RU2702852C1 - Method for using marker proteins of saprobic groups of indicator organisms for assessing ecological state of an environment - Google Patents

Abstract

FIELD: ecology.

SUBSTANCE: invention relates to a method for using marker proteins of saprobic groups of indicator organisms for assessing the ecological state of the environment. Method involves taking samples of organisms from the evaluated medium, performing analysis of amino acid sequences of marker proteins of indicator organisms to determine sections of amino acid sequences of marker proteins unique for each saprobic group of indicator organisms. For said analysis of amino acid sequences of marker proteins a sample of amino acid sequences of protein is created for each saprobic group of indicator organisms, method includes multiple alignment of marker proteins and determining the sections of amino acid sequences of marker proteins unique for each saprobic group of indicator organisms. Then, antibodies specifically recognizing these sections of amino acid sequences of marker proteins are obtained, enzyme immunoassay involves determining the number of marker proteins of each saprobic group of indicator organisms in a sample to which a saprobic group – antibodies – is obtained. Total number of marker proteins of all saprobic groups of indicator organisms is determined. Frequency of occurrence of each saprobic group of indicator organisms in the sample is calculated, for which the amount of marker protein of each saprobic group of indicator organisms is divided by the total number of marker proteins of all saprobic groups of indicator organisms in the sample. Total indicator weight (TIW) of saprobic groups of indicator organisms in the sample indicating saprobity of the water body is calculated, for which frequency of occurrence of each saprobic group of indicator organisms is multiplied by its – saprobic group – indicator weight equal to 0.1 for xenosaprobic group, 1.0 for oligosaprobic group, 2.0 for b-mesosaprobic group, 3.0 for a-mesosaprobic group, 4.0 for the polysaprobic group. Obtained results are summed up. Ecological state of the environment is evaluated as follows: if the TIW is less than 0.5, the environment is considered to be extremely clean and safe; with TIW 0.5 to 1.5, the environment is considered to be clean and safe, not requiring environmental protection measures; with TIW of 1.5 to 2.5, the environment is assessed for satisfactory purity requiring environmental protection measures whenever possible; at TIW 2.5 to 3.5, the environment is assessed as contaminated, in need of environmental protection measures aimed at elimination of certain types of pollutants and consequences of their action; with TIW of 3.5 or more, the environment is assessed as dirty and unsuccessful, in need of urgent implementation of integrated environmental protection measures.

EFFECT: invention can be used for assessment of ecological state of environment.

1 cl, 5 ex, 5 dwg

Description

The invention relates generally to the field of biology, mainly to the field of environmental monitoring, more specifically to methods for assessing the ecological state of the environment through the use of marker proteins of saprobic groups of indicator organisms isolated from the sample.

Further in the text, the applicant uses the terms that are necessary to facilitate an unambiguous understanding of the essence of the claimed materials and to exclude contradictions and / or disputed interpretations during the examination.

An antigen is any substance that, when ingested parenterally, elicits a specific specific immunological response, which manifests itself in the formation of specific antibodies [1].

Bioindication is an assessment of the quality of the environment by the state of its biota. Bioindication is based on observation of the composition and number of indicator species of indicator organisms - bioindicators.

Indicator organisms (bioindicators) - species, groups of species or communities, by the presence, degree of development, change in morphological, structural, functional, genetic characteristics of which judge the quality of water and the state of ecosystems [2].

Saprobity is a complex of physiological and biochemical properties of an organism, which determines its ability to dwell in water with one or another content of organic substances, that is, with one degree or another of pollution [3].

To assess the degree of pollution of water bodies with organic substances, five pollution zones were established: poly-, a-meso, b-meso, oligosaprobic and xenosaprobic [4].

Saprobionts are divided into three groups:

- Organisms of wastewater proper - polysaprobionts (p-saprobes);

- Organisms of heavily polluted waters - mesosaprobionts (two subgroups - α-mesosaprobes and β-mesosaprobes);

- Organisms of slightly polluted waters - oligosaprobes (o-saprobes)

- Pure water organisms - xenosaprobes (x-saprobes).

Biota is a historically established set of species of living organisms, united by a common area of distribution at present or in past geological epochs [5].

Hydrobionts - organisms that constantly live in the aquatic environment. [6 ] .

An organism is a living body with a combination of properties that distinguish it from nonliving matter, including metabolism, self-maintenance of its structure and organization, and the ability to reproduce them during reproduction, while preserving hereditary traits [7].

Zooplankton are aquatic animals that cannot withstand currents and are passively transported together with water masses [8].

Zoobenthos - includes organisms belonging to different systematic groups - arthropods, mollusks, worms [9].

Phytoplankton - a set of organisms that can freely carry out the process of photosynthesis freely drifting in the water column of organisms; phytoplankton is the primary producer of organic matter in a reservoir and serves as food for zooplankton and zoobenthos [10].

According to the applicant, attention should be paid to the following concepts described above, which are essential for the claimed technical solution - indicator organisms and saprobity. These concepts have different meanings and different purposes: saprobity is the ability of an organism to dwell in water with one or another content of organic substances, that is, with one degree or another of pollution, and indicator organisms characterize the quality of water and ecosystems and can exist only in one (or transitional) zone of saprobity.

The effectiveness of environmental measures depends on the timely receipt of reliable environmental monitoring data used to assess and diagnose the ecological state of the environment.

As you know, the assessment of the ecological state of the environment is performed in various ways using various methods, for example, physical, chemical, biological, using various representative indicators of the environment. One of the highly informative ways of assessing the ecological situation is the bioindication method, that is, the identification of the reaction of living organisms to changing environmental conditions, for example, pollution or purification of the environment [11].

 From the investigated prior art, the applicant has identified the source [Sladechek V. System of water quality from the biological point of view (Arch. Water quality assessment system ...) Arch. Hydrobiol. Ergeb. Limnol, 1973. - P.179-191] [12]. A brief essence of the known technical solution is the comparison of species in the sample with a list of indicator species and the assignment of species in the sample of indicator significance. The method involves the identification of aquatic organisms in the sample according to morphological (external) characteristics, for example, the configuration of the body, color, etc., visually using a microscope through manual manipulations, such as removing the body from the sample in the whole state and its visual identification. Assessment of the ecological state of the environment is carried out on the basis of the study of hydrobionts (indicator species) living in the studied environment.

 A disadvantage of the known technical solution is the high complexity and complexity of determining indicator types with the simultaneous use of optical instruments, reference books, the need to attract highly qualified specialists, which does not allow high efficiency to be used (known method) for its intended purpose.

 From the investigated prior art, the applicant has identified the source [Key to freshwater invertebrates of Russia and adjacent territories / ed. S.Ya. Tsalolykhina. - SPb .: Ed. "Science", 1994. - 394 p.] [13]. The essence of the known technical solution is a method for identifying an indicator species based on the identification and analysis of the morphological characteristics of an indicator organism, the idea of the method is identical to the analogue [12] above.

 The disadvantages of this method are the significant cost of identifying an indicator species and the dependence of the evaluation results on morphological characteristics of the qualifications and subjectivity of the expert. In addition, the known method has insufficient resolution and reliability of the results due to the existence of twin species having a similar external structure and existing in nature heteromorphic life cycle of some living organisms, that is, changes in the appearance of the determined organism that occur during life.

 From the investigated prior art, the applicant identified the source [Enzyme-linked immunosorbent assay (ELISA, ELISA). The essence, the principle of the method and the stages of the study. Analysis for antibodies, classes of antibodies, immune complex. http://www.polismed.com/articles-immunofermentnyjj-analiz-ifa-elisa-sut-princip-metoda-i-ehtapy-issledovanija.html] [14]. The essence of the known technical solution is the method of enzyme-linked immunosorbent assay (ELISA), which is used to determine the presence of antigens of pathogens of various infections in plants, animals, humans and other materials by identifying unique antigens characteristic of marker pathogens to marker proteins.

 The known technical solution is used by the applicant to obtain the amount of protein of saprobic groups of indicator organisms.

From the studied prior art, the applicant identified a patent for invention WO9946401 "Use of 14-3-3 proteins as sensitive biological markers for detecting pollution caused, inter alia, by polychlorinated biphenyl (PCB) and (xeno) oestrogen and method for quick determination of 14- 3-3 proteins ”[15] (“ The use of proteins 14-3-3 as sensitive biological markers for the detection of contamination caused, inter alia, by polychlorinated biphenyl (PCB) and (xeno) estrogen and the rapid determination of proteins 14-3- 3 "). The essence of the known technical solution is a method for detecting natural and anthropogenic environmental chemicals, in particular polychlorinated biphenyls and / or (xeno) estrogens in water bodies or in soil, characterized in that 14-3-3 proteins as biomarkers in bio-indicator organisms (vertebrates and invertebrates) corresponding to the one to be studied, exposed to water or soil. A method for determining and determining the concentration of protein (s) 14-3-3 (= ELISA), characterized in that microtiter plates are used for specific binding (and concentration) of proteins 14-3-3 to a peptide having a binding motif coated with 14-3 protein -3. For example, maleimide-activated microtiter plates can be used, to which the chemically synthesized peptide CAALPKINRSApSEPSLHR (pS phosphoserine), which binds with high affinity for proteins 14-3-3, is linked by the reaction of the sulfhydryl group of the N-terminal cysteine. After the extracts or biological fluids to be tested are added to the wells of a microtiter plate, the detection and quantification of the resulting protein complexes of peptide-14-3-3 occurs, for example, using labeled antibodies, such as an alkaline phosphatase conjugate antibody. Alternatively, microtiter plates coated with streptavidin can be used to which the biotinylated peptide binds at the time of binding of the 14-3-3 protein; incubation of the biotinylated peptide with the sample (formation of protein complexes of the peptide 14-3-3) is carried out before or after its binding to the plate. The ELISA detection limit is at least 10 ng 14-3 3 protein / ml and can be increased to less than 1 ng / ml, for example, by subsequently stopping the color reaction. The use of the ELISA method according to claim 2 for the determination and quantification of 14-3-3 protein in vertebrates and invertebrates exposed to natural and man-made environmental chemicals, polychlorinated biphenyls and / or (xeno) estrogens. The use of the ELISA method according to claim 2 for the detection and quantification of 14-3-3 protein in biological fluids (e.g. cerebrospinal fluid and serum) in humans and animals infected with prion pathogens.

Thus, the invention relates to the use of a new biological marker, namely, one or more proteins 14-3-3, for detecting anthropogenic stress in fresh water, sea water, drinking water or soil samples of natural and man-made chemical products obtained from the environment in particular polychlorinated biphenyl (PCB) and / or estrogen / xenoestrogen [(xeno) estrogen] and (2) a new method (ELISA method) for the quantitative and quantitative measurement of 14-3-3 proteins. The specified ELISA method can also be used to detect the appearance of 14-3-3 protein in physiological fluids of humans and animals infected with the pathogenic agent responsible for prion diseases.

A disadvantage of the known technical solution, in contrast to the claimed technical solution, according to the applicant, is that the known method was not used to assess the ecological state of the environment.

Closest to the claimed technical solution, selected by the applicant as a prototype , is the invention according to the patent RU 2661739 [16] “Method for the use of marker proteins for assessing the ecological state of the environment”. The essence of the known technical solution is the method of using marker proteins for assessing the ecological state of the environment, including the selection of samples of organisms from the medium being evaluated, followed by analysis of these samples, characterized in that the identification of the species composition of organisms from the sample is carried out using marker proteins of living organisms living in aquatic environment, and the assessment of the ecological state of the environment is carried out in accordance with the identified species composition of indicator organisms and the ratio in the sample, that is, enzyme-linked immunosorbent assay, determine the number of marker proteins of each of the indicator organisms in the sample to which antibodies are obtained, sum the number of marker proteins contained in the sample of indicator organisms, calculate the frequency of occurrence of indicator organisms in the sample by dividing the amount of marker protein of each organism by the total number of marker proteins of all organisms in the sample, the subsequent summation of the products of the frequency of occurrence of each indicator organ anism on its indicator weight and get the total indicator weight (SIW) of organisms in the sample, which assess the environmental state of the environment; with SIV less than 0.5, the environment is assessed as extremely clean and prosperous; with SIV from 0.5 to 1.5, the environment is assessed as clean and prosperous, not requiring environmental protection measures; with SIV from 1.5 to 2.5, the environment is estimated to be slightly polluted, in need of implementation of environmental measures as far as possible; with SIV from 2.5 to 3.5, the environment is estimated to be moderately polluted, in need of environmental protection measures aimed at eliminating certain types of pollutants and the consequences of their action; with SIV from 3.5 and more, the environment is assessed as dirty and dysfunctional, requiring urgent implementation of comprehensive environmental protection measures.

The disadvantage of the prototype, compared with the claimed technical solution, is the presence of a sign - identification of the species composition of organisms from the sample, which (identification) is carried out using marker proteins of living organisms living in the aquatic environment.

The indicated sign, according to the applicant, in comparison with the claimed technical solution, leads to a decrease in manufacturability when used as intended, which, as a result, leads to an increase in material costs, labor costs, time costs. That is, low manufacturability leads to an increase in costs, for example, for the individual selection and identification of each type of indicator organism in the sample. Moreover, in the claimed technical solution, the indicated action is absent, the applicant carries out the determination of saprobic groups of indicator organisms, which leads to higher manufacturability, providing increased efficiency when used as intended. Moreover, a formal reduction in the number of features leads in fact to a higher technical result, which, according to the applicant, meets the criterion of "inventive step".

The proof of the above is a detailed comparative analysis of the prototype with the claimed technical solution, which is shown in FIG. 5. Analysis of the Comparative Analysis Table shows that the claimed technical solution coincides with the prototype in 2 ways - by name and sampling and does not match in 7 ways out of 9. Moreover, 1 sign - identification of the species composition of organisms - is available in the prototype, is not in the claimed technical solution.

In general, the claimed technical solution compared with the prototype in the presence of fewer characters (8 versus 9 in the prototype) achieves a higher technical result, since it eliminates the need for a very laborious and costly process of identifying the species composition of organisms.

At the same time, no technology has been identified from the prior art that allows to achieve such a high level of results - the determination of saprobic groups of indicator organisms.

The claimed technical solution allows to resolve the seemingly insoluble contradiction at the filing date of the application - the determination of saprobic groups of indicator organisms, while spending the same amount of material resources as is required to identify one type of organism by prototype. This increases the availability of the claimed technical solution when used for its intended purpose, since it provides the opportunity to reduce (by an order of magnitude) costs while simplifying manufacturability. So, the cost of producing one antibody to one protein of one indicator organism on the market is at least 100 thousand rubles, while when assessing the ecological state of a reservoir, 10 to 100 or more organisms are analyzed (depending on the state of the reservoir). That is, it is possible to draw a conclusive conclusion that when applying the prototype method, the costs are from 1 million rubles. (in case of identification of 10 species of organisms) compared with 100 thousand rubles. according to the claimed technical solution (when determining 1 saprobic group of 10 species of organisms). Moreover, this effect is achieved due to the absence of one feature - identification of the species composition of organisms.

Thus, it seems possible to conclude that in the claimed technical solution a resolution of the contradiction was achieved in that, if necessary, to improve the quality of the results in cases of conventional design, they resort to either increasing the number of actions or costs, which complicates the technology. In the claimed technical solution, the applicant was able to improve the quality of the results while reducing the number of actions and resulting costs. This confirms the inventive step of the claimed technical solution, as it is not obvious to a person skilled in the art.

Based on the foregoing, in the applicant’s opinion, it can be concluded that the claimed technical solution does not fall under paragraph 77 of the Rules for the preparation, filing and consideration of applications for inventions, that is, it meets the patentability condition “inventive step”, since instead of identifying the species composition of organisms the applicant determines the saprobic group of indicator organisms, which gives an excess of the total technical effect.

Thus, taking into account the above analysis of the studied prior art, it is possible to draw a conclusive conclusion that the claimed technical solution has no analogues in terms of coinciding features, because from the studied prior art, no technical solutions have been identified that use the claimed combination of features presented in the independent claim of the alleged invention, whereby the applicant compiled the formula without a restrictive part, because the claimed technical solution partially coincides with the name and attribute for sampling from a reservoir, which is formal, because sampling is assumed in any way.

The purpose of the claimed technical solution is to develop a new method of using marker proteins of saprobic groups of indicator organisms to assess the environmental state of the environment by using the claimed combination of features that reduce the complexity of work, shorten the time for assessing the environmental state of the environment, increase the accuracy and reliability of the results of environmental monitoring, environmental assessment environmental conditions and conservation measures through that, instead of identifying the species composition of organisms, the applicant carries out the definition saprobity groups of indicator organisms.

The technical result of the claimed technical solution is a method of using marker proteins of saprobic groups of indicator organisms for assessing the ecological state of the environment, in which (the method) instead of identifying the species composition of organisms, the applicant determines the saprobic groups of indicator organisms.

The claimed technical solution is illustrated in Fig.1 - Fig.5.

Figure 1 shows Table 1, which shows the total indicator weight (SIW) of saprobic groups of indicator zooplankton organisms by protein cytochrome c oxidase subunit 1 (hereinafter - C1);

 Figure 2 shows Table 2, which shows the total indicator weight (SIW) of saprobic groups of indicator zoobenthos organisms by protein C1;

Figure 3 shows Table 3, which shows the total indicator weight (SIW) of saprobic groups of indicator phytoplankton organisms according to the protein of the chloroplast product of the ribulose 1-5 bifosfaat carboxylase Large subunit gene (hereinafter - rbcL).

Figure 4 shows Table 4, which shows the total indicator weight (SIW) for the СО1 protein of saprobic groups of indicator zooplankton organisms selected from Lake Maloe Glubokoe of the Republic of Tatarstan;

Figure 5 shows a table of comparative analysis of the prototype with the claimed technical solution.

The essence of the claimed technical solution is a method of using marker proteins of saprobic groups of indicator organisms for assessing the ecological state of the environment, which consists in taking a sample of organisms from the medium being evaluated, analyzing the amino acid sequences of marker proteins of indicator organisms to determine sections of the amino acid sequences of marker proteins unique to each saprobic group of indicator organisms receive antibodies that specifically recognize Using these sections of the amino acid sequences of marker proteins, an enzyme-linked immunosorbent assay determines the number of marker proteins of each saprobic group of indicator organisms in the sample to which antibodies are obtained to the saprobic group, the total number of marker proteins of all saprobic groups of indicator organisms is determined, and the frequency of occurrence of each saprobic group is calculated indicator organisms in the sample, for which the amount of marker protein of each saprobic group of indicator organisms is divided by the total the amount of marker proteins of all saprobic groups of indicator organisms in the sample, calculate the total indicator weight (SIW) of the saprobic groups of indicator organisms in the sample, indicating the saprobity of the reservoir, for which the frequency of occurrence of each saprobic group of indicator organisms is multiplied by its - saprobic group - indicator weight equal to 0.1 for the xenosaprobic group, 1.0 for the oligosaprobic group, 2.0 for the b-mesosaprobic group, 3.0 for the a-mesosaprobic group, 4.0 for the polysaprobic group, the results are summarized, estimate ayut ecological environment - at least 0.5 SIV environment evaluated extremely clean and safe; with SIV from 0.5 to 1.5, the environment is assessed as clean and prosperous, not requiring environmental protection measures; when SIV is from 1.5 to 2.5, the environment is assessed as satisfactory cleanliness, which requires the implementation of environmental measures as far as possible; with SIV from 2.5 to 3.5, the environment is assessed as polluted, in need of environmental protection measures aimed at eliminating certain types of pollutants and the consequences of their action; with SIV from 3.5 and more, the environment is assessed as dirty and dysfunctional, requiring urgent implementation of comprehensive environmental protection measures. Further, the applicant provides preliminary explanations to Examples 1-5 of a specific implementation of the claimed technical solution.

The applicants limited themselves to Examples with predominant use of the inhabitants of the aquatic environment - zooplankton, zoobenthos and phytoplankton because the ecological state of the reservoir located on the site unconditionally, reliably, in the volume necessary to restore the disturbed state of the environment, most objectively characterizes and reflects the ecological state of the surrounding non-aquatic reservoir - coastal and adjacent soil, vegetation and inhabitants of the soil, ambient air and inhabitants of the air and airborne environments. It characterizes the ecological state of the non-aquatic environment surrounding the body of water because the body of water on the ground is a place for collecting precipitation (for example, carrying information about the air environment of rain, snow, dust, water particles in the fog), drains of melt and other water flowing into the body of water, falling plant leaves washed away or shot down insects - all inflows into the reservoir, carrying with them (into the reservoir) substances that affect the environment. All these receipts (rain, dust, effluent, fog settling into the reservoir, meltwater, etc.) from the surrounding water reservoir first absorb information characterizing the individual components of the environment, for example, in the form of the presence (or absence) of environmental ill-being. Then, flowing down, falling down, settling into a body of water, affect the content of the aquatic environment and naturally affect the inhabitants of this aquatic environment. For this reason, the reservoir is the place of collection and the most complete concentration of all information about the surrounding water body. And the inhabitants of the reservoir, for example planktonic organisms, are the most convenient model objects of research that optimally and fully characterize the ecological state of the environment. Guided by this indisputable factor, the applicants limited themselves to a number of examples and cited the use of planktonic organisms, the ubiquitous inhabitants of water bodies, as examples.

 The use of aquatic environment inhabitants as indicator organisms has an informative advantage compared to the use of other environment inhabitants, which reflect the state of only the air or only soil component of the environment. Thus, the use of indicator organisms - inhabitants of a non-aquatic environment is less informative, and the results of the assessment are less reliable compared to the assessment of the ecological state of the environment using the inhabitants of a reservoir. For example, the use of ants makes it possible to assess the ecological state of mainly land, the use of bees - mainly the ecological state of land and air. But using ants and bees it is impossible to assess the ecological state of the water component of the natural environment. The inhabitants of the aquatic environment carry the most complete and reliable information about the ecological state of the environment. Therefore, the applicant limited himself to Examples using indicator organisms from a priority aquatic environment. When using inhabitants of non-aquatic environments, for example, soil, water-air and ground-air, the procedure is similar to the actions described in Examples 1, 2, 3, 4, 5.

In this case, the use of species other than the inhabitants of the reservoir, indicator organisms - inhabitants of the environment, for example, soil, air-water, air-ground environments, both individually and in any combination of them, also allows you to assess the ecological state of the environment according to the declared technical solution, but with less accuracy and, as a result, with less certainty.

Further, the applicant provides a general algorithm of actions that provides the opportunity to implement the claimed technical solution.

Step 1. Samples of organisms are taken from the evaluated aqueous medium by known methods.

Step 2. Organisms are analyzed to determine the sites of amino acid sequences of any marker proteins unique to saprobic groups of indicator organisms, resulting in antibodies that specifically recognize these sites of amino acid sequences of marker proteins.

Step 3. Determine the amount (in nanograms — ng) of marker proteins of each saprobic group of indicator organisms in the sample to which antibodies were obtained by the known method of immuno-enzyme analysis (ELISA).

Step 4. The total indicator weight (SIV) of the saprobic groups of indicator organisms is determined.

Step 5. Assess the ecological state of the environment (body of water or the object under study) by the calculated value of the total indicator weight of saprobic groups of indicator organisms in the sample. In accordance with the ecological and sanitary classification of land surface water quality according to hydrobiological indicators [17], with SIV less than 0.5, the environment is assessed as extremely clean and prosperous; with SIV from 0.5 to 1.5, the environment is assessed as clean and prosperous, not requiring environmental protection measures; when SIV is from 1.5 to 2.5, the environment is assessed as satisfactory cleanliness, which requires the implementation of environmental measures as far as possible; with SIV from 2.5 to 3.5, the environment is assessed as polluted, in need of environmental protection measures aimed at eliminating certain types of pollutants and the consequences of their action; with SIV from 3.5 and more, the environment is assessed as dirty and dysfunctional, requiring urgent implementation of comprehensive environmental protection measures.

Further, the applicant provides examples of the implementation of the claimed technical solution in accordance with the above Actions.

In total, the applicant examined 5 Examples of the use of marker proteins of indicator organisms, namely:

 - Example 1 considers the case of assessing the ecological state of the environment using marker protein СО1 of zooplankton indicator organisms.

- Example 2 considers the case of assessing the ecological state of the environment using the marker protein СО1 of other indicator organisms (other than zooplankton organisms) - zoobenthos organisms (zoobenthos).

- Example 3 considers the case of assessing the ecological state of the environment using another (different from СО1) marker protein - rbcL of other (different from zooplankton and zoobenthos) indicator organisms - phytoplankton.

- Example 4 considered a case showing the possibility of experimental obtaining absent in the databases of amino acid sequences of marker proteins of indicator species of organisms in order to further replenish international databases of amino acid sequences used to assess the ecological state of the environment.

- Example 5 considers the case of comparing the results of the claimed technical solution with the results of an expert assessment of the ecological state of the reservoir - Lake Maloe Glubokoe of the Republic of Tatarstan and the surrounding lake environment.

Example 1. Assessment of the ecological state of the environment using marker protein СО1 of zooplankton indicator organisms.

 Action 1. A sample of zooplankton organisms is taken from a reservoir located in the studied region in a known manner [Vinberg G.G. Guidelines for the collection and processing of materials in hydrobiological studies in freshwater bodies. Zooplankton and its products / Ed. G.G. Vinberg, G.M. Lavrentiev // Leningrad: GosNIORKH, ZIN AN SSSR, 1982. - 33 p.] [18], for example, using gauze net.

 Step 2. Organisms are analyzed to determine the sites of amino acid sequences of marker proteins that are unique for each saprobic group of indicator zooplankton organisms by marker protein — mitochondrial protein cytochrome c oxidase subunit 1 (СО1), for which the following steps are performed:

 - Create a sample of the amino acid sequences of the mitochondrial protein cytochrome c oxidase subunit 1 (СО1) from an international database, for example - TrEMBL / SwissProt on the UniProt website (http://www.uniprot.org/), for each saprobic group of indicator zooplankton organisms, for example - from [12] for each saprobity - from pure o-, contaminated with b -, to dirty p-;

- Carry out multiple alignment, for example, using the Clustal Omega program (http://www.ebi.ac.uk/Tools/msa/clustalo/), marker proteins, for example, cytochrome c oxidase subunit 1 (СО1);

- Identify sections of the amino acid sequences of the marker protein CO1, unique for each saprobic group of indicator zooplankton organisms;

- Determine the indicator weight of each saprobic group of indicator zooplankton organisms according to [12]:

• o-saprobity groups - one portion, for example - and a length of 5 minokislotnyh of STATCOM (hereinafter - aa), for example - aa at positions 31-35 ( FLGDE ) for the o-saprobic group of indicator organisms of the Macrotrachela genera - Macrotrachela multispinosa ACI46270, Macrotrachela papillosa ABV44838 and Synchaeta - Synchaeta grandis AFD23515, Synchaeta kitina AFD23607, Synchaeta tremula 1.0 = AC12;

• b-mesosaprobic group — one site, for example, 5 amino acid residues long, for example, a.o. at positions 31-35 ( YLGDE ) for the b-mesosaprobic group of indicator organisms of the genera Brachionus - Brachionus bidentatus AFQ31179, Brachionus diversicornis AFO72004, Brachionus plicatilis AAM47413 and Mytilina - Mytilina mucronata ABC02135, Mytilina ventricula 2.0 mqt37ha = 2.0

• p-saprobic group — one site, for example, 5 amino acid residues long, for example, aa at positions 154-158 ( FVWSV ) for the p-saprobic group of indicator organisms of the genus Chironomus — Chironomus plumosus BAR73215, Chironomus thummi AAD44526 (indicator weight = 4);

- Antibodies are obtained that specifically recognize the variable regions of the marker protein of each saprobic group of indicator zooplankton organisms in the sample, for example, for the cytochrome protein with oxidase subunit 1 (hereinafter - C1), for example:

• FLGDE for the o-saprobic group of indicator organisms of the genera Macrotrachela and Synchaeta;

• YLGDE for the b-mesosaprobic group of indicator organisms of the genera Brachionus and Mytilina;

• FVWSV for the p-saprobic group of indicator organisms of the genus Chironomus.

Antibodies are obtained according to the method of the antibody manufacturer, for example, Bialexa LLC (Russia) using standard animal research models, for example, the rabbit (http://www.bialexa.ru/).

 The resulting antibodies are stored in a storage facility, for example, in a refrigerator at a temperature of minus 30 ° C, stored and used to assess the environmental situation, moreover, without the need for re-performing the work under Step 2.

 Step 3. The quantity (in nanograms — ng) of marker proteins of each saprobic group of indicator zooplankton organisms in the sample to which antibodies were obtained was determined by the known method of immuno-enzyme analysis (ELISA) [Van Weemen B.K., Schuurs A.H. (1971). "Immunoassay using antigen-enzyme conjugates." FEBS Letters 15 (3): 232–236. doi: 10.1016 / 0014-5793 (71) 80319-8. PMID 11945853] [19], for which:

- separate the zooplankton organisms in the sample from water by centrifugation, for example, in a centrifuge SM-6MT, for example, in a test tube with a capacity of 50 ml;

- zooplankton organisms are homogenized in distilled water at a ratio of water: organisms by volume 1: 1 (one to one), for example, by grinding organisms in a mortar with clean crushed glass, and a homogenate is obtained;

- carry out centrifugation of the homogenate, for example, for 5 minutes at a speed of 14,000 rpm, and receive a supernatant (supernatant);

- place the supernatant in a new clean tube, and further procedures are carried out with it (supernatant);

- get the amount of proteins by the ELISA method [19], for example:

• СО1 protein of the o-saprobic group of indicator zooplankton organisms - 0.40 ng;

• СО1 protein of the b-mesosaprobic group of indicator zooplankton organisms - 0.05 ng;

• СО1 protein of the p-saprobic group of indicator zooplankton organisms - 0.05 ng.

The action is performed separately for each antibody to the marker protein of each saprobic group of indicator zooplankton organisms.

 Step 4. The total indicator weight (SIV) of the saprobic groups of indicator zooplankton organisms is determined, for which:

- Determine the total ∑ number of marker proteins of all saprobic groups contained in the sample indicator zooplankton organisms, for example, ∑ = (0.40 + 0.05 + 0.05) ng = 0.50 ng.

- Calculate the frequency f of occurrence of each saprobic group of indicator zooplankton organisms in the sample, for which the amount of marker protein of each saprobic group of indicator zooplankton organisms is divided by the total ∑ number of marker proteins of all saprobic groups of indicator zooplankton organisms in the sample.

For example:

• the frequency of occurrence of the o-saprobic group of indicator zooplankton organisms f = 0.40 / (0.40 + 0.05 + 0.05) = 0.40 / 0.50 = 0.80 = 0.8;

• the frequency of occurrence of the b-mesosaprobic group of indicator zooplankton organisms f = 0.05 / (0.40 + 0.05 + 0.05) = 0.05 / 0.50 = 0.10;

• the frequency of occurrence of the p-saprobic group of indicator zooplankton organisms f = 0.05 / 0.50 = 0.10;

- Calculate the total indicator weight (SIW) of the saprobic groups of indicator zooplankton organisms in the sample, indicating the saprobity of the reservoir, for which the frequency f of occurrence of each saprobic group of indicator zooplankton organisms is multiplied by its indicator group [12], the results are summarized:

SIW = 0.80 x 1.00 + 0.10 x 2.00 + 0.10 x 4.00 = 0.80 + 0.20 + 0.40 = 1.4.

 Step 5. Assess the ecological state of the environment (body of water or the object under study) by the calculated value of the total indicator weight of saprobic groups of indicator organisms in the sample. The assessment is carried out in accordance with the environmental and sanitary classification of the quality of land surface water according to hydrobiological indicators [17], described in detail in the above algorithm of actions for implementing the claimed technical solution (Step 5).

  The total indicator weight of the saprobic groups of indicator zooplankton organisms SIV = 1.4 (Table 1 in Fig. 1) (in the range of 0.5 <SIV <1.5), which according to the environmental sanitary classification [11] corresponds to the o-saprobic zone of the reservoir , i.e. the pond is in a very clean state.

According to the state of the reservoir, the surrounding (reservoir) environment is characterized (estimated) by a clean and prosperous one , which currently does not need to carry out environmental protection measures.

Thus, it can be seen from Example 1 that the applicant has achieved the set goals and the claimed technical result - a method for using marker proteins of saprobic groups of indicator organisms has been developed, according to which, by the example of zooplankton organisms, the ecological state of the environment surrounding the body of water has been assessed.

From the data given in Example 1, it can be seen that the three saprobic (o-, b-, p-) groups consist of a total of 12 species of indicator zooplankton organisms, namely:

- o-saprobic group consists of 5 types of indicator zooplankton organisms,

- b-mesosaprobic group consists of 5 organisms,

- p-saprobic group - from 2 organisms.

Antibodies are obtained for each of the three saprobic groups of zooplankton organisms, i.e. according to the claimed technical solution , 3 antibodies are obtained .

When comparing with the prototype, in which 12 antibodies are required for 12 species of indicator zooplankton organisms, it can be concluded that using the claimed technical solution to assess the ecological state of the environment, it is possible to save material, labor and time costs of at least than 4 times (12 antibodies get the prototype against 3 of the claimed). Moreover, in the claimed technical solution there is no very significant feature of the prototype - the identification of the species composition of organisms. That is, the reduction in the number of signs in the claimed technical solution led to the implementation of all the tasks and the declared technical results, which led to a significant reduction in total costs when used for their intended purpose - not less than 4 times.

Example 2. Assessment of the ecological state of the environment using marker protein CO1 of other (other than zooplankton organisms) indicator organisms - zoobenthos organisms (zoobenthos).

Zoobenthos - a collection of bottom animals that live on the ground and in the ground of marine and continental bodies of water.

 Action 1. A sample of zooplankton organisms is taken from a reservoir located in the studied region in a known manner [Methodological recommendations for the collection and processing of materials during hydrobiological studies in freshwater bodies of water. Zoobenthos and its products. / Ed. G.G. Vinberg, G.M. Lavrentieva. - L .: GosNIORKH, ZIN AN USSR. - 51 p.] [20], for example - using a bottom grab - a sample of zoobenthos organisms is taken.

 Step 2. Organisms are analyzed to determine the sites of amino acid sequences of marker proteins unique to each saprobic group of indicator zoobenthos organisms by the marker protein of the mitochondrial protein cytochrome c oxidase subunit 1 (СО1), for which the following steps are performed:

- Create a sample of the amino acid sequences of the variable mitochondrial СО1 protein from the international database, for example, TrEMBL / SwissProt on the UniProt website (http://www.uniprot.org/), for each saprobic group of indicator zoobenthos organisms, for example - [12] for each saprobity - from extremely pure x-, pure o-, to contaminated b-;

- Carry out multiple alignment, for example - using the Clustal Omega program (http://www.ebi.ac.uk/Tools/msa/clustalo/), marker proteins CO1;

- Identify sections of the amino acid sequences of the marker protein that are unique for each saprobic group of indicator zoobenthos organisms, for example, for the cytochrome protein with oxidase subunit 1 (СО1).

- Determine the indicator weight of each saprobic group of indicator zoobenthos organisms according to [12]:

• x-saprobic group — one site, for example, 5 amino acid residues long (hereinafter referred to as ao), for example, ao at positions 95-99 ( PLSAG ), for the x-saprobic group of organisms of the genera Protonemura - Protonemura meyeri AGZ03513 and Dinocras - Dinocras cephalotes AGU13981 (indicator weight = 0.1);

• o-saprobic group — one site, for example, 5 amino acid residues long, for example, aa at positions 95-99 ( PLSSN ) for the o-saprobic group of organisms of the Silo genus - Silo pallipes ANZ02284, Silo piceus ANY51527 and Wormandia subnigra ANY50378, Philopotamus montarus AII16166 (indicator weight = 1.0);

• b-mesosaprobic group — one site, for example, 5 amino acid residues long, for example, a.o. at positions 95-99 ( PLAGA ) for the b-mesosaprobic group of organisms of the Coenagrion genera - Coenagrion puella AMO65757 and Pyrrhosoma - Pyrrhosoma nymphula AMB62102 (indicator weight = 2.0).

- Antibodies are obtained that specifically recognize sections of the amino acid sequences of the marker protein that are unique to each saprobic group of indicator zoobenthos organisms in the sample, for example, for the CO2 protein, for example:

• PLSAG for the x-saprobic group of indicator organisms of the Protonemura and Dinocras genera;

• PLSSN for the o-saprobic group of indicator organisms of the genera Silo and Wormaldia;

• PLAGA for the b-mesosaprobic group of indicator organisms of the organisms of the genera Coenagrion and Pyrrhosoma.

Antibodies are obtained according to the method of the antibody manufacturer, for example, Bialexa LLC (Russia) using standard animal research models, for example, the rabbit (http://www.bialexa.ru/).

 The resulting antibodies are stored in a storage facility, for example, in a refrigerator at a temperature of minus 30 ° C, stored and used to assess the ecological state of the environment, moreover, without the need for repeated work under Step 2.

 Step 3. Determine the amount (in nanograms — ng) of marker proteins of each saprobic group of indicator zoobenthos organisms in the sample to which antibodies were obtained by the known method of immuno-enzyme analysis (ELISA) [Van Weemen B.K., Schuurs A.H. (1971). "Immunoassay using antigen-enzyme conjugates." FEBS Letters 15 (3): 232–236. doi: 10.1016 / 0014-5793 (71) 80319-8. PMID 11945853] [19]:

- separate zoobenthos organisms in the sample from water by centrifugation, for example, in a centrifuge SM-6MT, for example, in a test tube with a capacity of 50 ml;

- zoobenthos organisms are homogenized in distilled water at a ratio of water: organisms by volume 1: 1 (one to one), for example, by grinding (organisms) in a mortar with clean crushed glass, and a homogenate is obtained;

- carry out centrifugation of the homogenate, for example, for 5 minutes at a speed of 14,000 rpm, and receive a supernatant (supernatant);

- place the supernatant is placed in a new clean tube, and further procedures are carried out with it (supernatant);

- get the amount of proteins by the ELISA method [19], for example:

• СО1 protein of the x-saprobic group of indicator zoobenthos organisms - 0.10 ng;

• СО1 protein of the o-saprobic group of indicator zoobenthos organisms - 0.20 ng;

• СО1 protein of the b-mesosaprobic group of indicator zoobenthos organisms - 0.40 ng.

Step 3 is performed separately for each antibody to the marker protein of each saprobic group of indicator zoobenthos organisms.

 Step 4. Determine the total indicator weight (SIW) of saprobic groups of indicator zoobenthos organisms, for which:

- Determine the total ∑ number of marker proteins of all saprobic groups contained in the sample of indicator zoobenthos organisms, for example: ∑ = (0.10 + 0.20 + 0.40) ng = 0.70 ng.

- Calculate the frequency f of occurrence of each saprobic group of indicator zoobenthos organisms in the sample, for which the amount of marker protein of each saprobic group of indicator zoobenthos organisms is divided by the total ∑ number of marker proteins of all saprobic groups of indicator zoobenthos organisms in the sample.

For example:

 - the frequency of occurrence of the x-saprobic group of indicator zoobenthos organisms f = 0.10 / 0.70 = 0.14;

- the frequency of occurrence of the o-saprobic group of indicator zoobenthos organisms f = 0.20 / 0.70 = 0.29;

- the frequency of occurrence of the b-mesosaprobic group of indicator zoobenthos organisms f = 0.40 / 0.70 = 0.57.

- Calculate the total indicator weight (SIW) of the saprobic groups of indicator zoobenthos organisms in the sample, indicating the saprobity of the reservoir, for which the frequency f of occurrence of each saprobic group of indicator zoobenthos organisms is multiplied by its indicator group [12], the results are summarized:

SIW = 0.14 x 0.1 + 0.29 x 1.0 + 0.57 x 2.0 = 0.014 + 0.29 + 1.14 = 1.44

 Step 5. Assess the ecological state of the environment (body of water or the object under study) by the calculated value of the total indicator weight of saprobic groups of indicator organisms in the sample.

 The total indicator weight of the saprobic groups of indicator zoobenthos organisms in the sample is SIV = 1.44 (Table 2 in Fig. 2) (in the range of 0.5 <SIV <1.5), which corresponds to the o-saprobic zone of the reservoir. In accordance with the ecological and sanitary classification of land surface water quality according to hydrobiological indicators [11], according to the pollution index 1.44, the reservoir corresponds to the o-saprobic zone, i.e. the pond is in a very clean state.

According to the state of the reservoir, the surrounding (reservoir) environment is characterized (estimated) by a clean and prosperous one , which currently does not need to carry out environmental protection measures.

Thus, Example 2 also confirms the achievement of the set goals and the claimed technical result - a method has been developed by which the ecological state of the environment surrounding the body of water using zoobenthos organisms is assessed by marker protein CO1.

From the data given in Example 2, it can be seen that three saprobic groups (x-, o-, b-) of zoobenthos indicator organisms consist of a total of 8 species:

- x-saprobic group consists of 2 types of indicator zoobenthos organisms,

- the o-saprobic group consists of 4 species of organisms,

- b-mesosaprobic group - from 2 species.

Antibodies are prepared for each of the three saprobic groups, i.e. according to the claimed technical solution, 3 antibodies are obtained.

When comparing with the prototype, in which 8 antibodies are required for 8 species of indicator zoobenthos organisms, it can be concluded that using the claimed technical solution to assess the ecological state of the environment, it is possible to save material, labor and time costs of at least than 2.7 times (8 antibodies are obtained according to the prototype versus 3 in the claimed one). Moreover, in the claimed technical solution there is no very significant feature of the prototype - the identification of the species composition of organisms. That is, the reduction in the number of signs in the claimed technical solution led to the implementation of all the tasks and the claimed technical results, which led to a significant reduction in total costs when used for their intended purpose - not less than 2.7 times.

Example 3. Assessment of the ecological state of the environment using another (different from СО1) marker protein - rbcL of other (different from zooplankton and zoobenthos) indicator organisms - phytoplankton.

Phytoplankton - a set of organisms that can freely carry out the process of photosynthesis freely drifting in the water column of organisms; Phytoplankton is the primary producer of organic matter in the reservoir and serves as food for zooplankton and zoobenthos.

 Action 1. A sample of zooplankton organisms is taken from a reservoir located in the studied region in a known manner [V. Abakumov (Ed.) Guidelines for hydrobiological analysis of surface waters and bottom sediments. L .: Gidrometeoizdat, 1983. - 240 p.] [21], for example - using a bathometer - a sample of phytoplankton organisms is taken.

Step 2. Organisms are analyzed to determine the sites of amino acid sequences of marker proteins that are unique for each saprobic group of indicator phytoplankton organisms by marker protein — a fragment of the chloroplast product of the gene (protein) ribulose 1-5 bifosfaat carboxylase Large subunit (rbcL), for which the following are performed Steps:

- Create a sample of the amino acid sequences of the rbcL chloroplast protein from an international database, for example, TrEMBL / SwissProt on the UniProt website (http://www.uniprot.org/), for each saprobic group of indicator phytoplankton organisms, for example, from [12] for each (from clean to dirty) saprobity zone, for example - o-, b-, a-saprobity;

- Carry out multiple alignment, for example - using the Clustal Omega program (http://www.ebi.ac.uk/Tools/msa/clustalo/ ) , marker proteins, for example - rbcL;

- Identify sections of the amino acid sequences of the marker protein that are unique for each saprobic group of indicator phytoplankton organisms, for example, for the rbcL protein;

- Determine the indicator weight of each saprobic group of indicator phytoplankton organisms according to [12]:

• o-saprobic group — one site, for example, 5 amino acid residues long, for example, aa at position 273-277 ( YDTLL ) for the o-saprobic group of indicator organisms of the genera Rhopalodia - Rhopalodia gibba AQM56354 and Pinnularia - Pinnularia subcapitata var elongata AER42030 (indicator weight = 1.0);

• b-mesosaprobic group — one site, for example, 5 amino acid residues long, for example, a.o. at positions 145-149 ( YGTPL ) for the b-mesosaprobic group of indicator organisms of the genus Surirella - Surirella capronii AGE34586, Surirella biseriata AGE34629, Surirella minuta AEB91278, Surirella tenera AGE34623 (indicator weight = 2.0);

• a-mesosaprobic group — one site, for example, 5 amino acid residues long, for example, a.o. in position 22-25 ( DQYFA ) for the a-mesosaprobic group of indicator organisms of the Navicula genera - Navicula cryptocephala AEB91223 and Cryptomonas - Cryptomonas ovata CAJ19660 (indicator weight = 3);

- Antibodies are obtained that specifically recognize the variable regions of the marker protein of each saprobic group of indicator phytoplankton organisms in the sample, for example, for the rbcL protein, for example:

• YDTLL for the o-saprobic group of indicator organisms in the Rhopalodia and Pinnularia zones;

• YGTPL for the b-mesosaprobic group of indicator organisms of the genus Surirella;

• DQYFA for a-mesosaprobic group of indicator organisms of the genera Navicula and Cryptomonas;

 Antibodies are obtained according to the method of the antibody manufacturer, for example, Bialexa LLC (Russia) using standard research models (http://www.bialexa.ru/).

 The resulting antibodies are stored in a storage facility, for example, in a refrigerator at a temperature of minus 30 ° C, stored and used to assess the environmental situation, moreover, without the need for re-performing the work under Step 2.

 Step 3. The amount (in nanograms — ng) of the marker proteins of each saprobic group of indicator phytoplankton organisms in the sample to which antibodies were obtained using the known method of immuno-enzyme analysis (ELISA) is determined [Van Weemen B.K., Schuurs A.H. (1971). "Immunoassay using antigen-enzyme conjugates." FEBS Letters 15 (3): 232–236. doi: 10.1016 / 0014-5793 (71) 80319-8. PMID 11945853] [19]:

- Separate phytoplankton organisms in the sample from water by centrifugation, for example, in a centrifuge SM-6MT, for example, in a test tube with a capacity of 50 ml;

- phytoplankton organisms are homogenized in distilled water at a water: organisms ratio of 1: 1 in volume (one to one), for example, by grinding (organisms) in a mortar with clean crushed glass, and a homogenate is obtained;

- carry out centrifugation of the homogenate, for example, for 5 minutes at a speed of 14,000 rpm, and receive a supernatant (supernatant);

- place the supernatant in a new clean tube, and further procedures are carried out with it (supernatant);

- get the amount of proteins by the ELISA method [19], for example:

- protein rbcL o-saprobic group of indicator phytoplankton organisms - 0.05 ng;

- protein rbcL of the b-mesosaprobic group of indicator phytoplankton organisms - 0.15 ng;

- rbcL protein of the a-mesosaprobic group of indicator phytoplankton organisms - 0.50 ng.

Step 3 is performed separately for each antibody to the marker protein of each saprobic group of indicator phytoplankton organisms.

Step 4. The total indicator weight (SIV) of the saprobic groups of indicator phytoplankton organisms is determined, for which:

- Determine the total ∑ number of marker proteins of all saprobic groups contained in the sample of indicator phytoplankton organisms, for example: ∑ = (0.05 + 0.15 + 0.50) = 0.70 ng.

- The frequency f of occurrence of each saprobic group of indicator phytoplankton organisms in the sample is calculated, for which the amount of marker protein of each saprobic group of indicator phytoplankton organisms is divided by the total ∑ number of marker proteins of all saprobic groups of indicator phytoplankton organisms in the sample.

For example:

- the frequency of occurrence of the o-saprobic group of indicator phytoplankton organisms f = 0.05 / 0.70 = 0.07;

- the frequency of occurrence of the b-mesosaprobic group of indicator phytoplankton organisms f = 0.15 / 0.70 = 0.21;

- the frequency of occurrence of the a-mesosaprobic group of indicator phytoplankton organisms f = 0.50 / 0.70 = 0.72;

- Calculate the total indicator weight (SIW) of the saprobic groups of indicator phytoplankton organisms in the sample, indicating the saprobity of the reservoir, for which the frequency f of occurrence of each saprobic group of indicator phytoplankton organisms is multiplied by its indicator group [12], the results are summarized:

SIW = 0.07 x 1.0 + 0.21 x 2.0 + 0.72 x 3.0 = 0.07 + 0.42 + 2.16 = 2.65.

 Step 5. Assess the ecological state of the environment (body of water or the object under study) by the calculated value of the total indicator weight of saprobic groups of indicator phytoplankton organisms in the sample.

 The total indicator weight of the saprobic groups of indicator phytoplankton organisms in the sample SIV = 2.65 (Table 3 in Figure 3) (in the range 2.5 <SIV <3.5), which corresponds to the α-mesosaprobic zone of the reservoir. In accordance with the ecological and sanitary classification of land surface water quality according to hydrobiological indicators [11], according to the pollution index of 2.65, the reservoir corresponds to the α – mesosaprobic zone, i.e. the reservoir is in a polluted state.

 According to the state of the water body, the environment (water body) is characterized (estimated) by a polluted one, requiring environmental protection measures aimed at eliminating certain types of pollutants and the consequences of their action.

Thus, Example 3 also confirms the achievement of the goals and the claimed technical result - a method has been developed by which the ecological state of the environment surrounding the body of water using phytoplankton organisms is assessed using the rbcL marker protein.

From the data presented in Example 3, it can be seen that the three saprobic groups (o-, b-, a-) of phytoplankton indicator organisms consist of a total of 8 species:

- the o-saprobic group consists of 2 types of indicator phytoplankton organisms,

- b-mesosaprobic group consists of 4 types of indicator phytoplankton organisms,

- a-mesosaprobic group - from 2 species.

Antibodies are prepared for each of the three saprobic groups, i.e. according to the claimed technical solution, 3 antibodies are obtained.

When comparing with the prototype, in which 8 antibodies are required for 8 types of indicator phytoplankton organisms, it can be concluded that using the claimed technical solution to assess the ecological state of the environment, it seems possible to save material, labor and time costs of at least than 2.7 times (8 antibodies are obtained according to the prototype versus 3 in the claimed one). Moreover, in the claimed technical solution there is no very significant feature of the prototype - the identification of the species composition of organisms. That is, the reduction in the number of signs in the claimed technical solution led to the implementation of all the tasks and the claimed technical results, which led to a significant reduction in total costs when used for their intended purpose - not less than 2.7 times.

Example 4, showing the possibility of experimental obtaining absent in the databases of amino acid sequences of marker proteins of indicator species of organisms with the aim of further replenishing international databases of amino acid sequences used to assess the ecological state of the environment.

 In Example 4, the applicant considers the possibility of using the claimed technical solution even when the amino acid sequences of marker proteins of indicator species of organisms are not available in international databases used to assess the ecological state of the environment by experimentally obtaining them (amino acid sequences of marker proteins of indicator species of organisms) with the purpose of further replenishment of international databases, for example - TrEMBL / SwissProt (http://www.uniprot.org/).

  That is, in the case considered in Example 4, to assess the ecological state of the environment, it is first necessary to experimentally obtain the nucleotide sequences of a gene, for example, СО1 of indicator organisms, followed by their translation into the amino acid sequences of a marker protein, for example, СО1 of organisms, using the following steps:

 Step 1: A sample of organisms is taken from a reservoir located in the study region in a known manner.

 Step 2. Determine the species composition of the organisms in the sample — for example, by the variable mitochondrial gene cytochrome c oxidase subunit 1 (hereinafter - CO1), for which the following steps are performed:

- they look at a sample of organisms with an increase, for example, under an Axio Lab.A1 microscope (Carl Zeiss) and MBS-10 binoculars, separate an organism;

- each individual organism is placed in a separate container, for example, a test tube containing, for example, 50 microliters of a 7% aqueous solution of Chelex ion exchange resin and incubated, for example, for 20 minutes at a temperature of at least + 90 ° С, for example, at + 99 ° FROM. Get purified DNA in an aqueous solution;

 Step 3. An experimental nucleotide sequence of a gene, for example, СО1 of organisms is obtained, followed by translation into a amino acid sequence of a marker protein, for example, СО1 of organisms, for which:

- centrifuging the contents of the container with the selected organism is performed, for example, for 5 minutes at a speed of 14,000 rpm, a DNA-containing supernatant (supernatant) is obtained. The supernatant is placed in a new clean tube, and further procedures are performed with it (supernatant);

- carry out a polymerase chain reaction (hereinafter referred to as PCR) using the supernatant as a matrix. PCR is carried out, for example, in 25 μl of a reaction mixture containing a 5-fold qPCRmix-HS reaction mixture (Eurogen, Russia), bidistilled water, 0.4 mM forward and reverse primers, for example 5'-ggtcaacaaatcataaagatattgg-3 'and 5 '-taaacttcagggtgaccaaaaaatca-3'. Amplification is carried out, for example, by 35-fold repetition of the stages in the following sequence: denaturation (at plus 95 ° С, 30 sec), annealing (+59 ° С, 30 sec) and elongation (polymerization at +72 ° С, 120 sec), - using the well-known combination of primers - 5'-ggtcaacaaatcataaagatattgg-3 'and 5'-taaacttcagggtgaccaaaaaaatca-3' [Folmer O. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan inverteolates B. O. .Hoeh, R. Lutz, R. Vrijenhoek // Mol. Mar. Biol. Biotechnol. - 1994. - V.3. - P.294-299] [22]. Thus, an amount of a specific (specific) DNA fragment of the mitochondrial gene of cytochrome c oxidase subunit 1 (СО1) is obtained sufficient for further procedures.

A DNA fragment, for example, СО1, is a unique (one of a kind) gene fragment characteristic of each species among all types of living organisms, which provides a highly reliable, with an accuracy of 99.9%, determination of a specific organism to a species. Moreover, for practical use, it is sufficient to determine the organism with an accuracy of at least 90%, that is, the determination of the organism by variable gene fragments fully ensures the reliability of the results required for industrial use.

- PCR products are detected and purified, for example, by electrophoresis in a horizontal 1% agarose gel. As the electrode buffer solution for electrophoresis, for example, a single solution of 2-amino-2-hydroxymethylpropane-1,3-diol of acetic acid and ethylenediaminetetraacetic acid with a pH of 8.1 is used (hereinafter referred to as TAE). For the application of samples, a buffer coloring solution is used, for example, with the composition: 10 mM Tris-HCl, pH 7.8, 50 mM EDTA, 0.03% bromophenol blue, 0.03% xylene cyanol, 15% glycerol. A sample with a dye is mixed in a ratio of 4: 1 (8: 2 μl). Electrophoresis is carried out, for example, at a current strength of 50 mA, the gel is viewed in ultraviolet light, for example, on a Vilber Lourmat transilluminator (France). DNA (obtained by PCR, clause 1.2.1) is developed by adding ethidium bromide fluorescent in ultraviolet light to an agarose at a final concentration of 0.5 μg / ml. The developed DNA becomes visible in ultraviolet light, which confirms the presence of DNA in the sample.

- PCR amplification products are isolated from an agarose gel, for example, using a set of reagents from Eurogen (Novosibirsk, Russian Federation) according to the manufacturer's method, by adding 200 μg of an agarose gel containing a specific DNA fragment to 200 μl of a lysing solution (from a set), for example - mitochondrial gene cytochrome c oxidase subunit 1 (CO2). A mixture of lysis solution and agarose gel is incubated in a water bath at a temperature of + 55 ° C until the gel is completely dissolved. The resulting solution was transferred to a filter column inserted into a collecting column (from the Eurogen kit, Novosibirsk, RF), and incubated at room temperature for 120 sec. After that, the solution is centrifuged for 60 seconds at a rotation speed of 5000 rpm and the contents of the collecting column are drained. After centrifugation, a mixture of the DNA fragment with foreign substances remains in the filter column. The mixture (residue) is washed with a buffer solution, for example, the well-known Wash buffer.

- to wash the mixture, the filter column is inserted back into the collecting column, then 50 μl of the washing solution is added to the filter column and centrifuged, for example, for 60 seconds at 8000 rpm. The washing procedure is repeated at least two times to obtain pure DNA. After washing, pure DNA remains on the membrane of the filter column. The used collection column is replaced with a clean 1.5 ml tube. A clean DNA filter column is inserted into a clean tube. Then, the DNA is eluted by adding 30 μl of the buffer solution (from the kit) for elution to the filter column, and the DNA is washed off with the buffer solution in a test tube. After performing all the above procedures in vitro, a solution is obtained containing a specific DNA fragment of an individual organism, for example, the mitochondrial gene cytochrome c oxidase subunit 1 (СО1).

- Next, using the sequencing operation, the nucleotide sequence (nucleotide arrangement) of the DNA fragment is determined. The operation is carried out, for example, in 20 μl of a reaction mixture containing water, DNA, a 5-fold TMS buffer solution, a 2.5-fold Ready Reaction Premix and 3.3 mM primers. The sequencing operation is carried out, for example, 35-fold repetition. The result is decoded, for example, using an ABI Prism 310 Genetic Analyzer (Applied Biosystems, USA) according to the manufacturer's instructions [Watts D. Automated fluorescent DNA sequencing on the ABI PRISM 310 Genetic Analyzer / D. Watts, J.R. MacBeath // Methods Mol Biol. 2001. - R. 167, 153–170] [23]. Get sequential chromatograms.

- The sequencing results are processed, for example, with Lasergene 5.03 software package (DNA STAR, Inc., USA) and SeqMan software package for analysis of sequence chromatograms; the nucleotide sequence of a DNA fragment of an individual organism, for example, the mitochondrial gene cytochrome c oxidase subunit 1 (СО1), is obtained.

- Then, the obtained nucleotide sequence, for example, the СО1 gene of the organism, is converted (translated) into the amino acid sequence of the protein, for example, the organism СО1 protein, using the standard genetic code of a specialized computer program, for example, ORF Finder (http: //www.ncbi. nlm.nih.gov/projects/gorf).

 Step 4. Replenishment of international databases with experimentally obtained primary sequences of genes and proteins:

- The experimentally obtained sequence of the gene and the gene product of the protein is introduced into international databases, for example, TrEMBL / SwissProt on the UniProt website (http://www.uniprot.org/), for each indicator organism and is used for the purposes of the claimed method.

 Then, the same actions are carried out (Actions 1-5), as in Examples 1-3 according to the assessment of the ecological state of the environment (reservoir or the object under study) using the calculated value of the total indicator weight (SIW) of the organisms in the sample.

Using the manufacturer's instructions in Example 4, it is possible to obtain sequences of any marker genes and proteins of any organisms .

 In Example 4, the applicant has shown the possibility of using the claimed technical solution even when the amino acid sequences of marker proteins of indicator species of organisms are not available in international databases used to assess the ecological state of the environment, through their (amino acid sequences of marker proteins of indicator species of organisms) experimental with the aim of further replenishment of international databases.

 Example 5. Comparison of the results of the claimed technical solution with the results of an expert assessment of the ecological status of the reservoir - Lake Small Glubokoe of the Republic of Tatarstan and the natural environment surrounding the lake.

To confirm the reliability of the results obtained by the claimed technical solution, the applicant compares the results of the claimed technical solution with the results of the assessment of the condition of Lake Maloe Glubokoe based on materials [Ecology of Kazan. - Kazan: Publishing House of the Academy of Sciences of the RT, 2005. - 527 pp.] [9, Tab. 19, P.126] [24], in which (materials) experts assess the condition of Lake Maloe Glubokoe as satisfactory (moderately polluted).

 To this end, the applicant assessed the ecological state of Lake Maloe Glubokoe, located on the territory of Kazan, the Republic of Tatarstan and the natural environment of the lake, according to the claimed method using marker proteins for saprobic groups on the example of indicator species of zooplankton organisms.

Sequencing:

 Action 1. A sample of zooplankton organisms is taken from Lake Maloe Glubokoe located in the estimated environment.

 Action 2-3. Proceeding as described above (Example 1), saprobic groups of indicator species of zooplankton organisms are determined using marker proteins CO1, for example, a unique TTV site for an o-saprobic group is identified, including indicator species of Chydorus sphaericus ACJ01614 and Mesocyclops leuckarti ADQ90208; TTI for the b-mesosaprobic group, including indicator species Keratella cochlearis AMZ04798, Bosmina longirostris AVP51787, Simocephalus vetulus AHL83628, calculate the frequency content of each saprobic group of indicator species of zooplankton organisms in the sample and the total indicator weight (SIW) in Fig. 4, Table 4, Fig. 4. .

Step 4. The total indicator weight of SIV is calculated for the СО1 protein of saprobic groups of indicator zooplankton organisms in the SIV = 1,14 sample (Lake Small Glubokoe) (Table 4 in FIG. 4).

 Step 5. Assess the ecological status of the reservoir in this area - Lake Maloe Glubokoe based on the calculated value of the total indicator weight of saprobic groups of indicator zooplankton organisms in the sample.

 The total indicator weight of the saprobic groups of indicator zooplankton organisms in the sample SIV = 1.14 (Table 4 in Figure 4) (in the range of 0.5 <SIV <1.5) corresponds to the o-saprobity of the reservoir. In accordance with the ecological and sanitary classification of land surface water quality according to hydrobiological indicators [11], according to the pollution index 1.14, the reservoir corresponds to the o-saprobic zone, i.e. the pond is in a very clean state.

According to the state of the reservoir, the surrounding (reservoir) environment is characterized (estimated) by a clean and prosperous one , which currently does not need to carry out environmental protection measures.

Thus, from Example 5 it can be seen that the results of the assessment of the condition of Lake Maloe Glubokoe, obtained by the claimed method, completely coincide with the expert assessment according to [9], where the condition of Lake Maloe Glubokoe is also assessed as good, which, according to the applicant, confirms them (results of the claimed method) reliability.

 From the data given in Example 5 it is seen that two saprobic groups (o-, b-) consist of a total of 5 species of zooplankton indicator organisms, namely:

- the o-saprobic group consists of 2 types of indicator zooplankton organisms,

 - b-mesosaprobic group - of 3 species.

Antibodies are prepared for each of the two saprobic groups, i.e. according to the claimed technical solution , 2 antibodies are obtained .

When comparing with the prototype, in which 5 antibodies are required for 5 types of indicator zooplankton organisms, it can be concluded that using the claimed technical solution to assess the ecological state of the environment, it is possible to save material, labor and time costs of at least than 2.5 times (5 antibodies get the prototype against 2 of the claimed). Moreover, in the claimed technical solution there is no very significant feature of the prototype - the identification of the species composition of organisms. That is, the reduction in the number of signs in the claimed technical solution led to the implementation of all the tasks and the claimed technical results, which led to a significant reduction in total costs when used for their intended purpose - not less than 2.5 times.

From the results described in Examples 1-5, the following general conclusions can be drawn.

The applicant, in comparison with the prototype, achieved the goals and the claimed technical results:

- a method has been developed for assessing the ecological state of the environment using marker proteins of saprobic groups of indicator organisms, in which (the method) instead of identifying the species composition of organisms, the applicant determines the saprobic groups of indicator organisms. According to the claimed method, the ecological state of the environment surrounding the body of water was assessed (see Examples 1 to 5) ;.

- the complexity of the work has been reduced, the terms for assessing the ecological state of the environment have been reduced - see the conclusions in Examples 1, 2, 3, 5. Based on the results set out in the Examples, it is possible to draw a conclusive conclusion that as a result of using the claimed technical solution to assess the environmental environmental conditions it is possible to save material, labor and time costs when used as intended - depending on the number of species of indicator organisms in the saprobic group - the more types of indicators the beaten organisms in the group, the greater the effect of cost reduction. So, the cost of determining the saprobic group of indicator organisms according to the claimed technical solution, in which (the saprobic group) is, for example, from 2 to 10 (or more) species, is lower than the cost of the prototype, 2-10 ( and more) times.

- the accuracy and reliability of the results of environmental monitoring, assessment of the ecological state of the environment and environmental measures are improved by the fact that instead of identifying the species composition of organisms, the applicant determines the saprobic groups of indicator organisms, which yields an exceeding total technical effect (see Examples 1-5), so how a decrease in the volume of work is accompanied by a decrease in the probability of random errors that affect the accuracy and reliability of the results of the assessment of the ecological state I studied the environment.

Thus, the reduction in the number of features in the claimed technical solution compared to the prototype not only led to the implementation of all the tasks and claimed technical results, but also to a significant reduction in total costs (see Examples 1-5).

In addition to the above conclusions, it should be emphasized that the antibodies obtained by the applicant are stored in a repository and a database of antibodies to marker proteins of each saprobic group of indicator organisms is created. The database is used to assess the environmental situation, and without re-receiving antibodies. The antibodies recorded in the data bank against marker proteins of saprobic groups of indicator organisms are available for use by all users, including environmental assessors. This information is perceived, interpreted and used equally (unambiguously) by various specialists, regardless of their location and subjective influence of the expert - specialist. The use of an instrumentally obtained international database of antibodies to marker proteins for saprobic groups of indicator organisms, which is unified for all experts, makes the assessment of the ecological state of the environment independent of the subjectivity of the assessments of an expert, that is, the result of the evaluation is always objective, reliable, which compares favorably with subjective assessments using the prototype. The above confirms the applicants' achievement of their goals and the claimed technical result.

Based on the foregoing, according to the applicant, the method of assessing the ecological state of the environment using marker proteins is universal, does not depend on the geographical location of the object, has reliability at the level of the highest achievements of molecular genetics and bioinformatics, and the results of the method are more reliable compared to inventions, known from the prior art on the filing date of the application.

Thus, it seems possible to conclude that in the claimed technical solution a resolution of the contradiction was achieved in the sense that, if necessary, to improve the quality of the results in cases of conventional design, they resort to either increasing the number of actions or costs, which complicates the technology. In the claimed technical solution, the applicant was able to improve the quality of the results while reducing the number of actions and resulting costs. This confirms the inventive step of the claimed technical solution, as it is not obvious to a person skilled in the art.

The claimed technical solution meets the criterion of "novelty" presented to the invention, because since when determining the prior art no means were found that are characterized by features identical (that is, coinciding in the function they perform and in the form of execution of these features) to all the features presented in the independent claim, including the purpose of the assignment, when achieving higher compared with a prototype, technical results.

The claimed technical solution meets the criterion of "inventive step" for inventions, since no technical solutions have been identified that have features that match the distinguishing features of this invention, and the popularity of the distinctive features on the specified technical result has not been established. At the same time, the claimed technical solution ensured the resolution of contradictions that were unsolvable until the filing date of the application materials — the determination of saprobic groups of indicator organisms, while spending the same amount of material resources as needed to identify one type of organism by prototype. This increases the availability of the claimed technical solution, since its intended use provides the opportunity to reduce costs while reducing manufacturability.

The claimed technical solution meets the criterion of "industrial applicability" to the invention, because can be implemented on an industrial scale for environmental, agricultural, recreational activities (recreation), the activities of healthcare organizations, and moreover, through the use of well-known standard technical devices and equipment.

Used sources

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Claims (1)

The method of using marker proteins of saprobic groups of indicator organisms for assessing the ecological state of the environment, namely, that a sample of organisms is taken from the medium being evaluated, the amino acid sequences of marker proteins of indicator organisms are analyzed to determine sections of the amino acid sequences of marker proteins unique to each saprobic group of indicator organisms why create a sample of amino acid sequences of the protein for each saprobic group of indicator organisms, carry out multiple alignment of marker proteins and determine the sites of amino acid sequences of marker proteins unique to each saprobic group of indicator organisms, then antibodies are produced that specifically recognize these parts of the amino acid sequences of marker proteins, and the amount of marker proteins of each saprobic group of indicator is determined by immuno-enzymatic analysis. organisms in the sample, to which - the saprobic group - antibodies are obtained, determine the total number of marker proteins of all saprobic groups of indicator organisms, calculate the frequency of occurrence of each saprobic group of indicator organisms in the sample, for which the amount of marker protein of each saprobic group of indicator organisms is divided by the total number of marker proteins of all saprobic groups of indicator organisms in the sample, calculate the total indicator weight ( SIV) of the saprobic groups of indicator organisms in the sample, indicating the saprobity of the reservoir, for which the frequency of occurrence of each sa the test group of indicator organisms is multiplied by its - saprobic group - indicator weight equal to 0.1 for the xenosaprobic group, 1.0 for the oligosaprobic group, 2.0 for the b-mesosaprobic group, 3.0 for the a-mesosaprobic group, 4.0 for a polysaprobic group, the obtained results are summarized, the ecological state of the environment is assessed - if the SIV is less than 0.5, the environment is assessed as extremely clean and safe; with SIV from 0.5 to 1.5, the environment is assessed as clean and prosperous, not requiring environmental protection measures; when SIV is from 1.5 to 2.5, the environment is assessed as satisfactory cleanliness, which requires the implementation of environmental measures as far as possible; with SIV from 2.5 to 3.5, the environment is assessed as polluted, in need of environmental protection measures aimed at eliminating certain types of pollutants and the consequences of their action; with SIV from 3.5 and more, the environment is assessed as dirty and dysfunctional, requiring urgent implementation of comprehensive environmental protection measures.

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