CN106609299B - Kit for determining concentration of fetal free DNA in plasma of pregnant woman - Google Patents
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Abstract
The invention provides a kit for determining the concentration of fetal free DNA in the plasma of a pregnant woman, which comprises methylase for digesting the free DNA extracted from the plasma of the pregnant woman; a reagent for determining the concentration of free DNA extracted from the plasma of a pregnant woman; and a reagent for determining the concentration of free DNA extracted from the plasma of a pregnant woman after digestion with the methylase. When the kit provided by the invention is used for measuring the concentration of fetal free DNA in the plasma of the pregnant woman, the measured value and the actual value have good correlation. The primer group consists of an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, respectively.
Description
Technical Field
Specifically, the invention relates to a primer group for PCR amplification of human PDE9A gene, a kit containing the primer group and used for determining the fetal free DNA concentration in the plasma of pregnant women, and a PCR amplification method using the primer group.
Background
Since the phenomenon of the existence of fetal free DNA (cfDNA) in the plasma of pregnant women has been discovered, researchers have utilized this phenomenon to perform a variety of studies and applications, such as noninvasive prenatal DNA detection, fetal gender determination, fetal chromosomal abnormality detection, monogenetic genetic disease screening, pregnancy-related disease screening, and the like.
At present, methods for determining the concentration of fetal free DNA in the plasma of a pregnant woman by detecting SRY genes exist, but the SRY genes are unique genes of a male fetus, and the concentration of fetal free DNA cannot be detected for the pregnant woman pregnant with the female fetus. Later, scientists found that the genome of pregnant women and fetuses has great difference in methylation level through methylation sequencing and other means, so that some fetal-specific methylation genes are found, and the current methods for detecting the concentration of free DNA in fetuses are concentrated on detecting the methylation difference.
The PDE9A gene is located at 21q22.3 and is methylated in the peripheral blood of pregnant women and unmethylated in fetal tissues. Theoretically, fetal free dna (cffdna) in the plasma of pregnant women can be determined by a fetal-specific epigenetic marker, the unmethylated PDE9A gene (U-PDE 9A). U-PDE9A may also be used as a marker for non-invasive detection of trisomy 21. After digestion with methylationsensitive enzymes, the PDE9A gene from the fetus was digested and degraded, leaving only the PDE9A gene from the mother in the free DNA. By the method, the content of free DNA in the plasma of the pregnant woman can be effectively detected, the concentration of fetal free DNA in the plasma of the pregnant woman can be accurately detected, and guidance and theoretical basis are provided for the current noninvasive prenatal screening method.
However, as a cffDNA detection method having a clinical application value, it is necessary to ensure a good correlation (R) between a measured value and a true value2A value close to 1). Therefore, the theoretically possible methods described above are far from practical use.
Remarking: cffDNA, cell free total DNA, free fetal DNA; while cfDNA is cellfree DNA, free DNA.
Reference to the literature
[1]Presence of fetal DNA in maternal plasma and serum.Lancet1997.350:485-487.
[2]Cell-free fetal DNA plasma extraction and real-time polymerasechain reaction quantification.Methods Mol Med 2007.132:51-63.
[3]Quantification of Cell-free DNA in normal and complicatedpregnancies:overcoming biological and technical issues,PLoS One 2014.9(7):1-7.
[4]Cell-free Fetal DNA and Cell-Free Total DNA levels in spontaneousabortion with fetal chromosomal aneuploidy,PLoS One 2013.8:2.
[5]Non-invasive epigenetic detection of fetal trisomy 21in firsttrimester maternal plasma.PLoS One 2011.6(11):e27709.
[6]Non-invasive prenatal aneuploidy testing at chromosomes 13,18,21,X,and Y,using targeted sequencing of polymorphic loci.Prenat Diagn 2012.32(13):1233-1241.
Disclosure of Invention
In view of the problems of the prior art, the present invention aims to provide a kit for measuring the concentration of fetal free DNA in the plasma of a pregnant woman, which enables a good correlation between the measured value and the actual value of fetal free DNA.
The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that: the present invention has been accomplished by using a kit specifically for measuring the concentration of fetal free DNA in the plasma of a pregnant woman, which enables a good correlation between the measured value and the actual value of fetal free DNA obtained by the Q-PCR method.
Namely, the present invention comprises:
1. a primer group comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, respectively.
2. The primer set according to item 1, which is used for amplifying the human PDE9A gene.
3. The primer set according to item 2, wherein the human PDE9A gene is derived from maternal plasma free DNA.
4. A kit for amplifying a human PDE9A gene, comprising the primer set according to any one of items 1 to 3.
5. The kit according to item 4, further comprising a nucleotide sequence as set forth in SEQ ID NO: 3, and a standard for absolute quantification.
6. A method for amplifying a human PDE9A gene, which comprises performing PCR amplification using the primer set according to any one of items 1 to 3 as primers.
7. The method according to item 6, which uses free DNA extracted from plasma of pregnant women as a template.
8. The method according to item 6, which uses, as a template, free DNA extracted from plasma of a pregnant woman digested with methylase.
9. The method according to item 6, wherein the denaturation temperature of the PCR amplification is 93-98 ℃, preferably 94-96 ℃.
10. The method according to item 6, wherein the annealing temperature of the PCR amplification is 52-62 ℃, preferably 55-60 ℃.
11. The method of item 6, wherein the extension temperature of the PCR amplification is 72 ℃.
12. A kit for determining the fetal free DNA concentration in the plasma of a pregnant woman, comprising the primer set of claim 1.
13. The kit of claim 12, further comprising a nucleic acid sequence as set forth in SEQ ID NO: 3, and a standard for absolute quantification.
14. A kit for determining the fetal free DNA concentration in the plasma of a pregnant woman, comprising:
methylases for digesting free DNA extracted from the plasma of pregnant women.
15. The kit of item 14, further comprising:
a reagent for determining the concentration of free DNA extracted from the plasma of a pregnant woman; and
a reagent for determining the concentration of free DNA extracted from the plasma of pregnant women after digestion with the methylase.
16. The kit of item 14, further comprising the following primer sets:
the primer group consists of an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, respectively.
17. The kit of claim 14, further comprising a nucleic acid sequence as set forth in SEQ ID NO: 3, and a standard for absolute quantification.
18. The kit according to claim 14, wherein the methylase is one or more selected from BstUI, hhal and HpaII.
Effects of the invention
By using the kit for determining the concentration of fetal free DNA in the plasma of a pregnant woman of the present invention, it is possible to obtain a good correlation between the measured value and the actual value of fetal free DNA obtained by the Q-PCR method.
Drawings
FIG. 1 shows that there is a good correlation between the measured value and the actual value of fetal free DNA obtained by the Q-PCR method in the case of amplifying the human PDE9A gene using the primer set of the present invention.
FIG. 2 shows that the correlation between the measured value and the actual value of fetal free DNA obtained by the Q-PCR method was poor in the case of amplifying the human PDE9A gene using the primer set of the comparative example.
Detailed description of the invention
In one aspect, the present invention provides a primer set (primer set of the present invention) comprising an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is as set forth in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, respectively.
SEQ ID NO：1
5'-TCGGTGAGTGCGCGCTGC-3' (also known as PDE9A-FS)
SEQ ID NO：2
5'-CAGCCATCCCGAAAAGGCGA-3' (also known as PDE9A-RS)
The primer set can be used as a primer in PCR amplification against the human PDE9A gene. In this specification, the human PDE9A gene includes the complete human PDE9A gene and fragments thereof.
In another aspect, the present invention provides a kit for amplifying the human PDE9A gene (amplification kit of the present invention), which comprises the primer set of the present invention. The primer set of the present invention is necessary for the kit of the present invention, wherein the upstream primer and the downstream primer may be contained in different containers or may be contained in the same container. In addition to comprising the primer set of the invention, the kit of the invention may comprise other reagents, e.g., for PCR amplification, including but not limited to those selected from dNTPsS、MgSO4Taq enzyme, DMSO, Tween 20, betaine, fluorescent dye, PCR reaction buffer, standard for absolute quantitation, double distilled water (ddH)2O), etc., and can be appropriately selected by those skilled in the art as needed. In addition, the present invention also provides a kit for determining the fetal free DNA concentration in the plasma of a pregnant woman (the determination kit of the present invention), which comprises the primer set of the present invention or the amplification kit of the present invention. Here, as a standard for absolute quantification, it is preferable to use a standard whose nucleotide sequence is as shown in SEQ ID NO: 3, and a standard for absolute quantification. Preferably, the assay kit of the present invention may further comprise: a reagent for determining the concentration of free DNA extracted from the plasma of a pregnant woman; and/or reagents for determining the concentration of free DNA extracted from the plasma of pregnant women after digestion with the methylase. Here, the reagents for determining the concentration of free DNA extracted from plasma of pregnant women include, but are not limited to: dNTPS、MgSO4Taq enzyme, DMSO, Tween 20, betaine, fluorescent dye and PCR reaction buffer solution; the reagents for determining the concentration of free DNA extracted from the plasma of pregnant women after digestion with the methylase include, but are not limited to: dNTPS、MgSO4Taq enzyme, DMSO, Tween 20, betaine, fluorescent dye and PCR reaction buffer solution.
The primer set of the invention can be suitable for being used as a primer set for amplifying the human PDE9A gene in fluorescent real-time quantitative PCR. In the case of amplifying the human PDE9A gene using the primer set of the present invention, there can be a good correlation between the measured value and the actual value of fetal free DNA obtained by the Q-PCR method. Thus, in another aspect, the present invention provides a method for amplifying the human PDE9A gene (the amplification method of the present invention), which performs PCR amplification (e.g., fluorescent real-time quantitative PCR) using the primer set of the present invention as primers.
PCR (polymerase chain reaction) amplification is well known to those skilled in the art and is generally achieved by a certain PCR reaction procedure (temperature cycling). The PCR reaction procedure generally includes the steps of denaturation, annealing, extension, and the like.
For the amplification method of the present invention, the denaturation step may be performed at 93 to 98 deg.C (preferably 94 to 96 deg.C) for 0.1 to 10 minutes (preferably 0.2 to 2 minutes); the annealing step may be performed at 52 to 62 deg.C (preferably 55 to 60 deg.C) for 0.1 to 10 minutes (preferably 0.2 to 2 minutes); the extension step can be carried out at 72 ℃ for 0.1-10 minutes (preferably 0.5-5 minutes); the cycle consisting of the above denaturation, annealing and extension steps may be performed 10 to 50 times (preferably 25 to 45 times). Preferably, the template before being subjected to the above temperature cycle may be thermally pre-denatured at 93-98 deg.C (preferably 94-96 deg.C) for 1-60 minutes (preferably 5-20 minutes).
The amplification method of the present invention can be carried out using various samples as templates as long as the human PDE9A gene is contained in the samples. Preferably, the template may be free DNA extracted from plasma of pregnant women, or free DNA extracted from plasma of pregnant women digested with methylase. For example, in the case of determining the amount of fetal free DNA in the plasma of a pregnant woman by amplifying the human PDE9A gene based on fluorescent real-time quantitative PCR, the amount of fetal free DNA in the plasma of the pregnant woman can be determined by equally dividing the free DNA extracted from the plasma of the pregnant woman into two parts, one part being directly subjected to fluorescent real-time quantitative PCR (the amount of total free DNA in the plasma of the pregnant woman is determined), and the other part being subjected to methylase digestion and then to fluorescent real-time quantitative PCR (the amount of maternal free DNA in the plasma of the pregnant woman), and the difference between the two being the amount of fetal free DNA in the plasma of the pregnant.
Methods for extracting free DNA from the plasma of pregnant women are known to those skilled in the art. In addition, methods for methylase digestion of free DNA are also known to those skilled in the art, and methylases that can be used include, for example, BstUI, HhaI, HpaII, etc., but are not limited thereto.
Examples
The present invention will be described in further detail with reference to the following drawings and examples. It should be understood that the specific embodiments described herein are for the purpose of illustration and are not to be construed as limitations of the invention.
Example 1
The genome DNA of a pregnant woman (20 weeks of gestational age) and the genome DNA of a fetus in amniotic fluid (referred to as amniotic fluid DNA hereinafter) are respectively extracted, and after accurate quantification, the genome DNA is broken into fragments of about 200bp by an ultrasonic breaking instrument, so that the state of plasma free DNA is simulated.
The fragmented amniotic fluid DNA simulates fetal free DNA in plasma, the fragmented maternal genome DNA simulates maternal free DNA in plasma, the two are mixed according to a known ratio and mixed into samples with different fetal free DNA concentrations, then the primer group is adopted as an amplification primer of the human PDE9A gene, the fetal free DNA concentration is determined based on the Q-PCR method, and the measured value and the true value of the fetal free DNA concentration in the sample are shown in Table 1.
TABLE 1
| Sample(s) | True value of fetal free DNA concentration | Fetal free DNA concentration measurement |
| Sample1 | 2％ | 1.5％ |
| Sample2 | 6％ | 5.2％ |
| Sample3 | 9％ | 8.9％ |
| Sample4 | 13％ | 12.5 |
| Sample5 | ||
| 15％ | 15.4％ | |
| Sample6 | 17.2％ | 18％ |
| Sample7 | 19.5％ | 20％ |
The correlation between the measured value and the true value is shown in FIG. 1, and it can be seen that R is2Up to 0.997.
In this embodiment, each sample to be measured is divided into two parts, one part is directly subjected to fluorescence real-time quantitative PCR, and the other part is subjected to methylase digestion by a conventional method and then to fluorescence real-time quantitative PCR. The conditions for fluorescent real-time quantitative PCR were as follows:
reaction procedure:
95℃10min；(95℃15sec,60℃30sec,72℃for 30sec)45cycles。
the nucleotide sequence of the absolute quantitative standard used in the fluorescent real-time quantitative PCR is shown as SEQ ID NO: 3, respectively.
SEQ ID NO：3
5’-TGCCTCGGTGAGTGCGCGCTGCGGGCTCTGCCCGGTGACGCCACGCGGCCTCCTCGCCTTTTCGGGATGGCTGGGAG-3’
Comparative example 1
The determination of the fetal free DNA concentration in the sample was performed as in example 1, except that: as the amplification primers for the human PDE9A gene, the following primer sets were used in place of the primer set of the present invention. The measured values and the actual values of fetal free DNA concentration in the samples are shown in Table 2.
The primer group sequences are as follows:
FS：ACCGGCCTGCCTCGGTGAGTG
RS：GCCCCTCCCAGCCATCC
TABLE 2
| Sample(s) | True value of fetal free DNA concentration | Fetal free DNA concentration measurement |
| Sample1 | 2％ | 0.8％ |
| Sample2 | 6％ | 7.6％ |
| Sample3 | 9％ | 13％ |
| Sample4 | 13％ | 9.4 |
| Sample5 | ||
| 15％ | 11％ | |
| Sample6 | 17.2％ | 15.6％ |
| Sample7 | 19.5％ | 18.4％ |
The correlation between the measured value and the true value is not good, as shown in FIG. 2, R2Only 0.799. It can be seen that the detection effect of using the pair of primers is far lower than that of the primer pair provided by the invention.
It should be noted that any feature or combination of features described as part of one embodiment in this specification can be applied to other embodiments as well, without significantly departing from the spirit of the invention; further, the technical features described as the constituent elements of the different technical aspects may be combined in any manner to constitute the other technical aspects, without significantly departing from the gist of the present invention. The present invention also includes technical means obtained by combining the above cases, and these technical means are described in the present specification.
While the foregoing description shows and describes the preferred embodiments of the present invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as described herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Industrial applicability
According to the present invention, there are provided a primer set for amplifying the PDE9A gene which enables good correlation between a measured value and a true value of fetal free DNA, and a kit for measuring the fetal free DNA concentration in the plasma of a pregnant woman comprising the primer set.
Claims (9)
1. A kit for determining the fetal free DNA concentration in the plasma of a pregnant woman, comprising:
methylases for digesting free DNA extracted from plasma of pregnant women;
a reagent for determining the concentration of free DNA extracted from the plasma of a pregnant woman; and
a reagent for determining the concentration of free DNA extracted from the plasma of a pregnant woman after digestion with the methylase;
it also includes the following primer sets:
the primer group consists of an upstream primer and a downstream primer, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, respectively.
2. The kit of claim 1, further comprising a nucleic acid sequence as set forth in SEQ ID NO: 3, and a standard for absolute quantification.
3. The kit according to claim 1, wherein the methylase is one or more selected from BstUI, HhaI and HpaII.
4. A primer group comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO: 2, respectively.
5. A kit for amplifying the human PDE9A gene, comprising the primer set of claim 4.
6. The kit of claim 5, further comprising a nucleotide sequence set forth in SEQ ID NO: 3, and a standard for absolute quantification.
7. A method for amplifying the human PDE9A gene, which comprises performing PCR amplification using the primer set of claim 4 as primers.
8. The method of claim 7, which uses free DNA extracted from the plasma of pregnant women as a template.
9. The method of claim 7, which uses methylase digested free DNA extracted from maternal plasma as a template.
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| CN107217310A (en) * | 2017-08-01 | 2017-09-29 | 安诺优达基因科技(北京)有限公司 | A kind of library constructing method and kit detected for chromosome abnormality |
| CN111286529A (en) * | 2019-07-22 | 2020-06-16 | 常州市妇幼保健院 | Kit for prenatal screening of false positive by using free DNA of peripheral blood fetus |
| CN112899357A (en) * | 2021-01-05 | 2021-06-04 | 南京普济生物有限公司 | Method for detecting fetal fraction by using digital PCR and kit thereof |
| CN119799866A (en) * | 2025-02-06 | 2025-04-11 | 东莞博奥木华基因科技有限公司 | A method for preparing a standard sample of simulated plasma of pregnant women capable of enriching fetal concentration by fragment size |
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| Non-Invasive Epigenetic Detection of Fetal Trisomy 21 in First Trimester Maternal Plasma;Ji Hyae Lim等;《PLoS One》;20111123;1-8 * |
| Quantification of Cell-Free DNA in Normal and Complicated Pregnancies: Overcoming Biological and Technical Issues;Irina Manokhina等;《PLoS ONE》;20140702;1-7 * |
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