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

Abstract

Traditional detection methods for Enterobacteriaceae 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 FAMlabelled forward primer and a corresponding unlabeled primer. Set A was specific for E. coli and for some nonEnterobacteriaceae. SYBR Green-based real-time PCR confirmed the specificity of set B for the Enterobacteriaceae 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.

Quantitative detection of viable foodborne E. coli O157:H7, Listeria monocytogenes and Salmonella in fresh-cut vegetables combining propidium monoazide and real-time PCR

“…However, the application of the PMA approach for pathogen detection in food matrices has scarcely been evaluated. Moreover, there are other nucleic acid intercalating dyes such as reagent D (commercially available reagent from Biotecon) which has been applied for the detection of Enterobacteria in infant formula (Martinon, Cronin, & Wilkinson, 2011). This reagent contains a light sensitive substance which can only penetrate the cell membranes of dead cells.…”

A Comprehensive Evaluation of Enterobacteriaceae Primer Sets for Analysis of Host-Associated Microbiota

“…Molecular characterization of Enterobacteriaceae by PCR assays requires the use of taxonspecific primers; in the last eighteen years, to the best of our knowledge, 16 different primer sets have been designed, validated and published in scientific journals [15,[17][18][19][20][21][22][23][24][25][26][27]. However, a comprehensive evaluation of their performance, specificity and coverage has not yet been Pathogens 2022, 11, 17 2 of 11 carried out.…”

Development and optimization of an ELISA based method to detect Listeria monocytogenes and Escherichia coli O157 in fresh vegetables

“…The qPCR assays with PMA and EMA have been successfully optimized and tested to detect different bacteria, including E. coli O157:H7 and L. monocytogenes, in pure cultures and in foodstuffs. 11,12 The major disadvantages in the use of classical culture-based methods are the requirement of selective enrichment, plating and incubations, making these approaches time-consuming and laborious. Despite their reliability and sensitivity, such methods require several days (4-5 days) before results are available and are not ideal for short shelf life food products.…”