| Vibrio widely exists in seawater,sediments and marine animal body.It is a common pathogen infecting sea fish,shrimp,shellfish etc and pathogenic microorganisms mainly causing human acute gastroenteritis.And it has caused a great threat to human health.The traditional methods for detecting marine Vibrio exist time-consuming,tedious process and other shortcomings and can not meet the needs of marine monitoring.So we need to develop a rapid detection method with short time and simple operation.Gold immunochromatographic assay(GICA)is a newly developed rapid detection technology since the ninety's and has been applied in many aspects.The characteristics of short-time detection,simple operation,strong specificity make it play an important role in the modern food inspection work.Prepared antigen in this experiment of Vibrio parahaemolyticus(Vp)and Grimontia hollisae(Gh)by using formalin inactivated bacteria for immuning New Zealand rabbit to produce polyclonal antibody serum.The serum were purificated by using caprylic acid ammonium sulfate salting method.The titers of Vp polyclonal antibody,Gh polyclonal antibody1 and antibody 2 were determinated by ELISA method,and the results were 1:32000,1:64000,1:16000 respectively.The three antibodies'concentration determined by Bradford method were21.06,19.20,19.11 mg/mL respectively.All antibodies have a good purity by identificating with SDS-PAGE method and can be used for follow-up tests.Colloidal gold with a particle size of 20 nm was prepared using citric acid three sodium for conjugating with antibody.In this experiment,the optimal protein labeling of Vp polyclonal antibody and Gh antibody 1 were 27.6?g and 21.6?g respectively;the gold labeled probe diluent was 0.01 mol/L Tris-HCL(1%BSA,0.5%PEG 20000,5%sucrose,0.5%Tween-20);the sample pad treatment solution formula was 0.01 mol/L PB solution(1%BSA,0.5%Triton-100,1%tween-20,1%sucrose).Assembled into strips,the sensitivity of Vp strip is 10~3 cfu/mL.The sensitivity of Gh test strip was 10~5 cfu/mL.The two test strip has good specificity and stability after the test.And when the sample contains a mixed bacteria,the detection sensitivity is not affected.In this experiment,method that immunomagnetic beads separate and enrich bacteria was established.Experiment determine the optimal dosage of immunomagnetic beads was 60?L,the best capture time is 30 min.Used two methods:centrifugation and immunomagnetic beads to enrich Vibrio of samples,and analyzed their influence to sensitivity of strip.When used GICA detecting directly Vp in prawns,oysters,clams,squid samples,the sensitivity of strips were 10~5cfu/mL.Artificially polluted prawns,oyster samples(4 cfu/g)can be found to Vp after only8-hours culture using Vp strip.Detecting Gh in oyster samples by GICA,the sensitivity was 10~6cfu/mL.When IMS-GICA detected Vp in oyster samples,the sensitivity increased to 10~4 cfu/mL,and detected Gh,the sensitivity increased to 10~5 cfu/mL. |
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