| Xanthomonas axonopodis pv. Citri (Xcc) is one of the invasive pests, a bacterial citrous plant pathogen that causes citrus canker(Citrus bacterial canker disease, CBCD), which is the most serious citrus bacterial diseases of quarantine. The disease could infect a variety of rutaceae plants, including all the commercial citrus cultivars. Hamlin,sweet oranges and Limes are highly susceptible to the disease, and have very challenging of citrus canker. The bacterium usually multiply in lesions in citrous leaves, stems, root and fruit, with the long-distance transmission of illness seedlings and other propagating materials for national and international trade, Xanthomonas axonopodis pv. citri spread throughout the world. Citrus canker often leads to tree vigor decrease, defoliation, fruit covered with lesions, poor quality, fruit drop, entirely unmarketable, destroyed trees, seriously affecting the yield and quality of citrus, has received considerable political attention. Because of the lack of resistant citrus varieties and effects pharmacy, people still use the method of burn eradication method to to deal with susceptible plants. Currently work rely mainly on enhance the plant quarantine to prevention and control of the disease, cut off the spread of the epidemic source. With China's rapid economicdevelopment, international and national travel and trade of citrus products more frequently, increased the risk of invasive plant pests. In such a situation people urgent need some more better detection techniques,particularly the fast, high sensitive and easy field diagnostic method.Immunogold technique (ICG) based on the immunology and nano-particle technology is the new immune markers tools. Conjuncted on the antigen or antibody as tracer markers to reflect the antigen-antibody reaction. Colloidal gold particles (Au2+) in the colloidal gold solution disperse phase, which between l-150nm in diameter. The color of colloidal gold solution show the change from red to purple by different sizes of colloidal gold particles. Colloidal gold particles is non-toxicity, so it is very security. On the other hand, it be used as a probe can rapid and stable combine with biological molecular, does not change the biological activity of which. So Immunogold technique open a new perspectives in the development of immunology diagnosis for cell surface precise slice positioning positioning and daily routine diagnosis[11]. The advantage Immunogold technique of is rapid, high sensitive, specific, stable, and easy to operate, without any equipment, the results is intuitive and reliable, the basic staff easily master it. Thus in the electron microscope, immunoblotting, in vitro diagnostic kits the manufacture, drug residues, clinical diagnosis and other fields have been widely used.The aim of this study was main to find a gold-immunochromatographic test-strip kit using a colloidal gold-labelled monoclonal antibody which was developed for thedetection of Xanthomonas citri pv. Citri (Xcc). Using Preparate the genetic engineering recombinant antibodies by our lab(Gene Engineering Research Center of Chongqing University), Preparated the monoclonal antibody and polyclonal antibodies, compared the features of the three antibodies in practical application, then have research and development a Fast rapid diagnostic test gold card for the test of citrus canker pathogen, expected to provide a diagnosis of simple, fast, easy to use in grass-roots means detection. The main results are as follows:①Preparation and purification of the monoclonal antibody, polyclonal antibodies and recombinant antibodies, compared by indirect ELISA method to screen out the two specificity and good stability antibody (monoclonal antibody Xcc-2D8 and Xcc-2D6 titers were 1:128,000), for the research and development of colloidal gold-speed measurement card.②Using sodium citrate reduction method to made the colloidal gold particles, The color of colloidal gold solution is bright red, after centrifugate there are no black particles precipitation on tube wall. Scanned by UV 400-600nm, the width of absorption peak is small, show homogeneous particle distribution, the maximum absorption peak wavelength at 520nm.③The optimum range of pH value in which colloidal gold labeled antibody could stable conjunct is about 9.0~9.5. The optimum amount of labeled antibody add in the colloidal gold solution is about 20mg/mL.④Determine the pretreatment of various Chromatography materials.One of the antibody(Xcc-2D8)was selected to conjugate with colloidal gold as the detector antibody was spotted on a conjugate pad, and another(Xcc-2D6) was used as the capture antibody at the test line(T line). Goat anti-mouse IgG antibody was used as the capture antibody at the control line(C line). Assemble the colloidal gold-speed measurement card then sealed in dry place .⑤The entire test procedure only takes about 10 min without any equipment. The detection limit of the test strip for Xcc concentration as low as 1×103~4 cfu/mL scored as positive. We test 206 samples, the results were compared with those obtained by qPCR in order to examine the reliability. The two methods showed excellent correlation (kappa=95.15%).
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