The Establishment Of Immunochromatographic Electrochemical Analysis Method Of Erwinia Pyrifoliae

Erwinia pyrifoliae belongs to the genus Erwinia.It was first prevalent in South Korea in the 1990s.It is a particularly dangerous agricultural plant pest in our country(Shang Mingqing et al.,2009).With the global economic integration,our country's trade with South Korea,Japan and other places is constantly increasing,and pathogens may spread to my country through seedlings,fruits or other channels.Common detection methods take a long time and require high professional operation capability.The laboratory has initially established an IgM-based Erwinia pyrifoliae colloidal gold immunochromatographic test strip,but there are problems of poor stability and low specificity.Therefore,this paper takes Erwinia pyrifoliae as the research object,and prepares a more stable and specific method based on Erwinia pyrifoliae IgG monoclonal antibody colloidal gold immunochromatographic test strip method,which realizes the rapid and specific detection of Erwinia pyrifoliae.Based on the establishment of electrochemical immunoassay method,the sensitivity of the test strip is improved.Mainly include the following:(1)Preparation of antibodies:Antibodies are the core raw material for immunoassay.In this experiment,BALB/c mice were intraperitoneally injected with the whole bacteria of Enwinia pyrifoliae,and cell fusion was successfully performed.The hybridoma cell line 4AlC8 of bacterial monoclonal antibody was purified by ProteinG column,and the antibody subtype was identified.The antibody subtype was IgG3.(2)Establishment of immunochromatographic method:The colloidal gold was used as the label to mark the obtained I-G3.At this time,the gold-labeled antibody was the recognition antibody and the polyclonal antibody was the capture antibody.A double-antibody sandwich method was constructed.Colloidal gold immunochromatographic test strips were optimized for a series of parameters.Qualitative detection showed that the detection limit of the test strip for Erwinia pyrifoliae contained in the leaves was 1×107 CFU/mL,and there was no cross-reaction with Erwinia amylovory,which achieved rapid detection of Erwinia pyrifoliae Specific detection.(3)Establishment of immunochromatographic electrochemical analysis method:We prepared colloidal gold immunochromatographic test strips according to the double-antibody sandwich method.In order to further increase the minimum concentration of antigen detection and amplify the response signal of specific antigen and antibody,we combined the colloidal gold immunochromatographic test strip with electrochemical analysis technology.We label horseradish peroxidase(HRP)on the gold-labeled antibody complex.HRP reacts with the hydrogen donor o-phenylenediamine(OPD)to produce electrochemically active azo products.Carbon nanotubes are used to print electrodes.Wave voltammetry was used to measure the electrochemical signal to realize the sensitive detection of Pyricularia infestans in Asia.The immunochromatographic electrochemical analysis method we established can complete the detection of Pyricularia infestans within 15 minutes,with a minimum detection limit of 1×106 CFU/mL.