Study On Real-time PCR For The Detection Of Enterobacter Sakazakii In Infant Formula

Enterobacter sakazakii is considered an opportunistic pathogen and has been implicated in food diseases causing meningitis,bacteremia or necrotizing enterocolitis specially in neonates and infants. The fatality rates have reached above 20％～50％. Only powdered infant formula has been linked to the Enterobacter sakazakii-related outbreaks. Traditional detection method takes up 6～7 days to identify Enterobacter sakazakii and the sensitivity is lower. In this study, a Real-time PCR assay was developed to detect Enterobacter sakazakii form infant formula quickly and the time of detection was shortened.In this study, according to Enterobacter sakazakii(29544) strain 149 bp element, specific primers and a TaqMan probe were chose. The DNA fragment of 149 bp was amplified. PCR products were confirmed by DNA sequencing. The fragment of Enterobacter sakazakii was cloned into T vector and transformed into JM109. The positive recombinant plasmid was used as standard quantitative template to develop a Real-time PCR. Used the same way a internal control recombinant plasmid was made to coamplify with the target gene in the same PCR system. The PCR system was optimized including annealing temperature,the ratio of the primer and probes and the quantities of the intemal control. The reaction mixture consisted of 12.5μL Premix Ex Taq(2×), 0.5μL of each primer(10μM), 1μL probe(3μM), 1μL internal control probe(3μM), 1μL internal control recombinant plasmid DNA(10~4 copies/μL), 1μL target recombinant plasmid DNA, 8μL of double-distilled water, the whole system was 25μL; The reaction was run under the following conditions: DNA pre-denaturation at 95℃for 10 s; DNA denaturation at 95℃for 5 s, primer annealing and extension at 60℃for 20s, and for 45 cycles.There were 15 bacterial strains to be detected including 4 Enterobacter sakazakii strains and 11 other bacterial strains except for Enterobacter sakazakii strains in order to determine specificity of amplification of primers. The results of 4 strains of Enterobacter sakazakii were positive and those of 11 other strains were negative.The effect of 3 methods of extracting DNA form Enterobacter sakazakii in infant formula were compared after 6h' enrichment in mLST-BHI. A efficient extracting procedure was confirmed for extraction of Enterobacter sakazakii DNA form infant formula.The sensitivity of the Real-time PCR assay was 8.624 copies/μL, 2. 7 CFU/mL; The detection limit of artificially contamination was 14.117 copies/μL, 3.5 CFU/100g infant formula and the detection could be finished in one business day.In this study, real detection was made. The result indicates: the sensitivity of the Real-time PCR assay is 100％, the specificity is 99.0％, the coincidence rate is 99.0％.