Method and kit for detecting the presence of silver leaf disease
An antibody that is used to detect the presence of the Chondrostereum purpureum fungus in a plant sample, where the antibody specifically binds to the endolipogalacturonase enzyme produced by Chondrostereum purpureum (anti-endoPG from now). A kit and a method to detect fungal antigens, particularly, to detect the presence of the silverleaf disease (caused by the Chondrostereum purpureum fungus) in fruit trees, by means of detection of the endoPG enzyme through the binding of said antigen to the antibody, functionalized with gold nanoparticles, where the binding is detected via enzyme-linked immunosorbent assays (ELISA) or an immunochromatographic lateral flow assay.
1 . An antibody to detect presence of Chondrostereum purpureum fungus in a plant sample, wherein the antibody has a heavy chain defined by the amino acid sequence SEQ ID No. 2 and a light chain defined by the amino acid sequence SEQ ID No. 4, and wherein said antibody is conjugated with gold nanoparticles through an L-cysteine residue to form gold nanoparticles+L-cysteine+specific anti-endoPG monoclonal antibody complex (NpsAu+Lc+Ab complex) and specifically binds to enzyme endopolygalacturonase from Chondrostereum purpureum (anti-endoPG antibody), wherein the gold nanoparticles have an average diameter of 25 nm and a surface charge of ~19.9 mV.
2 . A method for detecting the Chondrostereum purpureum fungus presence in a plant tissue sample comprising the steps of:
a. preparing the vegetable or tree tissue sample of interest to obtain a supernatant from the sample,
b. establishing contact between the supernatants of the sample to be analyzed with the antibody of claim 1 , and
c. evaluating the specific union of the anti-endoPG antibody, where the sample contains the fungus if the antibody is specifically bound to the sample.
3 . The method for detecting the Chondrostereum purpureum fungus presence in a plant tissue sample in accordance with claim 2 , wherein, to prepare the sample, 1 g of plant tissue is grinded and crushed, left to macerate, and washed with an organic solvent and the supernatant of the sample is retained.
4 . The method for detecting the Chondrostereum purpureum fungus presence in a plant tissue sample in accordance with claim 2 , wherein the plant tissue corresponds to leaves, wood, roots, seeds.
5 . The method for detecting the Chondrostereum purpureum fungus presence in a plant tissue sample in accordance with claim 2 , wherein the specific union of the anti-EndoPG antibody is evaluated using a method selected from the group that consists on a lateral flow immunochromatography or ELISA procedure.
6 . A kit for the fast detection of the Chondrostereum purpureum fungus in a plant sample comprising:
a reactive strip composed of a nitrocellulose membrane where there are differentiated areas for the sample pad, test zone, control zone and absorption pad, where the test and control zone have the NpsAu+Lc+Ab complex with antibody of claim 1 ;
detection secondary antibodies; and
instructions of use.
7 . The kit for the quick detection of the Chondrostereum purpureum fungus in a plant sample in accordance with claim 6 , wherein the detection secondary antibodies are anti-rabbit IgG.
8 . The kit for the quick detection of the Chondrostereum purpureum fungus in a plant tissue sample in accordance with claim 7 , wherein the sample to be evaluated is a supernatant of a processed plant sample.
9 . The kit for the quick detection of the Chondrostereum purpureum fungus presence in a plant tissue sample in accordance with claim 8 , wherein the sample to be evaluated is prepared by grinding and/or crushing plant tissue, then left to macerate, washed with an organic solvent, and the supernatant of this sample is retained.
10 . The kit for the quick detection of the Chondrostereum purpureum fungus of claim 9 , wherein the plant tissue is leaves, wood, roots and/or seeds.