Dual priming oligonucleotide (DPO)-based multiplex PCR assay for specific detection of four diarrhoeagenic Escherichia coli in food

In this study, a dual priming oligonucleotide (DPO)-based multiplex PCR assay was developed for the specific detection of four foodborne diarrhoeagenic Escherichia coli (DEC) in food, including enterotoxigenic E.coli (ETEC), enteropathogenic E.coli (EPEC), enterohemorrhagic E.coli (EHEC) O157:H7 and enteroinvasive E.coli (EIEC). Species-specific DPO primers were designed based on rfbE, LT, ipaH and bfpA genes for EHEC O157:H7, ETEC, EIEC and EPEC respectively. Our optimized DPO-based multiplex PCR assay was able to simultaneously detect these DEC from pure cultures, spiked food or environmental sample with an analytical detection limit of <120CFUml(-1) (or g(-1)) for each at annealing temperature from 45 to 65 degrees C. A total of 336 bacterial strains including 51 target and 285 other bacterial strains were used to evaluate the specificity of the assay, and results showed that specific PCR products were only amplified in strains belonging to target bacteria. Applying the assay to 982 samples collected from food, clinical patients and environmental sources revealed that 73 samples were positive, which were confirmed by conventional culture-based assays combined with serological tests. Taken together, the DPO-based multiplex PCR assay developed in this study is a rapid, specific and reliable tool for efficient screening single or multiple DEC from food in laboratory diagnosis. Significance and Impact of the StudyThe high specificity of the DPO-based multiplex PCR assay developed in this study without false positive results indicates its great potential to be a rapid, reliable, practical and cost-effective method for the monitoring of diarrhoeagenic E.coli in food.