Immunochromatography - General Phytopathology

Immunochromatography

For the rapid analysis of phytopathogens, methods of immunochromatographic analysis, or devices with a "lateral flow" (Lateral Flow Assay, LFA). Often they are called & quot; test strips & quot ;. The test strip is consistently located:

- sample application area (& quot; filter membrane & quot;);

- membrane with antibodies (specific to the antigen to be determined), labeled particles of colored latex or colloidal gold Au ("membrane with conjugate");

is a nitrate-cellulose membrane with a region of antigen-specific antibodies (& quot; analytical line & quot;) and a site of anti-species antibodies that bind to regions of labeled antibodies characteristic of the animal in which they were obtained (& quot; control line & quot;) ;

absorbing zone from the absorbent material (Figure 5.4).

Fig. 5.4. The device of the test strip (according to A. N. Blintsov, Yu. F. Drygin)

The scheme of the immunochromatographic rapid test is as follows. The test sample is triturated in a special buffer solution, the solid particles are allowed to settle for several minutes. The supernatant is applied to the filter membrane, and then on the & quot; membrane with the conjugate & quot; it is mixed in a certain proportion with the conjugate-specific to the antigen-determined antibody associated with the colored particles of latex or colloidal gold. Particles of the complex "antigen-labeled conjugate" & quot; move with a fluid flow along the nitrocellulose membrane, in a specific place which (& quot; analytical line & quot;) are other antibodies also specific for the antigen. The molecules of these antibodies bind the particles of the moving complex, as a result of which the colored particles are concentrated in the test zone and a band visible from the eye is evidenced, indicating the presence of a detectable pathogen in the sample. When the & quot; antigen-labeled conjugate & quot; reaches the & quot; control line & quot ;, it binds all the labeled antibodies that reach it (both free and in combination with antigens). The appearance of a visible band near the & quot; control line & quot; indicates that the migration of the sample along the membrane has occurred normally, therefore, the analysis is performed correctly and its result is reliable. Thus, a positive result is indicated by the presence of two bands, and in the absence of detectable phytopathogen, only one (control) band appears in the sample, indicating that the test is working.

On the membrane of combined test strips intended for the determination of several pathogens simultaneously, antibodies of different specificity are present in several test zones (ie, several "analytical lines"), respectively, several test strips may appear as a result of the analysis, usually of different colors, corresponding to the pathogens present in the sample.

Identification of bacteria and fungi by fatty acid composition

MIDI (Microbial Identification Inc., USA) offers an automated method for identifying bacteria by the composition of fatty acids. The identification of bacteria by the composition of esters of fatty acids by gas-liquid chromatography has been used for more than 50 years. In bacteria, more than 300 fatty acids and related components were found. The type of fatty acids produced and the relative concentrations of individual fatty acids strictly depend on the genotype and are characteristic of a particular type of microorganism or even of its strain. Analysis of cell fatty acids allows differentiation and identification of genera, species and strains of various bacteria, mycobacteria and yeast, including phytopathogenic ones.

The composition of fatty acids is affected by the conditions of cultivation: the composition of media, temperature and time of incubation. Nevertheless, if these cultivation conditions are kept constant, the quantitative and qualitative composition of the fatty acids is well reproduced, stable and conservative. To standardize growing conditions, MIDI uses process automation. After growing and preparing the samples, the system automatically determines the composition of the fatty acid complex in the cells of the tested organisms by chromatography, and then compares it with the fatty acid profile of known microorganisms stored in the databases. Currently, the firm offers two identification systems: one for bacteria and yeast with a database containing information on more than 1500 species; and another for mycobacteria, which allows the determination of 25 species of mycobacteria and 35 species of other organisms. The database includes information on the composition of the fatty acid complex under different growing conditions. The analysis is carried out very quickly: the time of preparation of samples, the analysis of fatty acids and computer processing in the total takes no more than 15-30 minutes.

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