An automated fluorescent PCR method for detection of Shiga toxin-producing Escherichia coli in foods

• Published in: Applied and Environmental Microbiology 1998-11, Vol.64 (11), p.4210-4216
• Main Authors: Chen, S. (University of Guelph, Guelph, Ontario, Canada.), Xu, R, Yee, A, Wu, K.Y, Wang, C.N, Read, S, De Grandis, S.A
• Format: Article
• Language: English
• Subjects: ALIMENTOS; Animals; Automation - methods; Bacteria; BACTERIAL TOXINS; Bacterial Toxins - biosynthesis; Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; BIOLOGICAL CONTAMINATION; Cattle; CONTAMINACION; CONTAMINACION BIOLOGICA; CONTAMINATION; CONTAMINATION BIOLOGIQUE; DNA Primers; ESCHERICHIA COLI; Escherichia coli - classification; Escherichia coli - isolation & purification; Escherichia coli - pathogenicity; Fluorescence; Food contamination & poisoning; Food industries; Food Microbiology; FOODS; Fundamental and applied biological sciences. Psychology; Meat - microbiology; Meat Products - microbiology; MICROBIAL CONTAMINATION; Microbiology; PCR; POLYMERASE CHAIN REACTION; Polymerase Chain Reaction - methods; PRODUIT ALIMENTAIRE; Serotyping; Shiga Toxin 1; Shiga Toxin 2; Spectrometry, Fluorescence - methods; Swine; TOXINAS BACTERIANAS; TOXINE BACTERIENNE
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.64.11.4210-4216.1998

An automated fluorescence-based PCR system (a model AG-9600 AmpliSensor analyzer) was investigated to determine whether it could detect Shiga toxin-producing Escherichia coli (STEC). The AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to conserved target sequences in a 323-bp amplified fragment of Shiga toxin genes stx1, stx2, and stx(e). Using the Amplisensor assay, we detected 113 strains of STEC belonging to 50 different serotypes, while 18 strains of non-Shiga-toxin-producing E. coli and 68 strains of other bacteria were not detected. The detection limits of the assay were less than 1 to 5 CFU per PCR mixture when pure cultures of five reference strains were used and 3 CFU per 25 g of food when spiked ground beef samples that were preenriched overnight were used. The performance of the assay was also evaluated by using 53 naturally contaminated meat samples and 48 raw milk samples. Thirty-two STEC-positive samples that were confirmed to be positive by the culture assay were found to be positive when the AmpliSensor assay was used. Nine samples that were found to be positive when the PCR assay was used were culture negative. The system described here is an automated PCR-based system that can be used for detection of all serotypes of STEC in food or clinical samples