Method for non-invasive prenatal detection of 21-trisomy syndrome of fetus based on digital PCR

Abstract

The application discloses a detection method for noninvasive prenatal detection of fetus 21 trisomy syndrome based on digital PCR, and relates to the technical field of digital PCR.The application designs target region detection amplification primers and Taqman-MGB probes according to different sequences of human chromosome 21 genes, and designs specific common amplification primers and specific Taqman-MGB probes according to similar sequences.Based on digital PCR and improved human chromosome copy number variation detection technology, the application realizes accurate detection of low-proportion 21 trisomy syndrome positive samples, and greatly reduces the missed detection rate.Compared with second-generation sequencing and multiplex qPCR technology, the detection method has the advantages of simple operation, simple data analysis, short detection period, low cost and the like, and through the innovative use of similar sequence design technology, the detection method guarantees the high consistency of the amplification efficiency of the detection gene and the internal reference gene, and significantly improves the detection sensitivity and accuracy.

Description

Technical Field

[0001] This invention belongs to the field of digital PCR technology, and in particular relates to a method for detecting fetal trisomy 21 syndrome using non-invasive prenatal testing based on digital PCR. Background Technology

[0002] Chromosomal abnormalities are among the most common diseases. Currently, there are no effective treatments for these conditions; prevention, specifically prenatal screening and diagnosis, is crucial. Chromosomal abnormalities refer to numerical and / or morphological changes in chromosomes, which can occur on any chromosome. The most common autosomal aneuploidies are trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome).

[0003] Currently, clinical screening commonly uses serological testing to indirectly assess the risk of Down syndrome in the fetus. Pregnant women with high-risk results from serological tests usually undergo amniocentesis to confirm whether they are carrying a fetus with Down syndrome. While the former is a non-invasive test, it has a low detection rate and a high false positive rate. The latter, although accurate, carries a 1% miscarriage rate. Next-generation sequencing (NGS) technology can achieve an accuracy rate of 99.9%, but it is expensive, time-consuming, and requires trained specialists to prepare the library and perform bioinformatics data analysis.

[0004] Existing technologies such as multiplex qPCR and NGS involve the simultaneous amplification and detection of multiple targets, inevitably leading to differences in amplification efficiency at different sites within the multiplex PCR system, which can affect detection sensitivity and accuracy to some extent. To address these technical shortcomings of existing methods for detecting Down syndrome (trisomy 21), this application provides a non-invasive prenatal detection method for fetal Down syndrome based on digital PCR. Summary of the Invention

[0005] The purpose of this invention is to provide a non-invasive prenatal detection method for fetal trisomy 21 based on digital PCR. By effectively combining digital PCR technology and similar sequence technology, the amplification efficiency of the detection target and the internal reference target is strictly guaranteed, which improves the detection sensitivity and accuracy and can significantly reduce the false negative rate of positive fetuses.

[0006] To solve the above-mentioned technical problems, the present invention is achieved through the following technical solution: The present invention is a method for detecting fetal trisomy 21 syndrome based on digital PCR non-invasive prenatal testing, comprising the following steps:

[0007] Step S1: Design primers and probes based on similar sequences

[0008] Different regions of human chromosome 21 were used as detection targets, and similar sequences on human chromosomes 12, 15 and 1 were obtained as internal reference sequences. Common specific primers and specific Taqman-MGB probes for amplification and detection were designed for the target chromosomes and internal reference chromosomes, respectively.

[0009] Step S2: Design primers and probes based on conventional sequences

[0010] Using different regions of human chromosome 21 as detection targets and conventional sequences on human chromosome 1 as internal reference sequences, specific primers and Taqman-MGB probes for amplification and detection were designed for the target chromosome and internal reference chromosome, respectively.

[0011] Step S3: Reference Detection Based on Similar Sequences

[0012] DNA was extracted from plasma samples of negative pregnant women, and genomic gDNA was obtained from trisomy 21 positive cell lines. The fragments were then broken down by ultrasound to obtain a 200 bp fragment.

[0013] The concentration values ​​of the extracted negative plasma cfDNA and positive fragmented DNA were obtained by digital PCR amplification. The extracted negative plasma cfDNA and positive fragmented DNA were mixed at a certain concentration ratio to obtain different reference samples of negative, 1.5%, 2% and 2.5%. Based on step S1, a digital PCR amplification reaction system was constructed to detect the different reference samples.

[0014] Obtain the copy number of chromosome 21 of the test sample and the copy number concentration of the internal reference chromosome, calculate the ratio of the copy number of chromosome 21 of the test sample to the copy number of the internal reference chromosome of the sample, and determine whether the sample has an abnormal copy number of chromosome 21 based on the difference between the negative and positive sample ratios.

[0015] Step S4: Reference Detection Based on Conventional Sequences

[0016] Different reference samples of negative, 1.5%, 2% and 2.5% were prepared in the same manner as in step S3. Based on step S2, a digital PCR amplification reaction system was constructed to detect the different reference samples.

[0017] In a further preferred embodiment of the present invention, the nucleotide sequences of the specific primers for amplifying and detecting the chromosome 21 sequence are shown in SEQ ID NO: 1–SEQ ID NO: 2, SEQ ID NO: 5–SEQ ID NO: 6, SEQ ID NO: 9–SEQ ID NO: 10, and the nucleotide sequences of the specific Taqman-MGB probes for amplifying and detecting the chromosome 21 sequence are shown in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, and the specific Taqman-MGB probes are labeled with FAM fluorescent groups.

[0018] In a further preferred embodiment of the present invention, the nucleotide sequences of the common primers used for amplifying and detecting the chromosome 21 sequence and the internal reference chromosome sequence are shown in SEQ ID NO: 1-SEQ ID NO: 2, SEQ ID NO: 5-SEQ ID NO: 6, SEQ ID NO: 9-SEQ ID NO: 10, and the nucleotide sequences of the specific Taqman-MGB probes used for amplifying and detecting the internal reference chromosome sequence are shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, and the specific Taqman-MGB probes are labeled with HEX fluorescent groups.

[0019] A further preferred embodiment of the present invention is that the digital PCR amplification reaction system comprises: 4×dPCR Buffer: 10 μL; primers: 0.1-0.4 μL / reaction; probes: 0.03-0.2 μL / reaction; detection template: 20-25 μL; total volume: made up to 40 μL with deionized water; and the control program for 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] In a further preferred embodiment of the present invention, the copy number of the internal reference chromosome is the average copy number of chromosomes 12, 15 and 1 of the sample to be tested, and the primers and probes designed in steps S1 and S2 are diluted to 100 μM with deionized water before use.

[0021] In a further preferred embodiment of the present invention, the primer and probe design principles in step S1 are as follows: Primer 5.0 and Oligo 6 are used to design primers and probes for the chromosome 21 sequence and the internal reference sequence. The primer annealing temperature is 59-61℃, the annealing temperature of the probe and the perfectly matched template is 59-61℃, and the difference sites between the probe and the internal reference sequence must meet the requirement that the number of different bases is not less than 3, in order to ensure the detection specificity of the probe. The specificity of the primers and probes is detected by NCBI BLAST.

[0022] Furthermore, the primer and probe design principles in step S2 are as follows: Primer 5.0 and Oligo 6 are used to design primers and probes for the chromosome 21 sequence and internal reference sequence. The primer annealing temperature is 59-61℃, and the probe annealing temperature is 59-61℃. The specificity of the primers and probes is detected by NCBI BLAST.

[0023] In a further preferred embodiment of the present invention, the negative pregnant woman plasma sample obtained in steps S3 and S4 is eluted with 35 μL of deionized water after DNA is extracted using a magnetic bead-based cell-free DNA extraction kit; the genomic gDNA is obtained by extracting trisomy 21 positive cell lines using a blood / cell / tissue DNA extraction kit, and after mixing 10-20 μL of gDNA into the negative pregnant woman plasma, DNA is extracted using a magnetic bead-based cell-free DNA extraction kit and eluted with 50-150 μL of deionized water.

[0024] In a further preferred embodiment of the present invention, in step S3, after primers and probes are designed based on similar sequences, different reference samples of negative, 1.5%, 2%, and 2.5% are prepared and detected by digital PCR amplification reaction system. The similar sequence design technology can detect all of the 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 clearly distinguish between negative and positive samples.

[0025] In step S4, after primers and probes are designed based on conventional sequences, different reference samples of negative, 1.5%, 2%, and 2.5% are prepared and detected by digital PCR amplification reaction system. Conventional sequence design technology cannot detect the 2% and 2.5% reference samples, and there is a significant overlap with the ratio of negative reference samples, indicating that the use of conventional sequence + digital PCR design technology is prone to false negatives.

[0026] The present invention has the following beneficial effects: Compared with the existing technologies such as digital PCR and NGS, which can accurately detect Down syndrome, but are insufficient to effectively detect positive samples in a lower proportion of fetuses, resulting in a certain proportion of missed positive samples, the present invention, based on the effective combination of digital PCR technology and similar sequence technology, strictly ensures the consistency of amplification efficiency of the detection target and the internal reference target, improves detection sensitivity and accuracy, and can significantly reduce the missed detection rate of positive fetuses. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 This is a diagram showing the detection results of a reference sample in the similar sequence technology solution of Embodiment 2 of the present invention.

[0029] Figure 2 This is a diagram showing the test results of the reference sample using the conventional sequence technology solution in Comparative Example 2 of this invention. Detailed Implementation

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

[0031] Example 1: Design of Primers and Probes with Similar Sequences

[0032] Using different regions of human chromosome 21 as detection targets, and obtaining similar sequences from human chromosomes 12, 15, and 1 as internal reference sequences, common specific primers and their respective specific Taqman-MGB probes for amplification and detection were designed for both the target chromosomes and the internal reference chromosomes; specifically:

[0033] Sequences of different regions of human chromosome 21 were selected and downloaded from NCBI as detection targets. Similar sequences on human chromosomes 12, 15, and 1 were obtained through BLAT alignment on the UCSC website and used as internal reference sequences (i.e., reference regions). Common specific primers and specific Taqman-MGB probes for amplification and detection were designed for the target chromosome and internal reference chromosome sequences, respectively. The designed primers and probes were synthesized by a synthetic company and diluted to 100 μM with deionized water before use.

[0034] The primer and probe design principles in this embodiment are as follows: Primer 5.0 and Oligo 6 were used to design primers and probes for the chromosome 21 sequence and the internal reference sequence. The primer annealing temperature was around 60℃, and the annealing temperature of the probe and the perfectly matched template (T21 gene sequence) was around 60℃. The probe and the unmatched template (internal reference sequence) should have as many different sites as possible, requiring at least 3 different bases, to ensure the detection specificity of the probe. NCBI BLAST was used to detect the specificity of the primers and probes. Table 1 shows the sequence listing of specific primers and probes used in the similar sequence system, where underlined bases are different bases.

[0035] Table 1. Primers and probes with similar sequences

[0036]

[0037] Comparative Example 1: Design of Conventional Sequence Primers and Probes

[0038] Using different regions of human chromosome 21 as detection targets and conventional sequences from human chromosome 1 as internal reference sequences, specific primers and Taqman-MGB probes for amplification and detection were designed for the target chromosome and internal reference chromosome, respectively. Specifically:

[0039] Different regions of human chromosome 21 were selected and downloaded from NCBI as detection targets. Similarly, human chromosome 1 was selected as a reference region. Specific primers and Taqman-MGB probes for amplification and detection were designed for the target chromosome and internal reference chromosome sequences, respectively. The designed primers and probes were synthesized by a synthesis company and diluted to 100 μM with deionized water before use.

[0040] The primer and probe design principles in this comparative example are as follows: Primer 5.0 and Oligo 6 were used to design primers and probes for the chromosome 21 sequence and internal reference sequence. The primer annealing temperature was around 60℃, and the probe annealing temperature was around 60℃. NCBI BLAST was used to detect the specificity of the primers and probes. Table 2 shows the specific primers and probes used in the conventional sequence system.

[0041] Table 2. Conventional sequence primers and probes

[0042]

[0043] Example 2: Reference Detection of Similar Sequence Technology Scheme

[0044] Step (1): Mix the negative pregnant woman plasma samples obtained from the hospital and extract them using the magnetic bead method cell-free DNA extraction kit (version: MPG, catalog number: IVD5435), eluting with 35-80 μL of deionized water.

[0045] Step (2): Use the blood / cell / tissue DNA extraction kit (catalog number: DP304-02) to extract genomic DNA (gDNA) from the trisomy 21 positive cell line. Sonicate the gDNA to break it into small fragments of an average of 200 bp. Take 10-20 μL of gDNA and mix it into the plasma of a negative pregnant woman. Use the magnetic bead method cell-free DNA extraction kit (version: MPG, catalog number: IVD5435) to extract the DNA and elute with 50-150 μL of deionized water.

[0046] Step (3): The extracted negative plasma cfDNA and positive fragmented DNA were subjected to PCR amplification and detection according to the reaction system and procedure in Tables 3 and 4 to obtain the concentration value (copy / μL).

[0047] Table 3. System of digital PCR amplification reaction

[0048]

[0049] Table 4 Digital PCR Reaction Procedure

[0050]

[0051] Step (4): Mix negative cfDNA and positive fragmented gDNA according to the concentration ratio to obtain negative, 1.5%, 2%, and 2.5% reference samples. Prepare the reaction solution according to Table 3, and perform PCR reaction according to the reaction procedure in Table 4. Detect the negative, 1.5%, 2%, and 2.5% reference samples. The test results of the reference samples are as follows: Figure 1 As shown.

[0052] After designing primers and probes based on similar sequences, negative, 1.5%, 2%, and 2.5% reference samples were prepared and detected using a digital PCR amplification system. The copy number of chromosome 21 in the test sample and the copy number concentration of the internal reference chromosome were obtained. The ratio of the copy number of chromosome 21 in the test sample to the copy number of the internal reference chromosome in that sample was calculated. The difference in the ratio between negative and positive samples was used to determine whether the sample had an abnormal copy number of chromosome 21. The similar sequence design technology could detect all 2% and 2.5% reference samples, with no overlap with the negative reference sample ratio, indicating that the similar sequence + digital PCR design technology can clearly distinguish between negative and positive samples. In this application, the BLAT comparison results from the UCSC database showed that the similar sequence selected as the internal reference sequence had a similarity of 95.8% with chromosome 21, indicating that the similarity between the internal reference sequence selected in this application and chromosome 21 was greater than 90%, as shown in Table 5.

[0053] Table 5 UCSC Database BLAT Comparison Data Table

[0054]

[0055] Comparative Example 2: Reference Sample Testing Using Conventional Sequence Technology

[0056] Step (1): Mix the negative pregnant woman plasma samples obtained from the hospital and extract them using the magnetic bead method cell-free DNA extraction kit (version: MPG, catalog number: IVD5435), and elute with 35 μL of deionized water.

[0057] Step (2): Use the blood / cell / tissue DNA extraction kit (catalog number: DP304-02) to extract genomic DNA (gDNA) from the trisomy 21 positive cell line. Sonicate the gDNA to break it into small fragments of an average of 200 bp. Take 10-20 μL of gDNA and mix it into the plasma of a negative pregnant woman. Use the magnetic bead method cell-free DNA extraction kit (version: MPG, catalog number: IVD5435) to extract the DNA and elute with 150 μL of deionized water.

[0058] Step (3): The extracted negative plasma cfDNA and positive fragmented DNA were subjected to PCR amplification and detection according to the reaction system and procedure in Tables 3 and 4 to obtain the concentration value (copy / μL).

[0059] Step (4): Mix negative cfDNA and positive fragmented gDNA according to the concentration ratio to obtain negative, 1.5%, 2%, and 2.5% reference samples. Prepare the reaction solution according to Table 3, and perform PCR reaction according to the reaction procedure in Table 4. Detect the negative, 1.5%, 2%, and 2.5% reference samples. The test results of the reference samples are as follows: Figure 2 As shown.

[0060] After designing primers and probes based on conventional sequences, negative, 1.5%, 2%, and 2.5% reference samples were prepared and detected using a digital PCR amplification system. The copy number of chromosome 21 in the test sample and the copy number concentration of the internal reference chromosome were obtained. The ratio of the copy number of chromosome 21 in the test sample to the copy number of the internal reference chromosome in that sample was calculated. The difference in the ratio between negative and positive samples was used to determine whether the sample had an abnormal copy number of chromosome 21. Conventional sequence design techniques failed to detect the 2% and 2.5% reference samples, and there was a significant overlap in the ratio with the negative reference sample, indicating that the use of conventional sequence + digital PCR design techniques is prone to false negatives.

[0061] In this application, the nucleotide sequences of the specific primers used for amplifying and detecting the chromosome 21 sequence are shown in SEQ ID NO: 1 to SEQ ID NO: 2, SEQ ID NO: 5 to SEQ ID NO: 6, and SEQ ID NO: 9 to SEQ ID NO: 10, and the nucleotide sequences of the specific Taqman-MGB probes used for amplifying and detecting the chromosome 21 sequence are shown in SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 11. The specific Taqman-MGB probes are labeled with the FAM fluorescent group.

[0062] The nucleotide sequences of the common primers used for amplifying and detecting the chromosome 21 sequence and the internal reference chromosome sequence are shown in SEQ ID NO: 1-SEQ ID NO: 2, SEQ ID NO: 5-SEQ ID NO: 6, SEQ ID NO: 9-SEQ ID NO: 10. The nucleotide sequences of the specific Taqman-MGB probes used for amplifying and detecting the internal reference chromosome sequence are shown in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12. The specific Taqman-MGB probes are labeled with HEX fluorescent groups.

[0063] A digital PCR amplification reaction system was constructed for detecting DNA from the plasma sample to be tested. The composition of the digital PCR amplification reaction system was as follows: 4×dPCR Buffer: 10 μL; primers: 0.1-0.4 μL / reaction; probes: 0.03-0.2 μL / reaction; detection template: 20-25 μL; total volume: made up to 40 μL with deionized water. The control program for the digital PCR amplification reaction was as follows: 95℃: 2 min, 1 cycle; 95℃: 40 s, 1 cycle; 60℃: 40 s, 72℃: 40 s, 45 cycles.

[0064] Based on the digital PCR results in Example 2, the copy number of chromosome 21 in the test sample and the copy number concentration of the internal reference chromosome were obtained. The copy number of the internal reference chromosome was the average of the copy numbers of chromosomes 12, 15, and 1 in the test sample. The ratio of the copy number of chromosome 21 in the test sample to the copy number of the internal reference chromosome in the sample was calculated. The difference between the negative and positive sample ratios was used to determine whether the sample had an abnormal copy number of chromosome 21.

[0065] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0066] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. Use of a reagent set in the preparation of a fetal trisomy 21 cell-free DNA detection kit, characterized in that, The reagent set includes reagent set A, reagent set B, and reagent set C. Reagent set A includes detection targets based on different regions of chromosome 21, internal reference sequences based on similar sequences on chromosomes 12, 15, and 1, and corresponding designed shared specific primers and their respective specific Taqman-MGB probes. The nucleotide sequences of the specific primers used to amplify and detect the chromosome 21 sequence are shown in SEQ ID NO: 1–SEQ ID NO: 2, SEQ ID NO: 5–SEQ ID NO: 6, and SEQ ID NO: 9–SEQ ID NO:

10. The nucleotide sequences of the specific Taqman-MGB probes used to amplify and detect the chromosome 21 sequence are shown in SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO:

11. The nucleotide sequences of the shared primers used to amplify and detect the chromosome 21 sequence and the internal reference chromosome sequence are shown in SEQ ID NO: 1–SEQ ID NO: 2, SEQ ID NO: 5–SEQ ID NO: 6, and SEQ ID NO: 9–SEQ ID NO:

10. As shown in NO: 10, the nucleotide sequences of the specific Taqman-MGB probes used for amplifying and detecting the internal reference chromosome sequence are shown in SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 12; The reagent group B includes detection targets based on different regions of chromosome 21, internal reference sequences based on conventional sequences of chromosome 1, and corresponding designed specific primers and Taqman-MGB probes. The reagent group C includes different reference samples prepared by mixing negative plasma cfDNA and positive fragmented DNA based on concentration amplification, at concentrations of 1.5%, 2%, and 2.5%. A digital PCR amplification reaction system is constructed based on the above reagent group to detect the different reference samples.

2. The use of the reagent group according to claim 1 in the preparation of a fetal 21-trisomy free DNA detection kit, characterized in that, The specific Taqman-MGB probe used for amplifying and detecting chromosome 21 sequences is labeled with the FAM fluorescent group.

3. The use of the reagent group according to claim 1 in the preparation of a fetal 21-trisomy free DNA detection kit, characterized in that, The specific Taqman-MGB probe used for amplifying and detecting the internal reference chromosome sequence is labeled with the HEX fluorescent group.

4. The use of the reagent group according to claim 1 in the preparation of a fetal 21-trisomy free DNA detection kit, characterized in that, The digital PCR amplification reaction system consisted of: 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: add deionized water to 40 μL.

5. The use of the reagent group according to claim 1 in the preparation of a fetal 21-trisomy free DNA detection kit, characterized in that, The control program for the digital PCR amplification reaction is as follows: 95℃: 2 min, 1 cycle; 95℃: 40 s; 60℃: 40 s, 72℃: 40 s, 45 cycles.

6. The use of the reagent group according to claim 1 in the preparation of a fetal 21-trisomy free DNA detection kit, characterized in that, The primer and probe design principles in reagent group B are as follows: Primer 5.0 and Oligo 6 are used to design primers and probes for chromosome 21 sequence and internal reference sequence. The primer annealing temperature is 59-61℃, and the probe annealing temperature is 59-61℃. The specificity of the primers and probes is detected by NCBI BLAST.

7. The use of the reagent group according to claim 1 in the preparation of a fetal 21-trisomy free DNA detection kit, characterized in that, The method for preparing negative plasma cfDNA is as follows: obtain a negative pregnant woman's plasma sample, extract DNA using a magnetic bead-based cell-free DNA extraction kit, and then wash with 35 μL of deionized water; The positive fragmented DNA preparation method is as follows: Genomic gDNA is extracted from the trisomy 21 positive cell line using a blood / cell / tissue DNA extraction kit. After mixing 10-20 μL of gDNA into the plasma of a negative pregnant woman, DNA is extracted using a magnetic bead-based cell-free DNA extraction kit and eluted with 50-150 μL of deionized water.

8. The use of the reagent group according to claim 1 in the preparation of a fetal 21-trisomy free DNA detection kit, characterized in that, After designing primers and probes based on similar sequences, different reference samples of negative, 1.5%, 2%, and 2.5% were prepared and detected by digital PCR amplification reaction system. The similar sequence design technology could detect all of the 2% and 2.5% reference samples, and there was no crossover with the negative reference sample ratio, indicating that the similar sequence + digital PCR design technology can clearly distinguish between negative and positive samples. When primers and probes were designed based on conventional sequences, different reference samples of negative, 1.5%, 2%, and 2.5% were prepared and detected by digital PCR amplification reaction system. Conventional sequence design technology could not detect the 2% and 2.5% reference samples, and there was a significant overlap with the negative reference sample ratio, indicating that the use of conventional sequence + digital PCR design technology is prone to false negatives.

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