In this study, we developed a rapid on-site detection method using direct ultrafast PCR coupled with a microfluidic chip to identify the presence of chicken meat in processed ground meat products. Chicken-specific PCR primer targeting mitochondrial 16S rRNA gene was newly designed, and its specificity was confirmed against 17 other animal species and 4 different chicken meat samples from different countries of origin. The sensitivity of the chicken-specific ultrafast PCR was 0.1 pg of chicken DNA. To evaluate the limit of detection (LOD) of the direct ultrafast PCR method, different percentages of chicken meat mixed with pork or beef were prepared. The LOD of the direct ultrafast PCR method for the chicken meat/pork and chicken meat/beef mixtures was 0.1% for both raw meat and autoclaved meat. This method was used for 15 commercialized processed ground meat products. In this method, the target sequence was successfully amplified, and the presence of chicken meat in processed ground meat products was identified within approximately 25 min, including the time for sample preparation. Thus, our study shows that this developed direct ultrafast PCR method is a rapid and accurate method for on-site detection of chicken DNA in commercial food products.
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.