Real-time PCR targeting OmpA gene for detection of Cronobacter spp. in powdered infant formula

Abstract

Cronobacter spp. (formerly Enterobacter sakazakii), a pathogen commonly found in powdered infant formula (PIF), is a rare cause of invasive infection with a high mortality rate in neonates. In the present study, a realtime PCR assay was conducted to identify the pathogens in PIF using a TaqMan probe targeting the outer membrane protein A gene (ompA) of Cronobacter spp. The specificity of the PCR assay was tested against 25 strains of Cronobacter spp. and 38 non-Cronobacter bacterial species. The detection limits of this method are 1.0×10 2 copy/µL in standard plasmid, 1.1 CFU/100 g in PIF through 38 h of enrichment, and 2.8×102 CFU/mL in phosphate-buffered saline (pH 7.0). Based on the detection limits, real-time PCR is more sensitive than simplex and duplex PCR. These methods were successfully applied to actual samples, indicating that this real-time PCR assay can be used for the detection of Cronobacter spp. in PIF.

A new application of a sodium deoxycholate-propidium monoazide-quantitative PCR assay for rapid and sensitive detection of viable Cronobacter sakazakii in powdered infant formula

“…Mean cycle threshold (Ct) values of triplicate assays were plotted against the number of viable cells per gram for each PIF matrix. circumvent this, most studies use a DNA extraction kit, but this is costly, time consuming, and the LOD can only reach 10 2 cfu/mL (Dong et al, 2013). In this study, the DNA was extracted using the water-boiling method and the LOD was 3.3 × 10 2 cfu/mL in pure culture ( Figure 4A), and 4.4 × 10 2 cfu/g in PIF matrix ( Figure 4B).…”

Simultaneous detection and identification of pathogenic Cronobacter species by high‐resolution melting analysis in powdered infant formulas

“…As a powerful, fast and accurate method with high-throughput possibilities, HRM is simpler and more affordable than alternative approaches requiring post-PCR processing, enzyme restriction, gel electrophoresis and the use of labelled probes for SNP detection or TaqMan-probe-based real-time PCR (Sakaridis et al 2014). PCR assay targeting ompA (Dong et al 2013), palF (Soler et al 2012), rpoB (Li et al 2013) and cgcA (Carter et al 2013;Hu et al 2016) genes has been used for the detection of some Cronobacter spp. in many food matrices.…”