Development of a PCR Assay for Rapid Detection of Cronobacter sakazakii from Powdered Infant Formula Using a Target Sequence Identified by Comparative Genomic Analysis

Abstract

AbstractThe occurrence of outbreaks of Cronobacter sakazakii causing necrotizing meningitis in China highlights the need for strain characterization of this pathogenic species. In this study, SMM system was utilized to mine for new molecular markers of C. sakazakii. One C. sakazakii‐specific CDS (>ref|NC_009778.1|:c1077329‐1076055) with a length of 1,275 bp was used to design a primer set. A PCR assay was developed and optimized to detect C. sakazakii. The PCR assay amplified a 498 bp DNA product from all 43 C. sakazakii strains tested but not from 30 other bacteria strains. The detection limit was 7.11 pg/PCR (equivalent 1,422 genomic copies) when the genomic DNA of C. sakazakii ATCC 29544 was 10‐fold diluted. Three powdered infant formulas from different commercial brands were inoculated with the strain ATCC 29544 at the level of 56 cells contamination, and the target fragments (498 bp) were produced after samples were enriched for 6 h at 37C.Thirty food samples were detected simultaneously by this PCR assay and the conventional method, and a coincident detecting result was found between the PCR assay and the conventional method. This method provides a useful tool for rapid identification of C. sakazakii in food and environmental matrices.Practical ApplicationsThe gold standard based on the FDA‐recommended methods for isolation and identification of Cronobacter sakazakii from powdered infant formula is time consuming and labor intensive. A PCR assay has been established to detect C. sakazakii. This method provides a useful tool for rapid and accurate identification of C. sakazakii in food when a large number of food samples need to be rapidly isolated and identified.