*4.2.2 Multiplex PCR (mPCR)*

mPCR technology is more new-fashioned, which can simultaneously detect more pathogens than before, up to four or more pathogens [45–47]. Chen et al. simultaneously detected *S. enteritidis*, *S. flexneri*, and *E. coli* 157:H7 using five pairs of primers for invading protein (invA), 16S rDNA, invading plasmid antigen H (IPAH), Listeria hemolysin o (HlyA), and immunoglobulin (EAEA) genes [45]. The mPCR detection limit of mixed genomic DNA was 7.58 × 104 copies. Further improvements to mPCR by Gilmartin and O'Kennedy [46] promoted the process of a new GeXP PCR detection of four foodborne bacterial pathogens: *Salmonella*, *Yersinia*, *E. coli* 157:H7, and *Shigella*. The genome lab gene expression profiler (GeXP) gene analysis system can detect multiple pathogens in a single reaction with high throughput. Chimeric primers, universal primers and capillary electrophoresis with PCR products rather than agarose gel electrophoresis were involved in GeXP multiplex PCR amplification. Synthesis of amplicons with universal tags by chimeric primers containing genespecific sequences with universal tags at the 5′ end. Then, a universal primer will drive the remaining PCR reaction, which contains the same sequence of universal tags used by chimeric primers. Forward universal primer was covalently labeled with fluorescent dyes at the 5′ end for detection during capillary electrophoresis [47]. This method has higher sensitivity and is suitable for high-throughput analysis. Detection limits of Grignard PCR for *Salmonella*, *Yersinia*, *E. coli* 157:H7, and *Shigella*.

The characteristics of multiplex PCR are high efficiency, systematic and economic simplicity. High efficiency: a variety of pathogenic microorganisms in the same PCR reaction tube can be detected simultaneously, or multiple pathogens can be detected with multiple types of genes of interest. Systematic: mPCR is suitable for the detection of grouped pathogens. Economic simplicity: this will greatly economical of time, reagent and cost, and provide more accurate diagnostic information for clinical practice, because multiple pathogens are detected synchronously.
