| Acidovorax citrulli is a seedborn pathogen,which could cause diseases to many cucurbits,such as watermelon,melon,cantaloupe,pumpkin,cucumber and so on.The anti-drought ability of the pathogens is very strong,which make the pathogens survive on the seed surface for 35 years,and it could be spread long distances by seeds carrying bacteria.Pathogens could infect fruits,plants,and seeds.The resulting diseases have the characteristics of rapid onset,strong explosiveness,and high speed of transmission.Once infected,it will cause huge economic losses to the melon production,making the disease become one of the major diseases affecting the production of melon in China.At present,the commonly used methods for the detection of Acidovorax citrulli were PCR-based methods.The detection sensitivity of this method is relatively high,but it requires precise thermal cycler and a series of regants,making it usually takes 3 to 4 hours to obtain an accurate results.All the nentioned above lead to that this method is performed in a fully equipped laboratory which is not suitable for rapid field test of pathogens.The immunological detection methods,such as enzyme linked immunosorbent assay(ELISA)and protein array need longer incubation time and complicated steps.Rapid immunoassay methods,e.g.,immunochromatographic test strip,are simple in operation,short in detection time,and do not require the use of instruments,which have a good application prospect in the rapid screening of field pathogens.To establish a rapid immunoassay method for the detection of Acidovorax citrulli,monoclonal antibodies against the SD01 strain were firstly prepared.The specific research contents were as follows:BALB/c mice were immunized with wild type strain SD01,and cells were screened by selective medium and ELISA,following by cell fusion technique.Four monoclonal hybridoma cell lines secreting anti-SD01 strain antibody were successfully obtained.After preparation and purification of mouse ascites,four monoclonal antibodies were obtained and named as 6F,6D,7E,4F.The titers of four antibodies at 2 mg/mL detected by indirect ELISA were 1:12800,1:6400,1:3200,and1:6400,respectively and the antibody subtypes were all IgG 2a.Polyacrylamide gel electrophoresis verified that the purified antibodies had a light chain of about 25 kD,a heavy chain of about 50 kD,and no extra bands which indicated the purification methods of the antibody were efficient.Indirect ELISA was used to detect the binding of 4antibodies to 8 strains of Acidovorax citrulli and 6 strains of gene similar plant pathogens.The results showed that 6D and 4F could bind 8 strains of Acidovorax citrulli;6F could bind 6 strains of Acidovorax citrulli which could not bind to 00-1 and tw31;7E could bind 5 strains of Acidovorax citrulli which could not combine with ATCC 29625,00-1,and tw31.There was no cross reaction of 6D and 7E with 6 gene similar plant strains,while 6F had cross reaction with NCPPB 961 and ATCC 33996,and 4F had cross reaction with ATCC 33996 and ATCC 19307.Through the HRP enzyme labeling monoclonal antibodies 6D and 4F,a double antibodies sandwich ELISA method for the detection of Acidovorax citrulli was successfully established with a detection sensitivity of 10~5CFU/mL.All the results mentioned above showed that this experiment successfully prepared a monoclonal antibody 6D with strong specificity,followed by 4F,which could be used to establish rapid immunoassay methods.Since monoclonal antibodies 6F and 7E do not bind to other strains of Acidovorax citrulli,they are not suitable for the development of rapid immunoassay for the detection of Acidovorax citrulli.To further verify the detection performance of the prepared monoclonal antibody in practical application,the colloidal gold nanoparticles were selected as a tracer and the monoclonal antibody 6D was selected for colloidal gold labeling,following by fixed on the conjugate pad of the test strip.At the same time,monoclonal antibody 6D and goat anti-mouse IgG antibody were separately sprayed on the nitrocellulose membrane to form into the detection line and the control line to develop an antibody self-paired colloidal gold-based immunochromatographic test strip.The sensitivity,specificity,and stability of the test strip were evaluated.Meanwhile,the samples of watermelon plant carrying 8strains of Acidovorax citrulli were detected using the developed test strip,and the PCR method was used to verify the accuracy of test strips.The results showed that the colloidal gold immunochromatographic test strip with self-paired antibodies had good specificity,which could detect 8 strains of Acidovorax citrulli,and had no cross reaction with 6strains of gene similar plant pathogens.The detection sensitivity for pure cultured bacteria was 10~5 CFU/mL;the strip can be stored at room temperature for at least 6 months without losing the stability of the assay.The samples of watermelon plants carrying 8 strains of Acidovorax citrulli(concentration in 10~5 CFU/mL)were all detected to be positive.The verification results of the actual samples by the conventional PCR method were consistent with the test strip,which indicated that the developed self-paired colloidal gold test strip could be used for the rapid detection of field samples.At present,immunochromatographic test strip based on self-paired antibodies have not been reported at home or abroad.The method of self-paired antibodies is an extension of the double-antibodied sandwich mode.The successful application further demonstrated that this application model could be extended to the development of other immunological detection methods,which will become a new construction method and supplementary form for the development of immunological detection methods when specific monoclonal antibodies are very difficult to obtain.Colloidal gold as a tracer is the most commonly used colored marker in immunochromatographic analysis technology.To further simplify the prepared steps of immunochromatographic test strips and save costs,the topic has further developed a novel antigen-based fluorescence immunochromatographic test strip.In this study,fluorescein isothiocyanate(FITC)was used as a tracer,which was added to the bacterial medium to modify them.Bacteria could emit stable yellow-green fluorescence after cultured in the modified medium,making them to be a fluorescent probe during culture.The successful development of fluorescent immunoassay strip was based on the combination of fluorescent bacteria and unlabeled monoclonal antibody 6D immobilized on the test line.The fluorescent test strip developed in this method requires only one unlabeled antibody,which successfully simplifies the steps of the test strip.The preparation procedure saves costs by eliminating the use of labeled antibodies.The sensitivity,stability,and specificity of the test strip were evaluated.Thirteen watermelon plant samples,13 watermelon seed samples,and 6 watermelon diseased samples collected in the field or purchased from different markets were used to assess the detection ability of the test strip.The accuracy of the test strip for detecting real samples were also verified by conventional PCR method.The results showed that the detection sensitivity of the test strip was 10~6 CFU/mL,and the test results could be observed within 15 min.The test strip could detect 8 strains of Acidovorax citrulli and had no cross reaction with other 30 strains of pathogens.The strip could be stored at room temperature for at least 4 months without losing the stability.The results of real samples detected by the test strip were consistent with the results using the conventional PCR method,which indicated that the fluorescent strip have higher accuracy in the detection of real samples.In this study,the transfer of tracer from labeling antibodies to labeling antigens was achieved.The development of antigen-based fluorescent test strip resulted in the successful isolation of traditional antibody-labeled based double antibodies sandwich mode because only one unlabeled antibody was required.The mode,thus reduces the costs,makes the fluorescent test strip easier to popularize and apply.In order to improve the detection sensitivity of the prepared fluorescent immunochromatographic test strip,the FITC-conjugated monoclonal antibody 4F was further prepared,which was sprayed on the conjugate pad of the test strip to become a capture antibody.The modified culture medium was used to cultivate bacteria.An antigen and antibody dual labeled fluorescent strip based on a signal amplification system was successfully developed.In this study,fluorescent bacteria was as the first fluorescent probe,and FITC-labeled monoclonal antibody 4F was as the second fluorescent probe.Goat anti-mouse IgG antibodies and monoclonal antibody 6D was immobilized on both sides of the NC membrane to form into control line and detection line.The sensitivity,stability and specificity of the test strips were evaluated and the watermelon samples carrying 8 strains of Acidovorax citrulli were tested.The results showed that the test strip could detect 8 strains of Acidovorax citrulli,and had no cross-reaction with other 6 plant pathogens.The minimum detectable limit was 10~5 CFU/mL,which was observed with the naked eye.Compared with fluorescent immunochromatographic test strip based on FITC just labeled antigen or antibodies,the sensitivity of antigen and antibody dual labeled test strip was improved 10 times.It could be stored at room temperature for at least 4 months without losing the stability.The PCR results were consistent with the results of the test strip on simulated watermelon samples,which indicated that the developed dual labeled fluorescence immunochromatographic test strip could be used for rapid detection of actual samples.In this study,the use of FITC labeled antigen and antibody to develop a dual labeled fluorescence immunochromatographic test strip based on the signal amplification system,was successfully achieved signal amplification and the detection sensitivity could be increased by 10 times.In this study,four kinds of monoclonal antibodies against SD01 were prepared and immunological assays were developed to detect Acidovorax citrulli.On the basis of preparing a monoclonal antibody with strong specificity against the SD01strain,the colloidal gold nanoparticles as the colored markers and the FITC as the luminescent materials were selected for the development of the immunochromatographic test strips.On the basis of colloidal gold labeling monoclonal antibody 6D,self-paired immune bchromatographic test strip were successfully developed.Based on FITC labeling bacteria,an antigen labeled fluorescent immunochromatographic test strip were successfully developed.Based on FITC labeling bacteria and monoclonal antibody 4F,an antigen and antibody dual labeled immunochromatographic test strip was developed.The sensitivity,specificity,stability,and detection performance of the three kinds of test strip were evaluated.Compared with conventional ELISA and PCR methods,the three kinds of immunochromatographic test strips developed have shortened the detection time and simplified the operation steps.This simple,low cost and easy to operate method will play an important role in the monitoring of plant pathogens. |
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