A quantitative PCR assay for the detection and quantification of Shiga toxin-producing Escherichia coli (STEC) in minced beef and dairy products

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 × 10 6 to 40 CFU/mL for milk and from 10 7 to 10 2 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. ► A qPCR assay was developed to detect all known stx subtypes including stx2f. ► An internal amplification control was included to check for PCR inhibition. ► The detection limit of the method was < 4 STEC CFU per 25 g of minced beef or cheese. ► Less favourable STEC enrichment was observed for cheese compared to minced beef. ► The assay also accurately quantified STEC in milk and minced beef.