Simultaneous Identification of Seven Foodborne Pathogens and Escherichia coli (Pathogenic and Nonpathogenic) Using Capillary Electrophoresis–Based Single-Strand Conformation Polymorphism Coupled with Multiplex PCR

• DOI: 10.4315/0362-028x-72.6.1262
• Publication Date: 2016-12-01T17:56:30Z
• Authors: Mi-Hwa Oh; Se-Hee Paek; Gi Won Shin; Hae-Yeong Kim; Gyoo Yeol Jung; Sangsuk Oh
• Keywords: 0301 basic medicine; Staphylococcus aureus; Time Factors; POLYMERASE-CHAIN-REACTION; VIBRIO-PARAHAEMOLYTICUS; Clostridium perfringens; GEL-ELECTROPHORESIS; Food Contamination; CAMPYLOBACTER-JEJUNI; Polymerase Chain Reaction; Sensitivity and Specificity; Campylobacter jejuni; 03 medical and health sciences; Bacillus cereus; Species Specificity; FOOD; RNA, Ribosomal, 16S; RIBOSOMAL-RNA GENE; Escherichia coli; ASSAY; REAL-TIME PCR; LISTERIA-MONOCYTOGENES; Polymorphism, Single-Stranded Conformational; STAPHYLOCOCCUS-AUREUS; Gene Amplification; Temperature; Electrophoresis, Capillary; Reproducibility of Results; Salmonella enterica; 3. Good health; Consumer Product Safety; Food Microbiology; Vibrio parahaemolyticus

The objective of this study was to develop a novel technique for parallel analysis of eight important foodborne microbes using capillary electrophoresis-based single-strand conformation polymorphism (CE-SSCP) coupled with multiplex PCR. Specific primers for multiplex PCR amplification of the 16S rRNA gene were designed, corresponding to eight species of bacteria, including Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Salmonella enterica, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Bacillus cereus, for the species-specific identification and optimal separation of their PCR products in subsequent analysis by CE-SSCP. Multiplex PCR conditions including annealing temperature, extension time, the number of PCR cycles, and primer concentrations were then optimized for simultaneous detection of all target foodborne bacteria. The diagnostic system using CE-SSCP combined with multiplex PCR developed here can be used for rapid investigation of causative agents of foodborne illness. The simplicity and high sensitivity of the method may lead to improved management of safety and illness related to food.