Comparison of In-house and Commercial Real-time PCR Systems for the Detection of Enterobacteriaceae and their Evaluation Within an Interlaboratory Study Using Infant Formula Samples Alice Martinon & Ultan P. Cronin & Martin G. Wilkinson Received: 1 June 2010 / Accepted: 14 December 2010 # Springer Science+Business Media, LLC 2011 Abstract Traditional detection methods for Enterobac- teriaceae in foods are time-consuming and laborious. The current study assessed the specificity of three real-time PCR primer sets. Set A (IEC primers) targeted the conserved flanking regions of the 16S rRNA, the 16S- ITS-23S gene region. Set B (ENT primers) annealed to Escherichia coli 16S ribosomal RNA gene. The third set (C) used a D-LUX™ (Light Upon eXtension) single FAM- labelled forward primer and a corresponding unlabeled primer. Set A was specific for E. coli and for some non- Enterobacteriaceae. SYBR Green-based real-time PCR confirmed the specificity of set B for the Enterobacter- iaceae but also detected Vibrionaceae. In contrast, set C was poorly specific. However, set D including the forward LUX™ primer from set C and the reverse primer from set B had a specificity comparable to that of set B, but with higher sensitivity. This combined set was successfully applied to detect Enterobacteriaceae in infant milk formula and compared favourably with a commercial real-time PCR kit. Keywords Detection . Enterobacteriaceae . Real-time PCR . LUX™ primers . Infant formula milk Introduction The Enterobacteriaceae family is composed of widely studied microorganisms and includes several species such as Escherichia coli, Klebsiella pneumoniae or Salmonella Typhimurium which are responsible for food intoxications (Blood and Curtis 1995). Routine monitoring of Enter- obacteriaceae serves as a hygiene indicator within food processing plants and their presence typically signifies poor cleaning procedures for process surfaces or post-processing contamination of heat-processed foods. To date, most quality assurance laboratories use agar-based ISO proce- dures (de Boer 1998) in order to detect and quantify Enterobacteriaceae in food products or swab samples. Combined with enrichment steps, Violet Red Bile Glucose Agar (VRBGA) has been among the most popular media for detecting Enterobacteriacaeae in foods. However, this medium is recognised as having some shortcomings (Baylis 2006) as other strains like Aeromonas spp. can also grow. Consequently, the colonies that appear on VRBGA are qualified as presumptive with further confirmatory tests required. Overall, this method can take 5 to 7 days for a definitive result which is not satisfactory for allowing rapid product release. Therefore, it is of commercial interest to accelerate this procedure by investigating alternative rapid DNA-based methods. Detection of the Enterobacteriaceae by conventional PCR has been previously reported. Bayardelle and Zafarullah (2002) developed PCR protocols for detection of the most frequent species of the Enterobacteriaceae in blood, urine, and water samples using primer sets targeting the wec gene cluster involved in the synthesis of the enterobacterial common antigen. Real-time PCR has been widely accepted because of rapidity, sensitivity, reproduc- ibility and reduced carry-over contamination (Mackay et al. 2007). Real-time PCR protocols have also been developed and applied in food samples for the detection of Enterobacteriaceae (Nakano et al. 2003; Qiu et al. A. Martinon : U. P. Cronin : M. G. Wilkinson (*) Department of Life Sciences, University of Limerick, Castletroy, Limerick, Ireland e-mail: Martin.Wilkinson@ul.ie Food Anal. Methods DOI 10.1007/s12161-010-9188-7