CN112899357A - Method for detecting fetal fraction by using digital PCR and kit thereof - Google Patents
Method for detecting fetal fraction by using digital PCR and kit thereof Download PDFInfo
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Abstract
The invention provides a method for detecting fetal fraction by using digital PCR and a kit thereof, which extracts DNA from the peripheral blood of a pregnant woman by extracting the DNA in a sample; then, uniformly mixing the sample DNA, the multiple PCR reaction enzyme and a primer of a target gene, and carrying out digital PCR reaction, wherein the digital PCR reaction primer adopted for the target gene comprises a three-dimensional structure, the primer used by the digital PCR comprises a special nucleic acid sequence N with not less than ten basic groups, and the special nucleic acid sequence N only consists of adenine, guanine and thymine; the fetal fraction is detected by the fact that partial sequences of target genes have hypermethylation states or hypomethylation states in the peripheral blood of pregnant women and fetal placental cells. The invention provides a method for detecting fetal fractions by using digital PCR and a kit thereof, which adopt the technical means of ultra-multiplex digital PCR, have no non-specific amplification and good specificity, and have the advantages of prominent experimental effect, simple and convenient result analysis and short period.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for performing fetal fraction detection by using digital PCR and a kit thereof.
Background
Since the free fetal DNA is found in the peripheral blood of the pregnant woman, researchers carry out a great deal of research on the free fetal DNA, and develop a noninvasive prenatal detection technology based on a second-generation sequencing technology, and the technology is more accurate than the traditional serological screening of Down syndrome and safer than amniotic fluid puncture. The noninvasive prenatal detection technology is noninvasive, safe and accurate, is approved by doctors and pregnant women and is rapidly accepted clinically, so that the noninvasive prenatal detection technology is the most successful clinical application of the current next-generation sequencing technology. However, the accuracy of NIPT results is greatly affected by the proportion of fetal DNA in the maternal peripheral blood, and no suitable technique for detecting fetal fraction is currently available.
Therefore, there is a need to provide a new technical solution to overcome the technical obstacles existing in the existing fetal fraction detection.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for detecting fetal fractions by using digital PCR and a kit thereof, which apply special buffer solution and primers and realize the amplification of fetal specific gene loci in free DNA in maternal peripheral blood by a super-multiplex digital PCR path so as to realize the proportion calculation of fetal free DNA in maternal free DNA.
The invention provides a method for detecting fetal fraction by using digital PCR, which comprises the following steps:
step one, extracting DNA in a sample: extracting DNA from the peripheral blood of the pregnant woman to obtain sample DNA;
step two, uniformly mixing the sample DNA obtained in the step one, multiple PCR reaction enzyme and a primer of a target gene, and then carrying out digital PCR reaction;
wherein the partial sequence of the target gene has a hypermethylated state or a hypomethylated state in the peripheral blood of the pregnant woman and the fetal placental cells; the primer of the target gene comprises the following base sequences:
GGGUUGGGAAGAAACUGUGGCACUUCGGUGCCAGCAACCC；
in the digital PCR reaction, the primer of the target gene comprises a three-dimensional structure, the primer of the target gene comprises a special nucleic acid sequence N with no less than ten basic groups, and the special nucleic acid sequence N only consists of adenine, guanine and thymine.
According to the technical scheme, the amplification of the fetal specific gene locus in the free DNA in the maternal peripheral blood is realized through a super-multiplex digital PCR (polymerase chain reaction) path by using special primers, so that the detection of the fetal fraction is realized.
Preferably, in the method as described above, the partial sequence of the target gene differs in methylation state of the peripheral blood of the pregnant woman and the fetal placental cells. According to the technical scheme, the amplification of the fetal specific gene locus in the free DNA in the peripheral blood of the mother is realized through a super-multiplex digital PCR (polymerase chain reaction) path by using a special primer, the proportion calculation of the fetal free DNA in the maternal free DNA is realized, and the detection of the fetal fraction is realized.
Preferably, in the method as described above, the reaction solution used in the digital PCR reaction does not contain a cytosine triphosphate deoxynucleotide component. According to the technical scheme, the amplification of the fetal specific gene locus in the free DNA in the peripheral blood of the mother is realized through a super-multiplex digital PCR path by using a special buffer solution and a primer, the calculation of the proportion of the fetal free DNA in the maternal free DNA is realized, and the detection of the fetal fraction is realized.
Preferably, in the method as described above, the digital PCR in step two uses at least ten pairs of primers, and the detection of the target gene is performed simultaneously. According to the technical scheme, the amplification of the fetal specific gene locus in the free DNA in the peripheral blood of the mother is realized through a super-multiplex digital PCR path by using a special buffer solution and a primer, the calculation of the proportion of the fetal free DNA in the maternal free DNA is realized, and the detection of the fetal fraction is realized.
The invention provides a kit for fetal fraction detection by using digital PCR (polymerase chain reaction), which is used for completing noninvasive prenatal fetal chromosome polyploidy detection by using the digital PCR, wherein a primer used by the detection kit comprises a special nucleic acid sequence N with not less than ten basic groups, and the special nucleic acid sequence N only consists of adenine, guanine and thymine. According to the technical scheme, the amplification of the fetal specific gene locus in the free DNA in the peripheral blood of the mother is realized through a super-multiplex digital PCR (polymerase chain reaction) path by using a special primer, the proportion calculation of the fetal free DNA in the maternal free DNA is realized, and the detection of the fetal fraction is realized.
Preferably, in the kit as described above, the reaction solution used in the detection kit does not contain a cytosine triphosphate deoxynucleotide component. According to the technical scheme, the amplification of the fetal specific gene locus in the free DNA in the peripheral blood of the mother is realized through a super-multiplex digital PCR path by using a special buffer solution and a primer, the calculation of the proportion of the fetal free DNA in the maternal free DNA is realized, and the detection of the fetal fraction is realized.
Preferably, in the kit as described above, the primers used in the detection kit comprise a three-dimensional structure. According to the technical scheme, the amplification of the fetal specific gene locus in the free DNA in the peripheral blood of the mother is realized through a super-multiplex digital PCR path by using a special buffer solution and a primer, the calculation of the proportion of the fetal free DNA in the maternal free DNA is realized, and the detection of the fetal fraction is realized.
Preferably, in the kit as described above, the detection kit uses at least ten pairs of primers and simultaneously detects the target sequence. According to the technical scheme, the amplification of the fetal specific gene locus in the free DNA in the peripheral blood of the mother is realized through a super-multiplex digital PCR path by using a special buffer solution and a primer, the calculation of the proportion of the fetal free DNA in the maternal free DNA is realized, and the detection of the fetal fraction is realized.
The beneficial effects created by the invention are as follows:
the invention provides a fetal fraction detection method and a kit, which adopt a technical means of ultra-multiplex digital PCR, have no non-specific amplification and good specificity; has the advantages of outstanding operation, simple and convenient result analysis and short period.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to these drawings without inventive effort.
FIG. 1 is a diagram of the results of clinical sample detection with the ordinate of the FAM fluorescence channel and the abscissa of the HEX fluorescence channel; the FAM fluorescence channel corresponds to a ten-fold PCR detection on an autosome;
FIG. 2 is a diagram of the results of clinical sample detection with the ordinate of the FAM fluorescence channel and the abscissa of the HEX fluorescence channel; the FAM fluorescence channel corresponds to a ten-fold PCR detection on an autosome;
FIG. 3 is a schematic diagram of the structure of the primer partial sequence used in example 1.
Detailed Description
The experimental methods of the following examples, which are not specified under specific conditions, are generally determined according to national standards. If there is no corresponding national standard, it is carried out according to the usual international standards, to the conventional conditions or to the conditions recommended by the manufacturer.
The features mentioned with reference to the invention or the features mentioned with reference to the embodiments can be combined. All the features disclosed in this specification may be combined in any combination, and each feature disclosed in this specification may be replaced by alternative features serving the same, equivalent or similar purpose. Thus, unless expressly stated otherwise, the features disclosed are merely generic examples of equivalent or similar features.
In the present invention, all the technical features mentioned herein and preferred features may be combined with each other to form a new technical solution, if not specifically stated.
In the present invention, the term fetal fraction (total DNA fraction) as used herein refers to the percentage of fetal free DNA/total DNA in maternal plasma, unless otherwise specified.
In order to make the technical means, the original characteristics, the achieved purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments, but the invention includes but is not limited to the embodiments.
Example 1A method for fetal fraction detection Using digital PCR and kit therefor
This example mainly describes a method and kit for fetal fraction detection using digital PCR, which applies a special buffer solution and primers to amplify a fetal specific gene locus in free DNA in maternal peripheral blood via a super-multiplex digital PCR path, thereby calculating the proportion of fetal free DNA in maternal free DNA.
A method for fetal fraction detection using digital PCR, comprising the steps of:
1. sample processing
The peripheral blood sample of the pregnant woman was centrifuged at 1600g for 10 minutes at 4 ℃ and the plasma fraction was centrifuged again at 16000g for 10 minutes at 4 ℃ to further remove the residual blood cells.
2. DNA extraction
Free DNA was extracted from plasma using the QIAamp circulating nucleic Acid extraction kit (manufacturer: QIAGEN; catalog No.: 55114) according to the manufacturer's instructions.
3. Preparing a reagent:
digital PCR reaction solutions (1 reaction) were prepared according to the recipe in table 1:
TABLE 1 digital PCR reaction solution
| Components | Volume of |
| 10×dPCR Buffer | 3.5μL |
| Enzyme | 1.0μL |
| Primer and method for producing the same | 2.8μL |
| Detection template | 1.0～17.5μL |
| Total volume | Supplementing 30-35 μ L with ribozyme-free water |
The reaction solution adopted by the digital PCR reaction does not contain cytosine triphosphate deoxynucleotide components, is uniformly mixed for 30 seconds in a vortex mode, and is collected at the bottom of a tube and then is placed on ice for standby after instantaneous centrifugation. Among them, 10 XDPCR Buffer does not contain cytosine triphosphate deoxynucleotide (dCTP).
4. An oil phase mixture was prepared according to the formulation of table 2 (1 reaction):
TABLE 2 digital PCR reaction solution
| Volume of | |
| Oil phase A | 30μL |
| Oil phase B | 10μL |
| Total of | 40μL |
After mixing the reaction oil phases according to the formulation of table 2, vortex and mix them evenly for 30s, remove air bubbles by instantaneous centrifugation, and collect the liquid at the bottom of the tube and put it on ice for use.
5. Detection sample introduction
(1) Detection system
This example uses the U.S. Bio-rad digital PCR system with 2 fluorescence channels, which can process 12 samples in parallel, and the number of effective droplets per reaction system is 2 ten thousand.
6. Preparation of sample Gene standards
The sample DNA, the multiplex PCR reaction enzyme and the target gene primer were mixed uniformly under the conditions shown in Table 3, and then the digital PCR reaction was carried out to obtain a PCR product, and the experimental results shown in FIG. 1 were obtained. The ordinate is FAM fluorescence channel, and the abscissa is HEX fluorescence channel. FAM fluorescence channel corresponds to 10-fold PCR detection on one autosome.
TABLE 3 PCR reaction conditions
Before the experiment, the inventors designed 10-fold PCR primers covering the target gene locus by using the ION AMPLISEQ DESIGNER online design website, and the target gene of the designed primers is RASSF1A, as shown in Table 4. The nucleotide sequence "GGGUUGGGAAGAAACUGUGGCACUUCGGUGCCAGCAACCC (SEQ ID NO. 081)" was added to one end of the designed primer to obtain an initial nucleotide sequence for amplification. The initial nucleotide sequence for amplification designed by the primer and the probe aiming at the target gene locus is sent to the company of biological engineering (Shanghai) GmbH for synthesis, the synthesized nucleotide needs to be pretreated, and the pretreatment method comprises the following steps: adding the synthesized nucleotide sequence into a buffer solution B, heating to 65 ℃, preserving heat for 10 minutes, then cooling to 35 ℃, preserving heat for 30 minutes, and obtaining the full length of the nucleotide sequence for detection; wherein, the buffer solution B comprises the following components: 320mM NaCl, 7mM MgCl225mM Tris (pH 7.6), 1 μ M tea polyphenols.
TABLE 4 RASSF1A Gene detection primer Probe sequences
Fig. 1 is a diagram of the detection result of a clinical sample, with the ordinate of the FAM fluorescence channel and the abscissa of the HEX fluorescence channel. FAM fluorescence channel corresponds to 10-fold PCR detection on one autosome. Wherein, the design parameters of the digital PCR are used for preparing a PCR reaction system according to the tables 1 and 2. And loading the prepared reaction systems with different proportions onto a PCR system to form a micro-reaction unit. The samples were placed in a digital PCR instrument and PCR reactions were carried out according to the PCR reaction conditions in Table 3.
After the amplification is finished, the effective fluorescence positive points of the two channels are interpreted through computer analysis, and the result is analyzed, such as the experimental result shown in fig. 1.
The Fetal Fraction (FFA) in the sample can be calculated according to the following calculation:
1.genome equivalent counts(GE)＝FAM channel counts/10；
2.Fetal fraction(FF)＝Counts(HEX)/GE*100％；
3. the comparison of the detection result of this example with the existing detection means (since there is no existing mature commercial method, it can only be compared with the detection result of Y chromosome in the peripheral blood of the mother (the Y chromosome detection and fetal fraction determination method refers to CN 110914456A)).
TABLE 5 comparison of the results of the present example with those of the prior art
The P value of the two groups of data is 0.8952, which proves that the fetal fraction result is not different from the Y chromosome detection result, so that the fetal fraction detection accurately reflects clinical information.
Example 2 method for fetal fraction detection Using digital PCR and kit thereof
This example mainly describes a method and kit for fetal fraction detection using digital PCR, which applies a special buffer solution and primers to amplify a fetal specific gene locus in free DNA in maternal peripheral blood via a super-multiplex digital PCR path, thereby calculating the proportion of fetal free DNA in maternal free DNA.
A method for fetal fraction detection using digital PCR, comprising the steps of:
1. sample processing
The peripheral blood sample of the pregnant woman was centrifuged at 1600g for 10 minutes at 4 ℃ and the plasma fraction was centrifuged again at 16000g for 10 minutes at 4 ℃ to further remove the residual blood cells.
2. DNA extraction
Free DNA was extracted from plasma using the QIAamp circulating nucleic Acid extraction kit (manufacturer: QIAGEN; catalog No.: 55114) according to the manufacturer's instructions.
3. Preparing a reagent:
digital PCR reaction solutions (1 reaction) were prepared according to the recipe of table 6:
TABLE 6 digital PCR reaction solution
| Components | Volume of |
| 10×dPCR Buffer | 3.5μL |
| Enzyme | 1.0μL |
| Primer and method for producing the same | 2.8μL |
| Detection dieBoard | 1.0～17.5μL |
| Total volume | Supplementing 30-35 μ L with ribozyme-free water |
The reaction solution adopted by the digital PCR reaction does not contain cytosine triphosphate deoxynucleotide components, is uniformly mixed for 30 seconds in a vortex mode, and is collected at the bottom of a tube and then is placed on ice for standby after instantaneous centrifugation. Among them, 10 XDPCR Buffer does not contain cytosine triphosphate deoxynucleotide (dCTP).
4. An oil phase mixture was prepared according to the formulation of table 7 (1 reaction):
TABLE 7 digital PCR reaction solution
| Volume of | |
| Oil phase A | 30μL |
| Oil phase B | 10μL |
| Total of | 40μL |
After mixing the reaction oil phases according to the formulation of table 7, vortexing and mixing for 30s, removing air bubbles by instantaneous centrifugation, and collecting the liquid at the bottom of the tube and placing on ice for later use.
5. Detection sample introduction
(1) Detection system
This example uses the U.S. Bio-rad digital PCR system with 2 fluorescence channels, which can process 12 samples in parallel, and the number of effective droplets per reaction system is 2 ten thousand.
6. Preparation of sample Gene standards
The sample DNA, the multiplex PCR reaction enzyme and the target gene primer were mixed uniformly under the conditions shown in Table 8, and then subjected to digital PCR to obtain a PCR product, and the experimental results shown in FIG. 1 were obtained. The ordinate is FAM fluorescence channel, and the abscissa is HEX fluorescence channel. FAM fluorescence channel corresponds to 10-fold PCR detection on one autosome.
TABLE 8 PCR reaction conditions
Before the experiment, the inventors designed 10-fold PCR primers covering the target gene locus by using the ION AMPLISEQ DESIGNER online design website, and the target gene of the designed primers was HLCS, as shown in Table 9. The initial nucleotide sequence for amplification was obtained by adding nucleotide sequence "GGGUUGGGAAGAAACUGUGGCACUUCGGUGCCAGCAACCC" to one end of the designed primer. The initial nucleotide sequence for amplification designed by the primer and the probe aiming at the target gene locus is sent to the company of biological engineering (Shanghai) GmbH for synthesis, the synthesized nucleotide needs to be pretreated, and the pretreatment method comprises the following steps: adding the synthesized nucleotide sequence into a buffer solution B, heating to 70 ℃, preserving heat for 5 minutes, then cooling to 30 ℃, preserving heat for 25 minutes, and obtaining the full length of the nucleotide sequence for detection; wherein, the buffer solution B comprises the following components: 300mM NaCl, 5mM MgCl220mM Tris (pH 7.6), 5. mu.M tea polyphenols.
TABLE 9 HLCS Gene detection primer Probe sequences
Fig. 2 is a diagram of the detection result of a clinical sample, with the ordinate of the FAM fluorescence channel and the abscissa of the HEX fluorescence channel. FAM fluorescence channel corresponds to 10-fold PCR detection on one autosome. Wherein, the design parameters of the digital PCR are used to prepare PCR reaction systems according to tables 6 and 7. And loading the prepared reaction systems with different proportions onto a PCR system to form a micro-reaction unit. The samples were placed in a digital PCR instrument and PCR reactions were carried out according to the PCR reaction conditions in Table 3.
After the amplification is finished, the effective fluorescence positive points of the two channels are interpreted through computer analysis, and the result is analyzed, such as the experimental result shown in fig. 2.
The Fetal Fraction (FFA) in the sample can be calculated according to the following calculation:
1.genome equivalent counts(GE)＝FAM channel counts/10.
2.Fetal fraction(FF)＝Counts(HEX)/GE*100％
3. the comparison of the detection result of this example with the existing detection means (since there is no existing mature commercial method, it can only be compared with the detection result of Y chromosome in the peripheral blood of the mother (the Y chromosome detection and fetal fraction determination method refers to CN 110914456A)).
TABLE 10 comparison of the results of the present example with those of the conventional detection means
| Sample number | Fraction of fetus | |
| 1 | 9.21％ | 9.01％ |
| 2 | 10.30％ | 10.25％ |
| 3 | 12.47％ | 12.59％ |
| 4 | 7.45％ | 7.80％ |
| 5 | 9.34％ | 9.25％ |
| 6 | 16.20％ | 16.14％ |
| 7 | 10.32％ | 10.25％ |
| 8 | 8.17％ | 9.15％ |
| 9 | 20.15％ | 22.30％ |
| 10 | 7.31％ | 7.24％ |
| 11 | 10.19％ | 10.91％ |
| 12 | 10.29％ | 11.02％ |
| FgDNA | 3.29％ | NA |
| FgDNA | 2.65％ | NA |
| NIPT-PC | 3.52％ | NA |
| NIPT-NC | 4.24％ | NA |
The P value of the two groups of data is 0.9142, which proves that the fetal fraction result is not different from the Y chromosome detection result, so that the fetal fraction detection accurately reflects clinical information.
Example 3A method for fetal fraction detection Using digital PCR and kit therefor
This example describes a method and kit for fetal fraction detection using digital PCR, and the method and kit of example 1Except that the nucleotides after synthesis were pretreated as follows: adding the synthesized nucleotide sequence into a buffer solution B, heating to 69 ℃, preserving heat for 7 minutes, then cooling to 32 ℃, preserving heat for 27 minutes, and obtaining the full length of the nucleotide sequence for detection; wherein, the buffer solution B comprises the following components: 310mM NaCl, 6mM MgCl2，23mM Tris(pH 7.6)。
The comparison of the detection result of the present example with the conventional detection means (CN110914456A) revealed that the P value of the two sets of data for determining the fetal fraction was 0.8057, which confirmed that the fetal fraction result was not different from the Y chromosome detection result.
Example 4A method for fetal fraction detection Using digital PCR and kit therefor
This example mainly describes a method and kit for fetal fraction detection using digital PCR, which is different from example 2 in that the synthesized nucleotides need to be pretreated as follows: adding the synthesized nucleotide sequence into a buffer solution B, heating to 67 ℃, preserving heat for 6 minutes, then cooling to 33 ℃, preserving heat for 26 minutes, and obtaining the full length of the nucleotide sequence for detection; wherein, the buffer solution B comprises the following components: 310mM NaCl, 6mM MgCl223mM Tris (pH 7.6), 3. mu.M tea polyphenols.
The comparison of the detection result of the present example with the conventional detection means (CN110914456A) revealed that the P value of the two sets of data for determining the fetal fraction was 0.9256, which confirmed that the fetal fraction result was not different from the Y chromosome detection result.
The inventors determined the structure of each of the partial sequences of the detected nucleotides used in examples 1 to 4 by X single crystal diffraction experiments, and obtained different three-dimensional structures required for the experiments, wherein the structure of the partial sequence of the primer used in example 1 is shown in FIG. 3, and the fetal fraction detection required in examples 1 to 3 was accurately accomplished.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Nanjing Puji BioLimited
<120> method for fetal fraction detection by using digital PCR and kit thereof
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<213> Artificial sequence ("Artificial sequence")
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<212> DNA
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aggagggtgg gg 12
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<212> DNA
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<400> 11
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence ("Artificial sequence")
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gctcaggctc ccccgacatg 20
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<212> DNA
<213> Artificial sequence ("Artificial sequence")
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<212> DNA
<213> Artificial sequence ("Artificial sequence")
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<212> DNA
<213> Artificial sequence ("Artificial sequence")
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<210> 16
<211> 20
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 16
gctcaggctc ccccgacatg 20
<210> 17
<211> 14
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 17
ggagattgaa ggga 14
<210> 18
<211> 12
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 18
aggagggtgg gg 12
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 19
<210> 20
<211> 14
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 20
gctcaggctc cccc 14
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<211> 12
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 21
ggagattgaa gg 12
<210> 22
<211> 12
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 22
aggagggtgg gg 12
<210> 23
<211> 13
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 23
gagaggcctg gcg 13
<210> 24
<211> 14
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 24
gctcaggctc cccc 14
<210> 25
<211> 21
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 25
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<212> DNA
<213> Artificial sequence ("Artificial sequence")
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<212> DNA
<213> Artificial sequence ("Artificial sequence")
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<212> DNA
<213> Artificial sequence ("Artificial sequence")
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<212> DNA
<213> Artificial sequence ("Artificial sequence")
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<212> DNA
<213> Artificial sequence ("Artificial sequence")
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<210> 31
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<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 31
atccaacagc ttccgcaagt ac 22
<210> 32
<211> 20
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 32
<210> 33
<211> 18
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 33
ttgggcaggt aaaaggaa 18
<210> 34
<211> 17
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 34
agtttgtatg gaagatg 17
<210> 35
<211> 18
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 35
atccaacagc ttccgcaa 18
<210> 36
<211> 20
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 36
<210> 37
<211> 16
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 37
ttgggcaggt aaaagg 16
<210> 38
<211> 18
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 38
agtttgtatg gaagatgg 18
<210> 39
<211> 23
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 39
atccaacagc ttccgcaagt aca 23
<210> 40
<211> 18
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 40
ccagcgcgcc cagcgggt 18
<210> 41
<211> 28
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 41
ttgcaccacc aggcgggcca cgagaggg 28
<210> 42
<211> 13
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 42
aggggggtga agg 13
<210> 43
<211> 19
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 43
gccgcgcccc tgccacctc 19
<210> 44
<211> 30
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 44
cgccctctcg tggcccgcct ggtggtgcaa 30
<210> 45
<211> 20
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 45
<210> 46
<211> 13
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 46
aggggggtga agg 13
<210> 47
<211> 19
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 47
gccgcgcccc tgccacctc 19
<210> 48
<211> 22
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 48
cgccctctcg tggcccgcct gg 22
<210> 49
<211> 25
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 49
ttgcaccacc aggcgggcca cgaga 25
<210> 50
<211> 13
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 50
aggggggtga agg 13
<210> 51
<211> 19
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 51
gccgcgcccc tgccacctc 19
<210> 52
<211> 25
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 52
cgccctctcg tggcccgcct ggtgg 25
<210> 53
<211> 24
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 53
ttgcaccacc aggcgggcca cgag 24
<210> 54
<211> 13
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 54
aggggggtga agg 13
<210> 55
<211> 19
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 55
gccgcgcccc tgccacctc 19
<210> 56
<211> 24
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 56
cgccctctcg tggcccgcct ggtg 24
<210> 57
<211> 23
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 57
ttgcaccacc aggcgggcca cga 23
<210> 58
<211> 13
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 58
aggggggtga agg 13
<210> 59
<211> 19
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 59
gccgcgcccc tgccacctc 19
<210> 60
<211> 24
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 60
cgccctctcg tggcccgcct ggtg 24
<210> 61
<211> 25
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 61
ttgcaccacc aggcgggcca cgaga 25
<210> 62
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<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 62
aggggggtga agg 13
<210> 63
<211> 17
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 63
gccgcgcccc tgccacc 17
<210> 64
<211> 22
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 64
cgccctctcg tggcccgcct gg 22
<210> 65
<211> 22
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 65
ttgcaccacc aggcgggcca cg 22
<210> 66
<211> 13
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 66
aggggggtga agg 13
<210> 67
<211> 19
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 67
gccgcgcccc tgccacctc 19
<210> 68
<211> 22
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 68
cgccctctcg tggcccgcct gg 22
<210> 69
<211> 21
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 69
accaggcggg ccacgagagg g 21
<210> 70
<211> 13
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 70
aggggggtga agg 13
<210> 71
<211> 19
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 71
gccgcgcccc tgccacctc 19
<210> 72
<211> 24
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 72
ctcgtggccc gcctggtggt gcaa 24
<210> 73
<211> 23
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 73
ccaccaggcg ggccacgaga ggg 23
<210> 74
<211> 13
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 74
aggggggtga agg 13
<210> 75
<211> 19
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 75
gccgcgcccc tgccacctc 19
<210> 76
<211> 28
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 76
ccctctcgtg gcccgcctgg tggtgcaa 28
<210> 77
<211> 24
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 77
accaccaggc gggccacgag aggg 24
<210> 78
<211> 13
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 78
aggggggtga agg 13
<210> 79
<211> 19
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 79
gccgcgcccc tgccacctc 19
<210> 80
<211> 25
<212> DNA
<213> Artificial sequence ("Artificial sequence")
<400> 80
tctcgtggcc cgcctggtgg tgcaa 25
<210> 81
<211> 40
<212> RNA
<213> Artificial sequence ("Artificial sequence")
<400> 81
ggguugggaa gaaacugugg cacuucggug ccagcaaccc 40
Claims (10)
1. A method for fetal fraction detection using digital PCR, comprising the steps of:
step one, extracting DNA in a sample: extracting DNA from the peripheral blood of the pregnant woman to obtain sample DNA;
step two, uniformly mixing the sample DNA obtained in the step one, multiple PCR reaction enzyme and a primer of a target gene, and then carrying out digital PCR reaction;
wherein the partial sequence of the target gene has a hypermethylated state or a hypomethylated state in the peripheral blood of the pregnant woman and the fetal placental cells; the primer of the target gene comprises the following base sequences:
GGGUUGGGAAGAAACUGUGGCACUUCGGUGCCAGCAACCC；
in the digital PCR reaction, the primer of the target gene comprises a three-dimensional structure, the primer of the target gene comprises a special nucleic acid sequence N with no less than ten basic groups, and the special nucleic acid sequence N only consists of adenine, guanine and thymine.
2. The method of claim 1, wherein the partial sequence of the target gene is different in methylation status of the peripheral blood of the pregnant woman and the placental cells of the fetus.
3. The method according to claim 1, wherein the reaction solution used in the digital PCR reaction does not contain a cytosine triphosphate deoxynucleotide component.
4. The method of claim 1, further comprising: pretreating the primer of the target gene, wherein the pretreatment method comprises the following steps: and adding the primer containing the target gene into the buffer solution B, heating to 65-70 ℃, preserving heat for 5-10 minutes, cooling to 30-35 ℃, and preserving heat for 25-30 minutes.
5. The method of claim 2, wherein the digital PCR of step two uses at least ten pairs of primers and the detection of the target gene is performed simultaneously.
6. The method of claim 4, wherein the buffer B comprises 300-320 mM NaCl, 5-7 mM MgCl220-25 mM Tris (pH 7.6), 1-5 μ M tea polyphenols.
7. The kit for detecting the fetal fraction by using the digital PCR is characterized in that the detection kit completes the noninvasive prenatal fetal chromosome polyploidy detection by using the digital PCR, a primer used by the detection kit comprises a special nucleic acid sequence N with not less than ten basic groups, and the special nucleic acid sequence N only consists of adenine, guanine and thymine.
8. The kit according to claim 7, wherein the reaction solution used in the detection kit does not contain a cytosine triphosphate deoxynucleotide component.
9. The kit of claim 7, wherein the primers used in the assay kit comprise a three-dimensional structure.
10. The kit of claim 7, wherein the detection kit uses at least ten pairs of primers to simultaneously detect the target sequence.
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| CN109022541A (en) * | 2018-07-16 | 2018-12-18 | 苏州大学附属第二医院 | A kind of kit of concentration fetus dissociative DNA, method and its application |
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| CN102703430A (en) * | 2012-06-04 | 2012-10-03 | 昆明理工大学 | Method for extracting microorganism total DNA (Deoxyribonucleic Acid) in pu'er tea piling fermentation process |
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