RU2602366C2 - Method of producing dna primers and probes for minimally invasive prenatal pcr diagnosis of trisomy of 21st chromosome of fetus in blood of pregnant woman and diagnostic kit therefor - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present group of inventions relates to medicine. Disclosed is a method of obtaining DNA primers and probes for minimally invasive prenatal PCR diagnosis of trisomy of 21st chromosome in foetus in pregnant woman's blood, characterised by that it comprises selecting a site of differential methylation of foetal DNA 21st chromosome and DNA 21st chromosome of adult human, sensitive to endonucleases, synthesing direct and reverse primer corresponding to amplicon of length from 60 to 300 bps, as well as a probe which corresponds to said amplicon, performing real-time PCR of mixture of samples after restriction endonuclease processing thereof, selecting pair of primers and probes, providing efficiency of real-time PCR reaction higher than 90 % and linearity at variation of relative sample concentration higher than 90 %. Invention also discloses a kit and a minimally invasive method of prenatal PCR diagnosis in real time of trisomy of 21st chromosome in foetus in pregnant woman's blood.

EFFECT: present group of inventions provides effective agents and methods of determining trisomy of chromosome 21 of foetus in pregnant woman's blood.

16 cl, 15 dwg, 3 tbl, 1 ex

Description

FIELD OF THE INVENTION

The invention relates to medicine, in particular to molecular biological studies in the field of determination of aneuploidy, and can be used to determine the trisomy of the 21st chromosome of the fetus by the blood of a pregnant woman.

BACKGROUND

Chromosomal pathology is the cause of many congenital diseases, among which the most common is Down syndrome, caused by complete or partial trisomy on chromosome 21. The severity of the disease and the risk of specific clinical manifestations depends on many obscure factors, but even in mild forms, Down syndrome causes disability from the earliest the age of the child.

The use of invasive methods of prenatal diagnosis (transabdominal chorionbiopsy or placentobiopsy, transcervical chorionbiopsy or placentobiopsy, amniocentesis or cordocentesis) is most effective, since their results make it possible to accurately determine if the fetus has a hereditary pathology. However, despite the obvious advantages of early prenatal diagnosis, there are significant disadvantages associated, above all, with the occurrence of a threat of miscarriage. It was shown that uterine bleeding occurs in 10–12% of women, uterine tone increases in 3%, and spontaneous abortion occurs in 1.5–2% of cases. In many cases, if there is a violation of the technology for taking fetal material, the fetus and mother can become infected.

In recent years, non-invasive non-genetic methods of prenatal diagnosis of various hereditary pathologies have been intensively developed. The risk of complications during their implementation is significantly lower than when using invasive methods. Most often, non-invasive methods that do not require the extraction of fetal material are used to diagnose congenital malformations and chromosomal syndromes in the fetus. The accuracy of these methods, with their different combinations and at different stages of pregnancy, is 80–90%, while 10% of the results are false positive.

A number of approaches for the detection of trisomy based on the technology of genome-wide sequencing have been proposed, but they are characterized by high complexity and cost of research.

Thus, there is still a need for a method for diagnosing chromosomal syndromes that would be free from the aforementioned disadvantages of invasive methods and at the same time be superior to existing non-invasive and minimally invasive methods in terms of reliability of diagnostic results.

SUMMARY OF THE INVENTION

The following abbreviations are used in this text:

BSA is bovine serum albumin.

DNA is deoxyribonucleic acid.

PCR - polymerase chain reaction.

FAM / BHQ-1 is one of the common pairs of fluorescent dyes / fluorescence quenchers. Other pairs of fluorescent dyes / fluorescence quenchers, for example, FAM / Dabcyl, HEX / BHQ1, HEX / BHQ2, R6G / BHQ2, ROX / BHQ2, Cy5 / BHQ2, Cy5 / BHQ3, Cy5.5 / BHQ3, can be used in the present invention. , FAM / (BHQ2-dT) int., R6G / (BHQ2-dT) int.

EDTA - sodium ethylene diamine tetraacetate.

The objective of the present invention is to provide a method for determining the presence of trisomy of chromosome 21, which allows detecting DNA in human plasma at minimum concentrations, and a diagnostic kit for its implementation, which allows determining trisomy of chromosome 21 of the fetus by the blood of a pregnant woman.

The combined presence of the DNA of the expectant mother and the fetus in the maternal plasma suggests the development of DNA diagnostics, based largely on the use of genetic markers that would distinguish the DNA of the fetus and mother.

Most cell-based aneuploidy detection methods cannot be used, since nucleic acids circulate in the maternal plasma in extracellular form, therefore, the present invention proposes to use differences in methylation of different regions of the 21st chromosome, which allows differentiating fetal DNA and DNA mother and conduct a direct measurement of the number of copies of chromosome 21 in the fetus.

The technical result is the creation of a method for determining the presence of trisomy of chromosome 21, which allows detecting DNA in human plasma at minimum concentrations, and a diagnostic kit for its implementation, which allows determining trisomy of chromosome 21 of the fetus by the blood of a pregnant woman.

The above result was achieved due to the fact that the proposed method for producing DNA primers and probes for minimally invasive prenatal PCR diagnosis of trisomy of the 21st chromosome in the fetus by the blood of a pregnant woman is used:

(a) site-specific restriction endonuclease (s) sensitive to methylation,

(b) a sample of human hypermethylated or fetal DNA,

(c) a sample of hypomethylated or adult human DNA, characterized in that it comprises the following steps:

(1) selecting a site for differential methylation of fetal DNA of the 21st chromosome and DNA of the 21st chromosome of an adult that is sensitive to said endonuclease (s),

(2) synthesizing the forward and reverse primers corresponding to an amplicon from 60 to 300 pairs of nucleotides in length containing the differential methylation site (s) of the 21st chromosome, as well as a probe corresponding to this amplicon,

(3) carry out the polymerase chain reaction of a mixture of samples (b) and (c) after their treatment with restriction endonuclease (a), at least at three different concentration ratios of these samples,

(4) pairs of primers and probes are selected that provide a PCR reaction efficiency above 90% and linearity when the relative concentration of samples (b) and (c) changes above 90%.

Also, in another aspect, the present invention relates to a kit for minimally invasive prenatal PCR diagnosis of trisomy of the 21st chromosome in the fetus by the blood of pregnant women, containing at least one pair consisting of forward and reverse primers obtained in accordance with the above method, and at least one probe corresponding to them.

In developing methods for detecting the number of DNA copies using destructible oligonucleotide probes, the main problem is non-specific annealing of the probes, which can lead to significant systematic measurement errors. To eliminate this phenomenon, it is necessary to choose the most specific combinations of primers and probes and carefully optimize the concentration of oligonucleotides in the mixture. Also, limitations on the length of amplicons for fetal DNA, known from the literature, should be taken into account, since the length of its fragments in the bloodstream of the mother does not exceed 300 bp. With this in mind, the design of primers and probes was carried out for the selected regions, which made it possible to increase the specificity in silico and obtain the shortest amplicons.

Thus, preferably, when the kit includes primers, the amplicon size of which is an integer in the range of 60 to 300 nucleotides, preferably an integer in the range of 71 to 105 nucleotides.

Most preferred for inclusion in the kit are the following pairs of primers:

SEQ ID NO: 41 and SEQ ID NO: 42, and / or homologous to them, at least 95% of a pair of primers, and / or

SEQ ID NO: 71 and SEQ ID NO: 72, and / or homologous to them, at least 95% of a pair of primers, and / or

SEQ ID NO: 101 and SEQ ID NO: 102, and / or homologous to them, at least 95% of the pair of primers.

In one of the most preferred forms of execution, the above kit further includes the DNA of an individual with trisomy of the 21st chromosome for positive control. Positive control (s), allow (s) to control the quality of DNA extraction and reduce the likelihood of false negative results.

In yet another embodiment, the above kit further includes a thermostable DNA polymerase of 10 units / μl.

In another preferred embodiment, the above kit further includes an aqueous solution containing:

approximately 67 mm Tris-HCl, pH 8.8 at 25 ° C,

approximately 16.6 mM (NH4) 2S04,

approximately 6.7 mM MgCl2,

approximately 6.7 μm EDTA,

approximately 170 mcg BSA, and

a mixture of four basic dNTPs at a concentration of approximately 0.2 mM each.

In yet another preferred embodiment, the above kit further includes ampouled water.

In another aspect, the present invention relates to a minimally invasive method for prenatal PCR diagnosis of trisomy of the 21st chromosome in the fetus by the blood of a pregnant woman, in which they use:

(a) a blood sample from a pregnant woman;

(b) the above primers and probes;

(c) site-specific restriction endonuclease (s) sensitive to methylation, comprising the following steps:

(1) DNA is isolated from sample (a);

(2) treating the DNA isolated in step (1) with endonucleases (c);

(3) conduct a quantitative polymerase chain reaction of the reaction products obtained in stage (2) consisting of initial melting and a series of melting, annealing and synthesis cycles;

(4) determine the amount of PCR products by fluorescence spectrometry in the presence of probes (b).

In one of the preferred forms of implementation of the above diagnostic method, it is additionally used:

(d) a pair of primers and a probe to the hypermethylated fragment of the reference chromosome, which are obtained in accordance with the above method, while

(4) the ratio of signals from probes (b) and (d) is used as a diagnostic indicator.

Preferably, when the 13th chromosome is used as the reference chromosome.

It is especially preferred that a pair of primers and a probe having the sequences of SEQ ID NO: 131, SEQ ID NO: 132 and SEQ ID NO: 133, and / or at least homologous to them, be used as a pair of primers and probe (g); 95% sequence.

In a preferred embodiment of the method, the site-specific methylation sensitive restriction endonuclease is HpaII and / or BstFNI.

In another preferred embodiment of the method, during the PCR step

carry out initial melting for 2 min at a temperature of 95 ° C,

50–60 times a cycle is carried out, including melting at 94–96 ° C for 10–30 s, annealing and synthesis at 62–66 ° C for 30–60 min,

PCR products are detected during the annealing step.

In another preferred embodiment of the method, cleavage is carried out with 100 units of endonuclease for 16 hours.

In a particularly preferred embodiment of the method, the concentration of primers in the reaction mixture is 500 nM, the probes are 250 nM, and the annealing temperature is 60 ° C.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The results of experimental verification of different concentrations of the set of primers and probes for the ME1 region, along the X and Y axes are the values of the primer concentration, along the оси axis are the PCR efficiency values, in this example, the probe concentration used is 200 nM.

FIG. 2. Schedule of fluorescence accumulation for the ME1 region. DNA controls in the reaction mixture with the number of copies: 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10 ¹ .

FIG. 3. An example of the efficiency (E = 74%) of amplification for the ME1 region using real-time PCR. DNA controls in the reaction mixture with the number of copies: 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10 ¹ .

In FIG. Figures 4–15 present the results of an experimental verification of the methylation ratio for the ME1 – ME12 region, respectively.

DETAILED DESCRIPTION OF THE INVENTION

To assess the characteristics of the method of fluorescence detection, the concentration of primers and probes is varied in the range of 100-4000 nM in increments of 100 nM for primers and 50 ÷ 500 nM in increments of 50 nM for probes.

Evaluation of the amplification efficiency of various combinations of primer and probe concentrations is carried out with dilutions of DNA controls in the reaction mixture with the number of copies: 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² , 10 ¹ .

Samples of genomic DNA of Jurkat cell lines were used, unmethylated and methylated for all CpG islands (manufactured by Thermo Scientific), USA).

DNA concentration was measured on a Nano Vue spectrophotometer (HealthCare BioSciences AB, Sweden).

Endonuclease cleavage of maternal DNA is carried out with restriction endonucleases HpaII and BstFNI (SibEnzyme LLC, 100 units of endonuclease for 16 hours in the buffer recommended by the manufacturer).

Amplification is carried out on an ABI StepOnePlus thermal cycler (Applied Biosystems) in 20 μl of a reaction mixture of the following composition:

70 mm Tris-HCl, pH 8.8,

16.6 mm ammonium sulfate,

0.01% Tween-20,

2.0 mm magnesium chloride,

200 nM each dNTP, 100 ÷ 1000 nM primers, 50 ÷ 500 nM probes,

1.0 units of Taq DNA polymerase.

Amplification conditions for a DNA fragment: 95 ° C / 2 min — first cycle; 94 ° C / 10 s, 60 ° C / 90 s - 40 cycles.

The recorded accumulation of the fluorescence signal indicated that the sample contained DNA containing a hypermethylated region.

In FIG. Figure 1 shows the results of an experimental verification of different concentrations of a set of primer and probe pairs for the ME1 region, along the X and Y axes, the concentration of primer pairs, along the оси axis, the PCR efficiency values, in this example, the probe concentration used is 200 nM.

An example of the results of experimental verification of the characteristics of various sets of primers and probes is shown in Table 2 and in FIG. 1, graphs of fluorescence accumulation and calculation of PCR efficiency for the ME1 region are presented in figures 2, 3, 4 and 5.

Similar experiments are carried out for all regions and all variants of the concentration of primers and probes.

It was established experimentally for all variants of primer and probe concentrations for all kits that the optimal concentration of primers in the reaction mixture was 500 nM, probes 250 nM, and annealing temperature 60 ° C.

Based on the obtained values of the PCR parameters, we can conclude that all sets of primers and probes are suitable for analysis and demonstrate high efficiency values and linearity values close to unity R2, which allows using these sets of oligonucleotides to measure the number of copies of chromosome 21 (see table 3).

Efficiency and linearity are PCR parameters obtained from a series of dilutions of concentrations of 10 times. These parameters are determined as follows. Build a graph of the dependence (for example, as shown in Fig. 3) of the threshold cycle from the logarithm of concentration; carry out an approximating straight line (obtained, for example, by the least squares method); from the equation of the straight line y = ax + b calculate the efficiency equal to E = 10 ^{(-1 / a)} , while R ² is the correlation coefficient showing how accurately the experimental points are close to the approximating straight line, the value of the approximation confidence (for example, the average square of the deviation )

To assess the characteristics of the selected differentially methylated regions, we use the Wilcoxon-Mann-Whitney test, the calculation was based on the methylation degree parameter calculated by the following formula:

CM = (Mean normalized Ct of duplicate samples) / (Average normalized Ct of normal controls)

The value of SM≤1 was considered as the absence of trisomy.

The value of SM> 1 was considered as the presence of trisomy.

The average normalized Ct is calculated as the average of duplicates of the settings of one sample or control:

Norm Ct sample = E ^BXCT-mCt ,

Where:

BxCt is the threshold cycle of the sample before treatment with restriction endonucleases;

mCt is the threshold cycle of the sample after treatment with restriction endonucleases;

Ε - PCR reaction efficiency for a given pair of primers.

In figures 4-15 presents the values of SM for pairs of norm-trisomy (10 pairs) with minimum and maximum values, the upper and lower quartiles and average.

Based on the obtained values, it can be concluded that the following differential methylated regions are optimally included in the composition of the test system: ME4, ME7, ME10.

Using discriminant analysis and based on the values of the Wilks lambda statistics, the coefficients of the equation for discrimination were selected:

D = -3.461 + 0.842 X _ME4 +0.428 X _ME7 +0.845 Χ _ΜΕ10 ,

where D is the coefficient of discrimination,

for D> 0, the case is considered as trisomy,

when D≤0, the case is considered as the norm;

Χ is the relative content in the sample region of chromosome 21 normalized to the hypermethylated region for comparison of chromosome 13 according to the "delta-delta Ct" method, for example:

X _ME4 = E _ME4 ^{- (ME4Ct sample-ME4Ct control)} / E _HP1 ^{- (HP1Ct sample-HP1Ct control)} )

Claims (16)

1. A method of obtaining DNA primers and probes for minimally invasive prenatal PCR diagnosis of trisomy of the 21st chromosome in the fetus by the blood of a pregnant woman, which use:
(a) site-specific restriction endonuclease (s) sensitive to methylation,
(b) a sample of human hypermethylated or fetal DNA,
(c) a sample of hypomethylated or adult human DNA, characterized in that it comprises the following steps:
(1) selecting a site for differential methylation of fetal DNA of the 21st chromosome and DNA of the 21st chromosome of an adult that is sensitive to said endonuclease (s),
(2) synthesizing the forward and reverse primers corresponding to an amplicon from 60 to 300 pairs of nucleotides in length containing the differential methylation site (s) of the 21st chromosome, as well as a probe corresponding to this amplicon,
(3) carry out a real-time polymerase chain reaction of a mixture of samples (b) and (c) after they are treated with a restriction endonuclease (a) at least at three different concentration ratios of these samples,
(4) pairs of primers and probes are selected that provide real-time PCR reaction efficiency above 90% and linearity when the relative concentration of samples (b) and (c) changes above 90%. 2. A kit for minimally invasive prenatal PCR diagnostics in real time of the trisomy of the 21st chromosome in the fetus by the blood of pregnant women, containing at least one pair consisting of direct and reverse primers obtained in accordance with the method of claim 1, and at least one probe corresponding to them. 3. The kit according to claim 2, characterized in that the amplicon size is an integer in the range from 60 to 300 pairs of nucleotides, preferably an integer in the range from 71 to 105 pairs of nucleotides. 4. The kit according to claim 2, characterized in that it includes
a pair of primers SEQ ID NO: 41 and SEQ ID NO: 42, and / or at least 95% homologous to them by a pair of primers, and / or
a pair of primers SEQ ID NO: 71 and SEQ ID NO: 72, and / or at least 95% homologous to them by a pair of primers, and / or
a pair of primers SEQ ID NO: 101 and SEQ ID NO: 102, and / or at least 95% homologous to them by a pair of primers. 5. The kit according to claim 2, characterized in that it further includes an individual’s DNA with trisomy of the 21st chromosome for positive control. 6. The kit according to claim 2, characterized in that it further includes a thermostable DNA polymerase of 10 units / μl. 7. The kit according to claim 2, characterized in that it further includes an aqueous solution containing:
approximately 67 mm Tris-HCl, pH 8.8 at 25 ° C,
approximately 16.6 mM (NH ₄ ) ₂ SO ₄
approximately 6.7 mm MgCl ₂ ,
approximately 6.7 μm EDTA,
approximately 170 mcg BSA, and
a mixture of four basic dNTPs at a concentration of approximately 0.2 mM each. 8. The kit according to claim 2, characterized in that it further includes ampouled water. 9. A minimally invasive method of real-time prenatal PCR diagnosis of trisomy of the 21st chromosome in the fetus by the blood of a pregnant woman, which uses:
(a) a blood sample from a pregnant woman;
(b) primers and probes according to any one of paragraphs. 1 or 4;
(c) site-specific restriction endonuclease (s) sensitive to methylation,
characterized in that it provides for the following stages:
(1) DNA is isolated from sample (a);
(2) treating the DNA isolated in step (1) with endonucleases (c);
(3) conduct a quantitative polymerase chain reaction in real time of the reaction products obtained in stage (2) consisting of initial melting and a series of melting, annealing and synthesis cycles;
(4) determine the amount of PCR products by fluorescence spectrometry in the presence of probes (b). 10. The method according to p. 9, characterized in that it additionally uses:
(d) a pair of primers and a probe for a hypermethylated fragment of the reference chromosome obtained analogously to the method of claim 1, wherein
(4) the ratio of signals from probes (b) and (d) is used as a diagnostic indicator. 11. The method according to p. 10, characterized in that the reference chromosome is the 13th chromosome. 12. The method according to p. 11, characterized in that the pair of primers and probe (g) have the sequence SEQ ID NO: 131, SEQ ID NO: 132 and SEQ ID NO: 133, and / or at least 95% homologous to them sequence. 13. The method according to p. 9, characterized in that the site-specific restriction endonuclease, sensitive to methylation, is HpaII and / or BstFNI. 14. The method according to p. 9, characterized in that during the PCR stage, the initial melting is carried out for 2 minutes at a temperature of 95 ° C,
50-60 times spend a cycle including melting at 94-96 ° C for 10-30 s, annealing and synthesis at a temperature of 62-66 ° C for 30-60 minutes,
PCR products are detected during the annealing step. 15. The method according to p. 9, characterized in that in it, the cleavage is carried out with 100 units of endonuclease for 16 hours. 16. The method according to p. 9, characterized in that in it the concentration of primers in the reaction mixture is 500 nM, the probes are 250 nM, and the annealing temperature is 60 ° C.

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