In silico and in vitro validation of a duplex polymerase chain reaction assay for detecting Cronobacter sakazakii in powdered infant milk

Background: Cronobacter sakazakii causes life-threatening infections in neonates, primarily transmitted through contaminated powdered infant formula (PIF). In low- and middle-income countries, limited surveillance and diagnostic capacity hinder accurate detection of C. sakazakii, highlighting the need for rapid and affordable testing methods. Objective: To validate a duplex polymerase chain reaction (PCR) assay for rapidly detecting Cronobacter spp. and C. sakazakii in PIF using both in silico and in vitro approaches. Methods: This study was conducted in South Africa between March and August 2022. Seven gene targets and their primers were selected from published literature. To assess sensitivity and specificity, in silico PCR was performed using genome sequences of Cronobacter and non-Cronobacter species from the National Center for Biotechnology Information database. The best-performing primers were selected for an in vitro analysis using bacterial isolates and PIF samples from the Infection Control Service Laboratory. The specificity of the assay was assessed using eight foodborne pathogens, and further evaluated using PIF samples artificially contaminated with Cronobacter spp., Bacillus cereus, and Salmonella Typhi. Results: The best-performing primers, lpfA_1, fimG, and fimp1, showed 100% sensitivity and specificity. Duplex PCR assay successfully detected both Cronobacter spp. and C. sakazakii with no cross-reactivity with non-Cronobacter pathogens, and remained effective in the presence of contaminants such as B. cereus and Salmonella Typhi. Conclusion: The validated duplex PCR assay offers a rapid, specific, and affordable assay for detecting Cronobacter spp. and C. sakazakii in PIF. What this study adds: This assay combined in silico and in vitro validation of a rapid and affordable PCR assay for PIF screening in resource-limited settings.