WO2022071833A1 - Method for extracting nucleic acids from nail plates - Google Patents

Abstract

The invention relates to molecular biology. A method for extracting nucleic acids from nail plates is proposed, the method comprising lysis of a biological material in a buffer with guanidine thiocyanate, incubation for 20 min at 65°C, stirring, centrifuging at 13,000 rpm for 2 min; then precipitating the supernatant liquid with isopropanol, discarding the supernatant, washing the precipitate with alcohol solutions, and dissolving the precipitate containing nucleic acids in a buffer for elution. The invention provides for an increase in the purity and quantity of the nucleic acid preparation produced.

Description

METHOD FOR EXTRACTION OF NUCLEIC ACIDS FROM NAIL PLATES

Technical field

The invention relates to biotechnology, molecular biology, microbiology and medicine and is intended for the extraction of nucleic acids (NA) from biological material - nail plates, for subsequent analysis by polymerase chain reaction (PCR) or reverse transcription (RT) and PCR (RT-PCR) .

Prior Art

Widely used laboratory methods for the isolation of NA are described, for example, in J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 2001, NY, v. one; Short protocols in molecular biology, Wiley, 1995, or in Wilcockson J. The Use of Sodium Perchlorate in Deproteinization During the Preparation of Nucleic Acids // Biochem. J. 1973. V. 135. P. 559-561. Classical methods for the extraction of NA from "complex" initial samples, such as blood or tissues, include the lysis of biological material with detergents or chaotropic agents, sometimes in the presence of protein-degrading enzymes. This step is followed by several steps using organic solvents such as phenol and/or chloroform or ethanol.

At the present stage, the most effective methods for isolating and purifying NCs when working with "complex" biological material, such as nail plates, are recognized as sorption or precipitation.

Sorption methods of NA extraction provide a fairly high degree of purification from various types of biological material. The best known of the prior art is the method of isolation of NK, proposed by Boom et al. [US 5234809, priority date 07/01/1991]. This method includes the stage of cell lysis with a strong chaotropic agent, which destroys cell membranes and inactivates intracellular RNases, and subsequent sorption of NA on the carrier. NA reversibly binds to glass in the presence of a high concentration of chaotropic salts (for example, guanidine chloride, guanidine thiocyanate). Under these conditions, the binding of proteins to matrix does not occur. Chaotropic compounds are substances that disrupt the ordered structure of water and thereby bring membranes into a state of chaos (for example, urea, sodium iodide). Thus, in addition to binding, chaotropic agents ensure the destruction of cell membranes and cell lysis, followed by the release of NA. Impurities are washed off with chaotropic salt, and chaotropic salt - with 80% ethanol. The purified NA is removed from the glass with a low ionic strength buffer.

Despite the high degree of popularity of sorption techniques, their use can be associated with the loss of NC due to irreversible sorption on the carrier or in the course of several washings. In addition, the residual amount of the carrier (for example, silicon dioxide) in the final solution of NA can inhibit the enzymatic reactions of OT and/or NPR.

Precipitation (alcohol precipitation) involves the aggregation of NA (both DNA and RNA) in the presence of salt and alcohol. After precipitation with alcohol, NC is separated from the solution by centrifugation. The precipitate containing the target NA is repeatedly washed with alcohols and concentrated by centrifugation. At the final stage of the procedure, NA is dissolved in an aqueous buffer, often during incubation with heating to 55-65°C for better dissolution of the precipitate.

The advantage of this technology is the ability to work with "complex" samples, isolating both DNA and RNA equally, however, alcohol precipitation leads to the precipitation of not only NAs, but also proteins, which in turn makes it difficult to obtain a pure preparation. The simplest way to solve the problem with undesirable impurities is to reduce the initial volume of the analyzed sample, however, this approach is not applicable if it is necessary to obtain an NC preparation with a high degree of chemical purity and high concentration as a result.

From the prior art, a method is known for extracting NK by precipitation from such biological samples as blood, serum, plasma, other fluids (cerebrospinal fluid, saliva, semen and urine, etc.), blood products obtained from plasma or serum. This method is disclosed in patent EP1306446, priority date 02.08.2000, and includes the following steps: i) incubation of a biological sample with a reducing agent, co-precipitation and denaturing protein to degrade and denature proteins and other contaminants in a biological sample without the use of a protease; ii) direct alcohol precipitation. The method is implemented by using a kit for NA extraction, including a reducing agent, a co-recipient and a denaturing protein, while said kit does not contain a protease. In step i) the incubation takes place at 55-65°C, preferably about 60°C, for 5 minutes to 15 minutes, preferably about 10 minutes. Most preferably, the incubation is carried out at a temperature of about 60°C for about 10 minutes. In step ii) the reaction mixture obtained in step i) is combined with a lower alcohol to precipitate NC. Suitable alcohols include isopropanol and ethanol or the like. Preferably, isopropanol is added at a final concentration of 40% or more, or ethanol is added at a final concentration of 70% or more. Then the resulting mixture can be cooled to a temperature of minus 70°C to 4°C after adding a lower alcohol to improve the efficiency of salting out.

Despite the fact that this method is the most common for NA extraction by the precipitation method, it does not provide a sufficiently high yield of highly purified total DNA/RNA from nail plate samples.

From the prior art, a method is known for extracting DNA from the biological material of nails by the precipitation method, where nail flakes are introduced into a 1.5 ml tube with 100 μl of lysis buffer (50 mM TrisNO pH 9.5, 250 mM KC1, 60 mM NaHCCh) and incubated at a temperature 90-95°C 5-10 minutes. An equal volume of a mixture of chloroform and isoamyl alcohol (in a ratio of 24: 1) is added to the lysate, after which the entire mixture is stirred and centrifuged for 10 minutes at 9,000 g. The upper phase is removed and 0.1 volumes of 3M sodium acetate and 2 volumes of ethanol are added. After that, the tube is incubated for 2 hours at a temperature of minus 20°C. Then it is centrifuged for 10 minutes at 20,000 g in the cold, the DNA precipitate is washed from salts with 0.5 ml of 70% ethanol at room temperature for 10 minutes. The precipitated DNA is dissolved in 30 µl of ddHrO [Patent RU 2584035, priority date 12.02.2015]. This method was developed to isolate only DNA and is intended for extraction from samples of nail scales, while fragments of nail plates cannot be used as biological material. In addition, when implementing the above method, chloroform, a highly toxic substance, is used.

It is also known that the RIBO-prep reagent kit (RU No. FSR 2008/03147, TsNIIE, Russia) was used to isolate DNA from samples of the nail plates [First data on the prevalence in Russia of chromosomally integrated Human betaherpesvirus 6A/B, which is inherited. Domonova E.A., Silveistrova O.Yu., Goptar I.A., Kuleshov K.V., Nikiforova A.V., Matosova S.V., Shipulina O.Yu. Epidemiology and infectious diseases. Current Issues No. 4/2019, pp. 43-50]. DNA extraction was performed according to the instructions for use of the RIBO-prep kit for RNA/DNA extraction from clinical material [https://www.amplisens.ru/upload/iblock/a39/PH6o-npen%20(apxHB)210319.pdf ]. The instruction involves the following manipulations:

The solution for lysis is heated at a temperature of 65°C until the crystals are completely dissolved, the required number of 1.5 ml disposable tubes with tight-fitting lids is selected (including negative and positive isolation controls) and 10 µl of VKO is added to each tube. Then 300 μl of solution for lysis is added to the tubes, the tubes are labeled. In test tubes with solution for lysis and VKO, make 100 μl of prepared samples using tips with a filter. 100 µl of the negative control sample (NCC) is added to the negative control tube (CC), 90 µl of the CCO and 10 µl of the positive control sample (PCC) are added to the positive control tube (PC). The contents of the tubes are thoroughly mixed on a vortex, then centrifuged for 5 seconds on a microcentrifuge to remove drops from the inner surface of the lid and heated for 5 minutes at 65°C in a thermostat. Add 400 µl of the precipitation solution to the tubes, mix on a vortex, then centrifuge them on a microcentrifuge for 5 minutes at 13,000 rpm. Carefully remove the supernatant, without touching the precipitate, using a vacuum aspirator and a separate 200 µl tip for each sample. Add 500 µl of wash solution to the tubes, tightly close the lids, carefully wash the precipitate by turning the tubes 3-5 times, then centrifuge at 13,000 rpm for 1-2 minutes in a microcentrifuge. Carefully, without trapping the pellet, remove the supernatant using a vacuum aspirator and a separate 10 µl tip for each sample. Add 200 µl of wash solution to the tubes, tightly close the lids and carefully wash the precipitate by turning the tubes 3-5 times, then centrifuge at 13,000 rpm for 1-2 minutes in a microcentrifuge. Carefully, without trapping the pellet, remove the supernatant using a vacuum aspirator and a separate 10 µl tip for each sample. Place the tubes in a thermostat at 65°C for 5 minutes to dry the precipitate, add 50 µl of RNA buffer to the tubes, mix on a vortex, place in a thermostat at a temperature of 65°C for 5 minutes, periodically shaking on the vortex. Finally, the tubes are centrifuged at 13,000 rpm for 1 minute in a microcentrifuge. The supernatant contains purified RNA and DNA, the samples are ready for RT and NDP reactions.

Further extended laboratory studies showed that NA isolated from nail plate samples by the described extraction method did not have a sufficient degree of chemical purity and, in some cases, did not have an insufficient concentration for subsequent analysis by the PCR or RT-PCR method.

A common disadvantage of the above methods of NA extraction is the insufficient yield of DNA/RNA, the degree of their purification, which, in turn, reduces the quality of further molecular biological studies up to the impossibility of their conduct. In addition, in the overwhelming majority of cases, when performing the procedure for isolating NCs from nail plates, preliminary mechanical methods for homogenizing the samples under study are required.

Disclosure of invention

The problem to be solved by the claimed invention is is to obtain from samples of biological material - nail plates, a highly purified preparation of NK, free from inhibitors, with a high concentration, which ensures high analytical sensitivity of subsequent molecular biological studies (for example, including PCR, RT-PCR).

The technical result is achieved due to the fact that during the extraction of NC, the precipitation method is used, which includes the following stages: (i) lysis of biological material; (ii) precipitation; (iii) removing the supernatant and washing off the precipitate; (iii) dissolving the precipitate containing NA in the elution buffer, where 600 µl of lysis buffer is added to the nail plates at the lysis stage, then the contents of the tube are incubated at 65°C for 20 minutes, after which they are mixed and centrifuged at 13,000 rpm /min for 2 minutes, at the stage of precipitation, the resulting supernatant in a volume of 400 μl is added to separate tubes and the precipitation solution is added in an equal volume.

At the same time, 2 samples of nail plates with a size close to 2x10 mm are used for one study.

The efficiency of all extraction steps can be controlled by using:

- an internal exogenous control sample (IQR), which is an artificially constructed NA sequence, as well as:

- a negative control sample (NCO), which is a sterile solution that does not contain DNA and RNA,

- a positive control sample (PQS), which is an artificially constructed NA sequence of the desired target (for example, a fragment of a unique DNA sequence of a microorganism).

At the same time, VKO is added to each tube in a volume 50% more than the standard one (for example, recommended by the instructions for the reagent kit used) until the lysis buffer is added.

OKO is introduced into a test tube intended for negative extraction control in a volume of 100 μl. PKO is introduced into a test tube designed for positive extraction control, in a volume of 100 μl at a concentration increased by 1.5 times. After adding the lysis buffer, the tubes with OKO and PKO are thoroughly mixed on a vortex, droplets are precipitated from the inner surface of the lid by centrifugation for 5 seconds on a microcentrifuge, and only then incubated at a temperature of 65°C for 20 minutes.

The proposed method for the extraction of NA allows you to quickly and with a high yield (at least 2.08 and 1.92 times more for DNA and RNA, respectively, compared with the prototype) to simultaneously carry out the procedure for isolating and purifying NA from such "complex" types biological material such as nail plates. The resulting NA preparation can be used for molecular biological studies, including for PP-diagnostics.

The proposed solution is implemented as follows:

The collection of samples of the nail plates of the examined is carried out after removing the decorative varnish or gel polish (if there is a coating of the nail plate), thoroughly washing the hands, including the subungual spaces, and drying them with disposable napkins. Scissors cut off the free edge of the nail plate (3-4 samples) based on one study - 2 samples with a size close to 2x10 mm.

Samples of nail plates intended for PCR research are placed in disposable sterile plastic containers with a lid or Eppendorf type test tubes. When collecting samples of biological material, only disposable sterile instruments are used.

Extraction of total DNA/RNA is carried out by the precipitation method, for example, using a commercial kit of reagents. Extraction includes the following steps:

(i) lysis of biological material, in which:

(i) (a) select the required number of disposable polypropylene microtubes with tight-fitting lids.

If the study protocol provides for the use of an internal control sample, then in each test tube add VKO in a volume of 50% more than the standard one (for example, recommended by the instruction to the commercial reagent kit being used). Tubes are labelled.

(i) (b) each test tube is filled with the biological material to be tested - 2 samples of nail plates with a size close to 2x10 mm.

If the study protocol provides for the use of negative and positive controls for passing through the NA isolation stage, then 100 µl of CKO is added to the test tube intended for negative extraction control, and 100 µl of FKO at a concentration increased by 1.5 is added to the test tube intended for positive extraction control. times.

(i) (c) 600 µl of lysis buffer preheated to 65°C (until the crystals are completely dissolved) containing a chaotropic agent, for example, guanidine thiocyanate, is added to the test tubes. The biological material must be completely immersed in the solution.

Carrying out manipulations in the indicated order eliminates the possibility of insufficient immersion of the studied samples of biological material in the lysis buffer, minimizing errors at this stage of the preanalytical stage.

If the extraction protocol provides for the use of negative and positive controls for the passage of the NA isolation stage, then the tubes with OKO and PKO are thoroughly mixed on a vortex, centrifuged for 5 seconds in a microcentrifuge to remove drops from the inner surface of the cap.

(i) (d) the contents of the tubes obtained in step (i) (c) are incubated for 20 minutes at 65°C.

(i) (e) the contents of the tubes obtained in step (i) (d) are mixed and centrifuged for 2 minutes at a speed of 13,000 rpm to separate from the still incompletely lysed constituents of the biological material under study.

The result is a supernatant suitable for precipitation.

(j) precipitation, where:

(d) (a) select the required number of disposable polypropylene microtubes with tight-fitting lids. Tubes are labelled.

(ii) (b) 400 µl of carefully collected supernatant liquid obtained as a result of step (i) is added to each tube.

(ii) (c) Add 400 µl of precipitation solution containing isopropanol-isopropyl alcohol to the supernatant in each tube.

(ii) (d) the contents of the tubes obtained in step (ii) (c) are vortexed and centrifuged in a microcentrifuge for 5 minutes at 13,000 rpm.

The suspension obtained as a result of the implementation of stage (ii) is suitable for removing the supernatant and washing the precipitate.

(iii) removal of the supernatant and washing of the precipitate, in which:

(iii) (a) Remove the supernatant from the tube without touching the pellet and using a separate tip for each sample.

(iii) (b) Add 500 µl of a wash solution containing 70% ethanol to the pellet in the tubes, stopper the tube, and wash the pellet by inverting the tubes 3-5 times.

(iii) (c) centrifuge at 13,000 rpm for 1-2 minutes and, without capturing the pellet, withdraw the supernatant using a separate tip for each sample.

(iii) (d) add 200 µl of a wash solution containing 95% ethanol or a mixture of 70% acetone and 70% ethanol in equal proportions to the precipitate in the tube, stopper the tube and wash the precipitate by inverting the tubes 3-5 times;

(iii) (e) centrifuge at 13,000 rpm for 1-2 minutes and, without capturing the pellet, withdraw the supernatant using a separate tip for each sample;

(iii) (f) tubes with open lids are placed in a thermostat heated to a temperature of 65°C for 5 minutes to dry the precipitate.

As a result, a dried precipitate is obtained, suitable for subsequent elution in order to obtain a total DNA/RNA preparation.

(iiii) dissolving the precipitate containing NA in the elution buffer, at which:

- add 50 µl of elution buffer consisting of a sterile solution with a low salt content to a test tube with a dried precipitate, mix and heat for 5 minutes at a temperature of 65 °C, shaking occasionally.

- the tubes are centrifuged at 13,000 rpm for 1 minute.

At the output, a supernatant is obtained, which contains highly purified total DNA/RNA, ready for direct use in PCR or RT-PCR, and further molecular biological studies.

The claimed method for extracting nucleic acids from samples of nail plates makes it possible to improve the quality and quantity (percentage of NA output) of the resulting total DNA/RNA preparation without significantly increasing the duration of the procedure and increasing the cost of the process. In the claimed extraction method based on the principle of precipitation, in order to minimize the loss of total DNA / RNA and achieve the maximum possible removal of inhibitors of the PCR and RT-TTPR enzymatic processes, the volumes of reagents used, as well as the duration of the stages of incubation and centrifugation, are optimally selected. .

The creation of the claimed invention is based on a study carried out at the FBUN TsNIIE Rospotrebnadzor. The material for the study was 440 samples of nail plates of 100 people aged from 5 days to 80 years (median age 29 years), among which: 44.0% of men (44/100) and 56% of women (56/100). The study was conducted in accordance with the legislation of the Russian Federation, international ethical standards and regulatory documents of the Central Research Institute of Epidemiology of Rospotrebnadzor with the written consent of the patients.

Comparison of the extraction efficiency of NA according to the claimed method was carried out with commercially available kits of reagents based on a similar method for isolating total DNA/RNA, in which the extraction of NA from the analyzed samples of biological material was carried out simultaneously using 4 methods based on the precipitation method, namely: reagent kit "RIBO-prep" (FBUN Central Research Institute of Epidemiology of Rospotrebnadzor, RF) (hereinafter, reagent kit No. 1) in accordance with the manufacturer's instructions (standard protocol) and its modified protocol, according to the claimed method, as well as the kit of reagents "PROBA-NK" (LLC " NPO DNA-Technology, RF) (hereinafter - reagent kit No. 2) in accordance with the manufacturer's instructions (standard protocol) and its modified protocol, according to the claimed method. In this case, the reagent kit No. 1 (standard protocol) was taken as a prototype of the proposed method, and the reagent kit No. 2 (standard protocol) was the comparison method.

Determination of NA concentration was carried out by fluorimetric method using a Qubit® 3 fluorimeter (Thermo Fisher Scientific, USA). The NA concentration was measured using highly sensitive fluorescent probes capable of selectively binding to target DNA or RNA molecules to form a fluorescent complex. The fluorescence intensity of the complex was proportional to the analyte concentration in the range of 0.2-100 ng/µl and 5-100 ng/µl for the original DNA and RNA sample, respectively.

At the same time, the DNA concentration in the biological material samples and the absence of the effect of inhibitors on the passage of the enzymatic reaction were determined by real-time PCR with analysis of the obtained results in a quantitative format (AmpliSens® NNUb-screen-titer-FL, FBSI TsNIIE Rospotrebnadzor, RF). The calculation of the DNA concentration, estimated by the human P-globin gene, was performed in 1g copies/ml.

In addition, a study was made of the effect of inhibitors on the passage of RT-TTPR. The evaluation was carried out with respect to the effectiveness of the quantitative determination of exogenous VKO, which is an artificially constructed RNA sequence inserted into the phage ms2, at a concentration of 5x10 ⁵ -2x10 ⁶ copies / ml ("AmpliSense® NSU-Monitor-FL ”, FBUN TsNIIE Rospotrebnadzor, RF). The RNA concentration was calculated in lg copies/ml.

Amplification results were set up and analyzed on a Rotor-Gene Q instrument with a real-time fluorescent signal detection system (Qiagen, Germany). As a result of the study, the quantitative characteristics of NCs extracted from samples of nail plates (n=50) were determined using four different methods. The results of comparing the quantitative characteristics of NCs isolated from samples of the nail plates using various extraction methods are shown in Table 1. For comparative analysis, NC samples were used, obtained according to the methods taken as a prototype and comparison in standard and modified versions of the methods.

Table 1. Comparison of the quantitative characteristics of NCs isolated from nail plate samples using various extraction methods.

Quantitative characteristics of NA extracted by a modified method from samples of nail plates meet the requirements for samples intended for performing PCR and RT-PCR. This allows us to consider the claimed extraction method suitable for use in biomedical research related to the production of NK.

When determining the possibility of using the extraction method according to the claimed invention in practical work with samples of nail plates, a comparative assessment of the efficiency of NC extraction by PCR (n=30) and RT-PCR (n=30) was carried out. For comparative analysis NC samples were used, obtained according to the methods taken as a prototype and comparison in the standard protocol and the modified protocol according to the claimed method.

The claimed invention is illustrated by graphs - Fig. 1 and FIG. 2 where:

On FIG. 1 "Comparison of the concentration of DNA isolated from samples of the nail plates using different extraction methods" shows the results of the distributions of DNA concentrations, estimated by the human P-globin gene, in biological samples using four different methods, while the oblique shading column visualizes the results of the study , obtained by extraction using a standard protocol, and a column with horizontal shading - the results of the study obtained by performing extraction using a modified protocol according to the claimed method.

On FIG. 2 "Comparison of TKO RNA Concentration in Extraction of Nail Plate Samples by Different Methods" shows the concentration distributions of TKO, which is an artificially constructed RNA sequence inserted into the ms2 phage, in biological material samples using four different extraction methods, while the bar with oblique shading visualizes the results of the study obtained by carrying out the extraction using the standard protocol, and the column with horizontal shading - the results of the study obtained by carrying out the extraction using the modified protocol according to the claimed method.

As a result of the studies, it was found that the efficiency of DNA extraction, estimated by the human P-globin gene, when using the proposed method is higher (p<0.05) than when using standard research protocols for both reagent kit No. 1 and reagent kit No. 2. The lowest concentration of DNA was determined in NA samples extracted from samples of nail plates according to the methodology of the comparison method (average 2.7 1g DNA copies/ml). And the highest concentration of DNA - with the help of the proposed solution, amounting to an average of 5.2 and 5.3 1g DNA copies/ml, respectively, for modified protocols according to the claimed method of prototype research (reagent kit No. 1) and comparison (reagent kit 2). The results of the distributions of DNA concentrations, estimated by the human P-globin gene, in samples of biological material using four different methods are presented in Fig.1.

The RNA extraction efficiency study excluded RT-TTPR passage inhibition, as assessed by the efficiency of exogenous internal control amplification, using four different methods. The results of concentration distributions of WKO, which is an artificially designed RNA sequence inserted into the phage ms2, in biological material samples using four different extraction methods are shown in Fig.2. The lowest concentration of RNA was determined in NA samples extracted from samples of nail plates according to the methodology of the comparison method (average 4.33 1g RNA copies/ml). And the highest concentration of RNA - with the help of the proposed solution, averaging 5.01 and 5.28 1g RNA copies/ml, respectively, for modified protocols according to the claimed method of prototype research (reagent kit No. 1) and comparison (reagent kit 2).

Thus, the claimed invention allows for the extraction of total DNA/RNA from samples of biological material - nail plates, with a high concentration. The concentration of total DNA according to the claimed method is up to 89.1 ng/ml, and total RNA - up to 454.0 ng/ml, that is, the claimed method allows to increase the yield of the total DNA/RNA preparation by at least 1.92 times. The lack of data on the inhibition of enzymatic reactions of the passage of NRP and RT-TTPR allows the use of both DNA and RNA for further studies.

The solution proposed in the claimed invention is necessary and sufficient to achieve the task - extraction of DNA / RNA with a high degree of chemical purification, free from inhibitors, with a high concentration, suitable for PCR (RT-TTPR) and further molecular- biological research.

Brief description of the drawings

Fig. 1. Comparison of the concentration of DNA isolated from nail plate samples using different extraction methods.

Fig. Fig. 2. Comparison of the concentration of WKO RNA during the extraction of samples of the nail plates by various methods.

Embodiments of the invention

The following examples further illustrate the present invention, but are not intended to limit its application.

Example 1 This example demonstrates the possibility of using RNA extracted from samples of the nail plates in molecular biological studies.

A man (23 years old) was sent to the Department of Molecular Diagnostics and Epidemiology of the Central Research Institute of Epidemiology of Rospotrebnadzor to conduct molecular biological studies.

Samples of the nail plates were collected after thorough washing of the hands of the subjects, including the subungual spaces, and drying with disposable napkins. The free edge of the nail plate (n=4) was cut with disposable sterile scissors. The samples of biological material intended for analysis were placed in disposable sterile plastic test tubes of the Eppendorf type and labeled.

Before the extraction procedure, the studied biological samples were divided on the basis of one study - 2 samples of nail plates with a size close to 2x10 mm. Extraction of RNA from samples of the nail plates was carried out according to the method of the claimed invention. For all samples extracted from samples of biological material, the RNA concentration was determined using a Qubit® 3 fluorimeter (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The test results are presented in Table 2. Quantitative characteristics of RNA extracted from the samples of the nail plates of a man (23 years old). Further, RNA samples were stored at a temperature not exceeding minus 70°C until testing as part of further molecular biological research, avoiding repeated thawing-freezing, but not more than one year.

Table 2. Quantitative characteristics of RNA extracted from samples of the nail plates of a man (23 years old).

Thus, the quantitative characteristics of RNA extracted from samples of the nail plates according to the claimed method of the invention meet the requirements for samples intended for molecular biological studies. RNA samples are ready for further use.

Example 2 This example demonstrates the possibility of using DNA extracted from samples of the nail plates in the study of hereditary predisposition to various multifactorial diseases, such as bleeding disorders, using the TTPR-RT method.

The patient (female, 33 years old), as part of the pregravid preparation, was sent for a laboratory examination to exclude a hereditary predisposition to blood clotting disorders. From the anamnesis it is known: a woman (born in 1987) of the correct physique with obesity (BMI 39.9), BP 125/70 mm Hg, Kaya's pregnancy ended in a spontaneous abortion for a period of 8 weeks in 2013, in a first-line relative (mother) had blood clotting disorders - deep vein thrombosis of the legs, blood type A (II) Rh positive, kell negative, phenotype C+c+E+e+, no complaints. When applying for outpatient care to an obstetrician-gynecologist, the patient presented the results of a general clinical blood test (hemoglobin, g / l - 124.00; hematocrit,% - 39.40; platelets, 10 ⁹ /l - 264.00; absolute content: neutrophils, 10 ⁹ /l - 2.96; eosinophils, 10 ⁹ / l - 0.13; basophils, 10 ⁹ / l - 0.04; monocytes, 10 ⁹ / l - 0.40; lymphocytes, 10 ⁹ /l - 2.24; erythrocyte sedimentation rate, mm/h - 8.00), studies of coagulation hemostasis (INR - 1.154), urinalysis - no pathology.

To exclude a genetic predisposition to the development of thrombosis, a PCR study of DNA isolated from samples of the nail plates was performed, including a qualitative determination of genotypes by polymorphisms 20210 G>A (rsl799963) in the prothrombin gene (F2) and R534Q G>A (rs6025, Leiden mutation) in the proaccelerin gene (F5). The validity of the result of the analysis was evaluated relative to the data obtained in the study of DNA extracted from a sample of whole venous blood of the subject, according to similar parameters.

Samples of the nail plates were collected after thorough washing of the subject's hands, including the subungual spaces, and drying with disposable napkins. The free edge of the nail plate (n=2) was cut with disposable sterile scissors. Samples of biological material with a size close to 2x10 mm were placed in a disposable sterile plastic test tube of the Eppendorf type. Blood was taken on an empty stomach with a disposable needle (diameter 0.8-1.1 mm) into a disposable sterile tube with a volume of 4.0 ml with 6% EDTA by puncture of a peripheral vein after treatment of the puncture site with a skin antiseptic. Biological material samples intended for PCR were labeled. It was allowed to store samples of nail plates and whole venous blood at a temperature of 18-25°C and 2-8°C, respectively, within 72 hours from the moment of taking the biological material.

Isolation of DNA from the nail plates was carried out according to the claimed method of extraction, whole venous blood - using a set of reagents "RIBO-prep" (RU No. FSR 2008/03147, Federal Budgetary Institution of the Central Research Institute of Epidemiology of Rospotrebnadzor, RF). For all samples extracted from samples of biological material, the DNA concentration was preliminarily determined using a Qubit® 3 fluorimeter (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The test results are presented in Table 3. Quantitative characteristics of DNA extracted from samples of the nail plates and whole venous blood of a woman (33 years old). Table 3. Quantitative characteristics of DNA extracted from samples of nail plates and venous whole blood from a woman (33 years old).

Then, DNA samples were analyzed by real-time PCR using the AmpliSense® F2/F5-SNP-FL reagent kit (RU no. RZN 2019/8325, Central Research Institute of Epidemiology of Rospotrebnadzor, RF) according to the manufacturer’s instructions. Amplification results were set up and analyzed on a Rotor-Gene Q instrument with a real-time fluorescent signal detection system (Qiagen, Germany). The results of the laboratory examination of the patient are presented in Table 4. The results of the determination of genetic polymorphisms associated with the risk of thrombosis by RT-PCR.

Table 4. Results of determination of genetic polymorphisms associated with the risk of thrombosis by real-time PCR.

As follows from the table, a frequent ("wild type") variant of the genotype (GG genotype) was detected in the examined woman for each of the analyzed polymorphisms: rsl799963 in the prothrombin gene (F2) and rs6025 in the proaccelerin gene (F5). 100% coincidence of the results obtained for the compared types of biological material (samples of nail plates and whole venous blood) of the patient was established when studying hereditary predisposition to impaired blood clotting (development of thrombosis) by PCR-RT, risk alleles were not identified.

Thus, the quantitative and qualitative characteristics of DNA, extracted from samples of the nail plates, allow us to recommend this type of biological material for use in the study of the hereditary predisposition of patients to various multifactorial diseases by real-time PCR.

Example 3. This example demonstrates the possibility of using DNA extracted from samples of the nail plates in laboratory confirmation of hiHHV-6A status and deciphering the case of hereditary transmission of chromosomally integrated human herpes virus 6A (hiHHV-6A) in members of the same family.

From the anamnesis it is known: a boy (born in 2016) from his mother (born in 1986, somatically healthy), from the first pregnancy, which proceeded: I trimester - retrochorial hematoma, increased D-dimer, II trimester - without features, III trimester - anemia in pregnancy. Childbirth I spontaneous in time. The child was born with a weight of 3570 g, body length 54 cm, head circumference - 36 cm, chest circumference - 36 cm. Apgar score - 8/9 points. Neonatal period - without features. A laboratory examination of a child against the background of clinical health at the age of 1.5 months revealed changes in the hemogram: anemia (hemoglobin - 85 g/l), relative neutropenia (12.6%), thrombocytosis (506 thousand); in blood HHV-6A/B DNA at a concentration of 5.14 1g copies/10 ⁵ cells (RT-PCR) in the absence of virus-specific IgG class antibodies (ELISA) in blood serum. According to ultrasound - thymomegaly III degree. Based on the results of the clinical and laboratory examination, the boy was diagnosed with "Acute infection caused by human herpes virus 6?". It is recommended to conduct a clinical and laboratory examination of the mother of the child. The issue of prescribing specific antiviral therapy to the patient was considered. Further examination of the child, mother included laboratory confirmation or exclusion of active HHV-6A/B infection. As a result, HHV-6A/B DNA was detected by PCR-RT at a concentration of >5 1g copies/10 ⁵ cells in all types of biological material studied both in the child and in his mother (whole blood, blood plasma, oropharyngeal swab; urine child) without increasing the obtained values in dynamics. At the same time, the determined the virus DNA concentration correlated with the number of cells in the test sample, as estimated by the human P-globin gene. In the blood, the concentration of viral DNA was 5.23 1g copies/10 ⁵ cells in the mother, 5.24 1g copies/10 ⁵ cells in the child, 5.10 1g copies/10 ⁵ cells in the blood plasma (for both); smear from the oropharynx - 5.27 and 5.29 1g copies/10 ⁵ cells in mother and child, respectively; urine - 5.35 1g copies/10 ⁵ cells (child). Virus-specific antibodies of the IgG class were not found in the blood serum samples of patients by ELISA. A subsequent dynamic ELISA study of paired blood sera from mother and boy, taken with an interval of 14 days, did not reveal seroconversion of virus-specific AT-IgG, which indicated the absence of data indicating infection of patients with exogenous HHV-6A/B. It should be noted that the boy's mother did not complain at the time of the clinical and laboratory examination, she did not have any clinical symptoms of an active infection, the indicators of the general clinical and biochemical blood tests, and the general urinalysis were within the range of reference values.

The obtained laboratory examination data and the absence of clinical signs of active infection in patients were the basis for suspicion of their hiHHV-bA/B status.

To confirm or refute this assumption, an additional study of samples of the nail plates of the mother and child was carried out using the PCR-RT method. Samples of the nail plates were collected after thorough washing of the hands, including the subungual spaces, and drying with disposable wipes. The free edge of the nail plate (n=4) of the subjects was cut off with disposable sterile scissors, placed in disposable sterile plastic tubes of the Eppendorf type and labeled. It was allowed to store samples of nail plates at a temperature of 18-25°C for 72 hours from the moment of taking the biological material.

Before the extraction procedure, the studied biological samples were divided on the basis of one study for each patient separately - 2 samples of nail plates with a size close to 2x10 mm. Extraction of DNA from samples of nail plates was carried out according to the claimed method of the present invention. The concentration of total DNA extracted from nail plate samples was determined using a Qubit® 3 fluorometer (Thermo Fisher Scientific, USA) according to the manufacturer's instructions. The results of the study are presented in Table 5. Quantitative characteristics of DNA extracted from samples of the nail plates of a boy (2 months old) and his mother (31 years old).

Table 5. Quantitative characteristics of DNA extracted from samples of the nail plates of a boy (2 months old) and his mother (31 years old).

The concentration of HHV-6A/B DNA in the studied samples of the nail plates was determined by real-time PCR, taking into account the results of the analysis in a quantitative format (AmpliSens® NNUb-screen-titer-FL, FBSI TsNIIE Rospotrebnadzor, RF) according to the manufacturer's instructions. Amplification results were set up and analyzed on a Rotor-Gene Q instrument with a real-time fluorescent signal detection system (Qiagen, Germany). Concentration was calculated in logarithms of HHV-6A/B specific DNA copies per standard number (10 ⁵ ) of human cells estimated by the P-globin gene.

As a result of the study, hereditary hiHHH-bA was verified based on the determination of virus DNA by the TTPR-RV method in samples of whole venous blood and nail plates of the child and mother at a concentration of 5.24, 5.37 and 5.23, 5.63 1g DNA copies of HHV-6A/B/10 ⁵ cells, respectively. Species identification of the virus was carried out on the basis of data obtained using the massively parallel sequencing method (MiSeq, Illumina). The hiHHV-bA status of the child and mother was ascertained during the study of the total DNA of patients by the PNR method with specific primers complementary to the viral DNA region and human chromosomes, followed by the Sanger sequencing reaction. As a result of which two identical amplicons were obtained, including on one side a section of the HHV genome 6 A, on the other hand, a part of the 17p human chromosome, between which there was a region with telomeric repeats about 300 nucleotides long.

To decipher the case of hereditary transmission of chiHHH-bA, four conditionally healthy close relatives of the "mother-child" couple with chiHHH-bA status were examined: the child's father, mother's sister, maternal grandmother, maternal grandfather. At the time of the clinical and laboratory examination, the patients did not complain, they did not have any clinical symptoms of an active infection. Indicators of general and biochemical blood tests - within the range of reference values, general urinalysis - without pathology. Further molecular biological studies, carried out in a format similar to that described above, made it possible to identify and laboratory confirm the hiHHV-bA status in the maternal grandfather. The rest of the family members did not have a confirmed hiHHV-bA status.

Thus, the quantitative and qualitative characteristics of the DNA isolated from the sample of the nail plates according to the claimed extraction method make it possible to conduct laboratory confirmation of the hiHHV-bA/B- status of the subjects. This example demonstrates a case of detection and laboratory confirmation of hiHHV-bA status in members of the same family in three generations (from father to daughter and grandson). Laboratory confirmation of hHHV-bA status was the rationale for excluding active HHV-6A infection and for excessive antiviral therapy.

The claimed method for extracting nucleic acids from samples of nail plates is feasible using standard technical devices and equipment, and is necessary in various fields, for example, practical medicine (clinical and laboratory diagnostics, medical genetics), forensic, forensic practice (for example, within the framework of DNA testing). -personal identification), gene fingerprinting and DR-

Claims

CLAIM 1. A method for extracting nucleic acids from nail plates, including the stages of lysis of biological material using a lysis buffer with a chaotropic agent guanidine thiocyanate, precipitation using isopropanol, removal of the supernatant and washing the precipitate with 70% ethyl alcohol, 95% ethanol or a mixture of 70% acetone and 70% ethanol in equal proportions, dissolving the precipitate containing nucleic acids in an elution buffer consisting of a sterile solution with a low salt content, in which 600 μl of lysis buffer is added to the biological material at the lysis stage and incubated at a temperature of 65 ° C in for 20 min, after which it is stirred and centrifuged at 13,000 rpm for 2 min, and at the precipitation stage, the resulting supernatant in a volume of 400 μl is added to separate tubes and the precipitation solution is added in an equal volume. 2. Method according to and. 1, characterized in that for one study, 2 samples of nail plates with a size close to 2x 10 mm are used. 3. Method according to and. 1, characterized in that at the stage of lysis of biological material, an additional internal exogenous control sample is added in a volume increased by 50% from the standard one, until the lysis buffer is added. 4. Method according to and. 1, characterized in that at the stage of lysis of biological material, an additional negative control sample is added to a test tube intended for negative control of extraction in a volume of 100 μl, after which it is mixed and centrifuged for 5 s immediately before the start of incubation. 5. Method according to and. 1, characterized in that at the stage of lysis of biological material, an additional positive control sample is added to a test tube designed for a positive control of extraction in a volume of 100 μl at a concentration increased by 1.5 times, after which it is mixed and centrifuged for 5 s immediately before the start incubation. 23