A kit for detecting aneuploidy changes in the AZF region of the Y chromosome based on a multiplex PCR method

Abstract

The present invention discloses a kit for detecting aneuploidy changes in the AZF region of the Y chromosome based on a multiplex PCR method, and relates to the field of gene detection technology. The present invention provides a method and a kit for detecting sperm disomy rate and minor copy number changes of Y chromosome aneuploidy using multiplex PCR, with a detection sensitivity of about 1% and a detection lower limit of 5%, which is higher than the current industry sensitivity level of 20% to 30%. The method and kit disclosed in this patent can predict the clinical outcomes of subsequent auxiliary diagnosis by detecting minor changes in sperm aneuploidy, and avoid fertility risks in advance. The technology disclosed in the present invention achieves the detection purpose that cannot be achieved by current conventional technologies, and the disclosed technical parameters exceed the technical indicators of current conventional technologies by 4-30 times, which is extremely innovative.

Description

Technical Field

[0001] The present invention relates to the field of gene detection technology, and in particular to a kit for detecting aneuploidy changes in the AZF region of the Y chromosome based on a multiplex PCR method. Background Art

[0002] The human Y chromosome carries multiple genes responsible for testicular development, male characteristics, sperm formation and maintenance. The region where these genes are located is also called the AZF region. Among them, the long arm of the Y chromosome (Yq) where the AZF region is located contains many repeated regions and palindromic regions, and is prone to spontaneous recombination and partial or complete chromosome deletion during spermatogenesis. Current studies have shown that abnormal changes in the AZF region can lead to clinical symptoms such as male infertility, abnormal sperm count and morphology, recurrent miscarriage in women, and embryonic maldevelopment.

[0003] Research data reveals that a statistical analysis of nearly 40,000 Y chromosomes revealed a global prevalence of AZF microdeletions in infertile men of 7.5%. Furthermore, the symptoms caused by AZF deletions vary by ethnicity. For example, European men are less susceptible to Yq microdeletions, while American and East Asian men have the highest prevalence. The AZF region is further divided into four subregions: AZFa, AZFb, AZFc, and AZFd. Partial deletions at the AZFc locus, in particular, are associated with infertility, but the extent of their impact varies depending on ethnicity. These subregions are further divided into palindromic regions, such as b / b and gr / gr, based on their sensitivity to chromatin. Analysis of 17,000 Y chromosomes from men revealed that gr deletions are associated with infertility in Caucasian and Mongolian men, while b2 / b3 deletions are associated with infertility in African and Dravidian men.

[0004] Studies have shown that 2.5% to 7% of sperm exhibit chromosomal aneuploidy, affecting multiple chromosomes, including 13, 15, 21, 22, X, and Y. In 70 separate studies of 747 normal male semen samples, the proportions of individual sperm containing XX, XY, and YY chromosomes were 0.09%, 0.21%, and 0.12%, respectively. Overall, the cumulative disomy rate for all chromosomes in semen samples ranged from 3.99% to 4.28%. The highest rates of sperm chromosomal abnormalities were found in 50% of men with oligozoospermia, 33.3% of men with oligoasthenozoospermia, and 21% of men with oligoasthenozoospermia and teratozoospermia. High rates of sperm aneuploidy were also found in 36% of men with karyotypic abnormalities and 26% of men with meiotic abnormalities.

[0005] In a study comparing the clinical outcomes of intracytoplasmic sperm injection (ICSI) in men with different aneuploidy rates (as shown in the figure below), it was observed that 10 men with a change of only 1% to 5% in overall semen aneuploidy (a change of 0.12% in X / Y aneuploidy) were able to conceive normally, which was equivalent to a normal control; 9 men with a change of 3.91% in overall semen aneuploidy (a change of 0.67% in X / Y aneuploidy) underwent intracytoplasmic sperm injection (ICSI) and underwent at least 4 ICSI cycles to achieve biochemical pregnancy. The number of embryos transferred ranged from 2 to 11. Ten men with a 9.02% overall semen aneuploidy variation (1.29% X / Y aneuploidy variation) underwent intracytoplasmic sperm injection (ICSI). To achieve a biochemical pregnancy, they underwent more than four ICSI cycles, transferring 6 to 19 embryos. This study demonstrates that even small variations in chromosomal aneuploidy (4% to 8%, including 0.54% to 1.16% variations in sex chromosomes) can significantly impact pregnancy outcomes, requiring more ICSI cycles and a greater number of embryos transferred to achieve a biochemical pregnancy. The study found that a sperm disomy rate of less than 4.84% was associated with a 75.6% probability of retrieval of at least one euploid embryo. For every 1% reduction in the sperm disomy rate, the risk of not retrieval of a euploid embryo decreased by 2.071 times.

[0006] Given the importance of the AZF region, there are currently a variety of technical methods that can detect deletions and changes in the AZF region, such as the detection method for 6, 8 or even more STS sites established on the qPCR platform. This type of detection scheme is simple, easy to use and inexpensive, but in essence it is a point-to-surface approach and can only distinguish between presence and absence, with a high rate of missed detection. Therefore, capillary electrophoresis or sequencing schemes based on multiplex PCR or specific ligation methods have been further developed to further improve the accuracy of detection. A further scheme is the second-generation sequencing method based on target capture and enrichment of the AZF region, where the capture probe It is more dense and covers more sites, and can detect partial deletions, that is, some sperm in the same sample are missing the AZF region, while some sperm are not missing. In the test results, the copy number of Y is not 1, but an aneuploid value, further reducing the missed detection rate and providing more data for infertility biological research. However, the AZF region detection method based on second-generation sequencing technology is not sensitive to copy number changes and can only distinguish 20% to 30% copy number changes, that is, 0.7 to 1.3 copies are considered normal, and it cannot distinguish copy number changes below 10% or even 1%. Among the current representative patents, the technical parameters and features are shown in the following table:

[0007]

[0008] In summary, sperm chromosome aneuploidy, including Y chromosome aneuploidy, is significantly correlated with the clinical outcomes of assisted reproduction. Currently, there is an urgent need for a sperm aneuploidy detection kit with high throughput, relatively simple operation, short cycle, and conducive to clinical promotion. Summary of the Invention

[0009] To solve the above technical problems, the present invention provides a kit for detecting aneuploidy changes in the AZF region of the Y chromosome based on a multiplex PCR method.

[0010] The first object of the present invention is to provide a kit for detecting aneuploidy changes in the Y chromosome AZF region based on a multiplex PCR method, the kit comprising:

[0011] n1 resets the primers on the Y chromosome, denoted as Yplex,

[0012] n2 resets the primers on chromosome X, denoted as Xplex,

[0013] n3 reset the primers on the autosome, denoted as IPCplex,

[0014] n4 reset the primers in the PAR region, denoted as PARplex,

[0015] n5 reset the primers on the Indel type SNP, denoted as IndelPlex;

[0016] Among them, Yplex, Xplex, IPCplex, PARplex and IndelPlex are each divided into FP group, RPin group and RPout group. The specific reaction needs to include at least one of them. The optimal combination is to achieve the detection of 1% aneuploidy changes in the Y chromosome AZF region.

[0017] Furthermore, the FP group, RPin group, and RPout group satisfy:

[0018] When the salt concentration was 0.25 mol / L and the temperature was 55°C, the Gibbs free energy ΔG of the FP group, RPin group, and RPout group was in the range of -20.7073 kcal / mol≤ΔG≤-44.6782 kcal / mol.

[0019] Furthermore, the target detection limit, DNA input amount, PCR multiplicity, and required sequencing data volume conform to the following mathematical model:

[0020] The number of molecules that can be used to calculate aneuploidy changes is Cm = Di × Pl × 150;

[0021] Theoretical detection sensitivity S = Sqrt(Cm) / Cm;

[0022] Estimated detection limit SL = 8.01 / Sqrt(Cm);

[0023] Estimated sequencing data volume (M reads) = Di × 600 × Pl × MinRead;

[0024] Wherein 150, 8.01, and 600 are experimental experience coefficients of the present invention;

[0025] Where Di is the DNA input amount (ng), Pl is the PCR multiplex, MinRead is the expected minimum number of reads / multiplex (multiplex refers to the PCR multiplex), and Sqrt is the square root.

[0026] Furthermore, the sequence of the PARplex FP group is as follows:

[0027]

[0028]

[0029]

[0030] The sequence of the PARplex RPin group is as follows:

[0031]

[0032]

[0033]

[0034] The sequence of the PARplex RPout group is as follows:

[0035]

[0036]

[0037] Furthermore, the sequence of the Yplex FP group is as follows:

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044] The sequence of the Yplex RPin group is as follows:

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] The sequence of the Yplex RPout group is as follows:

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] Furthermore, the sequence of the IPCplex FP group is as follows:

[0059]

[0060]

[0061] The sequence of the IPCplex RPin group is as follows:

[0062]

[0063] The sequence of the IPCplex RPout group is as follows:

[0064]

[0065] Furthermore, the sequence of the IndelPlex FP group is as follows:

[0066]

[0067]

[0068] The sequences of the IndelPlex RPin group are as follows:

[0069]

[0070]

[0071] The sequence of the IndelPlex RPout group is as follows:

[0072]

[0073]

[0074] Furthermore, the sequence of the Xplex FP group is as follows:

[0075]

[0076]

[0077] The sequence of the Xplex RPin group is as follows:

[0078]

[0079]

[0080] The sequence of the Xplex RPout group is as follows:

[0081]

[0082] Furthermore, where H = one of the bases C, A, or T, HHHHHHHHHHHHHHH is called the UMI sequence. ΔG is regulated by the M1 sequence, which is TTCTT or TATCA.

[0083] Furthermore, the universal primer sequences are as follows:

[0084]

[0085] Furthermore, the FP, RPin, and RPout groups need to be used in conjunction with each other, and the characteristic is that the FP, RPin, and RPout of the same plex ID need to be used in conjunction with each other in the process to achieve the technical effect.

[0086] Furthermore, the typical operation process is:

[0087] S1. Select DNA samples for fragmentation, with fragment lengths primarily ranging from 150 to 250 bp. Sample types can include tissue DNA, peripheral blood DNA, cell-free DNA in body fluids, and genomic DNA from cell lines.

[0088] S2. Take 20 ng of fragmented DNA sample, mix the RPout group, FP group and universal PCR primer group ufp+urp, perform UMIPCR and universal PCR, and purify to obtain Product 1.

[0089] S3. Mix the RPin group primer and ufp primer (collectively called semi-nested PCR primers) into Product 1, perform semi-nested PCR on Product 1, and purify to obtain Product 2;

[0090] S4. Mix the index PCR primer group into Product 2, perform index PCR on Product 2, and purify to obtain Product 3; wherein, the index PCR primer group is a component in the commercial kit 《 Multiplex Oligos for (Dual IndexPrimers Set 1)》.

[0091] S5. Perform fluorescence quantification on Product 3, and after passing the qualification, perform on-machine sequencing, perform quality control on the off-machine data, perform bioinformatics analysis, and calculate the normalized P. After that, the P values of different target regions of different samples are comparable.

[0092] Further, after completing the typical operation process of this protocol, perform second-generation sequencing. After the off-machine data quality control is qualified, remove the adapters, perform alignment analysis, remove the UMI sequence types containing G bases and UMI sequence types with less than 3 UMI counts, and then calculate the number of UMI sequence types for each Plex. Use at least one of the IPCplex to perform normalization. The region included in the normalization method also needs to meet 0.02 Cm < UMI sequence type < 10 Cm. The normalization calculation formula is:

[0093] <00002�5>

[0094] Among them, the internal reference region can be part or all of the IPCplex, IndelPlex, PARplex groups, or part or all of the Xplex or Yplex groups.

[0095] ​​High-Fidelity DNA Polymerase (NEB); KOD-Plus-Neo (Toyobo); Secondary preference: High-Fidelity DNAPolymerase(NEB);TaKaRa LA (TaKaRa). DNA polymerase 2 preferred: PowerUp SYBR Green premix (thermofisher); KAPA HiFi HotStart ReadyMix PCR Kit (Roche); High-Fidelity PCR Kits (Bio-Rad); less preferred: 2× Max Master Mix (Dye Plus) (Norvozyme); DNA polymerase 3 preferred: PowerUp SYBR Green premix (thermofisher); KAPA HiFi HotStart ReadyMix PCR Kit (Roche); High-Fidelity PCR Kits (Bio-Rad); Second preferred: 2× Max Master Mix (DyePlus) (Novozymes).

[0096] The second object of the present invention is to provide the use of the above-mentioned kit in the preparation of reproductive detection products.

[0097] By means of the above solution, the present invention has at least the following advantages:

[0098] The present invention provides a method and kit for detecting sperm disomy rate and minor copy number changes of Y chromosome aneuploidy using multiplex PCR. The detection sensitivity is about 1%, and the detection limit is 5%, which is higher than the current industry sensitivity level of 20% to 30%. The method and kit disclosed in this patent can predict the clinical outcomes of subsequent auxiliary diagnosis by detecting minor changes in sperm aneuploidy, and avoid fertility risks in advance. The technology disclosed in the present invention achieves the detection purpose that cannot be achieved by current conventional technology. The disclosed technical parameters exceed the technical indicators of current conventional technology by 4-30 times, which is extremely innovative.

[0099] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0101] Figure 1 Assignment of primer region structure;

[0102] Figure 2 is the regional distribution of the Y chromosome;

[0103] Figure 3 is the position order of AZFa, AZFb, and AZFc genes in the AZF region;

[0104] Figure 4 Test results for patient 1;

[0105] Figure 5 Test results for patient 2;

[0106] Figure 6 Test results for patient 3;

[0107] Figure 7 Test results for patient 4;

[0108] Figure 8 Test results for patient 5;

[0109] Figure 9 Test results for patient 6. DETAILED DESCRIPTION

[0110] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0111] Example 1: Regional distribution of the kit design

[0112] like Figure 1 As shown, the XY aneuploidy panel consists of two parts:

[0113] 1. Autosomal part: The selected region on the autosome is used as the conventional internal reference position

[0114] The IPC regions are selected from multiple housekeeping gene regions, which are normally conserved and stable. The Indel regions, on the other hand, are iSNP sites with a frequency variation of 0.4 to 0.6 in East Asian populations. These Indel regions not only serve as internal reference positions but also allow for sample differentiation, contamination prevention, and determination of chimerism rates, providing intuitive evidence of individual differences.

[0115] 2. Sex chromosome part: The selection sites on the sex chromosome are located on the X chromosome and the Y chromosome respectively.

[0116] The homologous regions of the X chromosome and Y chromosome are PAR1 and PAR2. The X chromosome-specific region is the X region, and the Y chromosome-specific region is Figure 2 As shown, there are five regions, including Yp, SRY, AZFa, AZFb and AZFc. Among them, Yp is a specific region of the short arm of the Y chromosome; SRY is the sex-determining gene of males; the AZF region is located on chromosome Yq11 and is divided into AZFa, AZFb and AZFc regions. The genes on the chromosomes in these three regions respectively dominate different stages of the spermatogenesis process. The AZFa gene dominates the proliferation of spermatocytes; the AZFb gene (DAZsY134) is deleted, and the pathological diagnosis is germ cell maturation arrest; the gene defect of the AZFc region (DAZsY254) can cause azoospermia or extreme oligospermia. The arrangement of the AZF region is as follows Figure 3 shown.

[0117] Example 2: Performance of the kit on artificial standards

[0118] 1. DNA fragmentation and quantification.

[0119] DNA samples from both male and female individuals were fragmented into 150-bp fragments using Covaris ultrasound. Copy number quantification was performed using ddPCR. Based on the ddPCR copy number quantification results, the following standards were prepared: negative sample X:Y = 1:1 (male sample), 5% standard X:Y = 1.05:1, 10% standard X:Y = 1.1:1, 20% standard X:Y = 1.2:1, and 30% standard X:Y = 1.3:1.

[0120] For example: X:Y = 1:1 means male DNA; 1.05:1 means taking 20,000 copies of male DNA and adding 500 copies of female DNA.

[0121] 2. UMI PCR Process

[0122] The working concentration of the forward and reverse primers (FPout and RPout) in the UMI PCR system is 250 nM; the concentration of the universal primer set is 50 μM. Add 20 ng of standard DNA to the system. The reaction system is:

[0123] Vol / μL 5×HF buff 10 dNTP 1 phusion 1 FP+RPout 3 sample* 20ng water Variable total 50

[0124] The reaction conditions were: 98°C pre-denaturation for 30 seconds; one cycle of denaturation at 98°C for 10 seconds, annealing at 63°C for 30 minutes, and extension at 72°C for 15 seconds; one cycle of denaturation at 98°C for 10 seconds, annealing at 63°C for 25 minutes, annealing at 63°C for 5 minutes, and extension at 72°C for 15 seconds; two cycles of denaturation at 98°C for 10 seconds, annealing at 63°C for 15 seconds, and extension at 72°C for 15 seconds; and five cycles of denaturation at 98°C for 10 seconds, annealing at 71°C for 30 seconds. After PCR, the DNA was purified using 1.0× magnetic beads. During the second cycle at 63°C for 5 minutes, the PCR tube cap was opened and 1.55 μL of UFP+URP was added.

[0125] 3. Semi-nested PCR process

[0126] Vol / uL Power up 33 ufp+RPin 3 UMI PCR eluate 30 total 66

[0127] The reaction conditions were: 95°C for 3 minutes, denaturation at 95°C for 10 seconds, and annealing at 60°C for 30 minutes, for 2 cycles. After PCR, the DNA was purified using 1.0× magnetic beads.

[0128] 4. Index PCR Process

[0129]

[0130] After PCR was completed, the target fragment of 230-250 bp was purified using magnetic beads (0.8×, 0.2×).

[0131] 5. Library Quantification

[0132] DNA quality and concentration were tested using the dsDNA HS Assay Kit (ThermoFisher), and the procedure was performed according to the instructions.

[0133] 6. Sequencing and Bioinformatics Analysis

[0134] Sequencing was performed on the Illumina platform. After the data passed quality control, the adapters were removed and alignment analysis was performed. UMI sequences containing incorrect bases (including G) and UMI families with less than 3 UMI counts were removed. The number of UMI families was calculated, and the X / Y copy number variation was calculated. The formula for calculating copy number variation ploidy is:

[0135]

[0136] 7.QC summary

[0137]

[0138]

[0139] 8. X:Yploidy test results

[0140]

[0141] The results show that the kit disclosed in this patent can stably detect 5% copy number variation. Example 3 The kit detects the expression of super male XYY samples and super female XXY samples

[0142] 1. DNA fragmentation and quantification.

[0143] The DNA to be tested was sheared into 150 bp using Covaris ultrasound.

[0144] 2. The UMI PCR process is the same as in Example 2.

[0145] 3. The semi-nested PCR process is the same as in Example 2.

[0146] 4. The Index PCR process is the same as in Example 2.

[0147] 5. Library quantification was the same as in Example 2.

[0148] 6. The sequencing and bioinformatics analysis process was the same as in Example 2.

[0149] 7.Data Summary

[0150]

[0151]

[0152] 8.X:Yploidy Detection

[0153]

[0154] Samples A14576, A14674, A14752, A14822, A14537, and A14751 are known probands of Klinefelter syndrome. After normalization, their X:Y ratios ranged from 1.91 to 1.96, consistent with an X:Y ratio of 2:1 for Klinefelter syndrome. Samples A12315 and A13490 are known probands of hyperandrogenic syndrome. Their X:Y ratios ranged from 0.48 to 0.50, consistent with an X:Y ratio of 1:2. Sample A13973, also a proband of hyperandrogenic syndrome, was found to have an X:Y ratio of 0.8, meaning an X:Y ratio of 1:1.25, suggesting 25% mosaicism for hyperandrogenic syndrome.

[0155] Example 4: Kit for Detecting Samples from Oligospermia, Asthenozoospermia, and Teratospermia Patients

[0156] 1. DNA fragmentation and quantification.

[0157] Semen DNA was extracted using a blood / cell / tissue genomic DNA extraction kit (Tiangen), and DNA concentration was determined using a dsDNA HS Assay Kit (Thermo Fisher). The DNA to be tested was fragmented into 150 bp using Covaris ultrasound.

[0158] 2. UMI PCR process is the same as Example 2

[0159] 3. Semi-nested PCR process is the same as in Example 2

[0160] 4. Index PCR process is the same as in Example 2

[0161] 5. Library quantification is the same as in Example 2

[0162] 6. The sequencing and bioinformatics analysis process is the same as in Example 2

[0163] 7. The sequencing analysis results are shown in the following table:

[0164]

[0165] Testing of semen samples from patients 2-6 showed an X:Y ratio of approximately 1:1; the X:Y ratio of patient 1 was 1.16:1, which may indicate a certain proportion of X or XXY sperm mosaicism.

[0166] Example 5 Results of mFISH

[0167] The patient's semen sample is sent for FISH test: the test results are as follows: Figure 4-9 shown.

[0168] Results of fluorescence in situ hybridization using a centromere enumeration probe combination (CEP X / CEP Y / CEP18) with fixed sperm. Yellow arrows indicate sex chromosome-absent sperm (only one white signal for chromosome 18). Sperm with one white and one green signal are normal X sperm, and sperm with one white and one red signal are normal Y sperm. Otherwise, sperm with abnormal signals are abnormal. Blue represents the sperm nuclear region. Green, red, and white are the centromere probe signals for chromosomes X, Y, and chromosome 18, respectively.

[0169] Patient 1's test results are as follows Figure 4 As shown in the results of this experiment, the total proportion of sperm with normal X and Y chromosome signals in this sample was 96.73%, and the total proportion of sperm with abnormal X and Y chromosome signals was 3.27% (the average abnormality rate of autosomes in normal male sperm is 0.12%, and the average abnormality rate of sex chromosomes is 0.32%). The X:Y ratio is 1.22:1.

[0170]

[0171] Patient 2's test results are as follows Figure 5 As shown in the results of this experiment, the total proportion of sperm with normal X and Y chromosome signals in this sample was 98.93%, and the total proportion of sperm with abnormal X and Y chromosome signals was 1.07% (the average abnormality rate of autosomes in normal male sperm is 0.12%, and the average abnormality rate of sex chromosomes is 0.32%). The X:Y ratio is 0.94:1.

[0172]

[0173] Patient 3's test results are as follows Figure 6 As shown:

[0174] The results of this experiment found that the total proportion of sperm with normal X and Y chromosome signals in this sample was 98.45%, and the total proportion of sperm with abnormal X and Y chromosome signals was approximately 1.56% (the average abnormality rate of autosomes in normal male sperm is 0.12%, and the average abnormality rate of sex chromosomes is 0.32%). The X:Y ratio was 1.07:1.

[0175]

[0176] Patient 4's test results are as follows Figure 7 As shown: The experimental results showed that in this sample, the total proportion of sperm with normal X and Y chromosome signals was 99.18%, and the total proportion of sperm with abnormal X and Y chromosome signals was 0.82% (the average abnormality rate of autosomes in normal male sperm is 0.12%, and the average abnormality rate of sex chromosomes is 0.32%).

[0177]

[0178] Patient 5's test results are as follows Figure 8 As shown: The results of this experiment found that in the sperm sample, the total proportion of sperm with normal X and Y chromosome signals was 97.63%, and the total proportion of sperm with abnormal X and Y chromosome signals was 2.37% (the average abnormality rate of autosomes in normal male sperm is 0.12%, and the average abnormality rate of sex chromosomes is 0.32%).

[0179]

[0180] Patient 6 test results are as follows Figure 9 As shown: The results of this experiment found that in the sperm sample, the total proportion of sperm with normal X and Y chromosome signals was 98.68%, and the total proportion of sperm with abnormal X and Y chromosome signals was 1.32% (the average abnormality rate of autosomes in normal male sperm is 0.12%, and the average abnormality rate of sex chromosomes is 0.32%).

[0181]

[0182]

[0183] It can be seen that the mFISH results of this example are well consistent with those of Example 4.

[0184] Example 6 preferred solution

[0185] 1. DNA fragmentation and quantification.

[0186] The DNA to be tested was sheared into 150 bp using Covaris ultrasound.

[0187] 2. UMI PCR process is the same as Example 2

[0188] 3. Semi-nested PCR process is the same as in Example 2

[0189] 4. The index PCR process was the same as in Example 2. After the PCR was completed, the PCR product was purified using magnetic beads (0.7× discarded magnetic beads and 0.2× retained magnetic beads).

[0190] 5. Library quantification is the same as in Example 2

[0191] 6. The sequencing and bioinformatics analysis process is the same as in Example 2

[0192] 7. The results of the suboptimal and optimal sequencing analysis are shown in the following table:

[0193] lib_name total_reads Valid reads on-target_rate Second best choice 1 4241545 1557889 0.367293 Preferred 1 3882227 1926298 0.496184 Second best choice 2 3711732 1377741 0.371185 Preferred 2 4254762 2092118 0.491712

[0194] The second-best and best samples were manually prepared with a standard X:Y ratio of 1:1. The second-best and best samples were manually prepared with a standard X:Y ratio of 1.10:1. The on-target rate indicates that the optimal solution has a 10% higher on-target rate than the second-best solution.

[0195] 8.X:Yploidy Detection

[0196]

[0197]

[0198]

[0199] Judging from the homogenization results, both the suboptimal and optimal solutions can well detect the X:Y ratio of the artificially mixed standards. The optimal solution has a higher on-target rate than the suboptimal solution and is more cost-effective.

[0200] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A kit for detecting aneuploidy changes in the Y chromosome AZF region based on a multiplex PCR method, characterized in that: The kit includes: n1 resets the primer Yplex on the Y chromosome, n2 resets the primer Xplex on chromosome X, n3 reset the primers IPCplex on the autosome, n4 resets the primer PARplex in the PAR region, n5 reset the primers IndelPlex on the Indel type SNP; Among them, the M1 sequence is TTCTT or TATCA; Yplex, Xplex, IPCplex, PARplex, and IndelPlex each contain an FP group, an RPin group, and an RPout group. The sequence of the FP group contains a UMI sequence, and the UMI sequence is HHHHHHHHHHHHHHH, where H is one of C, A, and T bases. The sequence of the PARplex FP group is as follows: The sequence of the PARplex RPin group is as follows: The sequence of the RAPplex RPout group is as follows: The sequence of the Yplex FP group is as follows: The sequence of the Yplex RPin group is as follows: The sequence of the Yplex RPout group is as follows: The sequence of the IPC1pex FP group is as follows: The sequence of the IPCplex RPin group is as follows: The sequence of the IPCplex RPout group is as follows: The sequence of the IndlPlex FP group is as follows: The sequence of the IndelPlex RPin group is as follows: The sequence of the IndelPlex RPout group is as follows: The sequence of the Xplex FP group is as follows: The sequence of the Xplex RPin group is as follows: The sequence of the Xplex RPout group is as follows: o 2. The kit according to claim 1, wherein The FP group, RPin group, and RPout group described in each primer set satisfy: When the salt concentration was 0.25 mol / L and the temperature was 55℃, the FP group, RPin group and RPout group The Gibbs free energy ΔG of the group is in the range of -20.7073 kcal / mol≤ΔG≤-44.6782 kcal / mol.

3. The kit according to claim 2, wherein: ΔG is regulated by the M1 sequence.

4. Use of the kit according to any one of claims 1 to 3 in the preparation of a product for detecting abnormal sperm.

Images