Primer probe combination for detecting aneuploid abnormality of chromosome, kit and application

Abstract

The invention provides a primer probe combination for detecting aneuploid abnormality of chromosomes, a kit and application, and belongs to the technical field of chromosome abnormality detection. The nucleotide sequences of the primer probe combination are as shown in SEQ ID NO.1-SEQ ID NO.150. The primer probe combination or the kit disclosed by the invention can be used for rapidly and simultaneously detecting whether the number of the chromosome 21, the chromosome 18, the chromosome 13, the chromosome X or the chromosome Y is abnormal or not, and the primer probe combination or the kit is high in detection speed, high in flux, low in cost and accurate in detection result. The invention plays a positive role in detecting chromosome aneuploid abnormality and preventing and controlling the chromosome aneuploid abnormality. The primer probe combination or the kit disclosed by the invention is expected to be applied to the fields of prenatal screening, prenatal diagnosis, neonatal hereditary disease screening and the like, has the advantages of rapidness, high efficiency, accuracy and the like, and provides a reliable basis for clinical emergency decision-making.

Description

Technical Field

[0001] The present invention belongs to the technical field of chromosome abnormality detection, and in particular relates to a primer-probe combination, a kit and an application thereof for detecting chromosome aneuploidy abnormality. Background Art

[0002] Abnormal chromosome numbers (such as aneuploidy) and structural abnormalities (including inversions, translocations, deletions, and duplications) are the main causes of major birth defects, including fetal developmental delay and intellectual disability. Trisomy 21 (Down syndrome) is the most common chromosomal trisomy, affecting approximately 1 in 700 to 1 in 1000 newborns. Children with Down syndrome experience developmental delays in cognitive, language, and motor skills, and are often unable to fully care for themselves. Trisomy 18 (Edwards syndrome) affects approximately 1 in 6000 to 1 in 8000 newborns, often accompanied by severe organ malformations, such as heart and kidney abnormalities. Most children die shortly after birth, with only a few surviving beyond a year, resulting in a significant emotional impact on families. Trisomy 13 (Patau syndrome) affects approximately 1 in 12000 to 1 in 16000 newborns, resulting in severe malformations of the central nervous system and cardiovascular system. The fetus has a shorter lifespan, typically dying in utero or within a few days to weeks of birth. The incidence of 45,X (Turner syndrome) in females is approximately 1 in 2,500. The phenotype of patients varies widely, with short stature and gonadal dysgenesis being common features. Malformations of organs such as the heart and kidneys may also occur. Some patients may experience cognitive and psychological problems, such as learning difficulties and social anxiety. The incidence of 47,XXY (Klinefelter syndrome) in males is approximately 1 in 600 to 1 in 1,000. Patients primarily present with hypoplastic secondary sexual characteristics, azoospermia, or severe oligospermia. Some patients may also experience learning disabilities and delayed language development. Public awareness of Turner and Klinefelter syndromes is relatively low, and many patients are diagnosed in adolescence or adulthood, missing the optimal time for treatment. As adults, these patients face significant psychological pressure in education, employment, and marriage.

[0003] Prenatal screening and diagnosis are core technologies for preventing birth defects associated with chromosomal abnormalities and play a key role in detecting fetal chromosomal aneuploidies. Traditional serological screening primarily targets trisomy 21 and trisomy 18, but its detection indicators are often affected by multiple factors. For example, maternal age, weight, ethnicity, prevalence of conditions such as diabetes, and the presence of multiple pregnancies can affect the levels of serological markers (such as PAPP-A and β-hCG), leading to false-positive or false-negative results. Currently commonly used clinical techniques such as noninvasive prenatal testing (NIPT), karyotyping, chromosomal microarray analysis, and high-throughput sequencing offer high accuracy but still have limitations such as long testing cycles, complex procedures, and high costs. Noninvasive prenatal testing (NIPT) is based on the detection of cell-free fetal DNA (cffDNA) in maternal peripheral blood. NGS-NIPT uses high-throughput sequencing to analyze cffDNA and calculate fetal chromosome dosage, enabling the detection of fetal chromosomal abnormalities such as trisomy 21, trisomy 18, and trisomy 13. Compared with traditional serological screening, NGS-NIPT has higher accuracy, but the operation process is complicated and expensive, and professional bioinformatics analysis technology is required to support the interpretation of sequencing results. It has not yet been widely used as a first-line screening tool for fetal aneuploidy screening. The internationally recognized gold standard for the diagnosis of fetal chromosomal aneuploidy is still chromosome karyotype analysis, which is a detection method after chorionic villus sampling or amniocentesis. However, this technology has two major limitations: first, the cell culture cycle is as long as 2 to 3 weeks, which prolongs the diagnosis cycle and misses the optimal clinical intervention window; second, it has strict requirements on sample quality. It must meet sterile conditions and avoid contamination of maternal blood cells to ensure that the sample contains a sufficient number of living cells to meet in vitro culture requirements.

[0004] With the development of molecular diagnostics, digital PCR (dPCR) has emerged as a new generation of absolute nucleic acid quantification technology. Its core principle is to physically segment the traditional PCR reaction system into thousands to millions of independent microreaction units through microfluidics. Each unit contains only 0 or 1 target molecule template. After PCR amplification, positive signals are counted using a high-throughput fluorescence detection system, combined with a Poisson distribution algorithm, to achieve absolute quantification of the target molecule copy number in the original sample. However, the design of a kit using digital PCR technology to rapidly detect chromosomal aneuploidy abnormalities and achieve comprehensive testing in a single tube has not yet been reported. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide a primer-probe combination, a kit and an application for detecting chromosomal aneuploidy abnormalities. The primer-probe combination or the kit of the present invention can quickly and simultaneously detect whether chromosome 21, chromosome 18, chromosome 13, chromosome X or chromosome Y is abnormal, and the detection accuracy is high.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a primer probe combination for detecting chromosomal aneuploidy abnormalities, wherein the primer probe combination consists of a primer probe combination for chromosome 21, a primer probe combination for chromosome 18, a primer probe combination for chromosome 13, a primer probe combination for chromosome X, and a primer probe combination for chromosome Y.

[0008] The chromosome 13 primer-probe combination consists of a chromosome 13 primer and a chromosome 13 probe, the nucleotide sequences of the chromosome 13 primers are shown in SEQ ID NO.1 to SEQ ID NO.20, and the nucleotide sequences of the chromosome 13 probes are shown in SEQ ID NO.21 to SEQ ID NO.30;

[0009] The chromosome 18 primer-probe combination consists of a chromosome 18 primer and a chromosome 18 probe, the nucleotide sequences of the chromosome 18 primers are shown in SEQ ID NO.31 to SEQ ID NO.50, and the nucleotide sequences of the chromosome 18 probes are shown in SEQ ID NO.51 to SEQ ID NO.60;

[0010] The chromosome 21 primer-probe combination consists of a chromosome 21 primer and a chromosome 21 probe, the nucleotide sequences of the chromosome 21 primers are shown in SEQ ID NO.61 to SEQ ID NO.80, and the nucleotide sequences of the chromosome 21 probes are shown in SEQ ID NO.81 to SEQ ID NO.90;

[0011] The X chromosome primer probe combination consists of an X chromosome primer and an X chromosome probe, wherein the nucleotide sequences of the X chromosome primers are shown in SEQ ID NO.91 to SEQ ID NO.110, and the nucleotide sequences of the X chromosome probes are shown in SEQ ID NO.111 to SEQ ID NO.120;

[0012] The Y chromosome primer probe combination consists of a Y chromosome primer and a Y chromosome probe. The nucleotide sequences of the Y chromosome primers are shown in SEQ ID NO.121 to SEQ ID NO.140, and the nucleotide sequences of the Y chromosome probes are shown in SEQ ID NO.141 to SEQ ID NO.150.

[0013] Preferably, the chromosome 13 probe, chromosome 18 probe, chromosome 21 probe, chromosome X probe and chromosome Y probe are modified with fluorescent labels.

[0014] Preferably, the fluorescent label of the chromosome 21 probe is a FAM label; the fluorescent label of the chromosome 18 probe is a HEX label; the fluorescent label of the chromosome 13 probe is a Quasar705 label; the fluorescent label of the chromosome X probe is a Cy5 label; and the fluorescent label of the chromosome Y probe is a ROX label.

[0015] The present invention provides a kit for detecting chromosomal aneuploidy abnormality, which comprises the above-mentioned primer-probe combination.

[0016] Preferably, the kit further comprises a dPCR reaction system.

[0017] Preferably, the dPCR reaction system consists of the following components: 3×Maxuseful dPCR Buffer 10 μL, Taq DNA polymerase 0.5 μL, 10 μM upstream primer 6 μL, 10 μM downstream primer 6 μL, 10 μM probe 3 μL, final template concentration 12 ng, and ddH2O supplemented to 30 μL.

[0018] The present invention provides an application of the above primer-probe combination in the preparation of a product for detecting chromosomal aneuploidy abnormalities.

[0019] The present invention provides an application of the above primer-probe combination in the preparation of a product for detecting chromosomal diseases.

[0020] Preferably, the chromosomal disease includes one or more of Down syndrome, Edwards syndrome, Patau syndrome, Turner syndrome, Klinefelter syndrome, super female syndrome and super male syndrome.

[0021] Preferably, the samples used for the test include maternal peripheral blood plasma, early pregnancy cervical exfoliated cells, fetal chorionic villus tissue, amniotic fluid cells, abortion products or neonatal peripheral blood.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention provides a primer probe combination, a kit and an application for detecting chromosomal aneuploidy abnormalities. The primer probe combination or the kit of the present invention can quickly and simultaneously detect whether there is a numerical abnormality in chromosome 21, chromosome 18, chromosome 13, chromosome X or chromosome Y. The primer probe combination or the kit has a fast detection speed, high throughput, low cost and accurate test results. The present invention has a positive effect on the detection and prevention of chromosomal aneuploidy abnormalities. The primer probe combination or the kit of the present invention is expected to be applied in the fields of prenatal screening, prenatal diagnosis, neonatal genetic disease screening, etc., with the advantages of being fast, efficient and accurate, and providing a reliable basis for clinical emergency decision-making. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of super-multiplex digital PCR detection;

[0025] Figure 2 The left picture in the middle shows the result of detecting a normal karyotype (46,XX) using the detection method of the present invention, and the right picture shows the result of chromosomal karyotype diagnosis as a normal karyotype (46,XX);

[0026] Figure 3 The left picture in the middle shows the result of detecting a normal karyotype (46, XY) using the detection method of the present invention, and the right picture shows the result of chromosomal karyotype diagnosis as a normal karyotype (46, XY);

[0027] Figure 4 The left middle picture shows the result of detecting chromosome abnormality (47, XY, +21) using the detection method of the present invention, and the right middle picture shows the result of chromosome abnormality (47, XY, +21) diagnosed by chromosome karyotype;

[0028] Figure 5 The middle left picture shows the results of detecting sex chromosome abnormality (45, XO) using the detection method of the present invention, and the right picture shows the results of karyotype diagnosis of sex chromosome abnormality (45, XO);

[0029] Figure 6 The middle left picture shows the result of detecting Patau syndrome (T13) using the detection method of the present invention, and the right picture shows the result of karyotype diagnosis of Patau syndrome (47,XX,+13);

[0030] Figure 7 The left middle figure shows the result of using the detection method of the present invention to detect that the NGS-NIPT high risk (T21) is a normal karyotype (46, XY). The right middle figure shows the result of chromosome karyotype diagnosis that the NGS-NIPT high risk (T21) is a normal karyotype (46, XY).

[0031] Figure 8 The left middle picture shows the result of detecting super male syndrome (47, XYY) using the detection method of the present invention, and the right middle picture shows the result of karyotype diagnosis of super male syndrome (47, XYY);

[0032] Figure 9 The left picture in the middle shows the result of detecting Edward's syndrome (T18) using the detection method of the present invention, and the right picture shows the result of chromosomal karyotype diagnosis of Edward's syndrome (47, XY, +18);

[0033] Figure 10 The middle left picture shows the result of Turner syndrome (45, XO) diagnosed by the detection method of the present invention through abortion product tissue, and the right picture shows the result of Turner syndrome (45, XO) diagnosed by chromosome karyotype. DETAILED DESCRIPTION

[0034] The present invention aims to provide a super-multiplex digital PCR detection primer probe combination or kit with fast detection speed, high throughput, low cost and accurate detection results for Down syndrome (T21), Edwards syndrome (T18), Patau syndrome (T13), Turner syndrome (45,XO), Klinefelter syndrome (47,XXY), super-female syndrome (47,XXX) and super-male syndrome (47,XYY).

[0035] The present invention provides a primer probe combination for detecting chromosomal aneuploidy abnormalities, wherein the primer probe combination consists of a primer probe combination for chromosome 21, a primer probe combination for chromosome 18, a primer probe combination for chromosome 13, a primer probe combination for chromosome X, and a primer probe combination for chromosome Y.

[0036] The chromosome 13 primer-probe combination consists of a chromosome 13 primer and a chromosome 13 probe, the nucleotide sequences of the chromosome 13 primers are shown in SEQ ID NO.1 to SEQ ID NO.20, and the nucleotide sequences of the chromosome 13 probes are shown in SEQ ID NO.21 to SEQ ID NO.30;

[0037] The chromosome 18 primer-probe combination consists of a chromosome 18 primer and a chromosome 18 probe, the nucleotide sequences of the chromosome 18 primers are shown in SEQ ID NO.31 to SEQ ID NO.50, and the nucleotide sequences of the chromosome 18 probes are shown in SEQ ID NO.51 to SEQ ID NO.60;

[0038] The chromosome 21 primer-probe combination consists of a chromosome 21 primer and a chromosome 21 probe, the nucleotide sequences of the chromosome 21 primers are shown in SEQ ID NO.61 to SEQ ID NO.80, and the nucleotide sequences of the chromosome 21 probes are shown in SEQ ID NO.81 to SEQ ID NO.90;

[0039] The X chromosome primer probe combination consists of an X chromosome primer and an X chromosome probe, wherein the nucleotide sequences of the X chromosome primers are shown in SEQ ID NO.91 to SEQ ID NO.110, and the nucleotide sequences of the X chromosome probes are shown in SEQ ID NO.111 to SEQ ID NO.120;

[0040] The Y chromosome primer probe combination consists of a Y chromosome primer and a Y chromosome probe. The nucleotide sequences of the Y chromosome primers are shown in SEQ ID NO.121 to SEQ ID NO.140, and the nucleotide sequences of the Y chromosome probes are shown in SEQ ID NO.141 to SEQ ID NO.150.

[0041] In the present invention, the chromosome 13 probe, chromosome 18 probe, chromosome 21 probe, chromosome X probe, and chromosome Y probe are modified with fluorescent labels. The fluorescent label of the chromosome 21 probe is a FAM label; the fluorescent label of the chromosome 18 probe is a HEX label; the fluorescent label of the chromosome 13 probe is a Quasar 705 label; the fluorescent label of the chromosome X probe is a Cy5 label; and the fluorescent label of the chromosome Y probe is a ROX label. The chromosome 13 probe, chromosome 18 probe, chromosome 21 probe, chromosome X probe, and chromosome Y probe are modified with a quencher group. The quencher group of the chromosome 21 probe is BHQ1; the quencher group of the chromosome 18 probe is BHQ1; the quencher group of the chromosome 13 probe is BHQ3; the quencher group of the chromosome X probe is BHQ2; and the quencher group of the chromosome Y probe is BHQ2. Specifically, the chromosome 21 probe was labeled with FAM at its 5' end and with BHQ1 at its 3' end; the chromosome 18 probe was labeled with HEX at its 5' end and with BHQ1 at its 3' end; the chromosome 13 probe was labeled with Quasar705 at its 5' end and with BHQ3 at its 3' end; the chromosome X probe was labeled with Cy5 at its 5' end and with BHQ2 at its 3' end; and the chromosome Y probe was labeled with ROX at its 5' end and with BHQ2 at its 3' end.

[0042] The present invention uses a specific primer-probe combination designed to cover conserved regions on the long and short arms of target chromosomes (chromosomes 21, 18, 13, X, and Y). Furthermore, through bioinformatics analysis, it avoids regions known to be prone to mutations such as deletions and duplications. This optimized design significantly improves the specificity and accuracy of amplification of the target region, ensuring more reliable test results.

[0043] The present invention uses five sets of specific primer-probe combinations, which are precisely designed for chromosomes 21, 18, 13, X, and Y, respectively. This allows for flexible detection of the following chromosomal abnormality syndromes: selective detection for a single disease or single-tube full-scale testing to meet different clinical needs.

[0044] The specific primer-probe combination of the present invention can be used to accurately detect one or more of Down syndrome (T21, trisomy 21), Edwards syndrome (T18, trisomy 18), Patau syndrome (T13, trisomy 13), and sex chromosome abnormality syndromes, wherein the sex chromosome abnormality syndromes are one or more of Turner syndrome (45, XO), Klinefelter syndrome (47, XXY), super female syndrome (47, XXX) and super male syndrome (47, XYY).

[0045] The present invention provides a kit for detecting chromosomal aneuploidy abnormality, which comprises the above-mentioned primer-probe combination.

[0046] In the present invention, the kit also includes a dPCR reaction system. The dPCR reaction system consists of the following components: 10 μL of 3×Maximum Useful dPCR Buffer, 0.5 μL of TaqDNA polymerase, 6 μL of a 10 μM upstream primer, 6 μL of a 10 μM downstream primer, 3 μL of a 10 μM probe, a final template concentration of 12 ng, and ddH2O to 30 μL. The present invention does not specifically limit the source of the 3×Maximum Useful dPCR Buffer or TaqDNA polymerase; commercially available products in the art can be used.

[0047] The kit of the present invention has the advantages of fast detection speed, accurate detection results, low cost, etc., is easy to carry out in routine clinical laboratories, has extremely high application value in clinical practice, and can also provide a basis for prenatal diagnosis strategies.

[0048] The present invention provides an application of the above primer-probe combination in the preparation of a product for detecting chromosomal aneuploidy abnormalities.

[0049] The present invention provides an application of the above primer-probe combination in the preparation of a product for detecting chromosomal diseases.

[0050] In the present invention, the chromosomal disease includes one or more of Down syndrome, Edwards syndrome, Patau syndrome, Turner syndrome, Klinefelter syndrome, super female syndrome and super male syndrome.

[0051] In the present invention, the samples used for detection include maternal peripheral blood plasma, early pregnancy cervical canal exfoliated cells, fetal chorionic villus tissue, amniotic fluid cells, abortion products or neonatal peripheral blood.

[0052] When using primer-probe combinations or kits for rapid detection of chromosomal aneuploidy, the criteria are as follows:

[0053] The copy numbers of chromosomes 13, 18, 21, X, and Y are counted as N 13 、N 18 、N21 、N X 、N Y Considering that the number of sex chromosomes is unknown, the copy numbers of chromosomes 13, 18, and 21 are used as references. The copy number ratios of the target chromosomes are calculated by pairwise comparison, which is recorded as R 13 / 21 、R 13 / 18 、R 18 / 13 、R 18 / 21 、R 21 / 18 、R 21 / 13 、R X / 21 、R X / 18 、R X / 13 、R Y / 21 、R Y / 18 、R Y / 13 and R X / Y , and then calculate the Z-score (Z 13 、Z 18 、Z 21 、Z X 、Z Y ), if the Z-score (Z value) ≤ 1.96, the chromosome number of the sample to be tested is judged to be normal, that is, normal diploid; if 1.96 < Z value ≤ 2.58 (gray area), it is judged to be suspected positive and requires re-testing for confirmation or further karyotype diagnosis; if the Z value is greater than 2.58, the chromosome number of the sample to be tested is judged to be abnormal (confidence level of 99%).

[0054] 1. When the copy number ratio R is in the range of 0.9308 to 1.0814, the target chromosome is diploid (for example: R 21 / 18 =0.9492 and R 21 / 13 =1.0146, then chromosome 21 can be determined to be diploid);

[0055] 2. When the copy number ratio R is greater than 1.0814, the target chromosome is triploid (for example: R 21 / 18 =1.9212 and R 21 / 13 =1.6587, it can be determined that chromosome 21 is triploid, T21);

[0056] 3. When the copy number ratio R is less than 0.9308, the target chromosome is haploid (for example: R X / 21 =0.6107 and R X / 18 =0.5272 and R X / 13 =0.5061, then the X chromosome can be determined to be haploid);

[0057] 4. When the copy number ratio R is equal to 0, there is no target chromosome (for example: R Y / 21 =0 and R Y / 18 =0 and R Y / 13=0, it can be determined that the Y chromosome is missing).

[0058] The purpose of the present invention is to establish an efficient and accurate rapid detection method for common chromosomal aneuploidies based on a dPCR platform using the above-mentioned primer-probe combination or kit. The method shows excellent performance indicators in the detection of common T21, T18, T13 and sex chromosome abnormalities (45,XO, 47,XXY, 47,XXX, 47,XXY), with sensitivity and specificity both reaching 100% (Kappa=1). Compared with traditional diagnostic methods (such as amniotic fluid chromosome karyotyping), the super-multiplex dPCR technology established by the present invention has the following breakthrough advantages: (1) The detection cycle is significantly shortened to 4 hours, greatly improving the timeliness of diagnosis; (2) The operation process is simplified, making it easier to promote clinical standardization; (3) A single run can complete the detection of 48-96 samples, with the potential for large-scale screening; (4) It breaks through the sample quality limit and does not have strict requirements for sterile conditions; (5) It has strong anti-interference ability and can effectively overcome maternal cell contamination; (6) Only 1-2 mL of amniotic fluid or an equivalent amount of peripheral blood plasma is required to complete the detection, reducing the error introduced by culture, etc.

[0059] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0060] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0061] In the following examples, the 3×Maxuseful dPCR Buffer was from the Digital PCR Universal Kit (purchased from Shanghai Little Turtle Technology Co., Ltd.).

[0062] Example 1

[0063] A method for detecting chromosomal aneuploidy abnormality, comprising the following steps:

[0064] (1) Magnetic bead method for DNA extraction

[0065] The MagMAX cell-free DNA extraction kit (purchased from ThermoFisher, product number A29319) was used to extract nucleic acid DNA from the sample. The specific steps are as follows:

[0066] 1. Take an appropriate amount of sample, which is maternal peripheral blood plasma, early pregnancy cervical exfoliated cells, chorionic villus tissue, amniotic fluid cells, abortion product tissue cells or neonatal peripheral blood, centrifuge at 12000rpm for 10min, and retain maternal peripheral blood plasma, early pregnancy cervical exfoliated cell pellet, fetal chorionic villus tissue cells, amniotic fluid cells, abortion product tissue cells or neonatal peripheral blood mononuclear cells, add lysis buffer respectively, and mix thoroughly.

[0067] 2. Add 25 μL of proteinase K, mix thoroughly, and incubate at 56°C for 30 min.

[0068] 3. Add 2 mL of binding buffer, add 10 μL of magnetic beads, vortex mix for 1 minute, let stand at room temperature for 10 minutes, vortex mix again for 1 minute, let stand at room temperature for 10 minutes to allow the DNA to adsorb to the surface of the magnetic beads.

[0069] 4. Place the centrifuge tube on a magnetic rack and let it stand for 5 minutes to allow the magnetic beads to aggregate on the tube wall until the supernatant is clear. Carefully aspirate and discard the supernatant to avoid stirring the magnetic beads.

[0070] 5. Remove the centrifuge tube, add 1 mL of Washing Buffer I, and transfer the Washing Buffer I containing the magnetic beads to a 1.5 mL centrifuge tube.

[0071] 6. Perform magnetic separation using a magnetic stand for 1 minute and carefully discard the supernatant.

[0072] 7. Add 1 mL of Wash Buffer II and vortex to mix; perform magnetic separation using a magnetic stand for 1 min and carefully discard the supernatant.

[0073] 8. Use a 10 μL pipette tip to aspirate the wash solution II remaining at the bottom of the centrifuge tube, and dry the tube at room temperature with the lid open for 8 minutes to evaporate the residual ethanol (avoid excessive drying that affects DNA dissolution).

[0074] 9. Add 100 μL of elution buffer and vortex to mix.

[0075] 10. Perform magnetic separation again for 1 minute and transfer the supernatant to a new 1.5 mL centrifuge tube. The supernatant is the purified DNA sample.

[0076] 11. Measure the concentration of the obtained DNA sample and store it at -20℃ for future use.

[0077] (2) Primer and probe design

[0078] Using oligo7.6 software, multiple sets of specific primers and probes were designed for conserved sequences on chromosomes 21, 18, 13, X, and Y. The primers and probes were required to meet the following conditions: (1) the annealing temperature of all primers was 60±1°C, and the annealing temperature of the probes was 70±2°C; (2) the length of all primers was between 18 and 25 bp, and the length of the probes was between 25 and 35 bp; (3) the sequence amplified by the primers was 90 to 100 bp; (4) the Taqman probes were fluorescently labeled with FAM, VIC, Quasar705, Cy5, and ROX for chromosomes 21, 18, 13, X, and Y, respectively. The specific information of the primers and probes is shown in Table 1.

[0079] Table 1 Information of primers and probes

[0080]

[0081]

[0082]

[0083]

[0084]

[0085] Among them, the chromosome 21 probe in Table 1 was labeled with FAM at the 5' end and BHQ1 at the 3' end; the chromosome 18 probe was labeled with HEX at the 5' end and BHQ1 at the 3' end; the chromosome 13 probe was labeled with Quasar705 at the 5' end and BHQ3 at the 3' end; the chromosome X probe was labeled with Cy5 at the 5' end and BHQ2 at the 3' end; and the chromosome Y probe was labeled with ROX at the 5' end and BHQ2 at the 3' end.

[0086] (3) Digital PCR reaction system and reaction conditions

[0087] 1. Reagent preparation: Thaw the reagent at room temperature, vortex briefly to mix, and centrifuge briefly.

[0088] 2. Reaction system preparation: 3× Maxuseful dPCR Buffer 10μL, Taq DNA polymerase 0.5μL, 10μM upstream primer 6μL (final concentration 200nM), 10μM downstream primer 6μL (final concentration 200nM), 10μM probe 3μL (final concentration 100nM), template final concentration 12ng, ddH2O to 30μL.

[0089] 3. Sample loading: According to the instructions of the fully automatic sample processing system, select the injection mode and set the injection process (select the corresponding digital PCR chip, reagent well positions, oil tank group, and pipette tip group). After setting, start the injection. The sample injector automatically loads and evenly distributes the reaction system to tens of thousands of chip reaction wells, completely covering the entire chip.

[0090] 4. dPCR reaction: The reaction solution was divided into 20,000 nanoliter droplets using a droplet digital PCR system. The droplet reaction chip was placed in a PCR instrument for amplification. Each droplet contained at most one target DNA molecule. Amplification conditions were: 50°C for 10 minutes, 94°C for 5 minutes, followed by 45 cycles of 94°C for 10 seconds, 60°C for 60 seconds, and then incubation at 20°C.

[0091] (4) Signal detection

[0092] Based on the fluorescence signal intensity and fluorescence threshold of each microdroplet in each fluorescence channel after amplification, and by using statistical formulas to determine the ratio of single-positive microdroplets to co-positive microdroplets, precise quantitative detection and analysis of multiple target nucleic acid sequences can be achieved to determine the presence of aneuploid chromosomes. After digital PCR amplification, strict quality control standards are established, requiring a valid droplet rate greater than 85% for a single dPCR reaction. The copy number ratio between each chromosome is then calculated.

[0093] (V) Data Analysis

[0094] The copy numbers of chromosomes 13, 18, 21, X, and Y are counted as N 13 、N 18 、N 21 、N X 、N Y Considering that the number of sex chromosomes is unknown, the copy numbers of chromosomes 13, 18, and 21 are used as references. The copy number ratios of the target chromosomes are calculated by pairwise comparison, which is recorded as R 13 / 21 、R 13 / 18 、R 18 / 13 、R 18 / 21 、R 21 / 18 、R 21 / 13 、R X / 21 、R X / 18 、R X / 13 、R Y / 21 、R Y / 18 、R Y / 13 and R X / Y , and then calculate the Z value (Z 13 、Z 18 、Z 21 、Z X 、Z Y Z-score = (Ai-Mean) / SD, where Ai represents the copy number ratio of each sample to be tested, Mean represents the mean of each ratio, and SD represents the standard deviation. If the Z-score (Z-score) is ≤1.96, the chromosome number of the sample to be tested is judged to be normal, that is, normal diploid; if 1.96 < Z-score ≤2.58 (gray area), it is judged to be suspected positive and requires re-testing for confirmation or further karyotype diagnosis; if the Z-score is >2.58, the chromosome number of the sample to be tested is judged to be abnormal (confidence level of 99%).

[0095] 1. When the copy number ratio R is in the range of 0.9308 to 1.0814, the target chromosome is diploid (for example: R 21 / 18 =0.9492 and R 21 / 13 =1.0146, then chromosome 21 can be determined to be diploid);

[0096] 2. When the copy number ratio R is greater than 1.0814, the target chromosome is triploid (for example: R 21 / 18 =1.9212 and R 21 / 13 =1.6587, it can be determined that chromosome 21 is triploid, T21);

[0097] 3. When the copy number ratio R is less than 0.9308, the target chromosome is haploid (for example: R X / 21 =0.6107 and R X / 18 =0.5272 and R X / 13 =0.5061, then the X chromosome can be determined to be haploid);

[0098] 4. When the copy number ratio R is equal to 0, there is no target chromosome (for example: R Y / 21 =0 and R Y / 18 =0 and R Y / 13 =0, it can be determined that the Y chromosome is missing).

[0099] Schematic diagram of ultra-multiplex digital PCR detection is shown in Figure 1 .

[0100] Example 2

[0101] A primer probe combination for detecting chromosomal aneuploidy abnormalities consists of a primer probe combination for chromosome 21, a primer probe combination for chromosome 18, a primer probe combination for chromosome 13, a primer probe combination for chromosome X, and a primer probe combination for chromosome Y.

[0102] The chromosome 13 primer-probe combination consists of a chromosome 13 primer and a chromosome 13 probe, the nucleotide sequences of the chromosome 13 primers are shown in SEQ ID NO.1 to SEQ ID NO.20, and the nucleotide sequences of the chromosome 13 probes are shown in SEQ ID NO.21 to SEQ ID NO.30;

[0103] The chromosome 18 primer-probe combination consists of a chromosome 18 primer and a chromosome 18 probe, the nucleotide sequences of the chromosome 18 primers are shown in SEQ ID NO.31 to SEQ ID NO.50, and the nucleotide sequences of the chromosome 18 probes are shown in SEQ ID NO.51 to SEQ ID NO.60;

[0104] The chromosome 21 primer-probe combination consists of a chromosome 21 primer and a chromosome 21 probe, the nucleotide sequences of the chromosome 21 primers are shown in SEQ ID NO.61 to SEQ ID NO.80, and the nucleotide sequences of the chromosome 21 probes are shown in SEQ ID NO.81 to SEQ ID NO.90;

[0105] The X chromosome primer probe combination consists of an X chromosome primer and an X chromosome probe, wherein the nucleotide sequences of the X chromosome primers are shown in SEQ ID NO.91 to SEQ ID NO.110, and the nucleotide sequences of the X chromosome probes are shown in SEQ ID NO.111 to SEQ ID NO.120;

[0106] The Y chromosome primer probe combination consists of a Y chromosome primer and a Y chromosome probe. The nucleotide sequences of the Y chromosome primers are shown in SEQ ID NO.121 to SEQ ID NO.140, and the nucleotide sequences of the Y chromosome probes are shown in SEQ ID NO.141 to SEQ ID NO.150.

[0107] The specific nucleotide sequence information is shown in Table 1.

[0108] Example 3

[0109] Application of primer-probe combinations for detecting chromosomal aneuploidy abnormalities for normal karyotype (46,XX):

[0110] In this example, 1 mL of a peripheral blood sample with a known normal karyotype (46,XX) was used to perform chromosome karyotype detection using the primer-probe combination described in Example 2 and the method for detecting chromosome aneuploidy described in Example 1. G-banding was also used to diagnose the chromosome karyotype.

[0111] The copy number of each target chromosome is calculated by software, and the copy number of chromosomes 13, 18, 21, X, and Y is counted as N. 13 、N 18 、N 21 、N X 、N Y . Calculate the copy number ratios R1 and R2, and determine the Z value.

[0112] The copy number ratio of chromosome 13 is R1 = N 13 ÷N 21 ; R2=N 13 ÷N 18 .

[0113] The copy number ratio of chromosome 18 is R1=N 18 ÷N 21 ; R2=N 18 ÷N 13 .

[0114] The copy number ratio of chromosome 21 is R1=N 21 ÷N 18 ; R2=N 21÷N 13 .

[0115] The copy number ratio of chromosome X is R1 = N x ÷N 21 ; R2=N x ÷N 18 .

[0116] The copy number ratio of chromosome Y is R1 = N Y ÷N 21 ; R2=N Y ÷N 18 .

[0117] Among them, Z 13 =-0.18, Z 18 =1.69, Z 21 =0.18, Z X =-0.46, Y missing.

[0118] Table 2 Results of detecting normal karyotype (46,XX) using the detection method of the present invention

[0119]

[0120] According to Table 2 and Figure 2 The results show that the result determined by the detection method of the present invention is a normal diploid female, which is consistent with the karyotype diagnosis result (46,XX).

[0121] Example 4

[0122] Application of primer-probe combinations for detecting chromosomal aneuploidy abnormalities for normal karyotype (46, XY):

[0123] In this example, 2 mL of amniotic fluid sample with a known normal karyotype (46, XY) was used, and the primer-probe combination described in Example 2 was used to perform chromosome karyotype detection according to the method for detecting chromosome aneuploidy abnormalities described in Example 1. The chromosome karyotype was also diagnosed using the G-banding method.

[0124] The copy number of each target chromosome is calculated by software, and the copy number of chromosomes 13, 18, 21, X, and Y is counted as N. 13 、N 18 、N 21 、N X 、N Y . Calculate the copy number ratios R1 and R2, and determine the Z value.

[0125] The copy number ratio of chromosome 13 is R1 = N 13 ÷N 21 ; R2=N 13 ÷N18 .

[0126] The copy number ratio of chromosome 18 is R1=N 18 ÷N 21 ; R2=N 18 ÷N 13 .

[0127] The copy number ratio of chromosome 21 is R1=N 21 ÷N 18 ; R2=N 21 ÷N 13 .

[0128] The copy number ratio of chromosome X is R1 = N x ÷N 21 ; R2=N x ÷N 18 .

[0129] The copy number ratio of chromosome Y is R1 = N Y ÷N 21 ; R2=N Y ÷N 18 .

[0130] Among them, Z 13 =0.08, Z 18 =1.70, Z 21 =-0.16, Z X =-2.54, Z Y =-5.08.

[0131] Table 3 Results of detecting normal karyotype (46, XY) using the detection method of the present invention

[0132] chromosome Copy number / μL <![CDATA[Ratio R1]]> <![CDATA[Ratio R2]]> Z value Interpretation of results chromosome 13 14466.751 1.0073 0.9552 0.08 Diploid chromosome 18 14145.544 1.0546 1.0469 1.70 Diploid chromosome 21 14361.742 0.9482 0.9927 -0.16 Diploid X chromosome 7402.319 0.5154 0.4887 -2.54 haploid Y chromosome 7600.470 0.5292 0.5018 -5.08 haploid

[0133] Table 3 and Figure 3 The results showed that the result determined by the detection method of the present invention was a normal diploid male, which was consistent with the karyotype diagnosis result (46, XY).

[0134] Example 5

[0135] Application of primer-probe combinations for detecting chromosomal aneuploidy abnormalities: Detection of common chromosomal abnormalities (47, XY, +21):

[0136] In this example, 2 mL of amniotic fluid sample with a known common chromosomal abnormality (47, XY, +21) was used, and the primer-probe combination described in Example 2 was used to perform chromosome karyotype detection according to the method for detecting chromosomal aneuploidy abnormalities described in Example 1. The chromosome karyotype was also diagnosed using the G-banding method.

[0137] The copy number of each target chromosome is calculated by software, and the copy number of chromosomes 13, 18, 21, X, and Y is counted as N. 13 、N 18 、N 21 、N X 、N Y . Calculate the copy number ratios R1 and R2, and determine the Z value.

[0138] The copy number ratio of chromosome 13 is R1 = N 13 ÷N 21 ; R2=N 13 ÷N 18 .

[0139] The copy number ratio of chromosome 18 is R1=N 18 ÷N 21 ; R2=N 18 ÷N 13 .

[0140] The copy number ratio of chromosome 21 is R1=N 21 ÷N 18 ; R2=N 21 ÷N 13 .

[0141] The copy number ratio of chromosome X is R1 = N x ÷N 21 ; R2=N x ÷N 18 .

[0142] The copy number ratio of chromosome Y is R1 = N Y ÷N 21 ; R2=N Y ÷N 18 .

[0143] Among them, Z 13 =0.91, Z 18 =-0.82, Z 21 =12.67, Z X =-3.07, Z Y =-5.30.

[0144] Table 4 Results of detecting common chromosome abnormalities (47, XY, +21) using the detection method of the present invention

[0145]

[0146]

[0147] Table 4 and Figure 4The results showed that the result interpreted by the detection method of the present invention was Down syndrome (T21), which was consistent with the karyotype diagnosis result (47, XY, +21).

[0148] Example 6

[0149] Application of primer-probe combinations for detecting chromosomal aneuploidy abnormalities in detecting common sex chromosome abnormalities (45, XO):

[0150] In this example, 2 mL of amniotic fluid sample with known sex chromosome abnormality (45, XO) was used, and chromosome karyotype detection was performed according to the method for detecting chromosome aneuploidy abnormality described in Example 1 using the primer-probe combination described in Example 2. The chromosome karyotype was also diagnosed using the G-banding method.

[0151] The copy number of each target chromosome is calculated by software, and the copy number of chromosomes 13, 18, 21, X, and Y is counted as N. 13 、N 18 、N 21 、N X 、N Y . Calculate the copy number ratios R1 and R2, and determine the Z value.

[0152] The copy number ratio of chromosome 13 is R1 = N 13 ÷N 21 ; R2=N 13 ÷N 18 .

[0153] The copy number ratio of chromosome 18 is R1=N 18 ÷N 21 ; R2=N 18 ÷N 13 .

[0154] The copy number ratio of chromosome 21 is R1=N 21 ÷N 18 ; R2=N 21 ÷N 13 .

[0155] The copy number ratio of chromosome X is R1 = N x ÷N 21 ; R2=N x ÷N 18 .

[0156] The copy number ratio of chromosome Y is R1 = N Y ÷N 21 ; R2=N Y ÷N 18 .

[0157] Among them, Z 13=0.63, Z 18 =-0.02, Z 21 =-0.97, Z X =-8.58, Y missing.

[0158] Table 5 Results of detecting sex chromosome abnormalities (45, XO) using the detection method of the present invention

[0159]

[0160] According to Table 5 and Figure 5 The results show that the result interpreted by the detection method of the present invention is Turner syndrome (45, XO), which is consistent with the karyotype diagnosis result (45, XO).

[0161] Example 7

[0162] Application of primer-probe combinations for detecting chromosomal aneuploidy abnormalities in detecting fetal chromosomal aneuploidy in maternal plasma:

[0163] In this example, 2 mL of peripheral blood plasma was collected from a pregnant woman with a high-risk NGS-NIPT (T13) pregnancy, and chromosome karyotype detection was performed using the primer-probe combination described in Example 2 and the method for detecting chromosome aneuploidy abnormalities described in Example 1. The chromosome karyotype was also diagnosed using the G-banding method.

[0164] The copy number of each target chromosome is calculated by software, and the copy number of chromosomes 13, 18, 21, X, and Y is counted as N. 13 、N 18 、N 21 、N X 、N Y . Calculate the copy number ratios R1 and R2, and determine the Z value.

[0165] The copy number ratio of chromosome 13 is R1 = N 13 ÷N 21 ; R2=N 13 ÷N 18 .

[0166] The copy number ratio of chromosome 18 is R1=N 18 ÷N 21 ; R2=N 18 ÷N 13 .

[0167] The copy number ratio of chromosome 21 is R1=N 21 ÷N 18 ; R2=N 21 ÷N 13 .

[0168] The copy number ratio of chromosome X is R1 = N x ÷N 21 ; R2=N x ÷N 18 .

[0169] The copy number ratio of chromosome Y is R1 = N Y ÷N 21 ; R2=N Y ÷N 18 .

[0170] Among them, Z 13 =8.67, Z 18 =-2.99, Z 21 =-1.81, Z X =0.31, Y missing.

[0171] Table 6 Results of Patau syndrome (T13) detected by the detection method of the present invention

[0172]

[0173]

[0174] According to Table 6 and Figure 6 The results showed that the result interpreted by the detection method of the present invention was a female with Patau syndrome (T13), which was consistent with the karyotype diagnosis result (47, XX, +13).

[0175] Example 8

[0176] Application of primer-probe combinations for detecting chromosomal aneuploidy abnormalities in detecting fetal chromosomal aneuploidy in maternal plasma:

[0177] In this example, 2 mL of peripheral blood plasma was collected from a pregnant woman with a high-risk NGS-NIPT (T21) pregnancy, and chromosome karyotype detection was performed using the primer-probe combination described in Example 2 and the method for detecting chromosome aneuploidy abnormalities described in Example 1. The chromosome karyotype was also diagnosed using the G-banding method.

[0178] The copy number of each target chromosome is calculated by software, and the copy number of chromosomes 13, 18, 21, X, and Y is counted as N. 13 、N 18 、N 21 、N X 、N Y . Calculate the copy number ratios R1 and R2, and determine the Z value.

[0179] The copy number ratio of chromosome 13 is R1 = N 13 ÷N 21 ; R2=N13 ÷N 18 .

[0180] The copy number ratio of chromosome 18 is R1=N 18 ÷N 21 ; R2=N 18 ÷N 13 .

[0181] The copy number ratio of chromosome 21 is R1=N 21 ÷N 18 ; R2=N 21 ÷N 13 .

[0182] The copy number ratio of chromosome X is R1 = N x ÷N 21 ; R2=N x ÷N 18 .

[0183] The copy number ratio of chromosome Y is R1 = N Y ÷N 21 ; R2=N Y ÷N 18 .

[0184] Among them, Z 13 =-0.87, Z 18 =0.81, Z 21 =-1.35, Z X =-4.38, Z Y =-5.28.

[0185] Table 7 Results of detection using the detection method of the present invention

[0186] chromosome Copy number / μL <![CDATA[Ratio R1]]> <![CDATA[Ratio R2]]> Z value Interpretation of results chromosome 13 5333.65 0.9708 0.9425 -0.87 Diploid chromosome 18 5659.05 1.0301 1.0610 0.81 Diploid chromosome 21 5493.80 0.9708 1.0300 -1.35 Diploid X chromosome 2847.79 0.5184 0.5032 -4.38 haploid Y chromosome 2705.09 0.4924 0.4780 -5.28 haploid

[0187] According to Table 7 and Figure 7 The results show that the result interpreted by the detection method of the present invention is a normal diploid male, which is consistent with the karyotype diagnosis result (46, XY), but the NGS-NIPT result is a false positive for T21.

[0188] Example 9

[0189] Application of primer-probe combinations for detecting chromosomal aneuploidy abnormalities in detecting fetal chromosomal aneuploidy in maternal plasma:

[0190] In this example, 2 mL of peripheral blood plasma was collected from a high-risk pregnant woman with hyperandrogenic syndrome (47, XYY) using NGS-NIPT. The primer-probe combination described in Example 2 was used to perform chromosome karyotype detection according to the method for detecting chromosomal aneuploidy abnormalities described in Example 1. The chromosome karyotype was also diagnosed using the G-banding method.

[0191] The copy number of each target chromosome is calculated by software, and the copy number of chromosomes 13, 18, 21, X, and Y is counted as N. 13 、N 18 、N 21 、N X 、N Y . Calculate the copy number ratios R1 and R2, and determine the Z value.

[0192] The copy number ratio of chromosome 13 is R1 = N 13 ÷N 21 ; R2=N 13 ÷N 18 .

[0193] The copy number ratio of chromosome 18 is R1=N 18 ÷N 21 ; R2=N 18 ÷N 13 .

[0194] The copy number ratio of chromosome 21 is R1=N 21 ÷N 18 ; R2=N 21 ÷N 13 .

[0195] The copy number ratio of chromosome X is R1 = N x ÷N 21 ; R2=N x ÷N 18 .

[0196] The copy number ratio of chromosome Y is R1 = N Y ÷N 21 ; R2=N Y ÷N 18 .

[0197] Among them, Z 13 =-1.49, Z 18 =1.81, Z 21 =-2.26, Z X =-2.64, Z Y =0.17.

[0198] Table 8 Results of detecting hyperandrogenic syndrome (47, XYY) using the detection method of the present invention

[0199] chromosome Copy number / μL <![CDATA[Ratio R1]]> <![CDATA[Ratio R2]]> Z value Interpretation of results chromosome 13 251.895 0.9485 0.8565 -1.49 Diploid chromosome 18 294.087 1.0774 1.0675 1.81 Diploid chromosome 21 265.558 0.9030 1.0542 -2.26 Diploid X chromosome 132.504 0.4990 0.5260 -2.64 haploid Y chromosome 251.895 0.9308 0.9813 0.17 Diploid

[0200] According to Table 8 and Figure 8 The results show that the result interpreted by the detection method of the present invention is super male syndrome (47, XYY), which is consistent with the karyotype diagnosis result (47, XYY).

[0201] Example 10

[0202] Application of primer-probe combination for detecting chromosomal aneuploidy abnormalities in aborted products:

[0203] In this example, a mung bean-sized abortion product tissue sample that has been confirmed to be trisomy 18 (47, XY, +18) is taken, and the primer-probe combination described in Example 2 is used to perform chromosome karyotype detection according to the method for detecting chromosome aneuploidy abnormalities described in Example 1. The chromosome karyotype is also diagnosed using the G-banding method.

[0204] The copy number of each target chromosome is calculated by software, and the copy number of chromosomes 13, 18, 21, X, and Y is counted as N. 13 、N 18 、N 21 、N X 、N Y . Calculate the copy number ratios R1 and R2, and determine the Z value.

[0205] The copy number ratio of chromosome 13 is R1 = N 13 ÷N 21 ; R2=N 13 ÷N 18 .

[0206] The copy number ratio of chromosome 18 is R1=N 18 ÷N 21 ; R2=N 18 ÷N 13 .

[0207] The copy number ratio of chromosome 21 is R1=N 21 ÷N 18 ; R2=N 21 ÷N 13 .

[0208] The copy number ratio of chromosome X is R1 = N x ÷N 21 ; R2=N x ÷N 18 .

[0209] The copy number ratio of chromosome Y is R1 = N Y ÷N 21 ; R2=N Y÷N 18 .

[0210] Among them, Z 13 =-0.52, Z 18 =10.21, Z 21 =0.54, Z X =-2.38, Z Y =-4.41.

[0211] Table 9 Results of Edward's syndrome (T18) detected by the detection method of the present invention

[0212]

[0213] According to Table 9 and Figure 9 The results show that the result interpreted by the detection method of the present invention is a male with high risk of Edwards syndrome (T18), which is consistent with the karyotype diagnosis result (47, XY, +18).

[0214] Example 11

[0215] Application of primer-probe combination for detecting chromosomal aneuploidy abnormalities in aborted products:

[0216] In this example, a mung bean-sized abortion product tissue sample diagnosed as Turner syndrome (45, XO) was taken, and chromosome karyotype detection was performed using the primer-probe combination described in Example 2 and the method for detecting chromosome aneuploidy abnormalities described in Example 1. The chromosome karyotype was also diagnosed using the G-banding method.

[0217] The copy number of each target chromosome is calculated by software, and the copy number of chromosomes 13, 18, 21, X, and Y is counted as N. 13 、N 18 、N 21 、N X 、N Y . Calculate the copy number ratios R1 and R2, and determine the Z value.

[0218] The copy number ratio of chromosome 13 is R1 = N 13 ÷N 21 ; R2=N 13 ÷N 18 .

[0219] The copy number ratio of chromosome 18 is R1=N 18 ÷N 21 ; R2=N 18 ÷N 13 .

[0220] The copy number ratio of chromosome 21 is R1=N 21÷N 18 ; R2=N 21 ÷N 13 .

[0221] The copy number ratio of chromosome X is R1 = N x ÷N 21 ; R2=N x ÷N 18 .

[0222] The copy number ratio of chromosome Y is R1 = N Y ÷N 21 ; R2=N Y ÷N 18 .

[0223] Among them, Z 13 =-1.13, Z 18 =1.16, Z 21 =-0.66, Z X =-3.84, Y missing.

[0224] Table 10 Results of Turner syndrome (45, XO) detected by the detection method of the present invention

[0225] chromosome Copy number / μL <![CDATA[Ratio R1]]> <![CDATA[Ratio R2]]> Z value Interpretation of results chromosome 13 4377.150 0.9800 0.9606 -1.13 Diploid chromosome 18 4556.749 1.0202 1.0410 1.16 Diploid chromosome 21 4466.674 0.9802 1.0205 -0.66 Diploid X chromosome 2315.126 0.5183 0.5081 -3.84 haploid Y chromosome 1.683 0.0004 0.0004 - Missing

[0226] According to Table 10 and Figure 10 The results show that the result interpreted by the detection method of the present invention is Turner syndrome (45, XO), which is consistent with the karyotype diagnosis result (45, XO).

[0227] Example 12

[0228] Application of amniotic fluid chromosomal aneuploidy detection using primer and probe combinations for detecting chromosomal aneuploidy abnormalities:

[0229] In this example, 2 mL of amniotic fluid samples were collected from 23 cases of known common chromosomal abnormalities (5 cases of 47,XX,+21, 3 cases of 47,XX,+21, 2 cases of 47,XX,+18, 3 cases of 47,XX,+18, 2 cases of 47,XX,+13, 2 cases of 45,XO, 2 cases of 47,XXY, 2 cases of 47,XXX, and 2 cases of 47,XYY) and 7 cases of known normal chromosomes (3 cases of 46,XX and 4 cases of 46,XY). Chromosome karyotype detection was performed using the primer-probe combination described in Example 2 and the method for detecting chromosomal aneuploidy described in Example 1. The chromosome karyotype was diagnosed using the G-banding method.

[0230] The results in Table 11 show that the test showed excellent performance in the detection of common T21, T18, T13 and sex chromosome abnormalities (45,XO, 47,XXY, 47,XXX, 47,XXY), with both sensitivity and specificity reaching 100% (Kappa=1).

[0231] Table 11 Comparison results of the consistency between the primer-probe combination of the present invention and karyotype diagnosis

[0232] Chromosomal abnormalities Positive results of karyotype diagnosis Positive results of the present invention Kappa value 47,XX,+21 5 5 1 47,XY,+21 3 3 1 47,XX,+18 2 2 1 47,XY,+18 3 3 1 47,XX,+13 2 2 1 45,XO 2 2 1 47,XXY 2 2 1 47,XXX 2 2 1 47,XYY 2 2 1

[0233] Compared with traditional diagnostic methods (e.g., amniotic fluid chromosome karyotype detection), the ultra-multiplex dPCR technology established in the present invention has the following breakthrough advantages: (1) The detection cycle is significantly shortened to 4 hours, greatly improving the timeliness of diagnosis; (2) The operation process is simplified, making it easier to promote clinical standardization; (3) A single run can complete the detection of 48-96 samples, with the potential for large-scale screening; (4) It breaks through the sample quality limit and has no strict requirements for sterile conditions; (5) It has strong anti-interference ability and can effectively overcome maternal cell contamination; (6) Only 1-2 mL of amniotic fluid or an equivalent amount of peripheral blood plasma is required to complete the detection, reducing the error introduced by culture, etc.

[0234] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A primer-probe combination for detecting chromosomal aneuploidy, characterized in that: The primer probe combination consists of a primer probe combination for chromosome 21, a primer probe combination for chromosome 18, a primer probe combination for chromosome 13, a primer probe combination for chromosome X, and a primer probe combination for chromosome Y; The chromosome 13 primer-probe combination consists of a chromosome 13 primer and a chromosome 13 probe, the nucleotide sequences of the chromosome 13 primers are shown in SEQ ID NO.1 to SEQ ID NO.20, and the nucleotide sequences of the chromosome 13 probes are shown in SEQ ID NO.21 to SEQ ID NO.30; The chromosome 18 primer-probe combination consists of a chromosome 18 primer and a chromosome 18 probe, the nucleotide sequences of the chromosome 18 primers are shown in SEQ ID NO.31 to SEQ ID NO.50, and the nucleotide sequences of the chromosome 18 probes are shown in SEQ ID NO.51 to SEQ ID NO.60; The chromosome 21 primer-probe combination consists of a chromosome 21 primer and a chromosome 21 probe, the nucleotide sequences of the chromosome 21 primers are shown in SEQ ID NO.61 to SEQ ID NO.80, and the nucleotide sequences of the chromosome 21 probes are shown in SEQ ID NO.81 to SEQ ID NO.90; The X chromosome primer probe combination consists of an X chromosome primer and an X chromosome probe, wherein the nucleotide sequences of the X chromosome primers are shown in SEQ ID NO.91 to SEQ ID NO.110, and the nucleotide sequences of the X chromosome probes are shown in SEQ ID NO.111 to SEQ ID NO.120; The Y chromosome primer probe combination consists of a Y chromosome primer and a Y chromosome probe. The nucleotide sequences of the Y chromosome primers are shown in SEQ ID NO.121 to SEQ ID NO.140, and the nucleotide sequences of the Y chromosome probes are shown in SEQ ID NO.141 to SEQ ID NO.

150.

2. The primer-probe combination according to claim 1, characterized in that The chromosome 13 probe, chromosome 18 probe, chromosome 21 probe, chromosome X probe and chromosome Y probe are modified with fluorescent labels.

3. The primer-probe combination according to claim 2, characterized in that The fluorescent label of the chromosome 21 probe is FAM label; the fluorescent label of the chromosome 18 probe is HEX label; the fluorescent label of the chromosome 13 probe is Quasar705 label; the fluorescent label of the chromosome X probe is Cy5 label; the fluorescent label of the chromosome Y probe is ROX label.

4. A kit for detecting chromosomal aneuploidy, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also includes a dPCR reaction system.

6. The kit according to claim 5, characterized in that The dPCR reaction system consists of the following components: 10 μL of 3×Maxuseful dPCR Buffer, 0.5 μL of Taq DNA polymerase, 6 μL of 10 μM upstream primer, 6 μL of 10 μM downstream primer, 3 μL of 10 μM probe, final template concentration of 12 ng, and ddH2O added to 30 μL.

7. Use of the primer-probe combination according to any one of claims 1 to 3 in preparing a product for detecting chromosomal aneuploidy.

8. Use of the primer-probe combination according to any one of claims 1 to 3 in the preparation of a product for detecting chromosomal diseases.

9. The use according to claim 8, characterized in that The chromosomal disease includes one or more of Down syndrome, Edwards syndrome, Patau syndrome, Turner syndrome, Klinefelter syndrome, super female syndrome and super male syndrome.

10. The use according to claim 7 or 8, characterized in that The samples used for the test include maternal peripheral blood plasma, early pregnancy cervical exfoliated cells, fetal chorionic villus tissue, amniotic fluid cells, abortion products or neonatal peripheral blood.

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