Research On Rapid Detection Methods Of Salmonella Typhimurium Based On IMS And FICA

Salmonella is one of the most common zoonotic pathogens. Salmonella typhimurium(ST.)is an invasive bacteria always in the lead of salmonella infection, mainly affect the ileum andcolon, and even the entire gastrointestinal tract. It mainly causes enterocolitis, which is themost common in pediatric salmonella infection. The incubation period of this disease isusually range from8~48hours. It has the feature of urgent, often onset with diarrhea andfever. Physical differences lead to different priorities. Therefore, establishing asemi-quantitative detection method of ST., which is high rapid and sensitivity has greatsignificance in the clinical diagnosis of salmonellosis.In this paper, we prepared the polyclonal antibody of ST. by immuning a male NewZealand white rabbit with broken ST. antigen. Gathered the antiserum after4times immuneand1time strengthen immune, then adopt octylic acid ammonium sulfate precipitation topurificate, and measured its titer by indirect ELISA for10~6. Through SDS-PAGE experiment,there are some miscellaneous belts, but it has a clearly heavy chain of about50kD and a clearlight chain of about25kD.We also used indirect ELISA method to do the specificityexperiments of13kinds of bacteria. Pab only cross with E. coli O157: H7and several kindsof bacteria in the genus.In Fluorescent Immunochromatography platform, we use the immunology principle ofdouble antibodies sandwich, made Pab self-match, and conducted the optimization ofdipsticks in a series of conditions. We made dipsticks under the optimal conditions, and itsdetection limit is3.162×10~6cfu/mL, quantitative limit is4.082×106cfu/mL.After havingexperiments of three batch of dipsticks, the recovery rate of concentration logarithm isbetween96%~101%, the recovery rate of liquid concentration of high ST. bacteria liquidconcentration is between80%~120%, the recovery rate of the low ST. bacteria liquidconcentration is also above60%.And the variation coefficient of T, C and T/C value of sameplate and different plates are all within5%.In Immunomagnetic Separation platform, choosing the micron level magnetic beadsnamed XMag-COOH. After activated by EDC and NHSS and coupling of Pab, getting the ST.immune magnetic beads(IMB). Then determine the optimal coupling conditions are adding100μg Pab into0.5mg MB, coupling2hours in pH6.010mM MES buffer in37℃, closingIMB by1%BSA PBST. In enrichment experiments, adding0.5mg IMB into per ml bacterialiquid, capturing for45minutes under the condition of37℃. The best elution condition is eluting in pH2.60.2M Phosphate-Citrate buffer for5minutes, and using Tris-HCl to AdjustpH of the buffer system to about7.0, conducting the Fluorescent Immunochromatographyexperiment, and then measuring the elution recovery is about45%, then the actual totalmultiple of enrichment is about4.5to45times.This paper in view of establishing the joint semi-quantitative rapid detection method ofsalmonella typhimurium，which combined Fluorescent Immunochromatography technologywith Immunomagnetic Separation technology. In Fluorescence Immunochromatography testof simulation sample，the recovery rate is between60%to170%. In the joint detection ofsimulated sample，the recovery rate is between38%to55%, the actual enrichment ratio is upto55times, sensitivity can reach at least105cfu/mL.