Abstract：
:Shiga toxin (Stx)-producing Escherichia coli (STEC) are amongst major causes of food-borne infectious diseases and outbreaks. A new quantitative PCR (qPCR) assay was designed to detect all known stx gene subtypes in a single reaction, including the most distant variant stx2f. Performance of this assay was evaluated in combination with two different internal amplification controls (IAC), a competitive one specific for the assay and a universal IAC based on plasmid pUC19. The qPCR assay was 100% specific and showed analytical sensitivity of two STEC genome copies per reaction. The diagnostic approach proposed, combining enrichment, automated DNA extraction and qPCR detection, could reliably detect the presence of STEC in minced beef and cheese inoculated before enrichment at <4 CFU per 25 g. A comparative study performed on 240 minced beef and 113 raw milk cheese samples demonstrated that the method developed was as effective as two PCR screening assays used routinely in our laboratory to detect STEC. The new assay also proved to be appropriate for the direct quantification of STEC in milk and minced meat. It was found to be quantitative over a five log dynamic range, from 4 × 10⁶ to 40 CFU/mL for milk and from 10⁷ to 10² CFU/g for minced beef. In conclusion, the qPCR assay developed here represents a valuable tool for rapid detection and quantification of STEC in foods such as minced beef and dairy products as it ensures a high sensitivity and an optimal STEC diagnostic spectrum, taking into account the genetic stx variability observed in STEC population.
journal_name
Int J Food Microbioljournal_title
International journal of food microbiologyauthors
Derzelle S,Grine A,Madic J,de Garam CP,Vingadassalon N,Dilasser F,Jamet E,Auvray Fdoi
10.1016/j.ijfoodmicro.2011.07.039subject
Has Abstractpub_date
2011-11-15 00:00:00pages
44-51issue
1eissn
0168-1605issn
1879-3460pii
S0168-1605(11)00444-2journal_volume
151pub_type
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