Super-multiplex digital PCR (polymerase chain reaction) kit and primer group for detecting chromosome copy number

Abstract

The invention provides a super-multiplex digital PCR (Polymerase Chain Reaction) kit for detecting chromosome copy number, a primer group and a use method of the kit, and belongs to the technical field of gene detection. The kit comprises: 1) a primer probe composition for amplifying a p13-q22.2 region of a human chromosome 21; and (2) a primer probe composition used for amplifying q11.2 to q21.31 regions of the human No.18 chromosome, and 3) a primer probe composition for amplifying q13.1-q34 regions of the human chromosome 13, when the kit provided by the invention is used for detecting chromosome copy number variation through digital PCR detection, through copy number detection and analysis of 469 clinical samples, the coincidence rates of digital PCR and NGS positive and result negative reach 100% respectively, and the kit has the advantages of high sensitivity, good stability and simpler and quicker operation and interpretation.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection, and specifically relates to a super-multiplex digital PCR kit, a primer set and a method for using the kit for detecting chromosome copy number. Background Art

[0002] There is fetal cell-free DNA (cfDNA) in the maternal plasma of pregnant women. The length is about 75bp-250bp. Almost all of it comes from the trophoblast cells of the placenta. Its concentration is closely related to the gestational age and is stably present in the maternal peripheral plasma at a certain proportion (2%-30%). When the number of a chromosome in the fetus is abnormal, the cfDNA ratio of that chromosome will exceed the normal range. This phenomenon can be used for non-invasive prenatal testing (NIPT). By analyzing the cfDNA in maternal plasma, chromosomal abnormalities of the fetus, such as Down syndrome, Edwards syndrome, Patau syndrome, etc., can be detected. The advantage of NIPT is that it does not require invasive operations, such as amniocentesis or chorionic villus sampling, so it can reduce the risk and pain of miscarriage. The accuracy of NIPT is also high, with a sensitivity of more than 99% and a specificity of more than 99%.

[0003] For non-invasive prenatal testing, the current conventional method for chromosome copy number detection is NGS (second-generation high-throughput sequencing). However, this method has the following disadvantages: 1. High error rate: NGS technology will produce certain errors in the sequencing process, and these errors may affect the accuracy of the test results; 2. Complex data processing: NGS technology generates a large amount of data, which requires a lot of data processing and analysis, which requires high computing power and professional knowledge, resulting in a long wait for test results; 3. Expensive: Due to the complex process and the cost of instruments and equipment, the price of a single test of NGS remains high.

[0004] In recent years, some researchers have considered using PCR methods for NIPT testing, but traditional PCR technology is difficult to overcome technical problems such as nonspecific amplification and low sensitivity during its application, which greatly limits its scope of application. In order to avoid the impact of nonspecific amplification on the signal-to-noise ratio and specificity of the results, the number of amplifications in the existing PCR system is generally limited to 10. Therefore, current PCR technology is difficult to meet the high difficulty requirements of detection. Therefore, it is necessary to provide new technical solutions to overcome the technical problems existing in the existing technology.

[0005] Digital PCR (digital PCR) technology is a technology that performs specific amplification of two or more PCR fragments in one reaction system. Its reaction principle, operation process and instruments are the same as conventional PCR. Its advantage is that it can achieve one-time simultaneous amplification of different templates, greatly improve the detection throughput, and can achieve absolute quantitative amplification products. In addition, this technology is moderately priced, easy to detect, and highly accurate. There are literature reports on experimental methods for simultaneously detecting copy number variations of human chromosomes 21, 18, and 13 using digital PCR technology, but each chromosome is only set up with 10 sets of primer probe combinations at most, resulting in a high coefficient of variation, poor stability of the experimental system, and the problem that the algorithm for calculating chromosome copy number variation is relatively complex and difficult to judge. Summary of the invention

[0006] In view of the defects of the prior art, the present application provides a super-multiplex digital PCR kit and a primer set for detecting chromosome copy number, and an experimental method for detecting chromosome copy number variation by super-multiplex PCR using the primer combination and the kit.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0008] First, the present application provides a super-multiplex digital PCR kit for detecting chromosome copy number, the kit comprising three sets of primer probe compositions:

[0009] 1) a primer-probe combination for amplifying the p13-q22.2 region of human chromosome 21; and,

[0010] 2) a primer-probe combination for amplifying the q11.2-q21.31 region of human chromosome 18; and,

[0011] 3) A primer-probe combination for amplifying the q13.1-q34 region of human chromosome 13.

[0012] In this application, the sequence information of the human chromosome regions involved refers to the GRCh38 human genome version released by the Genome Reference Consorium in 2013.

[0013] Preferably, the above-mentioned kit also includes a digital PCR reaction solution; the digital PCR reaction solution includes a PCR reaction buffer, a dNTP mixture, a Taq enzyme and ddH2O.

[0014] Preferably, the nucleotide sequence of the primer probe combination used to amplify the p13-q22.2 region of human chromosome 21 is shown as SEQ ID NO.1 to SEQ ID NO.75; the nucleotide sequence of the primer probe combination used to amplify the q11.2-q21.31 region of human chromosome 18 is shown as SEQ ID NO.76 to SEQ ID NO.150; the nucleotide sequence of the primer probe combination used to amplify the q13.1-q34 region of human chromosome 13 is shown as SEQ ID NO.151 to SEQ IDNO.225.

[0015] The above-mentioned primer-probe combination design method is a conventional technology in the art, such as the literature "Dai P, Yang Y, Zhao G, et al. A dPCR-NIPT assay for detections of trisomies 21, 18 and 13 in a single-tube reaction-could it replace serum biochemical tests as a primary maternal plasma screening tool? [J]. Journal of Translational Medicine, 2022, 20 (1): 1-17.", "El Khattabi LA, Rouillac-Le Sciellour C, Le Tessier D, et al. Could digitalPCR be an alternative as a non-invasive prenatal test for trisomy 21: a proof of concept study [J]. PloS one, 2016, 11 (5): e0155009.", "Tong Y K, Jin S, Chiu RWK, et al. Noninvasive prenatal detection of trisomy 21 by an epigenetic-genetic chromosome-dosage approach[J].Clinical chemistry, 2010, 56(1):90-98.”

[0016] Secondly, the present application provides a method for using the above-mentioned super-multiplex digital PCR kit for detecting chromosome copy number, and the specific steps are as follows:

[0017] S1. Prepare PCR reaction system

[0018] The PCR reaction system includes three sets of primer-probe combinations designed for three chromosomes, and each set of probes is labeled with three different fluorescent groups; the PCR reaction system also includes sample DNA and PCR reaction solution;

[0019] S2. Multiplex PCR amplification

[0020] Add PCR reaction system and droplet generation oil to a droplet generation chip (commonly available in the market), place the droplet generation chip into a droplet generation instrument to generate droplets; after droplet generation is completed, transfer the droplets to a PCR plate and seal the plate; transfer the PCR plate to a PCR instrument for amplification;

[0021] S3. After the PCR amplification reaction is completed, the PCR plate is placed in a reader for detection, and the copy number of the target chromosome amplified by the primer combination is determined according to the detection result, specifically:

[0022] 1) In the same digital PCR test of the same sample, any one of the three chromosomes is used as the detected chromosome T, and the other two are used as reference chromosomes R1 and R2. The copy number CCN ratio between the detected chromosome T and the reference chromosomes R1 and R2 is calculated, i.e., RChrT / ChrR1 and RChrT / ChrR2. The calculation formula is as follows:

[0023]

[0024] 2) Calculate ZChrT / ChrR1 and ZChrT / ChrR2 values ​​based on the sample RChrT / ChrR1 and RChrT / ChrR2 and the average value of negative reference sample RChrT / ChrR:

[0025]

[0026] 3) Calculate the Z-score of the detected chromosome based on the ZChrT / ChrR1 and ZChrT / ChrR2 values:

[0027] If both ZChrT / ChrR1 and ZChrT / ChrR2 are greater than 0, the product of ZChrT / ChrR1 and ZChrT / ChrR2 is calculated and the absolute value is taken as the Z-score; otherwise, the product of ZChrT / ChrR1 and ZChrT / ChrR2 is calculated and the inverse of the absolute value is taken as the Z-score;

[0028] When Z-score≤6, it indicates that the chromosome copy number of the sample is normal; when 6<Z-score≤9, after re-testing, if the result is still 6<Z-score≤9, it indicates that the chromosome copy number of the sample is abnormal; when the Z-score value of the tested chromosome is>9, it indicates that the chromosome copy number of the sample is abnormal.

[0029] The above-mentioned method of labeling probes with fluorescent groups is a conventional technology in the field, as disclosed in the literature "Tan C, Chen X, Wang F, et al. A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies [J]. Analyst, 2019, 144 (7): 2239-2247.", "Lee SY, Kim S J, Han SH, et al. A new approach of digital PCR system for non-invasive prenatal screening of trisomy 21 [J]. Clinica Chimica Acta, 2018, 476: 75-80.".

[0030] Third, the present application provides a nucleotide sequence as shown in SEQ ID NO.1 to SEQ ID NO.225, which is used for detecting chromosome copy number. Super multiplex digital PCR primer probe composition.

[0031] Fourthly, the present application provides any of the following uses of the primer-probe combination having nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.225:

[0032] 1) Use in detecting chromosome copy number;

[0033] 2) Use in the preparation of reagents or kits for detecting chromosome copy number;

[0034] 3) Use in the preparation of diagnostic reagents or kits for diagnosing chromosome copy number.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) Accuracy: Through the copy number detection and analysis of 469 clinical samples, the consistency rate of the digital PCR and NGS results of the present invention reached 100%, indicating that the digital PCR technology of the present invention is accurate in the screening and detection of chromosome aneuploidy;

[0037] (2) Timeliness: The digital PCR of the present invention only requires three steps: droplet generation, amplification, and detection, and the detection can be completed in one day; while NGS requires five steps: library construction, enrichment, detection, analysis, and interpretation, and it takes at least five days to complete the experiment. For samples with urgent time, the digital PCR technology of the present invention has more advantages;

[0038] (3) Economical: The cost of the digital PCR reagents of the present invention is lower than that of NGS. At the same time, NGS detection usually requires 1 to 2 wild-type samples as controls, while the digital PCR of the present invention adopts the absolute quantitative principle and does not require wild-type control samples, thereby further reducing the detection cost. Therefore, the detection cost of the digital PCR of the present invention is about one-fourth of that of NGS, which is more suitable for promotion;

[0039] (4) Sensitivity: A major advantage of the digital PCR technology of the present invention is that it can detect trace samples, and the amount of samples detected is only half of that of NGS. Therefore, for trace samples, blood spots, single cells, etc., the digital PCR of the present invention is a better choice;

[0040] (5) Stability and simplicity: One embodiment of the present application reduces the coefficient of variation of the copy number ratio between the detected chromosome ChrT and the reference chromosome ChrR to below 1%, greatly improving the stability of the experimental system; and the present invention also provides a calculation method for determining whether there is a chromosome copy number variation in the amplified target chromosome, which is concise, efficient and easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is the total number of probes for R ChrT / ChrR The influence diagram of the above data is shown in Figure 1, where A: data of R21 / 18 calculated by digital PCR for a mixture of total chromosome primers and probe sets of 3, 15, 30, 45, 60, 69, and 75, and the coefficient of variation (CV) value decreased from 2.17% to 0.56%; B: data of R21 / 13 calculated by digital PCR for a mixture of total chromosome primers and probe sets of 3, 15, 30, 45, 60, 69, and 75, and the coefficient of variation (CV) value decreased from 2.66% to 0.39%; C: data of R18 / 13 calculated by digital PCR for a mixture of total chromosome primers and probe sets of 3, 15, 30, 45, 60, 69, and 75, and the coefficient of variation (CV) value decreased from 3.55% to 0.63%;

[0042] Figure 2 The figures are comparison diagrams of the test results of each channel before and after the adjustment of the number of primer probes, wherein Figures A and B are comparison diagrams of the FAM (Chr18) test channel before and after the adjustment.

[0043] Figure 3The figures are comparison charts of the test results of each channel before and after the adjustment of the number of primer probes, among which A and B are comparison charts of the VIC (Chr21) test channel before and after adjustment.

[0044] Figure 4 The figures are comparisons of the test results of each channel before and after the adjustment of the number of primer probes, where A and B are comparisons of the CY5 (Chr13) test channel before and after adjustment.

[0045] Figure 5 This is a graph showing the sensitivity evaluation results of the multiplex PCR method for detecting chromosome copy number variation;

[0046] Figure 6 This is the clinical sample validation result of the multiplex PCR method for detecting chromosome copy number variation.

[0047] Figure 7 This is a graph showing the results of chromosome copy number variation detection using PCR using different PCR detection systems. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. In the following embodiments, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0049] The experimental instrument used in the embodiment is Yongnuo Bio-MicroDrop-400 new droplet digital PCR, the system has 4 fluorescent channels, and a 96-well plate is used for detection. Each sample requires 2 reaction wells, and the effective number of droplets in each well reaction system is 50,000-100,000. 48 samples can be processed in parallel at a time. In specific implementation, other conventional commercially available PCR reaction instruments can also be used.

[0050] Example 1: Primer design and screening

[0051] The database of the National Center for Biotechnology Information (NCBI) of the United States was searched, and the online tools for nucleotide screening and primer screening provided on its website (https: / / www.ncbi.nlm.nih.gov) were used to select candidate primers and probes for human chromosomes 21, 18, and 13 (Chr21, Chr18, and Chr13) for multiplex PCR detection of chromosome copy number variation experiments. The Chr21, Chr18, and Chr13 primers and probes were divided into three groups and labeled with VIC, FAM, and Cy5 fluorophores, respectively, to distinguish the amplified signals. Super-multiplex PCR refers to a PCR technique that simultaneously amplifies multiple target sequences in the same reaction system. Compared with single-plex PCR, super-multiplex PCR requires the design of multiple pairs of primers, so primer design is a difficult point in super-multiplex PCR, and attention should be paid to factors such as primer length, primer specificity, and interactions between primers. After a series of evaluation experiments, it was finally determined that 25 sets of primers and probes were set up in each group, and the primer probe composition is shown in Table 1 below:

[0052] Table 1 Primer probe sequences

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061] Example 2: Super-multiplex PCR method for detecting chromosome copy number variation

[0062] 1. Sample processing

[0063] Peripheral blood samples (Shanghai Red House Hospital-Obstetrics and Gynecology Hospital Affiliated to Fudan University) were centrifuged at 1600 g for 10 min at 4°C, and the plasma portion was further centrifuged at 16000 g for 10 min at 4°C to further remove residual blood cells.

[0064] 2. DNA Extraction

[0065] Free DNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Extraction Kit (Manufacturer: QIAGEN; Catalog No.: 55114) according to the manufacturer's instructions.

[0066] 3. Preparation of reaction system:

[0067] According to the formula in Table 2, a digital PCR reaction premix (digital PCR Buffe, manufacturer: Guangdong Yongnuo Medical Technology Co., Ltd.; catalog number: S0200020101) was used to prepare a digital PCR reaction premix (1 reaction). The enzymes in Table 2 were DNA polymerases purchased from Beijing Read Micro Gene Technology Co., Ltd.

[0068] Table 2 Digital PCR reaction premix

[0069] Components volume 4× Digital PCR Buffer 5μL Enzyme (1500U / ml) 1.0μL Primer probe mixture (final concentration 10uM) 7μL Detection template (5-15ng) 1.0~7μL Total volume Make up to 20 μL with nuclease-free water

[0070] The above reaction solutions were mixed evenly, vortexed for 30 seconds, centrifuged instantaneously to collect the reaction solution at the bottom of the tube and placed on ice for later use.

[0071] 4. Sampling operation

[0072] Perform the injection operation according to the requirements of the operating instructions of Guangdong Yongnuo Medical's universal consumables for sample preparation for droplet digital PCR (item number: S0100010101 specification: 24 tests / box).

[0073] Equipment or consumables involved:

[0074] MicroDrop-400A sample preparation instrument and MicroDrop-400B biochip analyzer produced by Guangdong Yongnuo Medical Technology Co., Ltd., A200 / A300 PCR amplifier produced by Hangzhou Langji Scientific Instrument Co., Ltd., droplet detection oil (item number: S02000101), punctureable heat sealing film, PCR plate, microplate sealer (type S100), pipettes (2ul, 20μL, 200μL), DNase-free and RNase-free centrifuge tubes, DNase-free and RNase-free pipette filter nozzles, etc.

[0075] 5. Preparation of microdroplets

[0076] Place the droplet generation chip in the chip holder, add 50 μL of droplet generation oil to the oil phase well, add 20 μL of PCR reaction mixture with template added to the sample well, and then add 5 μL of sealant above the water phase of each sample well. After the oil phase and water phase are added, cover the droplet generation chip sealing pad and place the droplet generation chip in the sample preparation instrument for droplet generation;

[0077] After the droplets are generated, carefully transfer the generated droplets (about 45-55 μL) to the PCR reaction plate one by one;

[0078] After the droplets are transferred to the PCR reaction plate, cover it with a punctureable heat-sealing film and place it on a preheated microplate sealer for sealing.

[0079] 6. Thermal Cycling Reaction

[0080] Place the 96-well plate in a nucleic acid amplification instrument and perform thermal cycling reaction. The reaction program is shown in Table 3 below:

[0081] Table 3. Digital PCR reaction program

[0082]

[0083] 7. Droplet reading analysis

[0084] After the thermal cycling reaction is completed, the sample is transferred to a biological reader for reading and analysis to obtain the copy number of each fluorescent channel, that is, the copy number of Chr21, Chr18 and Chr13 of the measured sample is obtained respectively.

[0085] 8. Calculation of test results and determination of chromosome number abnormality

[0086] (1) Calculate the Z-score of Chr21, Chr18 and Chr13 of the sample:

[0087] The number of positive droplets of the target gene (C18-FAM fluorescent marker, C21-VIC fluorescent marker, C13-CY5 fluorescent marker) is calculated according to the fluorescent signals of different markers, and then the copy number of the target gene is measured according to the Poisson distribution principle. Based on formula (1), the copy number of each chromosome is automatically calculated by the instrument software:

[0088]

[0089] In the above formula, CCN is the copy number of chromosome (Chromosome Copy Number), n is the total number of effective droplets in the digital PCR reaction, d is the number of positive droplets, and V is the volume of a single droplet (μL).

[0090] Then use formula (2) to calculate the ratio between the corresponding chromosome copy numbers:

[0091]

[0092] In the above formula, R ChrT / ChrR is the CCN ratio between the tested chromosome (ChrT) and the reference chromosome (ChrR) from the same sample in the same digital PCR assay. 21 / 18 (or R21 / 13 , or R 18 / 13 , or R 18 / 21 , or R 13 / 18 , or R 13 / 21 ), ChrT represents Chr21 (or Chr21, or Chr18, or Chr18, or Chr13, or Chr13), and ChrR represents Chr18 (or Chr13, or Chr13, or Chr21, or Chr18, or Chr21).

[0093] According to the above R ChrT / ChrR , and further calculated the Z values ​​of chromosomes 21, 18, and 13 in the sample. ChrT / ChrR Value. Z ChrT / ChrR The calculation formula (3) of the value is as follows (the reference sample is a negative sample, and multiple negative samples are used as the reference sample library):

[0094]

[0095] According to the above Z ChrT / ChrR Value, further calculate the Z-score of the chromosome tested in the sample:

[0096] The Z-score calculation formula for C21 is: Chr21 / Chr18 and Z Chr21 / Chr13 are greater than 0, then calculate Z Chr21 / Chr18 and Z Chr21 / Chr13 Multiply and take the absolute value; otherwise, calculate Z Chr21 / Chr18 and Z Chr21 / Chr13 The product of and takes the opposite of the absolute value.

[0097] The Z-score calculation formula for C18 is: Chr18 / Chr21 and Z Chr18 / Chr13 are greater than 0, then calculate Z Chr18 / Chr21 and Z Chr18 / Chr13 Multiply and take the absolute value; otherwise, calculate Z Chr18 / Chr21 and Z Chr18 / Chr13 The product of and takes the opposite of the absolute value.

[0098] The Z-score calculation formula for C13 is: Chr13 / Chr21 and Z Chr13 / Chr18 are greater than 0, then calculate Z Chr13 / Chr21 and Z Chr13 / Chr18 Multiply and take the absolute value; otherwise, calculate Z Chr13 / Chr21 and Z Chr13 / Chr18 The product of and takes the opposite of the absolute value.

[0099] (2) Determine whether the chromosome number is abnormal based on the Z-score

[0100] Take Chr21 as an example:

[0101] Negative: When the Z-score of chromosome 21 is ≤6, it indicates that there is no significant difference between the chromosome 21 of the test sample and the normal chromosome 21 in the reference sample library, that is, the number of chromosome 21 is normal; gray zone sample: When the Z-score of chromosome 21 falls within the gray zone (6,9], the possibility of false negative due to low fetal DNA concentration (<4%) cannot be ruled out, so it is recommended to re-sample blood and re-test. If the re-test result is still in the gray zone, it is judged as copy number abnormality;

[0102] Positive: When the Z-score of chromosome 21 is greater than 9, it indicates that there is a significant difference between the chromosome 21 of the test sample and the normal chromosome in the reference database, that is, the copy number of chromosome 21 of the test sample is abnormal.

[0103] The judgment method for Chr18 and Chr13 is the same as the above-mentioned judgment method for Chr21 (Note: the Z-score range for judging negative and positive in the present invention is obtained by dividing according to the standard normal distribution diagram of the Z-score values ​​of the test results of a large number of clinical samples).

[0104] Example 3: Effect of the number of chromosome primers and probes on the CCN ratio of multiplex PCR results

[0105] Using R 21 / 18 , R 21 / 13 , R 18 / 13 As a verification example, a mixture of 1, 5, 10, 15, 20, 23, and 25 (total primer probe groups: 3, 15, 30, 45, 60, 69, and 75) was randomly selected from the primer probe groups of chromosome 21 / chromosome 18 / chromosome 13, and cfDNA was extracted from 32 normal human plasma samples (from Shanghai Red House Hospital-Fudan University Affiliated Obstetrics and Gynecology Hospital) and mixed evenly. The method of Example 2 was used to perform digital PCR detection and calculate the CCN value. The results are shown in Figure 2. Figure 1 As shown in the figure, the results show that as the total number of primers and probes increases, R ChrT / ChrR The coefficient of variation (CV) value of the two groups decreased from 2.17% / 2.66% / 3.55% to 0.56% / 0.39% / 0.63%, and there was a statistically significant difference (P<0.05).

[0106] Figure 2-Figure 4 This is a comparison chart of the test results of FAM (Chr18), VIC (Chr21), and CY5 (Chr13) channels (fluorescent groups) before and after adjusting the number of chromosome primers and probes. Figure 2-4 The A in the figure is the result of primer and probe amplification test with 10 groups. Figure 2-4Figures B in the figure are all primer and probe amplification test result graphs with a group number of 25. It can be seen that the positive and negative droplets amplified by the adjusted primers and probes have better discrimination and the scatter points are significantly reduced.

[0107] Example 4: Sensitivity evaluation of multiplex PCR detection of chromosome copy number variation

[0108] The analytical sensitivity of the digital PCR-NIPT detection method was evaluated by testing a group of simulated trisomic samples (simulating T21, T18 and T13 samples, adding DNA extracted from Chr21, Chr18 or Chr13 trisomic chorionic villus tissue provided by the Obstetrics and Gynecology Hospital Affiliated to Fudan University (simulating trisomic cffDNA) to the parturient cfDNA (10 / 5ng), respectively, with the addition amounts of 0% (normal), 3%, 5%, 10% and 20%, respectively, and the simulated trisomic samples were tested 8 times in a 20μL system. Repeated digital PCR-NIPT experiments). The method of Example 2 was used for digital PCR detection and analysis, and the results are shown in Figure 2. Figure 5 As shown, AC is the detection result of Chr21, Chr18, and Chr13 when the input volume is 10 ng; DF is the detection result of Chr21, Chr18, and Chr13 when the input volume is 5 ng. It can be seen that at different injection volumes, the cfDNA fraction that can be reliably detected by trisomy is 3%, because at this concentration, the positive detection rate of all trisomy is 100% (8 / 8).

[0109] Example 5: Copy number detection analysis of 469 clinical samples, comparison of the consistency of digital PCR and NGS results

[0110] The analytical sensitivity of the digital PCR-NIPT detection method was evaluated by testing a group of 469 clinical samples. The method of Example 2 was used to perform digital PCR detection and analysis. The results are as follows: Figure 6 As shown, AC are the test results of Chr21, Chr18, and Chr13. The positive and negative were judged according to the Z-score of 9, and compared with the NGS results, the detection coincidence rate of all positive samples was 100% (26 / 26), and the detection coincidence rate of negative samples was 100% (443 / 443).

[0111] Example 6: Instrument compatibility test

[0112] To verify the compatibility of the flat-plate digital PCR with the kit of this application, the experimental instrument used in this example is the Naica crystal fully automatic microdroplet chip digital PCR system developed by Stilla, France. The system has 6 fluorescent channels and uses Sapphire chips for detection. Compatibility was evaluated by 5 samples, and the method of Example 2 was used for digital PCR detection and analysis. The results are shown in Figure 2. Figure 7 As shown, AC are the detection results of FAM (Chr18), VIC (Chr21), and CY5 (Chr13) fluorescence channels (fluorescent groups) respectively. It can be seen that the polymerization degree and discrimination of the positive and negative droplet bands are good, and the sample detection compliance rate is 100%, which proves that the kit and detection method of the present application have good compatibility with the common PCR detection instruments on the market.

Claims

1. A super multiplex digital PCR kit for detecting chromosome copy number, characterized in that: The kit includes three sets of primer-probe combinations: 1) a primer-probe combination for amplifying the p13-q22.2 region of human chromosome 21; and, 2) a primer-probe combination for amplifying the q11.2-q21.31 region of human chromosome 18; and, 3) A primer-probe combination for amplifying the q13.1-q34 region of human chromosome 13.

2. The super-multiplex digital PCR kit for detecting chromosome copy number according to claim 1, characterized in that: The kit also includes a digital PCR reaction solution.

3. The super-multiplex digital PCR kit for detecting chromosome copy number according to claim 1, characterized in that: The nucleotide sequence of the primer probe combination used to amplify the p13-q22.2 region of human chromosome 21 is shown as SEQ ID NO.1 to SEQ ID NO.75; the nucleotide sequence of the primer probe combination used to amplify the q11.2-q21.31 region of human chromosome 18 is shown as SEQ ID NO.76 to SEQ ID NO.150; the nucleotide sequence of the primer probe combination used to amplify the q13.1-q34 region of human chromosome 13 is shown as SEQ ID NO.151 to SEQ ID NO.

225.

4. The super-multiplex digital PCR kit for detecting chromosome copy number according to claim 2, characterized in that: The digital PCR reaction solution includes a PCR reaction buffer, a dNTP mixture, a Taq enzyme and ddH2O.

5. A method for using the super-multiplex digital PCR kit for detecting chromosome copy number as described in any of claims 1 to 4, characterized in that: The specific steps are as follows: S1. Prepare PCR reaction system The PCR reaction system includes three sets of primer-probe combinations designed for three chromosomes, and each set of probes is labeled with three different fluorescent groups; the PCR reaction system also includes sample DNA; S2. Multiplex PCR amplification Using the PCR reaction system obtained in step S1 to generate droplets, PCR amplification is performed; S3. After the PCR amplification reaction is completed, the PCR plate is placed in a reader for detection, and the copy number of the target chromosome amplified by the primer combination is determined based on the detection result.

6. The method of use according to claim 5, characterized in that: In step S5, the step of determining the copy number of the target chromosome amplified by the primer combination according to the detection result refers to: 1) In the same digital PCR test of the same sample, any one of the three chromosomes is used as the detected chromosome T, and the other two are used as reference chromosomes R1 and R2. The copy number CCN ratio between the detected chromosome T and the reference chromosomes R1 and R2 is calculated, i.e., RChrT / ChrR1 and RChrT / ChrR2. The calculation formula is as follows: 2) Calculate ZChrT / ChrR1 and ZChrT / ChrR2 values ​​based on the sample RChrT / ChrR1 and RChrT / ChrR2 and the average value of negative reference sample RChrT / ChrR: 3) Calculate the Z-score of the detected chromosome based on the ZChrT / ChrR1 and ZChrT / ChrR2 values: If both ZChrT / ChrR1 and ZChrT / ChrR2 are greater than 0, the product of ZChrT / ChrR1 and ZChrT / ChrR2 is calculated and the absolute value is taken as the Z-score; otherwise, the product of ZChrT / ChrR1 and ZChrT / ChrR2 is calculated and the inverse of the absolute value is taken as the Z-score; When Z-score≤6, it indicates that the chromosome copy number of the sample is normal; when 6<Z-score≤9, after re-testing, if the result is still 6<Z-score≤9, it indicates that the chromosome copy number of the sample is abnormal; when the Z-score value of the tested chromosome is>9, it indicates that the chromosome copy number of the sample is abnormal.

7. A super-multiplex digital PCR primer-probe composition for detecting chromosome copy number, characterized in that: The nucleotide sequence of the primer-probe combination is shown in SEQ ID NO.1 to SEQ ID NO.

225.

8. Any of the following uses of the primer-probe combination having nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.225: 1) Use in detecting chromosome copy number; 2) Use in the preparation of reagents or kits for detecting chromosome copy number; 3) Use in the preparation of diagnostic reagents or kits for diagnosing chromosome copy number.

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