Detection method for noninvasive prenatal detection of fetal trisomy 21 syndrome based on digital PCR

Abstract

The invention discloses a detection method for noninvasive prenatal detection of fetal trisomy 21 syndrome based on digital PCR, and relates to the technical field of digital PCR. According to the invention, a target area detection amplification primer and a Taqman-MGB probe are designed according to different sequences of human chromosome 21 genes, and a specific common amplification primer and a specific Taqman-MGB probe are designed according to similar sequences of the human chromosome 21 genes. Accurate detection of a low-proportion trisomy 21 syndrome positive sample is realized, and the omission ratio is greatly reduced. Compared with next-generation sequencing, multiple qPCR technologies and the like, the detection method provided by the invention has the advantages of simplicity in operation, simplicity in data analysis, short detection period, low cost and the like, meanwhile, by innovatively using a similar sequence design technology, high consistency of amplification efficiency of a detection gene and a reference gene is ensured, and the detection accuracy is improved. The detection sensitivity and accuracy are obviously improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of digital PCR, and particularly relates to a detection method for non-invasive prenatal detection of 21 trisomy syndrome of a fetus based on digital PCR. BACKGROUND

[0002] Chromosomal abnormality disease is one of the main diseases, and there is currently no effective treatment technology method for the disease, and the key lies in prevention, that is, prenatal screening and diagnosis. Chromosomal abnormality disease refers to abnormality of the number and / or morphological structure of chromosomes, which can occur on each chromosome, and among them, 21 trisomy (Down syndrome), 18 trisomy (Edward syndrome) and 13 trisomy (Patau syndrome) are the most common autosomal aneuploidy.

[0003] At present, serology is generally used for clinical screening to indirectly assess the risk of a fetus suffering from Down syndrome, and pregnant women with high risk in serological detection generally further undergo amniocentesis detection to confirm whether they have a Down syndrome fetus. The former is a non-invasive detection, but the detection rate is low and the false positive rate is high, and the detection result of the latter is accurate, but causes a 1% abortion rate. The detection accuracy based on the second-generation gene sequencing (NGS) technology method can reach 99.9%, but the price is high, the time is long, and a specially trained person is needed to prepare the library and perform bioinformatics data analysis.

[0004] The multiplex qPCR and NGS technologies in the prior art all belong to simultaneous amplification and detection of multiple targets, and it is unavoidable that the amplification efficiency of different sites in the multiplex PCR system is different, which will affect the detection sensitivity and accuracy to some extent. In order to solve the technical defects existing in the above-mentioned 21 trisomy syndrome detection method, the application provides a detection method for non-invasive prenatal detection of 21 trisomy syndrome of a fetus based on digital PCR. SUMMARY

[0005] The purpose of the application is to provide a detection method for non-invasive prenatal detection of 21 trisomy syndrome of a fetus based on digital PCR, which effectively combines the digital PCR technology and the similar sequence technology, strictly ensures the consistency of the amplification efficiency of the detection target and the internal reference target, improves the detection sensitivity and accuracy, and can significantly reduce the missed detection rate of positive fetuses.

[0006] To solve the above technical problems, the application is implemented by the following technical scheme: the application is a detection method for non-invasive prenatal detection of 21 trisomy syndrome of a fetus based on digital PCR, which comprises the following steps:

[0007] Step S1, designing primers and probes based on similar sequences

[0008] Different regions of human chromosome 21 are taken as detection targets, and similar sequences on human chromosomes 12, 15 and 1 are taken as internal reference sequences, and common specific primers and respective specific Taqman-MGB probes for amplification and detection are designed for the target chromosomes and the internal reference chromosomes respectively;

[0009] Step S2, designing primers and probes based on conventional sequences

[0010] Different regions of human chromosome 21 are taken as detection targets, and conventional sequences on human chromosome 1 are taken as internal reference sequences, and specific primers and Taqman-MGB probes for amplification and detection are designed for the target chromosomes and the internal reference chromosomes respectively;

[0011] Step S3, reference product detection based on similar sequences

[0012] DNA is extracted from negative pregnant women plasma samples, and genomic gDNA is obtained from 21 trisomy positive cell lines, and 200 bp small fragments are obtained by ultrasonic disruption;

[0013] The extracted negative plasma cfDNA and positive disrupted DNA are subjected to digital PCR amplification to obtain concentration values, and the extracted negative plasma cfDNA and positive disrupted DNA are mixed according to a certain concentration ratio to obtain different reference products of negative, 1.5%, 2% and 2.5%, and a digital PCR amplification reaction system is constructed based on step S1 to detect different reference products;

[0014] The copy number of chromosome 21 and the copy number concentration of the internal reference chromosome of the sample to be tested are obtained, the ratio of the copy number of chromosome 21 of the sample to be tested to the copy number of the internal reference chromosome of the sample is calculated, and whether the sample is a chromosome 21 copy number abnormality is judged according to the difference in the ratio of the negative and positive samples;

[0015] Step S4, reference product detection based on conventional sequences

[0016] Different reference products of negative, 1.5%, 2% and 2.5% are prepared in the same way as step S3, and a digital PCR amplification reaction system is constructed based on step S2 to detect different reference products.

[0017] The nucleotide sequence of the specific primer for amplifying and detecting the chromosome 21 sequence is shown in SEQ ID NO: 1-2, SEQ ID NO: 5-6, SEQ ID NO: 9-10, the nucleotide sequence of the specific Taqman-MGB probe for amplifying and detecting the chromosome 21 sequence is shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, and the specific Taqman-MGB probe is labeled with a FAM fluorescent group.

[0018] The nucleotide sequence of the specific primer for amplifying and detecting the chromosome 21 sequence and the reference chromosome sequence is shown in SEQ ID NO: 1-2, SEQ ID NO: 5-6, SEQ ID NO: 9-10, the nucleotide sequence of the specific Taqman-MGB probe for amplifying and detecting the reference chromosome sequence is shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, and the specific Taqman-MGB probe is labeled with a HEX fluorescent group.

[0019] The digital PCR amplification reaction system is preferably composed of: 4x dPCR Buffer: 10 μL; primer: 0.1-0.4 μL / reaction; probe: 0.03-0.2 μL / reaction; detection template: 20-25 μL; total volume: supplemented with deionized water to 40 μL; and the control program of the digital PCR amplification reaction is: 95℃: 2 min, 1 cycle; 95℃: 40 s, 1 cycle; 60℃: 40 s, 72℃: 40 s, 45 cycles.

[0020] The copy number of the reference chromosome is the average of the copy numbers of the chromosome 12, the chromosome 15 and the chromosome 1 of the sample to be detected, and the primer and the probe designed in steps S1 and S2 are diluted to 100 uM with deionized water after synthesis.

[0021] The primer and the probe in step S1 are designed according to the following principles: Primer 5.0 and Oligo 6 are used to design the primer and the probe for the chromosome 21 sequence and the reference sequence, the primer annealing temperature is 59-61℃, the probe annealing temperature with the completely matched template is 59-61℃, the difference site of the probe and the reference sequence needs to satisfy that the number of difference bases is not less than 3, which is used to ensure the detection specificity of the probe, and the specificity of the primer and the probe is detected by NCBI BLAST.

[0022] Further, the principle of designing the primer and the probe in the step S2 is that: the primer and the probe are designed for the sequence of the chromosome 21 and the internal reference sequence by using Primer 5.0 and Oligo 6, the annealing temperature of the primer is 59-61℃, the annealing temperature of the probe is 59-61℃, and the specificity of the primer and the probe is detected by NCBI BLAST.

[0023] It is further preferred in the application that the negative pregnant woman plasma sample obtained in the steps S3 and S4 is eluted by 35 μL deionized water after the DNA is extracted by the magnetic bead method free DNA mass extraction kit; the genomic gDNA is extracted from the 21 trisomy positive cell line by the blood / cell / tissue DNA extraction kit, and after 10-20 μL gDNA is mixed into the negative pregnant woman plasma, the DNA is extracted by the magnetic bead method free DNA mass extraction kit and eluted by 50-150 μL deionized water.

[0024] It is further preferred in the application that in the step S3, after the primer and the probe are designed based on the similar sequence, different reference products of negative, 1.5%, 2% and 2.5% are prepared and detected by the digital PCR amplification reaction system, the similar sequence design technology can detect all the reference products of 2% and 2.5%, and there is no cross with the negative reference product ratio, which indicates that the similar sequence + digital PCR design technology can obviously distinguish the negative and positive samples.

[0025] In the step S4, after the primer and the probe are designed based on the conventional sequence, different reference products of negative, 1.5%, 2% and 2.5% are prepared and detected by the digital PCR amplification reaction system, the conventional sequence design technology cannot detect the reference products of 2% and 2.5%, and there is obvious cross with the negative reference product ratio, which indicates that the conventional sequence + digital PCR design technology is easy to cause missed detection.

[0026] The application has the following beneficial effects: compared with the existing technology based on digital PCR, NGS technology and the like, the application can realize the effective detection of positive samples with a lower fetal ratio, and can avoid the defects of missing detection of a certain proportion of positive samples, the effective combination of the digital PCR technology and the similar sequence technology in the application strictly ensures the consistency of the amplification efficiency of the detection target and the internal reference target, improves the detection sensitivity and accuracy, and can significantly reduce the missed detection rate of the positive fetus. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0028] Figure 1 The figure is a detection result of the reference product in the similar sequence technical solution of Embodiment 2 of the present application.

[0029] Figure 2 The figure is a detection result of the reference product in the conventional sequence technical solution of Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be described clearly and completely in the following with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0031] Embodiment 1 Similar sequence primer probe design

[0032] Different region sequences on human chromosome 21 are taken as detection targets, and corresponding similar sequences on human chromosome 12, 15 and 1 are obtained as internal reference sequences, and common specific primers and respective specific Taqman-MGB probes for amplification and detection are designed for the target chromosome and the internal reference chromosome, specifically:

[0033] Different region sequences on human chromosome 21 are selected and downloaded from NCBI as detection targets, and corresponding similar sequences on human chromosome 12, 15 and 1 are obtained as internal reference sequences (i.e. reference region) through BLAT comparison on the UCSC website, and common specific primers and respective specific Taqman-MGB probes for amplification and detection are designed for the target chromosome and the internal reference chromosome sequences, and the designed primers and probes are synthesized by a synthesis company and diluted to 100 μM with deionized water.

[0034] The design principles of the primers and probes in this example are: Primer 5.0 and Oligo 6 are used to design primers and probes for chromosome 21 sequences and reference sequences, the annealing temperature of the primers is about 60°C, the annealing temperature of the probes with the completely matched template (T21 gene sequence) is about 60°C, the difference sites of the probes with the unmatched template (reference sequence) are as many as possible, and the number of difference bases is required to be greater than or equal to 3, so as to ensure the detection specificity of the probes, and NCBI BLAST is used for primer and probe specificity detection. Table 1 is a sequence table of specific primers and probe sequences used in the similar sequence system, wherein the underlined bases are difference bases.

[0035] Table 1 Similar sequence primers and probes

[0036]

[0037] Comparative Example 1 Conventional sequence primer probe design

[0038] Different region sequences on human chromosome 21 are used as detection targets, and a conventional sequence on human chromosome 1 is selected as a reference sequence, and specific primers and Taqman-MGB probes for amplification and detection are designed for the target chromosome and the reference chromosome, specifically:

[0039] Different region sequences on human chromosome 21 are selected and downloaded from NCBI as detection targets, and human chromosome 1 sequences are also selected as reference regions, and specific primers and Taqman-MGB probes for amplification and detection are designed for the target chromosome and the reference chromosome sequences, and the designed primers and probes are synthesized by a synthesis company and diluted to 100 μM with deionized water.

[0040] The design principles of the primers and probes in this example are: Primer 5.0 and Oligo 6 are used to design primers and probes for chromosome 21 sequences and reference sequences, the annealing temperature of the primers is about 60°C, the annealing temperature of the probes with the completely matched template (T21 gene sequence) is about 60°C, the difference sites of the probes with the unmatched template (reference sequence) are as many as possible, and the number of difference bases is required to be greater than or equal to 3, so as to ensure the detection specificity of the probes, and NCBI BLAST is used for primer and probe specificity detection. Table 1 is a sequence table of specific primers and probe sequences used in the similar sequence system, wherein the underlined bases are difference bases.

[0041] Table 2 Conventional sequence primers and probes

[0042]

[0043] Example 2 Reference product detection of similar sequence technical solution

[0044] Step (1), the negative pregnant women plasma samples obtained from the hospital were mixed, and the free DNA bulk extraction kit (version: MPG, item number: IVD5435) was used to extract the DNA, and 35-80 μL of deionized water was used for elution.

[0045] Step (2), the 21 trisomy positive cell lines were extracted using the blood / cell / tissue DNA extraction kit (item number: DP304-02) to obtain genomic DNA (gDNA), which was broken into small fragments with an average size of 200 bp by ultrasonic treatment, and 10-20 μL of gDNA was mixed into the negative pregnant women plasma, and the free DNA bulk extraction kit (version: MPG, item number: IVD5435) was used for extraction, and 50-150 μL of deionized water was used for elution.

[0046] Step (3), the extracted negative plasma cfDNA and positive broken DNA were subjected to PCR amplification and detection according to the reaction system and procedure of Table 3 and Table 4, and the concentration value (copy / μL) was obtained.

[0047] Table 3 Digital PCR amplification reaction system

[0048]

[0049] Table 4 Digital PCR reaction procedure

[0050]

[0051] Step (4), the negative cfDNA and positive broken gDNA were mixed according to the concentration ratio to obtain negative, 1.5%, 2% and 2.5% reference materials, and the reaction solution was prepared according to Table 3, and the PCR reaction was carried out according to the reaction procedure of Table 4, and the negative, 1.5%, 2% and 2.5% reference materials were detected, and the reference material detection results are shown in Table 5. Figure 1

[0052] ​When the primer and probe are designed based on the similar sequence, different reference samples of 0, 1.5%, 2% and 2.5% are prepared and detected by the digital PCR amplification reaction system, the copy number of chromosome 21 and the copy number concentration of the internal reference chromosome of the sample to be tested are obtained, the ratio of the copy number of chromosome 21 to the copy number of the internal reference chromosome of the sample to be tested is calculated, and whether the sample is the copy number abnormality of chromosome 21 is judged according to the ratio difference between the negative and positive samples. Similar sequence design technology can detect all 2% and 2.5% reference samples, and there is no crossover with the negative reference sample ratio, indicating that the similar sequence + digital PCR design technology can obviously distinguish between negative and positive samples. In this application, the UCSC database BLAT comparison results show that the similarity of the selected similar sequence on chromosome 1 as the internal reference sequence to chromosome 21 reaches 95.8%, which indicates that the similarity of the selected internal reference sequence to chromosome 21 is greater than 90%, as shown in Table 5:

[0053] Table 5 UCSC database BLAT comparison data table

[0054]

[0055] Comparative Example 2 Reference sample detection by conventional sequence technology

[0056] Step (1), the negative pregnant woman plasma samples obtained from the hospital were mixed, and the magnetic bead method free DNA large extraction kit (version: MPG, item number: IVD5435) was used for extraction, and 35 μL deionized water was used for elution.

[0057] Step (2), the 21 trisomy positive cell line was extracted using the blood / cell / tissue DNA extraction kit (item number: DP304-02), and the genomic DNA (gDNA) was obtained, which was broken into small fragments with an average size of 200 bp by ultrasonic treatment, and 10-20 μL gDNA was mixed into the negative pregnant woman plasma, and the magnetic bead method free DNA large extraction kit (version: MPG, item number: IVD5435) was used for extraction, and 150 μL deionized water was used for elution.

[0058] Step (3), the extracted negative plasma cfDNA and positive broken DNA were subjected to PCR amplification and detection according to the reaction system and procedure of Table 3 and Table 4, and the concentration value (copy / μL) was obtained.

[0059] Step (4), the negative cfDNA and positive broken gDNA were mixed according to the concentration ratio, and the negative, 1.5%, 2% and 2.5% reference samples were obtained, and the reaction solution was prepared according to Table 3, and the PCR reaction was carried out according to the reaction procedure of Table 4, and the negative, 1.5%, 2% and 2.5% reference samples were detected, and the reference sample detection results are shown in Table 6. Figure 2 ​

[0060] When the primers and probes are designed based on the conventional sequence, different reference samples of 0, 1.5%, 2% and 2.5% are prepared and detected by the digital PCR amplification reaction system, the copy number of chromosome 21 and the copy number concentration of the reference chromosome of the sample to be tested are obtained, the ratio of the copy number of chromosome 21 to the copy number of the reference chromosome of the sample to be tested is calculated, and whether the sample is a chromosome 21 copy number abnormality is determined according to the ratio difference between the negative and positive samples. The conventional sequence design technology cannot detect the 2% and 2.5% reference samples, and there is a significant cross between the ratio of the negative reference sample, indicating that the conventional sequence + digital PCR design technology is prone to missed detection.

[0061] In the present application, the nucleotide sequence of the specific primer for amplifying and detecting the sequence of chromosome 21 is shown in SEQ ID NO: 1-2, 5-6, 9-10, the nucleotide sequence of the specific Taqman-MGB probe for amplifying and detecting the sequence of chromosome 21 is shown in SEQ ID NO: 3, 7, 11, and the specific Taqman-MGB probe is labeled with FAM fluorescent group.

[0062] The nucleotide sequence of the common primer for amplifying and detecting the sequence of chromosome 21 and the sequence of the reference chromosome is shown in SEQ ID NO: 1-2, 5-6, 9-10, the nucleotide sequence of the specific Taqman-MGB probe for amplifying and detecting the sequence of the reference chromosome is shown in SEQ ID NO: 4, 8, 12, and the specific Taqman-MGB probe is labeled with HEX fluorescent group.

[0063] The digital PCR amplification reaction system is constructed to detect the DNA of the plasma sample to be tested, and the composition of the digital PCR amplification reaction system is: 4xdPCR Buffer: 10 μL; primer: 0.1-0.4 μL / reaction; probe: 0.03-0.2 μL / reaction; detection template: 20-25 μL; total volume: supplemented with deionized water to 40 μL. The control program of the digital PCR amplification reaction is: 95℃: 2min, 1 cycle; 95℃: 40s, 1 cycle; 60℃: 40s, 72℃: 40s, 45 cycles.

[0064] According to the digital PCR result in embodiment 2, the copy number of chromosome 21 of the sample to be tested and the copy number concentration of the internal reference chromosome are obtained; wherein the copy number of the internal reference chromosome is the average value of the copy numbers of chromosome 12, chromosome 15 and chromosome 1 of the sample to be tested; the ratio of the copy number of chromosome 21 of the sample to be tested to the copy number of the internal reference chromosome of the sample is calculated respectively, and whether the sample is the chromosome 21 copy number abnormality is judged according to the ratio difference between the negative and positive samples.

[0065] In the description of the present specification, the description referring to the terms "one embodiment", "an example", "a specific example" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0066] The preferred embodiments of the application disclosed above are only used to help explain the application. The preferred embodiments do not describe all the details and limit the application to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of the present specification. The present specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the application, so that those skilled in the art can well understand and utilize the application. The application is limited only by the claims and their full scope and equivalents.

Claims

1. A method for detecting fetal trisomy 21 syndrome based on digital PCR non-invasive prenatal testing, characterized in that, It comprises the following steps: S1. Using different sequences on chromosome 21 as detection targets, selecting similar sequences on chromosome 12, 15 and 1 as internal reference sequences, designing common specific primers and respective specific Taqman-MGB probes; S2. Using different sequences on chromosome 21 as detection targets, selecting conventional sequences on chromosome 1 as internal reference sequences, designing specific primers and Taqman-MGB probes; S3. Selecting negative pregnant women plasma samples to extract DNA, extracting 21 trisomy positive cell lines to obtain genomic gDNA, and obtaining 200 bp small fragments by ultrasonic disruption; After the concentration of negative plasma cfDNA and positive disrupted DNA is amplified, different reference products of 1.5%, 2% and 2.5% are obtained, and the digital PCR amplification reaction system is constructed on the basis of step S1 to detect different reference products; The copy number of chromosome 21 and the copy number concentration of the internal reference chromosome of the sample to be tested are obtained, the ratio of the copy number of chromosome 21 to the copy number of the internal reference chromosome of the sample to be tested is calculated, and whether the sample is a chromosome 21 copy number abnormality is judged according to the ratio difference between the negative and positive samples; S4. According to the same way of step S3, different reference products of 1.5%, 2% and 2.5% are obtained, and the digital PCR amplification reaction system is constructed on the basis of step S2 to detect different reference products.

2. The method of claim 1, wherein the method is based on digital PCR. The nucleotide sequence of the specific primer for amplifying and detecting the sequence of chromosome 21 is shown in SEQ ID NO: 1-2, 5-6, 9-10, the nucleotide sequence of the specific Taqman-MGB probe for amplifying and detecting the sequence of chromosome 21 is shown in SEQ ID NO: 3, 7, 11, and the specific Taqman-MGB probe is labeled with FAM fluorescent group. 3.The method of claim 1, wherein the method is characterized by, The nucleotide sequence of the common primer for amplifying and detecting the sequence of chromosome 21 and the sequence of the internal reference chromosome is shown in SEQ ID NO: 1-2, 5-6, 9-10, the nucleotide sequence of the specific Taqman-MGB probe for amplifying and detecting the sequence of the internal reference chromosome is shown in SEQ ID NO: 4, 8, 12, and the specific Taqman-MGB probe is labeled with HEX fluorescent group.

4. The method of claim 1, wherein the method is based on digital PCR. The composition of the digital PCR amplification reaction system is: 4×dPCR Buffer: 10 μL; Primer: 0.1-0.4 μL / reaction; Probe: 0.03-0.2 μL / reaction; detection template: 20-25 μL; total volume: supplemented with deionized water to 40 μL.

5. The method of claim 1, wherein the method is based on digital PCR. The control procedure of the digital PCR amplification reaction is: 95℃: 2 min, 1 cycle; 95℃: 40 s, 1 cycle; 60℃: 40 s, 72℃: 40 s, 45 cycles.

6. The method of claim 1, wherein the method is based on digital PCR. The copy number of the internal reference chromosome in the step S3 is the average value of the copy numbers of the 12th, 15th and 1st chromosomes of the sample to be detected, and the primers and probes designed in the steps S1 and S2 are diluted to 100 uM with deionized water after synthesis.

7. The method of claim 1, wherein the method is based on digital PCR. The principle of designing the primers and probes in the step S1 is that the primers and probes are designed for the 21st chromosome sequence and the internal reference sequence by using Primer 5.0 and Oligo 6, the annealing temperature of the primers is 59-61℃, the annealing temperature of the probes to the completely matched templates is 59-61℃, and the difference sites of the probes to the internal reference sequence need to satisfy that the number of difference bases is not less than 3, which is used to ensure the detection specificity of the probes, and the specificity of the primers and probes is detected by NCBI BLAST.

8. The method of claim 1, wherein the method is based on digital PCR. The principle of designing the primers and probes in the step S2 is that the primers and probes are designed for the 21st chromosome sequence and the internal reference sequence by using Primer 5.0 and Oligo 6, the annealing temperature of the primers is 59-61℃, the annealing temperature of the probes is 59-61℃, and the specificity of the primers and probes is detected by NCBI BLAST.

9. The method of claim 1, wherein the method is based on digital PCR for non-invasive prenatal testing of fetal trisomy 21 syndrome. After the DNA is extracted from the negative pregnant woman plasma sample obtained in the steps S3 and S4 by using the magnetic bead method free DNA mass extraction kit, the DNA is eluted with 35 uL deionized water; the genomic gDNA is extracted from the 21-trisomy positive cell line by using the blood / cell / tissue DNA extraction kit, and after 10-20 uL gDNA is mixed into the negative pregnant woman plasma, the DNA is extracted by using the magnetic bead method free DNA mass extraction kit and eluted with 50-150 uL deionized water.

10. The method for detecting fetal trisomy 21 syndrome based on digital PCR non-invasive prenatal testing according to claim 1, characterized in that, In the step S3, after the primers and probes are designed based on similar sequences, negative, 1.5%, 2% and 2.5% different reference samples are prepared and detected by using the digital PCR amplification reaction system, the similar sequence design technology can detect all the 2% and 2.5% reference samples, and there is no cross with the negative reference sample, which indicates that the similar sequence + digital PCR design technology can obviously distinguish the negative and positive samples; In the step S4, after the primers and probes are designed based on conventional sequences, negative, 1.5%, 2% and 2.5% different reference samples are prepared and detected by using the digital PCR amplification reaction system, the conventional sequence design technology cannot detect the 2% and 2.5% reference samples, and there is obvious cross with the negative reference sample, which indicates that the conventional sequence + digital PCR design technology is easy to cause missed detection.

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