Development and Application of a Species-Specific PCR Assay for the Detection of the Emerging Food-Borne Pathogen...

Cronobacter sakazakii is an emerging opportunistic food-borne pathogen associated with a wide range of foods including seafood, meat, plant-based, and dairy products. Although seafood is a significant source of this pathogen, specific and sensitive methods that can differentiate C. sakazakii from other Cronobacter species are currently not available. In this study, a PCR assay using primers targeting fhuF gene was developed and evaluated for its specificity and sensitivity in detecting C. sakazakii. The designed primers exhibited high level of specificity for C. sakazakii and no cross reaction was observed with other Cronobacter species and related Enterobacterales. The sensitivity of detection was tested using pure culture of C. sakazakii and in shrimp samples artificially spiked with C. sakazakii. The fhuF-specific PCR assay could detect as low as 1.45 × 10³ cfu/ml in pure bacterial cultures and 2.9 cfu/g in spiked shrimp following four hours of enrichment. When applied to natural samples of fresh and dried seafood, the assay detected C. sakazakii in three samples following enrichment in a selective broth. The novel fhuF-specific PCR assay developed in this study provides a reliable and sensitive tool for the detection of C. sakazakii and will be useful in prevalence and risk assessment studies involving seafood.