RU2619898C1 - Method for biological materials preservation - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for biological materials preservation includes their immobilization on a dry carrier followed by drying. Large-pore chemically neutral hydrophilic material is used as a carrier for immobilization, the material is impregnated with a preservative solution containing 0.7 M sucrose, 1.5 M Tris-HCl pH 7.4 and 0.5 M ethylene diamine tetraacetic acid prior to biological material immobilization thereon.

EFFECT: increased safety of diagnostic and biologically significant properties of biological material for a long time and expanded range of preservation tools for biological materials, used later for laboratory analysis.

6 cl, 1 dwg, 2 tbl, 6 ex

Description

The invention relates to the field of molecular biology and biotechnology, in particular to a method for preserving biological materials (blood, urine, swabs, scrapings, plant suspensions, phytopathogens, etc.), which are further used for laboratory analysis.

Known methods for preserving and transporting biological materials require either freezing in a liquid nitrogen a suspension of biological material with a mixture of the compounds of the stabilizing composition in an aqueous solution to obtain solid frozen particles, granules, drops or threads, or enclosing the biological material in a non-aggressive metal capsule, or applying it to an absorbent paper sheet having a plurality of area samples, each area sample has a perforation extending substantially along its entire perimeter such that s capillary prevent overflow of sample liquid from one zone to another (RU 2573324, 20.01.2016; US 5516487, 14.05.1996; RU 2228617, 20.05.2004; WO 9957264, 11.11.1999). However, none of the conservation technologies gave a sufficient solution to the problem of long-term conservation of biological material while preserving their biologically and diagnostically significant properties. At the moment, it is relevant to create a simple method for preserving the diagnostically and biologically significant properties of biological material for a long time.

A known method of preserving biological materials by drying without freezing using a stabilizing mixture of substances comprising at least two components: one of the group comprising monosaccharides, disaccharides and zwitterion with a polar residue, the second substance of the stabilizing mixture is a zwitterion with a non-polar residue and is acetylphenylalanine ester, alanine, cysteine, glycine, leucine, methionine, tryptophan, valine, sarcosine. Stabilized biological materials are stable during storage at room temperature and are used for diagnostic and therapeutic purposes (RU 2191003, 10.20.2002). However, this method is complicated in execution and use.

A known method of preserving biological materials, ensuring the preservation of nucleic acids by obtaining a lysate by mixing the biological material with a preserving solution containing a guanidine compound in an amount sufficient to substantially completely denature the nucleases contained in the sample, as well as with components that prevent the degradation of nucleic acids, namely with a reducing agent SS protein bonds in an amount providing substantially complete restoration of SS bonds in lysate proteins with strong determinants a substance that does not form an insoluble compound with guanidine in an amount sufficient to form micelles in the lysate with at least one complexon of divalent and trivalent metal ions in an amount providing its molar concentration in the lysate, which is obviously higher than the expected maximum molar the concentration of ions of divalent and trivalent metals, and a buffer that maintains the pH of the lysate in the range from 6 to 8. The method ensures the preservation of nucleic acids during long-term storage of samples in not frozen state (RU 2322058, 04.20.2008). The disadvantages of the method are its complexity and low duration of storage of biological material, which was tracked only for 3, 7 and 14 days.

As the closest analogue, we considered a method for preserving biological materials by immobilizing them on a dry carrier, followed by drying. In accordance with this method, a human genetic sample or human genetic samples are stored in the process of self-storage at home (US 5856102, 01/05/1999). However, the authors have not shown the safety of biologically significant properties of the samples and the duration of their storage.

SUMMARY OF THE INVENTION

The technical result consists in increasing the safety of diagnostically and biologically significant properties of biological material for a long time and expanding the arsenal of means for preserving biological materials used in the future for laboratory analysis.

The technical result is achieved by preserving the biological materials by immobilizing them on a dry carrier, followed by drying, with the use of a large-porous chemically neutral hydrophilic material as a carrier for immobilization, which is preliminarily impregnated with a preservation solution containing 0 before immobilization of the biological material on it. 7 M sucrose, 1.5 M Tris HCl with a pH of 7.4 and 0.5 M ethylenediaminetetraacetic acid.

The dry carrier is impregnated with a preservative solution.

In this case, the preservation solution maintains a constant concentration of hydrogen ions, while maintaining a constant pH value.

In addition, drying is carried out under conditions that do not allow decomposition of the preservative solution.

The recovery of dried biomaterial is carried out by washing it off with water or a solvent.

The impregnation of large-pore chemically neutral hydrophilic material of the carrier with a preservative solution is carried out until it is completely saturated.

In addition, in the method, the preservation of biological materials to prevent the process of hemolysis and preservation of plant hypobiosis, the preservation solution contains 0.7 M sucrose. At the same time, it, impregnating a dry carrier, allows you to save material from plants and plant pathogens for a long time.

The implementation of the method is illustrated by the following examples.

Example 1

Before applying the reagent to a large-pore chemically neutral hydrophilic carrier material, a preservation solution with a certain concentration is prepared. A preservation solution was previously prepared by mixing sucrose, Tris HCl buffer and ethylenediaminetetraacetic acid. At the same time, 10 ml of the preservation solution contained Tris — 1.5 M, the pH was adjusted with HCl to pH 7.4, and sucrose — 0.7 M; 2 ml of 0.5 M EDTA (pH 7.4) was added to them.

A dry carrier with a width of 1 cm and a length of 2.5 cm embedded in a cardboard envelope was lowered into a container with a preserving solution containing 0.7 M sucrose, 1.5 M Tris HCl (pH 7.4) and 2 ml 0.5 M EDTA and kept in for two hours. Then the carrier was dried for 1.5 hours at room temperature.

100-150 μl of blood, that is, until completely absorbed, was added to the dried, large-pore chemically neutral hydrophilic material of the carrier, and dried for at least 1.5 hours at room temperature from 15 ° to 25 ° C.

Then, the cardboard envelope together with a desiccant (hydrogel) was packed in a zip bag and stored in a household refrigerator with a temperature of 2-5 ° C for at least five years until its main purpose (studies by polymerase chain reaction (PCR) and enzyme immunoassay (ELISA) )).

To study the biomaterials stored on the carrier, PCR and ELISA carried out their restoration in liquid form. To do this, using scissors, the carrier was carefully cut into three parts, placed in a 1.5 ml tube containing 300 μl of distilled water. The tube was left at room temperature for 10 minutes, periodically shaking on a vortex. Then the tube was centrifuged, the supernatant was taken and transferred to a clean tube. The obtained liquid sample was used in further work.

Using a commercial set of reagents for DNA / RNA isolation from various materials manufactured by BIOKOM LLC, DNA and RNA microorganisms were isolated from both membrane samples and liquid blood samples taken with a 3% EDTA solution. PCR was performed according to the protocol for amplification kits manufactured by BIOKOM LLC. ELISA was performed using a commercial IDEXX enzyme-linked immunosorbent reagent kit (USA); antibody titers in animal blood for salmonellosis were determined in both native and carrier-dried samples. The results of PCR analysis are shown in FIG. 1, where tracks: 1-3 - isolated Toxoplasma gondii DNA from liquid blood samples, 4-6 - isolated Toxoplasma gondii DNA from dry blood samples deposited on membranes soaked in a solution containing 0.7 M sucrose, 1.5 M Tris HCl ( pH 7.4) and 0.5 M EDTA. For all studied samples, the results obtained in ELISA for the determination of antibodies from dry membranes and native samples are correlated, which indicates the stability of canned dry samples.

Example 2

The study took potato and tomato leaves infected with X potato virus (PVX-Mushrooms), S potato virus (PVS-aphid), M potato virus (PVM-aphid), K tomato virus (TVK), L tomato virus (TVL). Juice was squeezed from the affected leaves of potatoes and tomatoes. A preservation solution was previously prepared by mixing sucrose, Tris HCl buffer and ethylenediaminetetraacetic acid. At the same time, 10 ml of the preservation solution contained Tris — 1.5 M, the pH was adjusted with HCl to pH 7.4, and sucrose — 0.7 M; 2 ml of 0.5 M EDTA (pH 7.4) was added to them.

A dry carrier with a width of 1 cm and a length of 2.5 cm, built into a cardboard envelope, was lowered into a container with a preserving solution for 10 seconds until it was completely saturated. The carrier was dried for 1.5 hours at room temperature. Immobilization was carried out by adding 100-150 μl of plant sap with phytopathogens to the dried, large-pore chemically neutral hydrophilic carrier material. After that, the carrier with the plant juice applied to it with phytopathogens, enclosed in paper cardboard, was dried for 1.5 hours at room temperature from 15 ° to 25 ° C. Then, the cardboard envelope together with a desiccant was packed in a zip bag and stored in a household refrigerator with a temperature of 2-5 ° C for 5 years until its main purpose (studies were performed by PCR and ELISA).

To study phytopathogens stored on a carrier by PCR and ELISA, they were restored to liquid form. To do this, using scissors, the carrier was carefully cut into three parts, placed in a 1.5 ml tube containing 300 μl of distilled water. The tube was left at room temperature for 10 minutes, periodically shaking on a vortex. Then the tube was centrifuged, the supernatant was taken and transferred to a clean tube. The obtained liquid sample was used in further work.

Using a commercial set of reagents for the isolation of DNA / RNA from various materials manufactured by BIOKOM LLC, potato and tomato DNA were extracted. Further, the polymerase chain reaction was carried out according to the protocol for the reagent kit PlantV-PCR-core, manufactured by BIOKOM LLC. Using the IMMUNOTEST-HVK reagent kit manufactured by BIOKOM LLC, potato X virus was determined in native and carrier-dried samples. The work was carried out according to the instructions for use with the IMMUNOTEST-KHV kit. The results of the work showed that the DNA of potatoes and tomatoes was detected from all samples, both native and dry media. The results of ELISA are presented in table. one

Thus, the obtained results show that this method of conservation is applicable to plants and phytopathogens and preserves the biological and diagnostic properties of the studied material.

Example 3

A study was made of the preservation of the biological properties of lactobacilli isolated from patients in urogenital smears. In this case, a preservation solution was preliminarily prepared by mixing sucrose, Tris HCl buffer and ethylenediaminetetraacetic acid. At the same time, 10 ml of the preservation solution contained Tris — 1.5 M, the pH was adjusted with HCl to pH 7.4, and sucrose — 0.7 M; 2 ml of 0.5 M EDTA (pH 7.4) was added to them. A dry carrier with a width of 1 cm and a length of 2.5 cm, embedded in a cardboard envelope, was lowered into a container with a solution containing 0.7 M sucrose, 1.5 M Tris Hcl (pH 7.4) and 0.5 M EDTA, and held for 10 seconds until its full impregnation. The carrier was dried for 1.5 hours at room temperature. Immobilization was carried out by introducing urogenital swabs onto the dried, large-pore chemically neutral hydrophilic carrier material until the material space was completely filled.

Then the cardboard envelope together with a desiccant was packed in a zip bag and stored in a household refrigerator with a temperature of 2-5 ° C for 5 years until its main purpose (microbiological study). A cut-off piece of carrier with a dry sample was placed in a test tube into which water was added to dissolve the dry sample. The tube was left in a rack at room temperature for 10 minutes and periodically shaken on a vortex. Then it was centrifuged for 40 seconds at 10 thousand rpm. The resulting suspension was transferred to a clean tube and used in microbiological examination of the material. Moreover, the number of viable lactobacilli varied within different limits. The results are presented in table 2.

Table 2. The number of viable cells in cultures of lactobacilli stored on a dry carrier, previously impregnated with a preservative solution.

The results show that this method of conservation allows you to save the biological properties of the materials deposited on a dry membrane.

Example 4

Juice was squeezed out of the leaves of potatoes and tomatoes, which was applied in an amount of 100-150 μl to a large-pore chemically neutral hydrophilic carrier material, previously impregnated with a preservation solution containing 0.7 M sucrose, 1.5 M Tris HCl with a pH of 7.4 in a final concentration with the addition of to them 2 ml of 0.5 M ethylenediaminetetraacetic acid, and dried at room temperature. After applying the biological material, the carrier was dried for 1.5 hours at room temperature from 15 ° to 25 ° C. Then a cardboard envelope with a carrier of biomaterial together with a desiccant was packed in a zip bag and stored in a household refrigerator with a temperature of 2-5 ° C for 5 years. To study the biomaterials of plants stored on a carrier by PCR and ELISA, they were restored to liquid form. To do this, using scissors, the carrier was carefully cut into three parts, placed in a 1.5 ml tube containing 300 μl of distilled water. The tube was left at room temperature for 10 minutes, periodically shaking on a vortex. Then the tube was centrifuged, the supernatant was taken and transferred to a clean tube. The obtained liquid sample was used in further work. Using a commercial set of reagents for the isolation of DNA / RNA from various materials manufactured by BIOKOM LLC, potato and tomato DNA were extracted. Next, the polymerase chain reaction was carried out according to the protocol for the reagent kit “Plant-PCR-core”, manufactured by BIOKOM LLC. All studied biological samples from leaves of potatoes and tomatoes, deposited on a carrier, followed by drying, showed a positive result when DNA was detected. The results are identical to the study of biological samples of potato leaves and tomatoes from a freshly obtained native suspension (plant sap). Thus, an isotonic preservative solution for plant tissues must necessarily contain sucrose. Experimental data show that 0.7 M sucrose used for blood preservation is also sufficient for long-term preservation of plant biomaterial (juice).

The concentrations of the reagents of the preserving solution are selected so that a constant concentration of hydrogen ions is maintained, that is, the pH of the mixture in the solution was kept at 7.4, since its change below 7.4 leads to alkalization, and above 7.4 to the oxidation of the biomaterial and accordingly to the destruction of its cells. And the use of the concentration of sucrose in the preservation solution (0.7 M) ensured the long-term preservation of the cells of biological material without damage. Examples 5 and 6 show that the concentration of reagents of the preservation solution ensures a constant concentration of hydrogen ions and long-term preservation of the biological and diagnostic properties of the cells of the biomaterial.

Example 5

Large-pore chemically neutral hydrophilic material impregnated with a preservation solution containing 0.7 M sucrose, 1.5 M Tris HCl with a pH of 7.4 at a final concentration and with the addition of 2 ml of 0.5 M ethylenediaminetetraacetic acid and immobilized blood drops on it and dried at room temperature temperature, weekly checked for pH. For this, a strip of litmus paper was moistened with distilled water, after which it was put directly onto a carrier with dry blood stains. The degree of color change was judged on the reaction. The pH value was stable (7.4) throughout the entire study period.

Example 6

Preservative solutions, which included sucrose 0.02 M, 0.2 M sucrose, 1.5 M Tris Hcl (pH 7.4) and 0.5 M EDTA (experimental solutions), allowed red blood cells to be stored for quite a long time without hemolysis, but were unsuitable for the recovery of dry samples into liquid form, i.e. formed a hard to break sediment. The reconstituted liquid form of dry blood samples has a consistency suitable for any nucleic acid isolation method, i.e., both on sorbent and alcohol precipitation, and especially for automated isolation systems where the consistency of the sample is of great importance. And it was possible only as a result of blood storage at a rather high concentration of sucrose in solution (0.7 M). The combined use of a buffer (1.5 M Tris-HCl (pH 7.4) and with 2 ml of 0.5 M EDTA) with sucrose for a dry vehicle was also effective. EDTA concentration (2 ml 0.5 M to 10 ml buffer) is the standard concentration used in buffer systems. But the concentration of Tris HCl (1.5 M) was selected experimentally, since in the course of the study it was revealed that its lower concentration (1 M, 0.5 M, 0.2 M) led to the oxidation of samples, and a large ( 2 M, 2.5 M) to alkalization.

Thus, the claimed method of preserving biological materials provides an increase in the safety of diagnostically and biologically significant properties of biological material for a long time by applying it to a large-pore chemically neutral hydrophilic carrier impregnated with a preservation solution that helps to prevent cell degradation, while maintaining a constant concentration of hydrogen ions and a constant pH value, as well as a protective effect that prevents the process of hemolysis and hypobiosis protected plants.

Claims (6)

1. A method of preserving biological materials, including immobilizing them on a dry carrier, followed by drying, characterized in that a large-porous chemically neutral hydrophilic material is used as a carrier for immobilization, which is preliminarily impregnated with a preservative solution containing 0 before immobilization of the biological material onto it. 7 M sucrose, 1.5 M Tris-HCl with a pH of 7.4 and 0.5 M ethylenediaminetetraacetic acid. 2. The method according to p. 1, characterized in that the dry carrier is impregnated with a preservative solution. 3. The method according to p. 1, characterized in that the preservative solution maintains a constant concentration of hydrogen ions, while maintaining a constant pH value. 4. The method according to p. 1, characterized in that the drying is carried out under conditions that do not allow the decomposition of the preservative solution. 5. The method according to p. 1, characterized in that the restoration of the dried biomaterial is carried out by washing it off with water or a solvent. 6. The method according to p. 1, characterized in that the impregnation of large-pore chemically neutral hydrophilic material of the carrier with a preservative solution is carried out until it is completely saturated.

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