A test system is described and expanded upon for mass field immunochromatography assay on porous membrane carriers for rapid diagnostics of potato virus X (PVX) in potato leaf tissue and sprout extracts using colloidal gold nanoparticles as a marker. Sensitivity of the assay developed for PVX identification is found to be comparable to the sensitivity of solid-phase sandwich-ELISA. Complete assay time does not exceed 15 min, and the lower limit of the PVX detection in non-clarified leaf extract is 2 ng/ml. A single measurement requires 0.1-0.2 ml (3-5 drops) of tested solution only (extracted from 10-20 mg of potato leaf tissue or sprouts). The simplicity and reliability of the method makes it especially efficient in direct rapid monitoring of many infected potato specimens in the field, as verified by field trials of 360 clones of 28 domestic and foreign cultivars of potato. A diagnostic kit for routine analyses of potato viral infections both in the laboratory and in the field is described and expanded upon.
A highly sensitive express immunochromatography method for molecular diagnosis of plant virus infections was elaborated on the example of a model object - tobacco mosaic virus (TMV). The analysis time does not exceed 5 min, and the lower limit of TMV detection in non-clarified leaf extract (2-4 ng/ml) is comparable with the sensitivity of the enzyme-linked immunosorbent assay of the virus. A single measurement requires 0.1-0.2 ml tested solution (extract from 10-20 mg of leaf material). The sensitivity of TMV determination in the leaf tissue extract was increased by more than one order of magnitude using signal enhancement by silver and is 0.1 ng/ml. In this case, analysis time did not exceed 25 min. The simplicity of this method makes it especially convenient in express diagnosis of numerous analyzed specimens. The prototype of a diagnostic kit for serial analyses of plant viral infections both in laboratory and field conditions was elaborated.
A new ELISA method is proposed for differential quantitative determination of free (indolyl-3-acetic acid; IAA) and bound (indolyl-3-acetyl-L-aspartate) forms of natural auxins. There is similarity in results obtained by this and some traditionally used methods. The standard error of determination of the active form of IAA by our method is 1.5-2.0 times less than that using the traditional method. The method of quantitative differential determination of the main natural auxins does not require preliminary sample preparation, and this shortens assay time. The developed method has been used for practical determination of different forms of endogenous IAA in wheat and dandelion ovaries subjected to minimal treatment. This method can be used to investigate changes in the ratio of various hormonal forms of auxins that differ in their physiological activity in reproductive organs of angiosperms at various stages of reproduction.
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