Simultaneous identification of seven foodborne pathogens and Escherichia coli (pathogenic and nonpathogenic) using capillary electrophoresis-based single-strand conformation polymorphism coupled with multiplex PCR.
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.
Oh
Mi-Hwa
MH
Department of Food Science and Technology, College of Engineering, Ewha Womans University, Seoul 120-750, Korea. ssoh71@ewha.ac.kr
Paek
Se-Hee
SH
Shin
Gi Won
GW
Kim
Hae-Yeong
HY
Jung
Gyoo Yeol
GY
Oh
Sangsuk
S
D001409:Bacillus cereus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D016123:Campylobacter jejuni / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D003016:Clostridium perfringens / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D003257:Consumer Product Safety; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D015203:Reproducibility of Results; D019779:Salmonella enterica / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013211:Staphylococcus aureus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D013696:Temperature; D013997:Time Factors; D014736:Vibrio parahaemolyticus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification
D012336:RNA, Ribosomal, 16S