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Title: Development of Multiplex Real-time PCR with Internal Amplification Control for Simultaneous Detection of Salmonella and Cronobacter sakazakii in Powdered Infant Formula.

Author
item HYEON, JI-YEON - Konkuk University
item PARK, CHAN-KYU - Konkuk University
item CHOI, IN-SOO - Konkuk University
item Holt, Peter
item SEO, KUN-HO - Konkuk University

Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/24/2010
Publication Date: N/A
Citation: N/A

Interpretive Summary: Industry and regulatory laboratories continue to search for simple and rapid methods to detect human pathogens in food samples. A real time PCR assay was developed for the simultaneous detection of Salmonella and Cronobacter sakazakii (CS) in powdered infant formula (PIF). Also included in the assay was an internal amplification control which helped provide assurance that negative values were due to absence of organism and not to inhibition of reaction because of the infant formula components. The assay was able to detect as few as 1000 Salmonella and CS organisms/ml in artificially contaminated PIF, without any methods to enrich growth of the organisms. If the samples were subjected to enrichment procedures for 12 hours or longer to increase the growth of the organisms in the PIF prior to running the PCR, the assay could detect the organisms at initial level of 0.1 organisms per gram of formula These results indicate that the multiplex real-time PCR assay is an effective method for the rapid and simultaneous detection of Salmonella and CS.

Technical Abstract: Contamination of powdered infant formula (PIF) by the bacteria Cronobacter sakazakii and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive multiplex real-time PCR assay for the simultaneous detection of C. sakazakii and Salmonella in PIF. In addition, an internal amplification control (IAC) was also included for exclusion of false negative results in this study. The quantitative detection range for pure cultures in this optimized multiplex real-time PCR assay was 103 to 108 CFU/ml for both Salmonella and C. sakazakii. When our established multiplex real-time PCR system was applied to artificially contaminated PIF, the detection limit was 103 CFU/ml for Salmonella and C. sakazakii without enrichment. The commercial PIF was then inoculated with Salmonella and C. sakazakii at 10, 1 and 0.1 CFU per gram of formula and the single enrichment broth samples were analyzed by multiplex real-time PCR after enrichment for 9, 12, and 24 h. Twelve h post-enrichment, we could detect Salmonella and C. sakazakii at initial inoculation levels of approximately 0.1 CFU/g in PIF. Additionally, stable fluorescent IAC signals could be assessed between 29 and 34 cycles of PCR amplification. Results from this study showed that the multiplex real-time PCR assay is an effective method for the rapid and simultaneous detection and quantification of C. sakazakii and Salmonella in PIF.