ISO 16140 Certification of a New Alternative to Detect Cronobacter spp. in Infant Formula and Environmental Samples

Introduction: The iQ-Check™ Cronobacter method is based on the real PCR principle for Cronobacter spp. detection in infant formula and environmental samples. An ISO 16140 method comparison study was conducted, by analyzing 171 samples in the relative accuracy, sensitivity and specificity part and showing equivalent performances between the alternative method and the ISO/TS 22964 methods. Depending on the tested (matrix/strain) pairs, the relative detection limits of the Real-Time PCR method spp method vary from 0.5 and 1.6 CFU/25g, those of the ISO standard from 0.5 to 1.5 CFU/25g. The selectivity and specificity of the alternative method was assessed by testing 52 target strains and 31 non-target strains.

Purpose: An independent inter-laboratory study was conducted at ADRIA, to compare the alternative method precision to the ISO/TS 22964 one, as part of the NF Validation approval process and according to the ISO 16140 standard.

Methods: The iQ-Check™ Cronobacter spp. 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, the 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: The alternative method was evaluated in a ring trial involving 13 laboratories. Probiotic infant formula was contaminated with the wild C. sakazakiiAd 940 strain. 8 blank samples, 8 samples contaminated at a fractional recovery level (0.8 cells/g) and 8 highly contaminated samples (20.6 cells/g) were sent to each collaborator. At the fractional recovery and high inoculation levels, the sensitivity values of the standard method were, respectively, 52% and 100%, those of the alternative method 55% and 99%.

Significance: This ISO 16140 study clearly shows that the iQ-Check™ Cronobacter method is a reliable alternative method for Cronobacter spp. detection in infant formula and environmental samples, offering important economic savings by reducing time to result and handling time.