The Development Of Rapid Detection System Of Common Foodbone Pathogenic Bacteria

ObjectiveTo separate and/or enrich the common foodbone pathogenic bacteria, labmade magnetic beeds were developed. After coating with chitosan, the magneticbeeds were connected with glutaraldehyde. The coated magnetic beeds wereexpected to be used in the preparation of immunomagnetic beeds in thefollowing research. At the same time, the rapid detection method of Bacilluscereus group using TaqMan real time PCR was established. Then the reactionsystem was optimized and applied in the simulative sample.Methods1 The preparatipn and coating of magnetic beeds1.1 The preparation of magnetic heedsUnder the basic environment supplied by ammonia, Fe²⁺ and Fe³⁺ reactwith OH^- and produce Fe(OH)₂ and Fe(OH)₃ respectively. Fe(OH)₂ and Fe(OH)₃are unstable and decompose into FeO and Fe₂O₃, which are the maincomponants of Fe₃O₄. The magnetic beeds were. washed with distilled water until the pH is 7.0, and then was separated in magnetic separating Shelf.1.2 The coating of magnetic beedsThe glutaraldehyde was resolved in the 1ï¼…acetic acid. After coating withchitosan, the magnetic beeds were connected with glutaraldehyde, which used ascross linker in the coated partles.2 The estabishment of rapid detection method for Bacillus cereus group usingTaqMan real time PCRChoose a suitable gene for the detection of Bacillus cereus group. And thendesign a pair of primers and TaqMan probe using professional software PrimerExpress 2.0 and Primer Primier 5.0.Compare the advantage and disadvantage of CTAB method with Silica GelModel^TM Genome DNA extraction method and their influence factors. Choosethe better one as the preparation method for DNA standards in initial stage ofthis study.The real time PCR reaction condition was optimized, such as annealingtemparature and the concentration of primers, Mg²⁺, dNTPs, buffer and TaqDNA polymease. The sensitivity and specificity of the established methods wereestimated. In the end, this rapid detection method was applied in the detection ofsimulative rice samples contaminated with Bacillus cereus and quantified thereal number of these bacteria.Results1 The magnetic beads were prepared successfully. The diameter of thenagnetic beads was in naometer grade under the observation of transmissionelectron microscope. The magnetic beads are homogeneous in the liquid and can ba separated in the magnetic field very well. The volume of the coatedmagneticbeads increased and was suitable for the connection with antibody in order topreparing the immunomagnetic beads.2 16S-23S rDNA gene was chosen as the target gene through comparing thedifferent characteristic of several genes in Bacillus cereus group. After analyzingthe alignment of different ITS genes in GenBank, a pair of primers and TaqManprobe were designed as follow: Forward Primer: 5'-GAGTCCATTTAGGCCCACCATAC-3' Reverse Primer: 5'-AGCTTGGCGACTGGAGGACGC-3' TaqMan Probe: 5'-FAM-CAAGGCAGGCGCTCTCCCAGCTGAG-TAMRA-3'The result of comparison of CTAB method with Silica Gel Model^TMGenome DNA extraction method indicated that the Silica Gel ModelTMGenome DNA Extraction method had the advantage of high extractionefficiency and good purity of DNA genome. It is suitale for applying in theinitial reseach stage to prepare the DNA standards.After optimiztation, we have established the rapid detection method ofBacillus cereus group using TaqMan real time PCR successfully. The optimizedreal time PCR. reaction conditions are shown as the following table. Theestimated rusults for this method show that the sensitivity of this reaction hasreached 22cfu/25μl reaction system and 172fg target gene. This method coulddetect four bacteria including Bacillus cereus, Bacillus thuringiensis, Bacillusmycoides, Bacillus weihenstephanensis, in the Bacillus cereus group and showedgood specificity. ConclusionsIn this study, the nanomimeter garade lab made magnetic beads wereprepared and coated successfully. This production plays a basis for thepreparation of immunomagnetic beads in the following research. And it's alsorespected to be used in the separation of common foodbone pathogenic bacteriadirectly. Furthermore, a rapid detection method for Bacillus cereus group usingTaqMan real time PCR was proved to be sensitive and specific. It could beapplied to the rapid diagnosis in food poisoning caused by Bacillus cereusgroup.