CN110923306A - Primer, probe, kit and method for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR (polymerase chain reaction) - Google Patents
Primer, probe, kit and method for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention provides a primer, a probe, a kit and a method for noninvasive prenatal screening of 21-trisomy syndrome based on digital PCR. The invention designs an amplification primer and a taqman probe according to the gene sequence of the human chromosome 21, and detects a target gene; simultaneously, designing an amplification primer and a taqman probe according to the gene sequences of the human chromosome 2 and chromosome 8, and detecting a reference gene; and obtaining the detection result of whether the number of the 21 st chromosome of the sample to be detected is abnormal or not based on the digital PCR technology and data analysis. Compared with the second-generation sequencing, the detection method provided by the invention is simple to operate, does not need steps such as library building and sequencing, is simple and easy to learn in data analysis, is high in large-scale detection speed and low in detection cost, and is convenient to develop in a conventional clinical laboratory.
Description
Technical Field
The application relates to the technical field of digital PCR, in particular to a primer, a probe, a kit and a method for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR.
Background
China has become a high-incidence country of birth defects, the number of newly-increased birth defect children is as high as 120 ten thousand per year on average, and accounts for about 4-6% of the total number of newly-increased people per year, wherein the chromosome abnormality disease is one of the main diseases. No effective treatment for this type of disease is currently available, and the key point is prevention, i.e. prenatal screening and diagnosis.
Chromosomal abnormality disease refers to an abnormal number of chromosomes and/or a change in morphological structure, which may occur on each chromosome. Among them, three autosomal aneuploidies, trisomy 21 (down syndrome), trisomy 18 (edward syndrome) and trisomy 13 (pado syndrome), are most common, and the incidence rates in newborns are 1/800, 1/8000 and 1/25000, respectively.
Currently, there are several methods for detecting chromosomal aneuploidy diseases, amniocentesis is commonly used to diagnose fetal genetic abnormalities, but amniocentesis is an invasive test that may cause infection and abortion. In recent years, methods for detecting fetal DNA in blood of pregnant women based on second-generation sequencing so as to realize noninvasive screening of genetic abnormalities are widely applied, but have the problems of high detection cost, long detection process and the like.
Disclosure of Invention
In view of the problems in the background art, the present application aims to provide primers, probes, kits and methods for noninvasive prenatal detection of trisomy 21 syndrome based on digital PCR technology.
In order to achieve the above object, a first aspect of the present application provides a primer combination for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR, comprising: a primer designed according to the detection site of human chromosome 21 has a nucleotide sequence shown as SEQ ID NO: 1 to SEQ ID NO: 12 is shown in the specification; primers are designed according to the detection sites of human chromosomes 8 and 2 respectively, and the nucleotide sequences of the primers are shown as SEQ ID NO: 19 to SEQ ID NO: shown at 26.
A second aspect of the present application provides a probe set for noninvasive prenatal detection of fetal trisomy 21 syndrome based on digital PCR, comprising: the Taqman probe is designed according to the detection site of human chromosome 21, and the nucleotide sequence of the Taqman probe is shown as SEQ ID NO: 13 to SEQ ID NO: 18 is shown in the figure; the Taqman probe is designed according to the detection sites of human chromosomes 8 and 2 respectively, and the nucleotide sequence of the Taqman probe is shown as SEQ ID NO: 27 to SEQ ID NO: shown at 30.
Preferably, in the probe combination, a Taqman probe designed according to the detection site of human chromosome 21 is labeled by FAM, HEX or ROX; marking a Taqman probe designed according to the detection site of the human chromosome 8 by CY 5; a Taqman probe designed according to the detection site of human chromosome 2 is marked by Alexa Fluor 700.
A third aspect of the present application provides a kit for noninvasive prenatal detection of fetal trisomy 21 syndrome based on digital PCR, comprising: a specific primer and a Taqman probe which are designed according to the detection site of the human chromosome 21 and are used for amplifying and detecting a target gene; specific primers and Taqman probes for detecting reference genes are designed according to the detection sites of human chromosomes 8 and 2 respectively; wherein, the nucleotide sequences of the specific primer and the Taqman probe for amplifying and detecting the target chromosome are shown as SEQ ID NO: 1 to SEQ ID NO: 18 is shown in the figure; the nucleotide sequences of the specific primers and the Taqman probe for amplifying and detecting the reference chromosome are shown as SEQ ID NO: 19 to SEQ ID NO: shown at 30.
Preferably, in the kit for noninvasive prenatal detection of fetal trisomy 21 syndrome based on digital PCR, a Taqman probe designed according to a detection site of chromosome 21 of human is labeled by FAM, HEX or ROX; marking a Taqman probe designed according to the detection site of the human chromosome 8 by CY 5; a Taqman probe designed according to the detection site of human chromosome 2 is marked by Alexa Fluor 700.
The detection sites of human chromosomes 21, 8 and 2 selected in the embodiments of the present invention, and the specific primers and Taqman probes designed to amplify and detect the target chromosome and the reference chromosome are shown in table 1 below. The design principle of the primers and the probes is as follows: primer express3.0.1 is adopted to carry out primer and probe design on the screened sequence, the annealing temperature of the primer is about 59 ℃, the annealing temperature of the probe is about 68 ℃, dimer and hairpin structures are not easy to form, the GC content is normal, the amplification range of the detection is 65-100bp, and the specificity detection of the primer and the probe is carried out.
TABLE 1 specific primers and Taqman probes
A fourth aspect of the present application provides a digital PCR-based noninvasive prenatal detection method for fetal trisomy 21 syndrome, comprising the steps of:
(1) obtaining free DNA from the peripheral blood of a pregnant woman to be detected as genome DNA to be detected;
(2) selecting a detection site of human chromosome 21, and designing a specific primer and a Taqman probe for amplifying and detecting a target chromosome; selecting detection sites of human chromosomes 8 and 2, and respectively designing specific primers and Taqman probes for amplifying and detecting reference chromosomes; wherein, the nucleotide sequences of the specific primer and the Taqman probe for amplifying and detecting the target chromosome are shown as SEQ ID NO: 1 to SEQ ID NO: 18 is shown in the figure; the nucleotide sequences of the specific primers and the Taqman probe for amplifying and detecting the reference chromosome are shown as SEQ ID NO: 19 to SEQ ID NO: 30 is shown in the figure;
(3) preparing a digital PCR amplification reaction system, taking the to-be-detected genome DNA in the step (1) as a template, and performing digital PCR amplification and detection by using the specific primer and the Taqman probe in the step (2);
(4) respectively obtaining the copy number a of the chromosome 21 of the sample to be detected according to the fluorescent signals of the digital PCR21And the copy number b of the reference chromosome; wherein the copy number b of the reference chromosome is the copy number b of the No. 2 chromosome of the sample to be detected2And copy number b of chromosome 88Average value of (d);
(5) calculating the copy number a of chromosome 21 of the sample to be detected21Y is the percentage of the copy number b of the reference chromosome of the sample21；
(6) Calculating the Z-score of No. 21 of the sample to be tested:
wherein, y0The average of the copy number a21 of chromosome 21 of a trisomy syndrome negative sample as a percentage of the copy number b of a reference chromosome of the sample; sigmayStandard deviation of copy number a21 of chromosome 21 as a percentage of copy number b of the reference chromosome of the sample, which is a negative sample for trisomy syndrome;
(7) and judging whether the number of the chromosome 21 of the sample to be detected is abnormal or not according to the Z-score value of the chromosome 21 of the sample to be detected.
Preferably, in the detection method provided in the embodiments of the present application, (Z-score)21When the number is less than or equal to 1.96, the number of the No. 21 chromosome of the sample to be detected is normal; when 1.96 < (Z-score)21When the detection time is less than or equal to 3, re-detecting; when (Z-score)21>And 3, indicating that the number of the 21 chromosome in the sample to be detected is abnormal.
Preferably, in the detection method provided in the embodiments of the present application, the Taqman probe designed based on the detection site of human chromosome 21 is labeled with FAM, HEX or ROX; marking a Taqman probe designed according to the detection site of the human chromosome 8 by CY 5; a Taqman probe designed according to the detection site of human chromosome 2 is marked by Alexa Fluor 700.
Preferably, in the detection method provided by the embodiment of the present application, the system of the digital PCR amplification reaction is composed of: 10 × dPCR Buffer: 3.5 mu L; enzyme: 1.0 μ L; primers and probes: 2.8 mu L; detecting the template: 1.0-17.5 muL; total volume: supplementing 30-35 μ L with ribozyme-free water.
Preferably, in the detection method provided in the embodiments of the present application, the procedure of the digital PCR amplification reaction is: 50 ℃ C: 10 minutes and 1 cycle; 90 ℃ C: 10 minutes and 1 cycle; 95 ℃: 20 seconds, 59 ℃: 40s, 45 cycles; keeping at 25 ℃.
Compared with the prior art, the invention provides a digital PCR-based noninvasive prenatal screening method and a kit for 21-trisomy syndrome. According to the invention, an amplification primer and a taqman probe are designed according to a gene sequence of chromosome 21, and a target gene is detected; meanwhile, an amplification primer and a taqman probe are designed according to the gene sequences of the No. 2 and No. 8 chromosome genes, and a reference gene is detected. The length of maternal episomal DNA in plasma is about 166bp, the length of fetal episomal DNA is about 146bp, the invention designs 6 detection sites on a target chromosome, and designs 2 detection sites on a reference chromosome, thus the design can improve the detection accuracy. The main standard of screening the detection sites is sequence conservation without repeated sequences, and the interval of the detection sites is more than 500 bp.
The method is based on the digital PCR technology, has simple operation compared with the second-generation sequencing, does not need steps of library building, sequencing and the like, has simple and easy-to-learn data analysis, high large-scale detection speed, is convenient for developing in a conventional clinical laboratory, has the cost which is half of that of the second-generation sequencing method, and is a novel method for noninvasive prenatal DNA detection.
Drawings
FIG. 1 is a flow chart of a detection method according to an embodiment of the present application;
FIG. 2 is a standard normal distribution plot of y-values for a trisomy 21 syndrome negative sample according to an embodiment of the present application.
Detailed Description
Embodiment A method validation
1. Preparation of samples to be tested
(1) Experimental group samples:
respectively mixing the plasma of a patient with trisomy 21 with the plasma of a normal person uniformly according to the proportion of 16%, 8%, 4%, 2% and 1% to obtain trisomy 21 mixed samples with different mutation frequencies.
(2) Control group samples: i.e. the reference sample, is a large number of 1000 known normal pregnant woman samples (negative).
Sample DNA was extracted using a plasma free DNA extraction kit for use.
2. Preparing a reagent:
a digital PCR reaction solution (recommended digital PCR reaction system, 1 reaction) was prepared according to the following formula:
| components | Volume of |
| 10×dPCR Buffer | 3.5μL |
| Enzyme | 1.0μL |
| Primers and probes | 2.8μL |
| Detection template | 1.0～17.5μL |
| Total volume | Supplementing 30-35 μ L with ribozyme-free water |
And (3) uniformly mixing the reaction solution, uniformly mixing the reaction solution by vortex for 30 seconds, and collecting the reaction solution on the bottom of the tube by instantaneous centrifugation and then placing the reaction solution on ice for later use.
3. Preparing an oil phase mixed solution according to the following proportion (1 reaction):
| components | Dosage of |
| Oil phase A | 30μL |
| Oil phase B | 10μL |
| Total of | 40μL |
Mixing the reaction oil phases according to the formula shown in the table, uniformly mixing the mixture for 30s by vortex, removing bubbles by instantaneous centrifugation, collecting the liquid at the bottom of the tube, and placing the liquid on ice for later use (note: the mixed oil phase mixture is used within 30 min).
4. Chip sample introduction
(1) Detection system
In the embodiment, a digital PCR system biodata blue of Shanghai small sea turtle science and technology Limited is used, the system is a 5-channel fluorescence channel, 96 samples can be processed in parallel, and the number of effective liquid drops of a chip is 20 ten thousand.
1 biochip per sample assay was used: in table 1, SEQ ID NO: 1 to SEQ ID NO: 4. SEQ ID NO: 13. SEQ ID NO: 14 primer probes located on FAM channel, SEQ ID NO: 5 to SEQ ID NO: 8. SEQ ID NO: 15. SEQ ID NO: 16 primer probe is located in HEX channel, SEQ ID NO: 9 to SEQ ID NO: 12. SEQ ID NO: 17. SEQ ID NO: 18, a primer probe is positioned in a ROX channel and used for detecting chromosome 21; SEQ ID NO: 19 to SEQ ID NO: 22. SEQ ID NO: 27. SEQ ID NO: 28 primer probe is positioned in CY5 channel, and detects chromosome 8; SEQ ID NO: 23 to SEQ ID NO: 26. SEQ ID NO: 29. SEQ ID NO: the 30 primer probe is positioned in an Alexa Fluor700 channel and detects chromosome 2.
(2) Sample introduction operation
According to the requirements of the digital PCR sample injection instrument using operation instructions, starting a chip sample injection program to finish the chip sample injection.
5. Chip thermal cycling reaction
The chip is placed in a chip groove of a nucleic acid amplification instrument for thermal cycle reaction.
Reaction procedure:
6. chip reading analysis
After the thermal cycling reaction is finished, the chip is moved to a biochip reader, reading and analyzing are carried out according to the requirements of the biochip reader using the operation instruction, and the copy number of each fluorescence channel is obtained, namely the copy number a of the chromosome 21 of the sample to be detected is respectively obtained21And the copy number b of the reference chromosome; wherein the copy number b of the reference chromosome is the copy number b of the No. 2 chromosome of the sample to be detected2And copy number b of chromosome 88Average value of (d);
calculating the copy number a of chromosome 21 of the sample to be detected21Y is the percentage of the copy number b of the reference chromosome of the sample21。
In addition, y providing a negative sample of trisomy 21 syndrome (i.e., a reference sample)0And σy:
y0＝0.9944，σy＝0.006647。
Then, the Z-score of sample No. 21 to be tested was calculated:
wherein, y0Copy number a of chromosome 21 as negative sample for trisomy syndrome21An average of the percentage of the copy number b of the sample reference chromosome; sigmayCopy number a of chromosome 21 as negative sample for trisomy syndrome21Standard deviation as a percentage of the copy number b of the sample reference chromosome;
and judging whether the number of the chromosome 21 of the sample to be detected is abnormal or not according to the Z-score value of the chromosome 21 of the sample to be detected.
21 three-body mixed sample detection result
Note: chromosome copy number is the sum of copy numbers of each relevant channel
In addition, the y values of 1000 negative samples were analyzed by the Kolmogorov-Smirnov test and the shapero test, and the result showed that the y value of chromosome 21 satisfied the standard normal distribution (as shown in FIG. 2), so that the Z-score test was used to verify whether there was a significant difference between the positive and negative samples.
The result meets the standard normal distribution through the statistics of the Z-score value of the reference sample, and the probability that the Z-score of the trisomy negative sample is less than or equal to 1.96 is about 98 percent; the probability of Z-score between (1.96, 3) is 1.14% -2.14%, the probability of Z >3 is 0. under a standard normal distribution, the probability of Z-score ≦ 1.96 is 97.49%, the probability of Z-score between (1.96, 3) is 2.37%, and the probability of Z-score >3 is 0.14%, so the Z-score value of the trisomy negative sample is substantially consistent with the standard normal distribution.
7. Determination of results
And (3) judging a negative sample: when the Z-score of the chromosome 21 is less than or equal to 1.96, the chromosome 21 of the detection sample has no significant difference from the normal chromosome in the reference sample library, namely the number of the chromosome 21 is normal;
judging a gray area sample: when the Z-score of chromosome 21 falls in the gray zone (1.96, 3), because the possibility of false negative caused by too low fetal DNA concentration (< 4%) cannot be eliminated, the re-examination after re-blood sampling is recommended, and the re-examination result is still in the gray zone and is judged to be positive;
and (3) judging a positive sample: when the Z-score >3 of chromosome 21 indicates that there is a significant difference between chromosome 21 of the test sample and normal chromosomes in the reference database, i.e., there is an abnormality in the number of chromosome 21 of the test sample, it is predicted that trisomy 21 syndrome will occur.
In conclusion, the system can accurately detect the maternal plasma free DNA containing 4 percent of fetal DNA and above. A flow chart of a detection method according to an embodiment of the application is shown in the attached figure 1.
EXAMPLE two sample detection assay
1. Preparation of samples to be tested
3 plasma samples of 21 trisomy syndrome patients confirmed by the water goat puncture are respectively taken, and the sample numbers are 1-3. Sample DNA was extracted using a plasma free DNA extraction kit for use.
2. The reagent preparation is carried out according to the instruction.
3. Chip sample introduction
(1) Detection system
In the embodiment, a digital PCR system biodata blue of Shanghai small sea turtle science and technology Limited is used, the system is a 5-channel fluorescence channel, 96 samples can be processed in parallel, and the number of effective liquid drops of a chip is 20 ten thousand.
(2) Sample introduction operation
According to the requirements of the digital PCR sample injection instrument using operation instructions, starting a chip sample injection program to finish the chip sample injection.
4. The chip thermal cycle reaction and detection analysis are the same as above.
5. Result of sample detection
Note: chromosome copy number is the sum of copy numbers of each relevant channel
Samples No. 1-3 have Z-score >3, namely the number of chromosome 21 is abnormal, and the 21-trisomy syndrome is predicted to be suffered. The detection result is stable with the expectation.
Variations and modifications to the above-described embodiments may occur to those skilled in the art based upon the disclosure and teachings of the above specification. Therefore, the present application is not limited to the specific embodiments disclosed and described above, and some modifications and variations of the present application should fall within the scope of the claims of the present application. In addition, although specific terms are used herein, they are used in a descriptive sense only and not for purposes of limitation.
SEQUENCE LISTING
<110> Jiangsu Shengji Gene science and technology Co., Ltd
<120> primers, probes, kit and method for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR
<130>191569CN-CH-I
<160>30
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aactgctcct tttccaaggc ctttcctg 28
<210>19
<211>21
<212>DNA
<213> Artificial sequence
<400>19
gagcagggtg agatcgactg a 21
<210>20
<211>24
<212>DNA
<213> Artificial sequence
<400>20
agaatatcac agcagcccct taac 24
<210>21
<211>24
<212>DNA
<213> Artificial sequence
<400>21
gccattggtt attactgcac attc 24
<210>22
<211>24
<212>DNA
<213> Artificial sequence
<400>22
gcaaagtgga taaatgctga ctca 24
<210>23
<211>23
<212>DNA
<213> Artificial sequence
<400>23
tgcctctgaa aagctggaag tag 23
<210>24
<211>23
<212>DNA
<213> Artificial sequence
<400>24
actaaaatgg gctgtgctcc tta 23
<210>25
<211>25
<212>DNA
<213> Artificial sequence
<400>25
atatatctgt tccaaacccc atctg 25
<210>26
<211>23
<212>DNA
<213> Artificial sequence
<400>26
tgggccagga gaaatcttat acc 23
<210>27
<211>24
<212>DNA
<213> Artificial sequence
<400>27
aggaacaagc ctggcacaga gccc 24
<210>28
<211>30
<212>DNA
<213> Artificial sequence
<400>28
acagcctcac ttcagactgt cttcttccca 30
<210>29
<211>28
<212>DNA
<213> Artificial sequence
<400>29
ctcttctctc cttcccgctt cttgcagt 28
<210>30
<211>29
<212>DNA
<213> Artificial sequence
<400>30
taattctatc ccttctccac tccctgggc 29
Claims (10)
1. A primer combination for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR (polymerase chain reaction), which is characterized by comprising:
a primer designed according to the detection site of human chromosome 21 has a nucleotide sequence shown as SEQ ID NO: 1 to SEQ ID NO: 12 is shown in the specification;
primers are designed according to the detection sites of human chromosomes 8 and 2 respectively, and the nucleotide sequences of the primers are shown as SEQ ID NO: 19 to SEQ ID NO: shown at 26.
2. A probe combination for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR, comprising:
the Taqman probe is designed according to the detection site of human chromosome 21, and the nucleotide sequence of the Taqman probe is shown as SEQ ID NO: 13 to SEQ ID NO: 18 is shown in the figure;
the Taqman probe is designed according to the detection sites of human chromosomes 8 and 2 respectively, and the nucleotide sequence of the Taqman probe is shown as SEQ ID NO: 27 to SEQ ID NO: shown at 30.
3. The probe combination for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR of claim 2, wherein Taqman probes designed according to the detection site of human chromosome 21 are labeled with FAM, HEX or ROX; marking a Taqman probe designed according to the detection site of the human chromosome 8 by CY 5; a Taqman probe designed according to the detection site of human chromosome 2 is marked by Alexa Fluor 700.
4. A kit for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR (polymerase chain reaction), which is characterized by comprising:
a specific primer and a Taqman probe which are designed according to the detection site of the human chromosome 21 and are used for amplifying and detecting a target gene;
specific primers and Taqman probes for detecting reference genes are designed according to the detection sites of human chromosomes 8 and 2 respectively;
wherein,
the nucleotide sequences of the specific primer and the Taqman probe for amplifying and detecting the target chromosome are shown as SEQ ID NO: 1 to SEQ ID NO: 18 is shown in the figure; the nucleotide sequences of the specific primers and the Taqman probe for amplifying and detecting the reference chromosome are shown as SEQ ID NO: 19 to SEQ ID NO: shown at 30.
5. The digital PCR-based noninvasive prenatal detection kit for fetal 21-trisomy syndrome according to claim 4, wherein:
marking a Taqman probe designed according to a detection site of a human chromosome 21 by FAM, HEX or ROX; marking a Taqman probe designed according to the detection site of the human chromosome 8 by CY 5; a Taqman probe designed according to the detection site of human chromosome 2 is marked by Alexa Fluor 700.
6. A method for noninvasive prenatal detection of fetal 21-trisomy syndrome based on digital PCR is characterized by comprising the following steps:
(1) obtaining free DNA from the peripheral blood of a pregnant woman to be detected as genome DNA to be detected;
(2) selecting a detection site of human chromosome 21, and designing a specific primer and a Taqman probe for amplifying and detecting a target chromosome; selecting detection sites of human chromosomes 8 and 2, and respectively designing specific primers and Taqman probes for amplifying and detecting reference chromosomes;
wherein, the nucleotide sequences of the specific primer and the Taqman probe for amplifying and detecting the target chromosome are shown as SEQ ID NO: 1 to SEQ ID NO: 18 is shown in the figure; the nucleotide sequences of the specific primers and the Taqman probe for amplifying and detecting the reference chromosome are shown as SEQ ID NO: 19 to SEQ ID NO: 30 is shown in the figure;
(3) preparing a digital PCR amplification reaction system, taking the to-be-detected genome DNA in the step (1) as a template, and performing digital PCR amplification and detection by using the specific primer and the Taqman probe in the step (2);
(4) respectively obtaining the copy number a of the chromosome 21 of the sample to be detected according to the fluorescent signals of the digital PCR21And the copy number b of the reference chromosome;
wherein the copy number b of the reference chromosome is the copy number of chromosome 2 of the sample to be detectedb2And copy number b of chromosome 88Average value of (d);
(5) calculating the copy number a of chromosome 21 of the sample to be detected21Y is the percentage of the copy number b of the reference chromosome of the sample21；
(6) Calculating the Z-score of No. 21 of the sample to be tested:
wherein,
y0copy number a of chromosome 21 as negative sample for trisomy syndrome21An average of the percentage of the copy number b of the sample reference chromosome;
σycopy number a of chromosome 21 as negative sample for trisomy syndrome21Standard deviation as a percentage of the copy number b of the sample reference chromosome;
(7) and judging whether the number of the chromosome 21 of the sample to be detected is abnormal or not according to the Z-score value of the chromosome 21 of the sample to be detected.
7. The digital PCR-based noninvasive prenatal detection of fetal 21-trisomy syndrome according to claim 6,
when (Z-score)21When the number is less than or equal to 1.96, the number of the No. 21 chromosome of the sample to be detected is normal; when 1.96 < (Z-score)21When the detection time is less than or equal to 3, re-detecting; when (Z-score)21>And 3, indicating that the number of the 21 chromosome in the sample to be detected is abnormal.
8. The digital PCR-based noninvasive prenatal detection method for fetal 21-trisomy syndrome according to claim 6, wherein Taqman probes designed according to the detection site of human chromosome 21 are labeled with FAM, HEX or ROX; marking a Taqman probe designed according to the detection site of the human chromosome 8 by CY 5; a Taqman probe designed according to the detection site of human chromosome 2 is marked by Alexa Fluor 700.
9. The digital PCR-based noninvasive prenatal detection of fetal 21-trisomy syndrome according to claim 6, wherein the digital PCR amplification reaction is composed of: 10 × dPCR Buffer: 3.5 mu L; enzyme: 1.0 μ L; primers and probes: 2.8 mu L; detecting the template: 1.0-17.5 muL; total volume: supplementing 30-35 μ L with ribozyme-free water.
10. The digital PCR-based noninvasive prenatal detection of fetal 21-trisomy syndrome according to claim 6, wherein the digital PCR amplification reaction is programmed to: 50 ℃ C: 10 minutes and 1 cycle; 90 ℃ C: 10 minutes and 1 cycle; 95 ℃: 20 seconds, 59 ℃: 40s, 45 cycles; keeping at 25 ℃.
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| CN112899358A (en) * | 2021-01-06 | 2021-06-04 | 南京普济生物有限公司 | Noninvasive prenatal fetal chromosome aneuploidy detection method and kit thereof |
| CN114921459A (en) * | 2022-05-16 | 2022-08-19 | 西北工业大学 | Primer combination for noninvasive prenatal screening of common fetal chromosomal abnormalities based on digital PCR technology |
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| CN105695567A (en) * | 2015-11-30 | 2016-06-22 | 北京昱晟达医疗科技有限公司 | Kit, primers, probe sequence and method for detecting fetus chromosome aneuploid |
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| CN104087672A (en) * | 2014-07-14 | 2014-10-08 | 钦州市妇幼保健院 | Kit for quickly detecting number of human chromosomes 21 by multiplex real-time fluorescence quantitative PCR (polymerase chain reaction) technique |
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| CN114921459A (en) * | 2022-05-16 | 2022-08-19 | 西北工业大学 | Primer combination for noninvasive prenatal screening of common fetal chromosomal abnormalities based on digital PCR technology |
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