Development Of Rapid Detection Kit For Common Pathogenic Bacteria Of Food Poisoning

The people's health was affected by the bacterial food poisoning seriously. And it is very important to detect the pathogen rapidly and accurately. Traditional detecting is based on such methods as germiculture, morphology, physiological biochemistry, etc. And the operation is complicated and time consuming. To study the rapid detection is an important work.Five kinds of antiserum were prepared by immunizing the rabbits in this study. It contained the most pathogenicity serotypes of Samonella, Escherichia coli O157 and Shigella, except the Vibrio paraharmolyticue and Staphylococcus aureu. The polyclonal antibody was purified by saturated ammonium and affinity chromatography. And the activity and purity were satisfied to prepare the immunogold.Three kinds of colloid gold with different diameter were prepared by trisodim cicitrate reduction. According to the exosyndrome of color, scanning curve and scanning electron microscope, the colloid gold with 15nm in diameter was used to label antibody. The optimun pH for labeling was 8.2, and the amount of antibody was 18mg/L. Immunogold(colloid gold labeled antibody complex) was purified at high speed and low temperature and it was proved alive by using dot immunogold filtration assay.Nitrocellulose membrane, gold conjugate pad, sample pad and absorbing pad were assembled to immunochromatographic test strip. The gold conjugate pad was freeze dried after soaked immunogold to polyester membrane. The HF13504 of Millipore corporation were used to prepare the test strip, in which the sheep anti-rabbit IgG coated at 0.25mg/mL as control line, the polycolne antibody coated at 0.2mg/mL as the test line. The materials with high hydroscopicity were used as the sample pad and absorbent pad.The positive strains were detected with 100% positive result by the five kinds of strips. And the negative strains were detected without nonspecific reaction except the Escherichia coli O157 strip. The sensitivity of five kinds of strips was different but it was very common with the limited of 10~6cfu/mL~10~7cfu/mL. The result could be read clearly in 5 minutes. If sealed in plastic bag with drier, the strip can be stored 4 months in ordinary temperature (20℃~25℃).The sensitivity is improved by using the immunomagnetic nanoparticles. The target microorganism can be isolated from 1mL sample by using the immunomagnetic nanoparticles in 15min. And then the target is seprated from the particle by 60℃ heating 30min. At last the microorganism is detected by using the strip. "The rapid detection kit forcommon bacterial food poisoning" is made of strips assisted with immunomagnetic nanoparticles. The kit is used to detect the Escherichia coli 0157 from beef. The sensitivity increased 1 magnitude degree compared with using the strip detection only.In a word, five kinds of strips are prepared to detect Samonella, Vibrio paraharmolyticue, Escherichia coli 0157, Shigella and Staphylococcus aureu. And "the rapid detection kit for common pathogenic bacteria of food poisoning" is made of strips assisted with immunomagnetic nanoparticles. The kit is used to detect the Escherichia coli 0157 from beef. And it shows that the kit is time saving, sensitive, easy to be justified and suitable for at grassroot level and epidemiology investigate. These key techniques and the data will help us research and develop a great kit to detect more pathogen.