Detection of enterohemorrhagic Escherichia coli O157:H7 by using multiplex real-time PCR technology

• Main Authors: ching-Fang Hsu, 許瀞方
• Other Authors: Tzn-Ming Pan
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/17555476995842352456

碩士 === 國立臺灣大學 === 微生物與生化學研究所 === 92 === Real-time polymerase chain reaction (PCR) assays have been developed for detection and quantification of pathogens in recent years. A multiplex real-time PCR assay was designed and evaluated on the ABI 7700 sequence detection system (TaqMan®) to detect enterohemorrhagic Escherichia coli O157:H7 in pure culture, feces and food samples. Two sets of primers and fluorescent probes were used for amplification and real-time detection of a 192-bp region of the rfbO157 gene encoding E. coli O157:H7-specific O-antigen, and 170-bp segment of stx2 gene encoding Shiga-like toxin 2. Analysis of 217 bacterial strains demonstrated that the multiplex real-time PCR assay successfully distinguished E. coli O157:H7 serotype from non- E. coli O157:H7 serotypes and provided accurate profiling of genes encoding O-antigen and Shiga-like toxin 2. Bacterial strains lacking these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for the two genes were linear over DNA concentrations corresponding from 103 to 109 CFU/mL of E. coli O157:H7 in pure culture and milk sample. The real-time PCR allowed construction of standard curves that facilitated quantification of E. coli O157:H7 in feces, apple juice, milk and ground beef sample. Detection sensitivity of the real time PCR assay ranged from 104 to 109 CFU/g (or 104 to 109 CFU/mL) of feces or apple juice without enrichment. Detection sensitivity of the real time PCR assay ranged from 105 to 109 CFU/g of ground beef without enrichment.After enrichment of milk and apple juice samples in modified Tryptic Soy Broth, the detection of levels were from 100 to 104 CFU/mL. The real-time PCR assay for rfbO157 and stx2 proved to be a rapid test for detection of E. coli O157:H7 in food matrices and could also be used for quantification of E. coli O157:H7 in foods or fecal samples.