Rapid and Visual Determination of Cronobacter sakazakii in Powdered Infant Formula Using Competitive Annealing Mediated Isothermal Amplification (CAMP)

Abstract

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen that causes serious infections, particularly in infants. Neonatal infections with this pathogen have an 80% mortality rate. One mechanism that C. sakazakii infects infants is through contaminated powdered infant formula (PIF). Here, based on the 16S rRNA gene, competitive annealing-mediated isothermal amplification (CAMP) is reported for the determination of C. sakazakii to reduce the risk of infection. The established CAMP method was able to accurately identify all target bacterial strains and did not yield false positive results with non-target bacterial strains. Furthermore, accelerating primers were designed and included in the CAMP reaction to enhance the amplification efficiency. The sensitivity of the assay for C. sakazakii in spiked PIF was 1.9 × 103 CFU/g. The CAMP assay with enrichment accurately detected the presence of C. sakazakii in PIF even at an initial inoculation level of 1 CFU/g. Additionally, to make the procedure simpler, hydroxynaphthol blue was employed as a visual indicator. For positive results, the reaction solution turns blue instead of violet, while the negative samples remain violet, which is suitable for rapid analysis in resource-limited settings.