Rapid Detection and Identification of Foodd-Borne Bacterial Pathogens by Multiplex PCR and Restriction Endonuclease Digestion

• Main Authors: Chung-Hsing Hwang, 黃中興
• Other Authors: Chung-Lung Cho
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/16304518326196407602

碩士 === 國立中山大學 === 生物科學系研究所 === 89 === 英文摘要 Multiplex PCR amplification of 16S rRNA gene、virA、tpl、and H1d genes was developed enabling simultaneous detection in Escherichia coli，an indicator of fecal contamination and food-borne microbial pathogens，Shigella flexneri、Citrobacter freundii、Salmonella typhi、Vibrio cholerae、Vibrio parahaemolyticus、and Staphylococcus aureus。Each of the nine pairs of oligonucleotide primers was found to support PCR amplifications of only its targeted gene。The optimized multiplex PCR reaction utilized a primer annealing temperature of 59 ℃and used agarose gel electrophoresis for detection of the PCR-amplified products。Selection of appropriate target genes、oligonucleotide primers 、PCR reaction、and cycling parameters resulted in the amplification of four target genes simultaneously in a single PCR reaction with the sensitivity of detection was 102 CFU after 32 cycles。Multiplex PCR amplification followed by differential PCR for E. coli / Shigella， and Citrobacter / Salmonella，sequenced for the PCR-amplified products of 16S rRNA gene of the seven pathogens in this study，and used restriction endonuclease AfaI to confirm the PCR-amplified products of V. cholerae，V. parahaemolyticus and Staphylococcus aureus，has been shown to be an sensitive，specific，and rapid method to detect food-borne bacterial pathogens。