Study On Polymerase Chain Reaction Assay For Detection Of Enterobacter Sakazakii In Powder Infant Formula Milk

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 powered infant formula has been linked to the Enterobacter sakazakii and the sensitivity is lower. In this study, the use of PCR amplification with FTA filters was developed to detect Enterobacter sakazakii form infant formula quickly and the time of detection was shortened.In this study, took use of E. sakazakii of special sequences of conservative 16S rRNA to design a special primers. In the PCR process, Mg²⁺ concentration, annealing temperature and PCR circles are optimized to determine the optimal PCR. The reaction mixture consisted of 50μL: 6μL 10×PCR buffer, 4μL mixture of dNTPs, 2.5μL Mg²⁺ (25 mM), 2μL of 10μmol forward primer, 2μL of 10μmol reverse primer, 0.25μL (5U /μL) Taq enzyme, ddH₂O 23.25μL. The reaction was run under the following conditions: Cool start, DNA pre-denaturation at 95℃for 5 min, DNA denaturation at 95℃for 1 min, primer annealing at 61℃, run 35 cycles. The PCR products were examined by 2% agarose gel electrophoresis. The result of sequencing compared with target gene sequence showed the homology got to 100%, so PCR amplified products were certified. There were 16 bacterial strains to be detected including four strains of E. sakazakii and 14 other bacterial strains except for E.sakazakii strain in order to determine specificity of amplification of primers. The results showed four strains belonging to positive results after primers extending, the results were negative for other strains. In this study result proved that the primers used in PCR was specific to detection E. sakazakii.The Sensitivity of PCR-based assay with FTA filters as templates was 7×10¹ cfu/mL.The Detection limit of E. sakazakii in reconstituted infant formula by using FTA filter for DNA template preparation after an enrichment step of 4 h was 7×10⁰ cfu/100g.The whole experimental procedure can be completed only 6 hours, shorter 12-22 h than the traditional biochemistry detection. The effect of six methods of extracting DNA from Enterobacter sakazakii in infant formula were compared, an efficient extraction procedure was confirmed for extraction of Enterobacter sakazakii DNA from infant formula. Enterobacter sakazakii DNA was extracted using FTA filter. The detection time of PCR method is significantly shorter than the other methods.In this study, real detection was made. The result indicates: the sensitivity of the method is 100%, the specificity is 98.2%, and the coincidence rate is 98.2%. Preparation of PCR templates with using FTA filters more sensitive and was able to efficiently remove inhibitors that could affect the PCR reaction. The results of this study demonstrate that a filter-based method can efficiently be used to detect food-borne bacterial pathogens in food.