spiked dumpling. The assay could detect VBNC state of S. aureus without interference of dead cells and other bacteria.