- in the DNA extraction step, since the users can choose between the standard and easy protocol;
- by storing the enrichment broth at 5 ± 3 °C for 3 days, before running the PCR analysis.
Purpose: An independent study was conducted to validate this new method in comparison to the ISO/TS 22964 reference method, as part of the NF Validation approval process and according to the ISO 16140 standard.
Methods: The method protocol includes an overnight enrichment in BPW supplemented with vancomycin. An additional sub-culture is done in BPW for 4h ± 1h for infant formula analysis. After the DNA extraction step, real-time PCR is run with a Bio-Rad automate. The presumptive positive results are confirmed by direct streaking onto RAPID’Sakazakii Agar for infant formula, and after a subculture in mLST prior to streaking for environmental samples
Results: 171 infant formula and environmental samples were analyzed in the relative accuracy, sensitivity and specificity study, which concludes to equivalent performances between the new method Cronobacter spp and the ISO/TS 22964 methods. Depending on the tested (matrix/strain) pairs, the relative detection limits of the Cronobacter spp new method vary from 0.5 et 1.6 CFU/25 g, those of the ISO standard from 0.5 to 1.5 CFU/25 g. The selectivity and specificity of the alternative method was assessed by testing 52 target strains and 31 non-target strains.
Significance: The new method is a reliable alternative method for Cronobacter spp detection in infant formula and environmental samples, and offers important economic savings by reducing time to result and handling time.