RU2600442C1 - Method for determining risk of developing of arterial hypertension - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and aims at determining the risk of developing of arterial hypertension. Venous blood is sampled, recovering the genetic material, carrying out a polymerase chain reaction (PCR) in real time and determining of insertion/deletion (I- and D-alleles) of Alu-fragment of gene of angiotensin converting enzyme. Risk of developing of arterial hypertension in operating conditions of increasing of noise level higher than maximum allowable at genotype of I/I gene of angiotensin converting enzyme is estimated below 50 %, if the genotype I/D - 50-70 %, if the genotype D/D - higher than 70 %.

EFFECT: invention provides increasing of determination speed of risk of developing of arterial hypertension at persons who exposed to noise, and simplifying of the method with simultaneous increase of degree of its reliability.

1 cl, 3 dwg, 3 ex

Description

The invention relates to medicine and can be used to determine the risk of developing hypertension.

A known method for determining the risk of developing arterial hypertension, including venous blood sampling, isolation of genetic material, polymerase chain reaction (PCR) with specific primers, analysis of amplification products by agarose gel electrophoresis, and based on this, the insertion / deletion of the Alu fragment of the angiotensin converting gene enzyme (see Goncharova L.N., Snegovsky V.A., Kuzovenkova O.N. “Insertion-deletion polymorphism of the angiotensin-converting enzyme gene in individuals with essential arterial hypertension ”// Kazan Medical Journal. - 2009. - No. 4 (volume 90). - P. 564-566).

However, this method due to the use of PCR with electrophoretic detection of amplification products has a low throughput due to complexity, complexity and duration (after amplification, this method has an additional electrophoresis step). Also, this method requires the use of reagents that are harmful to the medical personnel conducting them, and requires higher costs for mass research. In addition, the known method has low reliability due to the high risk of contamination of the amplification mixture and amplification products. All of the above makes it difficult to determine the risk of developing arterial hypertension in people exposed to noise, for example, during medical examinations or periodic medical examinations of a large number of people working in conditions of exceeding the noise level above the maximum permissible for short (short) time periods.

The objective of the present invention is to simplify the method for determining the risk of developing arterial hypertension in people exposed to noise, and to increase its reliability during preliminary or periodic medical examinations of a large number of people working in conditions of exceeding the noise level above the maximum permissible for short (short) periods of time.

In the present invention, the real-time PCR method is used, which has several advantages over the PCR method with electrophoretic detection of amplification products, namely, simplifying and increasing the speed of obtaining results due to the exclusion of an additional stage of electrophoresis, increasing the degree of reliability of the results in connection with process automation , eliminating the risk of contamination of the amplification mixture and amplification products, this method does not require the use of reagents that are harmful to honey Iqing personnel conducting them.

The technical result of the invention is to simplify the method and increase the rate of determining the risk of developing hypertension in persons exposed to noise, while increasing its reliability.

This task is achieved by the fact that in the known method for determining the risk of developing hypertension, including venous blood sampling, isolation of genetic material, polymerase chain reaction (PCR) with specific primers, analysis of amplification products by real-time fluorescence detection, and based on this determination of the insertion / deletion of the Alu fragment of the angiotensin converting enzyme gene; specific forward and reverse steps are used in the preparation of the amplification mixture insertion / deletion measures and two TaqMan-type probes on the I- and D-alleles, the direct primer being gga gac cac tec cat cct ttc, 14045-14067 according to AF118569, 13351-13372 according to EU 332840, the reverse primer is atg tgg cca tca cat tcg tca gat, 14499-14522 according to AF 118569, 13515-13537 according to EU 332840, probe on I-allele - (5′TAM) cca cag gcg ccc gec act ac (3′BHQ1), 14235-14252 according to AF 118569 with the FAM fluorophore, with the probe on the I-allele located inside the insert (14094-14381 according to AF 118569), the probe on the D-allele - (5′HEX) get gec tat asa gtc act ttt atg tgg t (3′BHQ1) , 13391-13417 according to EU 332840 with HEX fluorophore, after preparing the amplification mixture, the polymerase chain reaction is carried out in real time with fluorescence detection of accumulation of amplification products and the intensity of the fluorescence signals obtained by PCR, the genotype of the angiotensin converting enzyme gene is determined: homozygote for the I-allele (genotype I / I), heterozygous (genotype I / D) or homozygote for the D-allele (genotype D / D), while the risk of developing arterial hypertension in workers with noise levels exceeding the maximum permissible for the I / I genotype of the angiotensin converting enzyme gene is below 50%, for the I / D genotype - 50-70%, and for the D / D genotype - above 70%.

Studies on patent and scientific and technical information sources have shown that the proposed method is unknown and does not follow explicitly from the studied prior art, i.e. meets the criteria of "novelty" and "inventive step".

The proposed method can be used during preliminary and periodic medical examinations of industrial workers in medical units and diagnostic laboratories, equipped with standard equipment manufactured by domestic or foreign industry. Therefore, the claimed method is affordable and practically applicable.

Arterial hypertension is a multifactorial disease, where a complex mechanism of phenotype formation is revealed, which is accompanied by the interaction of genetic factors with environmental factors. Chronic exposure to noise can contribute to the development of hypertension by activating the hypothalamic-pituitary-adrenal and sympathetic nervous systems. Another system for regulating blood pressure is the renin-angiotensin-aldosterone system, the key enzyme of which is the angiotensin-converting enzyme, the level and activity of which is associated with the insertion / deletion of the Alu fragment of the angiotensin-converting enzyme gene. In this connection, the chronic effect of noise, together with the deletion of the Alu fragment of the angiotensin-converting enzyme gene, can significantly increase the risk of developing hypertension due to the simultaneous activation of various systems involved in the development of hypertension.

The proposed method is illustrated in the drawing.

In FIG. 1 shows an example of a graph of DNA accumulation obtained using a CFX 96 amplifier (Bio Rad) in a PCR reaction according to the proposed method, corresponding to a 1-allele homozygous (genotype 1L) gene of the angiotensin converting enzyme.

In FIG. 2 shows an example of a graph of DNA accumulation obtained using a CFX 96 amplifier (Bio Rad) in a PCR reaction according to the proposed method, corresponding to the heterozygote (genotype I / D) of the angiotensin converting enzyme gene.

In FIG. Figure 3 shows an example of a graph of DNA accumulation obtained using a CFX 96 amplifier (Bio Rad) in a PCR reaction according to the proposed method, corresponding to the homozygous D-allele (D / D genotype) of the angiotensin converting enzyme gene.

The proposed method is as follows.

Venous blood sampling is performed in patients. Blood is examined in the claimed manner: from 100 μl of whole blood, the sorbent DNA is isolated by a single-tube method using the DNA-sorb-B isolation kit (TU9398-071-01897593-2008) in accordance with the instructions for the kit used.

A series of dilutions in a TE buffer to a final concentration of (1 ÷ 3) ng / μl is prepared from the obtained DNA preparation (with a volume of 50 μl). This DNA solution is used to set up an amplification reaction with specific primers.

An amplification mixture is then prepared to detect the insertion / deletion of the Alu fragment of the angiotensin converting enzyme gene. The volume of the amplification mixture is 20 μl, using a standard buffer for the polymerase chain reaction containing 1 unit of Taq polymerase activity. To amplify the studied genetic locus, specific oligonucleotide primers with probes of the TaqMan type are used on the I- and D-alleles of the angiotensin-converting enzyme gene.

The amplification reaction is carried out according to the following program:

According to the proposed method, the tubes with the amplification mixture are kept at + 95 ° C for 5 minutes, then 40 cycles: at 95 ° C - 20 s, at 64 ° C - 30 s. Amplification products are detected via FAM and HEX channels. In this case, for example, a CFX 96 amplifier (Bio Rad) is used. Amplification products are detected on each amplification cycle. Based on these data, the amplifier constructs the fluorescence signal accumulation curves according to the signal specified for the samples.

The results of PCR according to the proposed method are presented in FIG. 1 ÷ 3.

Moreover, with the genotype I / I of the angiotensin-converting enzyme gene for workers in conditions of exceeding the noise level above the maximum permissible risk of hypertension is less than 50%, with the genotype I / D of the angiotensin-converting enzyme gene for workers in conditions of exceeding the noise level is above the maximum permissible risk of developing arterial hypertension hypertension is 50-70%, with the D / D genotype of the angiotensin-converting enzyme gene in people working in conditions of exceeding the noise level above the maximum permissible risk of developing arterial th hypertension is higher than 70%.

Example 1

Patient P., 56 years old. Profession - aircraft commander, flight experience - 35 years, total flight time - 15 108 hours 28 minutes. He worked under conditions of intense noise, monthly equivalent noise levels exceeded the remote control from 1.75 to 31.02 dBA (see SN 2.2.4 / 2.1.8.562-96 “Noise at workplaces, indoor, residential, public buildings and residential areas "). Periodic medical examinations were carried out annually; the development of arterial hypertension was not revealed in P.'s employee.

The patient’s blood was tested by the claimed method: sorbent DNA was isolated from 100 μl of whole blood using a single-tube method using the DNA-sorb-B isolation kit (TU9398-071-01897593-2008), in accordance with the instructions for the kit used.

A series of dilutions in TE buffer was prepared from the obtained DNA preparation (50 μl) to a final concentration of (1 ÷ 3) ng / μl. This DNA solution was used to set up an amplification reaction with specific primers.

An amplification mixture was then prepared to detect the insertion / deletion of the Alu fragment of the angiotensin converting enzyme gene. The volume of the amplification mixture was 20 μl, and a standard buffer for the polymerase chain reaction containing 1 unit of Taq polymerase activity was used. Specific oligonucleotide primers with probes of the TaqMan type on the I- and D-alleles of the angiotensin converting enzyme gene were used to amplify the studied genetic locus.

The amplification reaction was carried out according to the following program:

According to the proposed method, the tubes with the amplification mixture were kept at + 95 ° C for 5 minutes, then 40 cycles: at 95 ° C for 20 s, at 64 ° C for 30 s. We used a CFX 96 amplifier (Bio Rad), and amplification products were detected using the FAM and HEX channels. Amplification products were detected on each amplification cycle. Based on these data, the amplifier constructed the accumulation curves of the fluorescent signal from the signal specified for the samples. A plot of DNA accumulation with primers for determining the insertion / deletion of the Alu fragment of the angiotensin converting enzyme gene is shown in FIG. one.

Output:

Patient P. revealed a homozygous I / I genotype of the angiotensin-converting enzyme gene, indicating a less than 50% risk of developing hypertension under conditions of exposure to noise levels above the maximum permissible level. This is confirmed by the fact that after 35 years of work, the employee was diagnosed with arterial hypertension under conditions of exposure to noise levels above the maximum permissible level.

Example 2

Patient Sh., 41 years old. Profession: molder of manual (and machine) molding of a foundry site, experience - 17 years. He worked under the conditions of intense noise during an 8-hour working day, exceeding the remote control from 10.15 to 22.3 dBA (see SN 2.2.4 / 2.1.8.562-96 “Noise at workplaces, indoor, residential, public buildings and residential areas "). Periodic medical examinations were conducted annually, grade 2 arterial hypertension was diagnosed after 17 years of work. He is constantly taking antihypertensive drugs.

The patient’s blood was tested by the claimed method: sorbent DNA was isolated from 100 μl of whole blood using a single-tube method using the DNA-sorb-B isolation kit (TU9398-071-01897593-2008), in accordance with the instructions for the kit used.

A series of dilutions in TE buffer was prepared from the obtained DNA preparation (50 μl) to a final concentration of (1 ÷ 3) ng / μl. This DNA solution was used to set up an amplification reaction with specific primers.

An amplification mixture was then prepared to detect the insertion / deletion of the Alu fragment of the angiotensin converting enzyme gene. The volume of the amplification mixture was 20 μl, and a standard buffer for the polymerase chain reaction containing 1 unit of Taq polymerase activity was used. Specific oligonucleotide primers with probes of the TaqMan type on the I- and D-alleles of the angiotensin converting enzyme gene were used to amplify the studied genetic locus.

The amplification reaction was carried out according to the following program:

According to the proposed method, the tubes with the amplification mixture were kept at + 95 ° C for 5 minutes, then 40 cycles: at 95 ° C for 20 s, at 64 ° C for 30 s. We used a CFX 96 amplifier (Bio Rad), and amplification products were detected using the FAM and HEX channels. Amplification products were detected on each amplification cycle. Based on these data, the amplifier constructed the accumulation curves of the fluorescent signal from the signal specified for the samples. A plot of DNA accumulation with primers for determining the insertion / deletion of the Alu fragment of the angiotensin converting enzyme gene is shown in FIG. 2.

Output:

Patient S. revealed a heterozygous I / D genotype of the angiotensin-converting enzyme gene, indicating a 50-70% risk of developing hypertension under conditions of exposure to noise levels above the maximum permissible level. This is confirmed by the fact that after 17 years of work, this employee was diagnosed with arterial hypertension of the 2nd degree under conditions of exposure to noise levels above the maximum permissible level.

Example 3

Patient I., 38 years old. Profession - co-pilot of an aircraft, flight experience - 7 years, total flight time - 7765 hours 40 minutes. He worked under conditions of intense noise in excess of the remote control during the entire period of flight operation from 1.15 to 16.49 dBA (see SN 2.2.4 / 2.1.8.562-96 “Noise at workplaces, in rooms, residential, public buildings and in residential areas "). Periodic medical examinations were conducted annually, grade 2 arterial hypertension was diagnosed after 7 years of work. He is constantly taking antihypertensive drugs, but the level of blood pressure is poorly controlled.

The patient’s blood was tested by the claimed method: sorbent DNA was isolated from 100 μl of whole blood using a single-tube method using the DNA-sorb-B isolation kit (TU9398-071-01897593-2008), in accordance with the instructions for the kit used.

A series of dilutions in TE buffer was prepared from the obtained DNA preparation (50 μl) to a final concentration of (1 ÷ 3) ng / μl. This DNA solution was used to set up an amplification reaction with specific primers.

An amplification mixture was then prepared to detect the insertion / deletion of the Alu fragment of the angiotensin converting enzyme gene. The volume of the amplification mixture was 20 μl, and a standard buffer for the polymerase chain reaction containing 1 unit of Taq polymerase activity was used. Specific oligonucleotide primers with probes of the TaqMan type on the I- and D-alleles of the angiotensin converting enzyme gene were used to amplify the studied genetic locus.

The amplification reaction was carried out according to the following program: According to the proposed method, the tubes with the amplification mixture were kept at + 95 ° C for 5 minutes, then 40 cycles: at 95 ° C for 20 s, at 64 ° C for 30 s. We used a CFX 96 amplifier (Bio Rad), and amplification products were detected using the FAM and HEX channels. Amplification products were detected on each amplification cycle. Based on these data, the amplifier constructed the accumulation curves of the fluorescent signal from the signal specified for the samples. A plot of DNA accumulation with primers for determining the insertion / deletion of the Alu fragment of the angiotensin converting enzyme gene is shown in FIG. 3.

Output:

Patient I. revealed a homozygous D / D genotype of the angiotensin-converting enzyme gene, indicating a more than 70% risk of developing hypertension under conditions of exposure to noise levels above the maximum permissible level. This is confirmed by the fact that after 7 years of work, this employee was diagnosed with arterial hypertension of the 2nd degree under conditions of exposure to noise levels above the maximum permissible level.

Thus, the proposed method provides an increase in the speed and reproducibility of determining the risk of developing arterial hypertension during preliminary or periodic medical examinations of a large number of people working in conditions when the noise level is above the maximum permissible level. It also allows you to simplify the process of determining the risk of developing hypertension in people exposed to noise, to increase its reliability.

Using the proposed method will allow to develop preventive measures to prevent the development of arterial hypertension, taking into account the individual characteristics of the body, to timely assess the value of various environmental factors on the health of an individual. It can be widely used in occupational medicine, therapy, cardiology and human ecology.

Claims (1)

A method for determining the risk of developing arterial hypertension, including venous blood sampling, isolation of genetic material, polymerase chain reaction (PCR) with specific primers, analysis of amplification products by real-time fluorescence detection, and based on this, the determination of the insertion / deletion of the Alu gene fragment angiotensin-converting enzyme, characterized in that in the preparation of the amplification mixture using specific forward and reverse primers on the sides of insertion / de two TaqMan probes on the I- and D-alleles, the forward primer being gga gac cac tcc cat cct ttc, 14045-14067 according to AF118569, 13351-13372 according to EU332840, the reverse primer is atg tgg cca tca cat tcg tca gat , 14499-14522 according to AF118569, 13515-13537 according to EU332840, probe for I-allele - (5′FAM) cca cag gcg ccc gcc act ac (3′BHQ1), 14235-14252 according to AF118569 with FAM fluorophore, while the probe The I-allele is located inside the insert (14094-14381 according to AF118569), the probe on the D-allele is (5′HEX) gct gcc tat asa gtc act ttt atg tgg t (3′BHQ1), 13391-13417 according to EU332840 with HEX fluorophore, after preparation of the amplification mixture, a polymerase chain reaction is carried out in real time with fluorescence detection of the amplification products and the intensity of the fluorescence signals obtained as a result of PCR determines the genotype of the angiotensin converting enzyme gene: homozygous for the I-allele (genotype I / I), heterozygous (for the I / D genotype) or homozygous for the D-allele (the D / D genotype), at the same time, the risk of developing arterial hypertension in workers with noise levels exceeding the maximum permissible for the I / I genotype of the angiotensin converting enzyme is below 50%, for the I / D genotype - 50-70%, and for the D / D genotype - above 70% .

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