Sensitive and specific detection of E. coli, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium in milk by microchip electrophoresis combined with multiplex PCR amplification

Abstract

The rapid and sensitive detection of foodborne bacteria is used to prevent bacterial infectious diseases and food poisoning. Here, we report a simple and sensitive strategy to test three foodborne pathogenic bacteria (E. coli, L. monocytogenes, and S. Typhimurium) by using multiplex PCR based on microchip electrophoresis coupled with laser-induced fluorescence (MCE-LIF). Three pairs of primers for E. coli, L. monocytogenes, and S. Typhimurium were designed using BLAST in this assay. The PCR products of the three foodborne pathogens were used to optimize the MCE separation conditions. Under the optimal conditions, the separation of the PCR products of three foodborne pathogenic bacteria was obtained within 135 s with limits of detection (S/N = 3) of 2.1 ng μL−1, 1.8 ng μL−1, and 2.4 ng μL−1, respectively. The levels of detection were as low as 82 CFU mL−1 for E. coli, 68 CFU mL−1 for L. monocytogenes, and 85 CFU mL−1 for S. Typhimurium. This assay also facilitated the simultaneous detection of the three pathogenic bacteria based on multiple PCR in artificially contaminated milk samples. This detection strategy has the advantages of shorter analysis times, smaller amounts of reagents, and high selectivity and sensitivity. Thus, this detection strategy is more budget-friendly and can ultimately be used for routine food microbial analysis.