Development And Application Of Assays For Rapid Detection Of Enterobacter Sakazakii(Cronobacter Spp.) In Food Samples

Enterobacter sakazakii (Cronobacter spp.) is an opportunistic pathogen and is linked with life-threatening infections in neonates. Clinical symptoms of Enterobacter sakazakii infection include necrotizing enterocolitis, bacteremia and meningitis with high fatality rates in neonates and infants. The organism has been isolated from a wide variety of foods and environments. The accurate identification and rapid detection of Enterobacter sakazakii is of great importance. Detection assays based on PCR technology has been applied widely in the detection of foodborne pathogen for its high sensitivity, specificity and rapidness, and has a wide application foreground.In this study new detection systems were developed, and the following work was carried out.1. Traditional duplex PCR assay for the detection of Enterobacter sakazakii(Cronobacter spp.)Considering the genetic diversity and classification complexity of Enterobacter sakazakii, single PCR detection assay can give a false-positive result. In this study duplex PCR assay was developed based on its16S rRNA gene and partial macromolecular synthesis (MMS) operon. Among all the bacterial strains used in this study, duplex PCR amplified two specific products from4strains of Enterobacter sakazakii but not from others. In pure culture, the sensitivity of single PCR for former gene was6.3x101CFU/mL, while for the later was6.3×103CFU/mL; whereas for duplex PCR, it was6.3×103CFU/mL. The detection limit for duplex PCR assays was100CFU/mL or/g for different levels of Enterobacter sakazakii inoculated into food samples (milk powder, milk and chicken meat) after24h enrichment. The detection limit of the duplex PCR, however, remained unaffected in the presence of Salmonella typhimurium in milk powder in another analysis. So, the methods described in our study can be adopted to detect Enterobacter sakazakii in food samples with higher sensitivity and specificity. 2. Real-time PCR assay with an internal amplification control for the detection of Enterobacter sakazakii (Cronobacter spp.)In this study, a Taqman real-time PCR assay incorporating an internal amplification control (IAC) was developed and evaluated for specific detection of Enterobacter sakazakii in foods. An IAC, amplified from the pTG-19vector, was constructed using composite primer method, which was heterologous to the target gene, probe was designed. Previously reported macromolecular synthesis (MMS) operon sequence was selected for specificity, and67bacterial strains, including four strains of Enterobacter sakazakii, were evaluated. All Enterobacter sakazakii strains were successfully identified, however no cross-reactivity was observed with non-Enterobacter sakazakii strains. Detection limit of the assay in pure culture and milk powder without enrichment was1.2×103CFU/mL (1.2×101CFU/assay). After24h enrichment in broth, as few as100CFU/mL or g of Enterobacter sakazakii could be detected in artificially contaminated food samples (milk powder, sterilized milk and chicken meat). The detection limit of the real-time PCR assay however remained unaffected in the presence of108CFU/mL Salmonella typhimurium in another analysis. The adjusted real-time PCR assay can be adopted to rapidly detect Enterobacter sakazakii in food samples with high specificity and sensitivity, and can prevent false negative results by using the IAC.3. Application of rapid detection assaysIn attempt to evaluate the accuracy of the rapid detection assays, and to understand the distbiution of Enterobacter sakazakii in food samples, rapid detection assays and traditional culture method were used to investigate the presence of Enterobacter sakazakii in92food samples. Our results showed that one pork samples out of the total were observed positive by all three detection methods. The identity of the rapid assays was98.9%(91/92) with the traditional culture method, and no missed diagnosis was found.