A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef

Paula K. Witham, Carl Yamashiro, Kenneth J. Livak, Carl A. Batt

Research output: Contribution to journalArticle

97 Citations (Scopus)

Abstract

A detection system based on the PCR has been developed for Escherichia coli strains which harbor the Shiga-like toxin genes. This quantitative detection system involves the 5'→3' nuclease activity of Thermus aquaticus DNA polymerase, which cleaves an internal oligonucleotide probe that has been labeled with both a fluorescent reporter dye (6-carboxy-fluorescein [FAM]) and a quencher dye (6-carboxytetramethyl-rhodamine [TAMRA]). Parameters which affected the performance of the assay included primer probe distance, probe concentration, and probe target sequence homology. The optimized assay format includes two PCR primers that generate a 497-bp amplicon specific for the sltI gene with the fluorogenic probe located 19 bp from the upstream PCR primer. When the distance between the upstream PCR primer and the probe was reduced from 190 to 19 bp, ΔRQ values increased from approximately 1.5 to 3.0. The ΔRQ for Shiga-like toxin I probe 102 reached a maximum of 4.15 at concentrations between 25 and 50 nM. The assay is sensitive and can detect approximately 10 ± 5 CFU per PCR. As few as 0.5 CFU of Shiga-like toxin I- producing E. coli per g could be detected in ground beef with only 12 h of enrichment in modified E. coli broth.

Original languageEnglish (US)
Pages (from-to)1347-1353
Number of pages7
JournalApplied and Environmental Microbiology
Volume62
Issue number4
StatePublished - Apr 1 1996
Externally publishedYes

Fingerprint

Shiga-like toxins
Shiga Toxins
ground beef
toxin
probe
assay
Escherichia coli
Polymerase Chain Reaction
Shiga Toxin 1
dyes
gene
Thermus aquaticus
assays
Genes
3'-nucleotidase
oligonucleotide probes
genes
nucleases
DNA-directed DNA polymerase
fluorescein

ASJC Scopus subject areas

  • Biotechnology
  • Environmental Science(all)
  • Microbiology

Cite this

Witham, P. K., Yamashiro, C., Livak, K. J., & Batt, C. A. (1996). A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef. Applied and Environmental Microbiology, 62(4), 1347-1353.

A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef. / Witham, Paula K.; Yamashiro, Carl; Livak, Kenneth J.; Batt, Carl A.

In: Applied and Environmental Microbiology, Vol. 62, No. 4, 01.04.1996, p. 1347-1353.

Research output: Contribution to journalArticle

Witham, PK, Yamashiro, C, Livak, KJ & Batt, CA 1996, 'A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef', Applied and Environmental Microbiology, vol. 62, no. 4, pp. 1347-1353.
Witham, Paula K. ; Yamashiro, Carl ; Livak, Kenneth J. ; Batt, Carl A. / A PCR-based assay for the detection of Escherichia coli Shiga-like toxin genes in ground beef. In: Applied and Environmental Microbiology. 1996 ; Vol. 62, No. 4. pp. 1347-1353.
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