Evanescent Wave Fiber Optic Biosensor For The Detection Of Salmonella And Salmonella Enteritis

Salmonella is one of the world’s most common pathogen of the outbreaks of water-borne and foodborne disease, disseminating by contaminated water and food, have a very important significance in public health area. Salmonella infection has become a worldwide public health problem, and people pay more and more attention to its harmfulness. At present, salmonella detection is still the traditional separation method, and it will take5to7days. This process is cumber some and time-consuming, and it is not conducive to the prevention and control of salmonella infection. Fiber optic biosensor can meet the needs of the rapid detection of pathogenic bacteria, and it can supply a new method for the rapid detection of salmonella.Objective1. As fiber-optic evanescent wave biosensors can make on-site rapid screening, we had initially established rapid detection platform of salmonella, and provide technical platform for the rapid detection of other common pathogenic bacteria.2. Using hybridoma technology, prepare the monoclonal antibody of salmonella enteritis, and identificate the specificity, concentration, titer and subtype of the antibody.3. Using the monoclonal antibody of salmonella enteritis by our laboratory, relying on the rapid detection of fiber-optic biosensor, to establish the rapid detection platform of salmonella enteritis.Methods1. Study the rapid detection method of fiber-optic evanescent wave biosensor for salmonella, and according to the principle of double antibody sandwich method we had established the platform for rapid detceion of salmonella, we conjugated the detection antibody with flourescent dye of Alexa fluor647, and stored the labeled antibody at-20℃. The fibers were coated with streptavidin after washed by isopropanol, and the biotin-labeled capture antibodies were more effectively connected to the fibers by avidin-biotin reaction. On the basis, we optimized the conditions, including the optimal concentrations of capture antibody and detection antibody. Then under the optimical condition, we determine the detection sensitivity and specificity, and we confirmed sensitivity and specificity of the method in practical samples by detecting artificial infection samples. And preliminary established salmonella rapid detection method.2. Using small doses of immunization programs for a long period. Inactivate salmonella enteritis as immunogen, and immune12mice with different doses through intravenous injection, the immune dose were2.5×108,5×107,1×107,5×106cfu separately. After the whole immune cycle, we fuse murine Sp2/0cells with spleen cells from mice immunized with salmonella enteritis and subclone for3to4cycles. The ascites containing monoclonal antibodies against salmonella enteritis was gained via inoculation of the hybridoma cells into the abdominal cavity of BALb/C mice. Adopting the traditional saturated ammonium sulfate purification method to purificate the ascites, and identificate the specificity, concentration, titer and subtype of the antibody.3. Study the rapid detection method of fiber-optic evanescent wave biosensor for salmonella enteritis. According to the principle of double antibody sandwich method, we had established the platform for rapid detection of salmonella enteritis. We optimized the conditions, including the best response times of capture antibody and target, reaction system and detection antibody, the optimal concentrations of capture antibody and detection antibody. Then under the optimical condition, we determine the detection sensitivity and specificity.Results1. The Fiber-optic evanescent wave biosensor automatically completes the detection within40minutes for salmonella. Under the best condition, the sensitivity of the method could be5.5×103cfu/mL. It had a better specificity to the salmonella, which the detection results were all negative for the other non-salmonella.7tipical strains serotypes of salmonella were all positive. And we got the100%positive results for the environmental water samples that the concentrations of the salmonella were4.2×10cfu/mL or more. Double TSB enrichment experiments show that the salmonella concentration is50cfu/mL, after6h enrichment we can. reach a positive signal value.2. One hybridoma cell line excreting monoclonal antibodies against salmonella enteritis coded4G11was obtained by fusing murine Sp2/0cells with spleen cells from BALb/C mice immunized with salmonella enteritis. After the purification, the antibody concentration is1.972mg/mL, the antibody titer is8000, and the antibody subclass is IgG3.3. Using the monoclonal antibodies prepared by our laboratory as capture antibody, the Fiber-optic evanescent wave biosensor automatically completes the detection within60minutes for salmonella enteritis. The best response times of capture antibody and target was10min, the best response times of reaction system and detection antibody was20minutes, the optimal dilution degrees of capture antibody is1000, and the optimal detection antibody80μg/mL. Under the best condition, the sensitivity of the method could be3.0×103cfu/mL. It had a better specificity to the salmonella enteritis, which the detection results were all negative for the othe7strains serotypes of salmonella and4strains of non-salmonella.ConclusionsThis study established the method of rapid detection for salmonella in water with the fiber-optic evanescent wave biosensor. It will provide a method of rapid detection for salmonella pollutted water, and a technical platform for the detection of other common pathogenic bacteria. At the same time, using self-made salmonella enteritis monoclonal antibody we established the method of rapid detection for salmonella enteritis, provided a new method for the detection salmonella epidemic superiority serotype.