Design and standardization of four multiplex polymerase chain reactions to detect bacteria that cause gastrointestinal diseases

Gastrointestinal diseases and diarrhea are the major cause of morbidity and mortality in children in the developing world, including Mexico. In order to diagnose acute diarrhea and dysentery, four-multiplex polymerase chain reaction (mPCR) sets were designed and standardized in this study. Main virulence genes of relevant enteropathogens, that is,Escherichia coli pathotypes, Shigella spp., Salmonella enterica, Yersinia enterocolitica,Campylobacter jejuni and Aeromonas spp., were detected. Sixteen primer pairs were designed using extensive in silico analysis. As predicted in vitro, specific amplicons were obtained and four multiplex PCR sets were standardized. Our sixteen primer pairs and four multiplex PCR results demonstrate the simultaneous amplification of different pathogens according to the type of diarrhea in the same reaction. In the future, these methods might be used as rapid, sensitive and specific epidemiological and diagnostic tools.   Key words: Gastrointestinal diseases, diarrhea, multiplex polymerase chain reaction, design, standardization, bioinformatics.