A highly sensitive multiplex lateral flow immunoassay for simultaneous detection of Listeria monocytogenes, Salmonella Typhimurium and Escherichia coli O157:H7

In this study, a sensitive, fast and reliable multiplex lateral flow immunoassay based on multiple PCR and gold nanoparticles (AuNPs) was developed. Genomic DNA of Listeria monocytogenes , Salmonella Typhimurium and Escherichia coli O157:H7 was extracted by a simple boiling method. Three pairs of primers were designed and labeled according to specific gene fragment of the three strains for multiple PCR. The PCR products were then conjugated with AuNPs and detected by multiplex lateral flow strip, on which the test lines loaded with anti-biotin antibody, anti-FITC antibody and anti-digoxin antibody corresponding to the labels of primers, respectively. Results showed the limit of detection of L. monocytogenes , S. Typhimurium and E. coli O157:H7 in pure culture were 1.0×10 1 CFU mL − 1 , 1.0×10 2 CFU mL − 1 and 1.6×10 2 CFU mL − 1 , respectively, without culture enrichment. In addition, the lateral flow immunoassay showed good specificity, no reaction to each other or no cross-reactivity with other tested foodborne bacteria were observed. The detection took less than 4 h including PCR amplification, AuNPs conjugation and strip detection. Furthermore, the developed method was applied for the detection of food samples (chicken breast), which was verified by plate count method. The recoveries ranged from 92.7–112.1%, with the coefficient of variation less than 8.73%, revealing the feasible and reliable application of this method in practical sample. Therefore, the developed multiplex lateral flow strip is sensitive, accurate and visualized, which is applicable to simultaneous detection of the three foodborne pathogenic bacteria in food sample.