D-3833

RealBest-Genetics Hemostasis

FOR PROFESSIONAL USE

PAI-1/ITGB3

Real-time PCR test kit for the determination of single-point polymorphisms of the PAl-1 and ITGB3 genes of the blood coagulation system, by detecting melting curves

The kit contains reagents for 48 tests, including control samples.

The "RealBest-Genetics Hemostasis PAl-1/ITGB3" test kit detects 5G(-675J4G in the PA/-1 gene (encoding the endothelial inhibitor of plasminogen activator, serpin 1) and T1565C in the /7TGB3 gene (encoding the fibrinogen receptor part, encodes integrin beta-3) is used for the differential determination of single-point nucleotides using real-time polymerase chain reaction (PCR) and melting curve analysis.

USER'S GUIDE

1. USE OF ORDINANCE

The results of genotyping are taken into account in the overall evaluation of the risk of thrombophilia (inherited tendency to thrombophilia).

2. WHO CONTENTS

• Control sample "Normal homozygous" (CS1) - 1.cs, 1 ml;
• Control sample "Mutant homozygous" (CS2) - 1 tsp, 1 ml;
• PCR master mix (RMM), lyophilized - 48 cs;
• "Genetics" sample diluent - 2 pcs, each 12 ml injection glass;

The kit also contains PCR optical quality film - 1 sheet.

3. PRINCIPLE OF THE METHOD

The principle of the method is based on the amplification of the selected human DNA region, then the formation of hybrid duplexes of the PCR product and a specific DNA probe, and the detection of the melting curves of the duplexes.

A DNA probe to detect the 5G(-675)4G polymorphism complements the normal PAl-1 gene; the DNA probe to detect the T1565C polymorphism complements the mutant ITGB3 gene. Thus, the melting point is different for two allelic variants. As a result, the genotype of each polymorphism can be identified (normal homozygous, heterozygous, mutant homozygous).

The amplification step consists of repeated cycles; denaturing the DNA template, annealing the primers to specific sites on the DNA template, and extending the primers with Tag polymerase.

After the amplification step, the temperature is lowered and the specific fluorescently labeled DNA probe hybridizes with the quencher -labeled amplicon, leading to a decrease in the fluorescent signal. The melting step during which the DNA probe is released, results in an increase in fluorescence. A mismatched duplex has a lower melting point than a fully complementary duplex.

4. SPECIFICATIONS

4.1. For each polymorphism in CS1, a melting peak corresponding to the "normal homozygous" genotype must be detected.

4.2. In CS2, a melting peak corresponding to the "mutant homozygous" genotype must be detected for each polymorphism.

4.3. 5G(-675)4G in the PAI-1 gene and / ITGB3

5. WARNINGS AND SAFETY PRECAUTIONS

The kit may only be used by qualified personnel.

When handling the kit, follow the national safety regulations for working with pathogens.

For in vitro use only.

Wear disposable gloves, a lab coat, and eye protection when handling samples and kit components.

In order to avoid contamination, the stages of PCR and DNA removal must be spatially separated from each other.

Use only disposable pipette tips with filters.

Never use the same pipette tip for different samples.

Do not use the kit after the expiration date.

Do not use kit components from different sources in the same experiment.

Do not combine reagents from different batches or from different vials of the same batch.

For reliable results, strictly follow the instructions for use that come with the kit.

Dispose of unused reagents and waste in accordance with national, federal, state, and local regulations.

6. REQUIRED BUT NOT SUPPLIED ADDITIONAL MATERIALS AND EQUIPMENT

• DNA extraction kit;
• Laminar safety box;
• Refrigerator;
• Microcentrifuge;
• Vortex mixing;
• Disc mixer, which provides a rotation speed of 1200 revolutions per minute;
• Real-time PCR cycler;
• Biohazardous waste container;
• Disposable pipette tips with aerosol spray;
• Semi-automatic, variable volume pipettes;
• Racks for 2 ml and 0.2 ml tubes;
• Disposable medical, non-sterile, dust-free gloves;
• Scissors.

7. SAMPLE PREPARATION

The test must be performed on DNA samples extracted from clinical material using one of the DNA extraction kits listed on page 1, in accordance with its instructions for use. If using an extraction kit containing magnetic particles, keep the tubes with the extracted NA in a magnetic rack.

7.1. DNA extraction with the "RealBest extraction 100" extraction kit

Attention! DNA extraction should be performed without the addition of an internal control (IC).

DNA extraction can be performed with the help of extraction kits: "RealBest-Genetics DNA-express", "RealBest extraction 100" (manufacturer: AO Vector-Best), or other kits intended for DNA extraction from whole blood and buccal swabs.

Each group of samples subjected to the DNA extraction procedure should contain a "Normal homozygous" (CS1) and a "Mutant homozygous" (CS2) control sample from this kit.

For samples CS1 and CS2, the same DNA extraction procedure as for the positive control sample must be performed (see the instructions for use of the extraction kit).

7.2. DNA extraction using the "RealBest-Genetics DNA-express" extraction kit

Attention! Immediately put the remaining tubes back into the foil bag, squeeze out the air, and seal tightly with a pinch.

The extracted DNA is stored at (2-8)°C for a maximum of 24 hours.

8. PROCEDURE

8.1. Preparation of kit components

Before the analysis, take out the PCR master mix (RMM) in readiness, sealed in the package (18-26) °C for at least 30 minutes. Open the package and use scissors to cut the required number of tubes with RMM (counting test samples and control samples: 1 CS1 and 1 CS2). Cut the tubes together with the cover film.

After opening the package for the first time, RMM must be stored for a maximum of 3 months (2-8)°C.

After first opening the tubes, store CS1 and CS2 for up to 1 month at (2-8)°C, or in 50 ul aliquots at minus (18-24)°C for up to 3 months.

After opening the vial for the first time, the "Genetics" sample diluent must be stored at (2-8)°C for a maximum of 1 month.

8.2. Label the tubes for each test and control sample with RMM.

8.3. Carefully remove the cover sheet leaving the RMM in the tube.

8.4. Add 50 μl of the appropriate extracted DNA solution to each tube using a new pipette tip fitted with a filter. Seal the tubes tightly with PCR optical grade foil. Start the PCR no later than 30 minutes after adding the DNA.

Attention! The optical film must remain clean (free from labels and marks).

Attention! Labels should be placed on the side of the pipes.

8.5. Shake the PCR plate for 1 minute on a plate shaker at 1200 rpm.

8.6. Place the tubes in the real-time PCR cycler.

8.7. Program 1 real-time PCR cycler for 1 specific DNA

Stage 1: 50°C - 2 minutes;
Stage 2: 95°C - 2 minutes;
Stage 3: 50 cycles (94°C - 10 sec; 60°C - 20 sec);
Stage 4: "melting curve" from 27°C to 75°C in steps of 1°C for 5 seconds (fluorescence is measured at each step);
Stage 5: 10°C - 1 min.

8.8. Select the detection channels in the melting curves:

The "FAM" channel is used to locate the 5G(-675)4G polymorphic location of the PA/I-1 gene;
The "HEX" channel is used to define the T1565C polymorphic site in the ITGB3 gene.

8.9. For CS1, the program must:

detect a melting peak in each of the "FAM" and "HEX" channels corresponding to the "normal homozygous" genotype and determine the melting temperatures.

8.10. For CS2, the program must:

detects a melting peak corresponding to the "Mutant Homozygous" genotype on the "FAM" and "HEX" channels and determines the melting temperatures.

Run the program.

9. DATA ANALYSIS AND INTERPRETATION

9.3. Analysis of the 5G(-675)4G polymorphisms detected in the PAI-1 gene and the T1565C polymorphisms detected in the ITGB3 gene.

9.3.1. For each test sample, one (homozygous) or two (heterozygous) melting peaks should be detected in the "FAM" and "HEX" channels and the melting temperatures should be determined.

9.3.2. A genotype can be considered "normal homozygous" if a melting peak is observed and the melting temperature differs by no more than 2°C from the value obtained for CS1.

9.3.3. The genotyping result is "mutant homozygous" if a melting peak is observed and the melting temperature differs by no more than 2°C from the value obtained for CS2.

9.3.4. The genotyping result is "heterozygous" if two melting peaks are detected and the melting temperature of the first peak differs by no more than 2°C from the value obtained for CS1 and the melting temperature of the second peak differs by no more than 2°C from CS2 with the value obtained for (see the appendix).

9.3.5. The genotyping result can be considered doubtful if the melting temperature of the hybridization product differs by more than 2°C from the temperature values obtained for CS1 and CS2. In case of an unequivocal result, repeat the analysis of the test sample starting with the DNA removal step or sampling.

9.4. The diagnostic value of the test:

Hereditary predisposition is one of the most important factors in the development of thrombophilia. The tendency to the development of diseases such as thrombosis, thromboembolism, ischemia and infarction of organs should appear if hereditary factors are combined with exogenous factors (smoking, taking oral contraceptives and some other drugs, pregnancy, long-term immobilization, affecting large veins interventions) and chronic diseases (obesity, diabetes, heart disease, malignant tumors).

As a rule, without genetic analysis, it is difficult to reveal the hereditary tendency before the disease develops. It is impossible to identify a single "responsible" gene, since thrombophilia is influenced by the combined effect of several polymorphisms of different genes related to the hemostatic system.

Serpin 1, an endothelial inhibitor of plasminogen activator, inhibits the conversion of plasminogen to plasmin, which is necessary for the lysis of fibrin clots and the dissolution of the extracellular matrix. The 5G(-675)4G polymorphism in the gene promoter enhances PAI-1 expression, leads to a decrease in the thrombolytic activity of plasmin and is associated with ischemic heart disease.

The 4G/4G genotype is associated with an increased risk of developing gestosis, preeclampsia, placental dysfunction, hypoxia and delayed fetal development. The risk of gestosis is increased in patients with the 4G/4G genotype. The 5G/5G genotype can lead to a decrease in the risk of myocardial infarction.

The ITGB3 gene encodes the integrin beta-3 glycoprotein, which is a part of the platelet receptor involved in platelet adhesion and aggregation, as well as several signaling interactions. The T1565C polymorphism results in a Leu33Pro amino acid residue change in the protein, which results in increased platelet adhesion. Patients suffering from myocardial infarction or unstable angina carry the 1565C variant twice as often as the control group. Carriers of the 1565C allele may develop acute coronary syndrome at an earlier age (before the age of 60).

Those who carry both the 1565C polymorphism of the ITGB3 gene and the 4G polymorphism of the PAI-1 gene have an increased risk of developing the disease (especially in men). The tendency to develop ischemic disease is also increased if diabetic patients carry the 1565C polymorphism. In women, the 1565C/C genotype can be associated with a high risk of inguinal hernia after menopause. The 1565C and 1565/C genotypes occur 2 times and 4 times more often in women suffering from repeated miscarriages, respectively, compared to the control group.

9.5. Literature:

1. N.V. Pizova, Thrombophilia: genetic polymorphisms and vascular catastrophes. // M. IMA-PRESS, 2013, 248 P.
2. G.A. Tsvetovskaia, E.D. Chikova, G.I. Lifshits, et al., Genetic risk factors for thrombophilia in women of reproductive age in the West Siberian region // Fundamentalnyie issledovaniya (Fundamental Research, in Russ.), 2010, No. 10, P. 72-79.
3. E.G. Shchepotina, M.A. Prasolova, M.K. Ivanov, New reagent kit for the determination of single nucleotide polymorphisms in genes of the folate cycle and blood coagulation system // Novosti Vektor-Best (Vektor-Best News, in Russ.), 2013, Vol. 68, No. 2, P. 2-8.
4. I. Belykh, S. Petrikov, Y. Kotovshchikova, I.G. Peregudova, G.I. Kostiuchenko, The role of genetic polymorphisms in the formation of venous thromboembolism // Meditsina i obrazovanie v Sibiri (Medicine and Education in Siberia, in Russ.), 2012, No. 4.

10. QUALITY CONTROL

AO Vector-Best's ISO 13485 certified quality management system requires quality control to be performed on each batch produced to ensure consistent product quality.

The shelf life of the kit is 12 months from the date of manufacture.

11. STORAGE AND DELIVERY

1. Store the set in the manufacturer's packaging (2-8) at 2-8°C during the entire shelf life.
2. The kit must be transported at (2-8)°C. Transport at a temperature of no more than 26°C is acceptable for a maximum of 10 days.
3. The manufacturer is responsible for the non-compliant properties of the product, with the exception of those defects that arose as a result of violation of the instructions for use, transport and storage conditions, the activities of third parties or force majeure.
4. The safety and quality of the products are guaranteed during the entire shelf life.
5. The manufacturer is obliged to replace the product at his own expense if the technical and functional characteristics of the product do not comply with the normative and technical documentation, and these disadvantages are caused by a hidden material defect or manufacturing defect.
6. The manufacturer hereby guarantees that the manufactured products meet the requirements of the normative and technical documentation.

Version: e08.09.23