Abstract: The objective of this study was to establish a multiplex polymerase chain reaction (PCR) detection method for Listeria monocytogenes, Salmonella typhimurium and E. coli O157:H7 on fresh-cut cantaloupe. Three pairs of specific primers were designed according to the inlA gene of L. monocytogenes, the invA gene of Salmonella typhimurium, and the wzy gene of E. coli O157:H7. The primer concentration, annealing temperature, Mg2+ concentration and dNTP concentration were optimized. The results showed that the PCR reactions were performed in a total volume of 25 μL consisting of 2.5 μL of 10 × PCR buffer, 3.5 μL of 25 mmol/L MgCl2, 2 μL of 2.5 mmol/L dNTPs, 1 μL of DNA template, 0.3 μL of exTaq DNA polymerase, 1 μL of 5 μmol/L upstream and downstream primers for inlA and invA and 2 μL of 5 μmol/L upstream and downstream primers for wzy made up to 25 μL with ddH2O. The reaction conditions were as follows: pre-degeneration at 95 ℃ for 3 min followed by 32 cycles of degeneration at 94 ℃ for 30 s, annealing at 53.9 ℃ for 30 s and extension at 72 ℃ for 30 s and a final extension step at 72 ℃ for 10 min. The sensitively of the multiplex PCR was 2.7 × 104 CFU/g for L. monocytogenes, 3.3 × 104 CFU/g (E. coli O157:H7) and 3.8 × 104 CFU/g Salmonella typhimurium inoculated onto freshcut cantaloupe. This method can lay the basis to detect and control these pathogenic microorganisms on fresh-cut cantaloupe.

Key words: multiplex PCR, rapid detection, foodborne pathogen, fresh-cut cantaloupe