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

Food safety emergencies are high frequency in. recent years. Enterobacter sakazakii has attracted worldwide attention since 1961, Enterobacter sakazakii is a food-borne bacteria, which can cause sepsis in infants, necrotizing, enterocolitis, or meningitis, especially for neonate or immunobabies, and lead to apparent severe symptoms even dead.This paper summarized the PCR detection methods of E. sakazakii in powder infant formula milk.In this study, took use of E. sakazakii of special sequences of conservative 23S rDNA according to the result of BLAST to design a special primers. In the PCR process, DNA concentration, Mg²⁺ concentration, template volume, annealing temperature and PCR circles are optimized to determine the optimal PCR. The reaction mixture Consisted of 25uL: 2.5uL 10×PCR buffer(10 mmol/L Tris-HCl[pH 8.3], 50 mmol/L KC1, 0.1%[wt/vol] Gelatin. 0.5% Tween 20), 25 mmol/L Mg²⁺ 3.0uL. 2.0uL mixture of dNTPs, 0.5uL of 10 uM forward primer, 0.5uL of 10 uM reverse primer, 0.25uL (5U uL^-1) Taq enzyme, FTA cards as the template, ddH₂O 16.25uL. The reaction was run under the following conditions: Cool start; DNA pre-denaturation at 94℃for 5 min, DNA denaturation at 94℃for 1 min, primer annealing at 55.6℃, and DNA extention at 72℃for 1 min, run 34 cycles; the final extention was performed at 72℃for 10 min. The PCR products were examined by 2% agarose gel electrophoresis. PCR product was cloned into the pMD-18T plasmid to sequencing. The result of sequencing compared with target gene sequence showed the homology got to 100%, so PCR amplified products were certified. Five standard strains, one wild strain of E.sakazakii and 19 strains of Gram-positive bacteria and other negative bacteria in this study were detected by PCR, the results showed six 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 in infant formula powder, meanwhile, the sensitivity got to 10^-9.With PCR detection process was designed by seven different types of genomic DNA extraction methods, by the comparing their sensitivity, work process and detection limits found out one most simple and effective method of DNA extraction. Seven of genomic DNA extraction methods got different detection limit. Each method based on the different detection limit polluted again infant formula powder to find detection enrichment time of each method, the result showed these methods save much time and avoid culture of over night, which is time wasting. The results of DNA extraction methods show that the use of FTA filter to pyrolysis E. sakazakii cell membrane to release DNA method is better than the other six DNA extraction methods, the whole experimental procedure can be completed only 8 hours, shorter 12～22 h than the traditional biochemistry detection.Currently, country standard method about the detection of E. sakazakii has not formed, the typical traditional method FDA BAM experiment, DFI method, OK method and TITEK^TMmade comparisons, the test results of 35 samples were as follows: the sensitivity of PCR is 80.0% and the specificity of PCR is 96.7%, the coincidence rates of PCR is 94.3%, detection time of DNA made by FTA cards method is 8 h, DNA extraction kit and common pyrolysis method takes 11～12h, reagent pyrolysis, bacteriolyase, common reagent and proteinase K method about take 12～13h; FDA BAM method is with the detection sensitivity of 80.0% and the specificity of 100%,the coincidence rates of 97.1%, the detection time of 5～7 days; the sensitivity of DFI method is 80%, the specificity of DFI is 100%, the coincidence rates of DFI is 97.1%,the detection time need 4～5d; the sensitivity of OK method is 80.0% the specificity of OK is 93.3%,the coincidence rates of OK is 91.4%, the detection time need 4～5d. The result shows that the sensitivity of four methods are the same, the specificity of PCR is less than the two methods of BAM and DFI, which all higher the method of OK, the coincidence rates of PCR is slightly lower than other BAM and DFI method but still higher than OK method. In all, the detection time of PCR method is significantly shorter than the other three methods. In all, take use of FTA filter can detect E. sakazakii from IFP in 8 h, sensitivity get to 1.5CFU/100 g in the 2h enrichment time.