Primer combination product, kit and method for detecting thalassemia gene and application

Abstract

The invention discloses a primer combination product, a kit and a method for detecting thalassemia genes and application. The primer combination product comprises primer pairs for detecting the thalassemia gene mutation and a reference genome taking a GRCh37 version human nuclear genome as a standard, and the primer pairs for detecting the thalassemia gene mutation comprise 670 primer pairs. When being used for detecting thalassemia genes, the primer combination product has the advantages of wide detection range, high accuracy, high sensitivity and short detection time.

Description

Technical Field

[0001] The present application relates to the field of molecular biology technology, and in particular to a primer combination product, a kit, a method and an application for detecting thalassemia genes. Background Art

[0002] Thalassemia (abbreviated as thalassemia) is a common hemolytic single gene genetic disease with the widest distribution and the largest cumulative population in the world. Thalassemia is usually divided into 4 types: α, β, δβ and δ. Among them, α thalassemia (abbreviated as α thalassemia) and β thalassemia (abbreviated as β thalassemia) are the most common. α thalassemia is mainly caused by the copy number variation (CNV) of α-globin gene (HBA1 and HBA2 gene). The most common α thalassemia deletion type is- SEA , -α 4.2 and -α 3.7 A few are caused by single nucleotide variants (SNVs) and small insertions / deletions (indels) in the α-globin gene. Common SNVs include α CS α, α QS α and α Westmead α. β-thalassemia is mainly caused by mutations in the β-globin gene (HBB gene), including SNVs and indels. A small number of β-thalassemias are caused by large deletions in the β-globin gene cluster. Common β-thalassemia gene SNVs and indels are IVS-II-654 (C>T), IVS-II-5 (G>C), CD71-72 (+A), CD43 (G>T), CD41-42 (-TCTT), etc.

[0003] The clinical phenotypes of thalassemia patients are diverse. Mild cases may develop anemia, while more severe cases may die before adulthood, and the most severe cases may die in utero. Currently, there is still a lack of ideal means for the treatment of thalassemia, and preimplantation genetic testing for single genes (PGT-M) is an effective method to block the family inheritance of thalassemia. PGT-M detects genetic markers in a certain range upstream and downstream of the pathogenic gene region, combines the genetic relationship between family members and the presence of pathogenic genes, and constructs a haplotype to distinguish between pathogenic and normal embryos, avoiding the transplantation of embryos carrying pathogenic genes into the mother.

[0004] Common methods for thalassemia PGT-M detection include the following: SNP chip technology, multiplex PCR technology, probe capture technology, whole genome sequencing, etc. Among them, SNP chip technology has a high cost, a long detection time, and poor direct detection effect on pathogenic variants; some multiplex PCR technologies have a limited detection range and can only detect α-thalassemia, unable to complete β-thalassemia detection, kinship identification, and gender identification; capture probe technology has a complex detection process, a long detection time, and a high cost; whole genome sequencing can obtain the whole genome information of the sample, but requires a high amount of sequencing data, is expensive, has a low sequencing depth, and complex data.

[0005] Therefore, there is an urgent need for an efficient, accurate, and wide-detection-range method and product for detecting thalassemia pathogenic genes. Summary of the Invention

[0006] Based on this, it is necessary to provide a primer set, kit, method, and application for detecting thalassemia genes.

[0007] In the first aspect of the present application, a primer combination product is provided. The primer combination product includes primer pairs for detecting thalassemia gene mutations, with the human nuclear genome in the GRCh37 version as the standard reference genome. The primer pairs include 670 primer pairs.

[0008] In some embodiments, the primer combination product further includes primer pairs for kinship identification, with the human nuclear genome in the GRCh37 version as the standard reference genome. The primer pairs for kinship identification include 150 primer pairs.

[0009] In some embodiments, the primer combination product further includes primer pairs for gender identification, with the human nuclear genome in the GRCh37 version as the standard reference genome. The primer pairs for gender identification include 29 primer pairs.

[0010] In the second aspect of the present application, a kit for detecting thalassemia genes is provided. The kit includes the primer combination product described in the first aspect of the present application.

[0011] In some embodiments, the kit further includes PCR amplification reagents; optionally, the PCR amplification reagents include one or more of PCR buffer, DNA polymerase, and dNTPs.

[0012] In some embodiments, the kit further includes one or more of tag primers, DNA extraction reagents, and DNA purification reagents.

[0013] In the third aspect of the present application, a library construction method for detecting thalassemia genes is provided, including the following steps:

[0014] Extract the genomic DNA of the sample to be tested;

[0015] Using the genomic DNA as a template, perform PCR amplification with the primer combination product described in the first aspect of the present application or the kit described in the second aspect of the present application to obtain the first-round amplification product;

[0016] Using the first-round amplification product as a template, perform PCR amplification with the tag primer to obtain the second-round amplification product; and,

[0017] Construct a library for detecting thalassemia genes based on the second-round amplification product.

[0018] In some embodiments, the sample to be tested is selected from one or more of blood samples, tissue samples, and embryo samples.

[0019] In some embodiments, the step of extracting the genomic DNA of the sample to be tested further includes: using the genomic DNA of the embryo sample as a template to perform whole-genome amplification.

[0020] The fourth aspect of the present application provides the application of the primer combination product described in the first aspect of the present application in the preparation of a product for detecting thalassemia.

[0021] By using the aforementioned primer set in combination with multiplex PCR and high-throughput sequencing for detection, it is possible to achieve an integrated and efficient detection of copy number variations (CNVs), single nucleotide variations (SNVs), and insertion / deletion variations (indels) on common thalassemia-related pathogenic genes with only one experiment. It can directly detect variations and indirectly analyze the variation information of the embryo through the haplotype typing results. The two results can be mutually verified, thereby improving the accuracy of the results and reducing the verification work;

[0022] Moreover, the detection method provided by the embodiments of the present application can also perform accurate gender identification and kinship identification, has the advantages of comprehensive, efficient, and accurate detection, saves the detection cost, can be used in fields such as pre-implantation thalassemia detection of embryos, prevents the birth of thalassemic children, reduces their birth defects, and is conducive to popularization and application in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments and implementations of the present application and to more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for description in the embodiments or implementations. Obviously, the following drawings are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 Schematic diagram of the process of multiplex PCR sequencing in an embodiment of the present application;

[0025] Figure 2 Schematic diagram of the principle of family haplotype typing in an embodiment of the present application;

[0026] Figure 3 Schematic diagram of the coverage of α-thalassemia pathogenic genes in an embodiment of the present application;

[0027] Figure 4 Schematic diagram of the coverage of β-thalassemia pathogenic genes in an embodiment of the present application;

[0028] Figure 5 β of sample P01 in family 4 in an embodiment of the present application IVS-Ⅱ-654 Sanger detection result of locus. Specific embodiments

[0029] To facilitate the understanding of the present application, the present application will be described more comprehensively below with reference to the relevant drawings. Preferred embodiments of the present application are shown in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0031] In the present application, "optionally", "optional", "option" mean optional, that is, any one selected from two parallel options of "yes" or "no". If "optional" appears in a technical solution for multiple times, without special explanation and without contradiction or mutual restriction relationship, each "optional" is independent of each other.

[0032] In the present application, "preferred", "better", "more preferably", "it is advisable" are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of the present application.

[0033] As used in this application, the terms "have", "contain", "include" and "comprise" are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also include actions, conditions under which actions occur, timing, states, etc.

[0034] In this application, in the technical features or technical solutions described in open language, the closed technical features or technical solutions composed of the listed contents are included, and the open technical features or technical solutions containing the listed contents are also included.

[0035] In this application, for units related to data ranges, if the unit is only attached after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0036] In this application, for a method process involving multiple steps, unless there are clear different descriptions in this article, the execution of these steps has no strict order limit, and it can be executed in other orders than the described one. Moreover, any step may include multiple sub-steps or multiple stages. These sub-steps or stages do not necessarily need to be executed at the same time, but can be executed at different times, and their execution order does not necessarily need to be sequential, but can be executed alternately or simultaneously with other steps or a part of the sub-steps or stages of other steps.

[0037] In this application, the exemplary descriptions such as "in some embodiments" or "in one embodiment" can cover but are not limited to the following meanings: These solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0038] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive listing and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0039] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of the selectable numerical values within the numerical interval is considered continuous, and includes the two numerical endpoints of the numerical interval (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. The "numerical values" in the numerical interval can be any quantitative values, such as numbers, percentages, ratios, etc. The "numerical interval" allows for a broad inclusion of numerical interval types such as percentage intervals, ratio intervals, ratio value intervals, etc.

[0040] The common methods for thalassemia PGT-M detection are as follows: SNP chip technology, multiplex PCR technology, probe capture technology, whole genome sequencing, etc. There are research reports providing a method for constructing family haplotypes to identify affected embryos and normal embryos with CNVs microdeletion microduplication syndrome, using illumina SNP chip technology for whole genome SNP genotype detection to construct haplotypes; there are research reports providing a method for constructing family haplotypes for detecting α-thalassemia, using multiplex PCR technology to amplify SNP loci tightly linked within 1 Mb upstream and downstream of HBA1 to achieve family linkage analysis; there are research reports using capture probes for haplotype linkage analysis of Duchenne muscular dystrophy (DMD) families, which can predict whether the transplanted embryos carry homozygous deletions of the DMD gene; there are research reports that through whole genome sequencing, the copy number variations (CNVs) of the target region and chromosomal aneuploidy detection of embryos can be directly detected at the same time. In addition, haplotype linkage analysis of effective SNPs in the CNVs region and its upstream and downstream can achieve the detection of direct site detection combined with indirect family linkage analysis.

[0041] However, these methods have some deficiencies. Among them, SNP chip technology has a high cost, a long detection time, and a poor direct detection effect on pathogenic variants; multiplex PCR technology has a limited detection range and can only detect α-thalassemia, and cannot complete the detection of β-thalassemia, kinship identification, and gender identification; capture probe technology has a complex detection process, a long detection time, and a high cost; whole genome sequencing can obtain the whole genome information of the sample, but requires a high amount of sequencing data, is expensive, has a low sequencing depth, and the data is complex.

[0042] Based on this, the embodiments of the present application at least provide a primer combination product, a kit, a method and an application for detecting thalassemia genes.

[0043] In some embodiments, the present application relates to a method of multiplex PCR sequencing. This method uses the DNA after whole genome amplification of blastocyst trophoblast cells and the genomic DNA of both of its parents and / or related relatives as templates, simultaneously amplifies multiple target regions to obtain amplicon products. After the first round of magnetic bead purification, adapter PCR reaction is carried out to introduce the adapter sequences of next-generation sequencing to both sides of the amplicon products, obtaining an amplicon library. After the second round of magnetic bead purification, the library is quantified, and a platform is selected for on-machine sequencing according to the adapter type, with the average sequencing depth ≥ 500×. Its detection process is as Figure 1 shown.

[0044] In some embodiments, after sequencing is completed, data analysis is carried out using the supporting analysis software to directly detect mutation sites. In addition, through a known and clear family pedigree map, using genetic laws and SNP linkage analysis methods, the haplotypes of the pathogenic site regions of both husband and wife are determined, and then through linkage analysis, it is judged whether the embryo sample carries pathogenic variants, so as to determine the genotype of the embryo. The principle of haplotype typing is as Figure 2 shown.

[0045] In some embodiments, the primer set described in the present application is a primer designed for thalassemia pathogenic sites, tightly linked SNP sites within 2M upstream and downstream of the pathogenic genes, sex identification SNP sites and kinship identification SNP sites. The primers designed according to the target regions are 849 multiplex in a single tube, and the length range of the amplicon products is 101 - 281bp, with an average length of 188bp. Among them, 652 pairs of primers, 18 pairs of primers, 150 pairs of primers and 29 pairs of primers are designed for the SNP sites upstream and downstream of the pathogenic genes, thalassemia pathogenic sites, kinship identification SNP sites and sex identification SNP sites respectively. The thalassemia pathogenic sites include common CNVs, SNVs and indels. For example, the α-thalassemia pathogenic sites include -α 3.7 , -α 4.2 , -- - SEA , -- THAI , α CS α, α QS α, α wsα, CD13 (GCC>TCC), CD30 (-GAG), etc., and the β-thalassemia pathogenic loci include IVS-II-654 (C>T), IVS-II-5 (G>C), CD71-72 (+A), CD43 (GAG>TAG), CD41-42 (-CTTT), IVS-I-1 (G>T), CD27 / 28 (+C), CD26 (GAG>AAG), CD17 (AAG>TAG), 5′UTR Cap+39 (C>T), -28 (A>G), -29 (A>G), etc. Figure 3 and Figure 4 is a schematic diagram of the coverage of thalassemia pathogenic genes.

[0046] In the first aspect of the present application, a primer combination product is provided, which includes primer pairs for detecting thalassemia gene mutations. Using the human nuclear genome in the GRCh37 version as the standard reference genome, the primer pairs for detecting thalassemia gene mutations include 670 primer pairs, and the binding positions of these 670 primer pairs are shown as follows:

[0047]

[0048]

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060] In some embodiments, the primer combination product further includes primer pairs for kinship identification. Using the human nuclear genome in GRCh37 version as the standard reference genome, the primer pairs for kinship identification include 150 primer pairs, and the binding positions of the 150 primer pairs are shown as follows:

[0061]

[0062]

[0063]

[0064]

[0065] In some embodiments, the primer combination product further includes primer pairs for sex identification. Using the human nuclear genome in GRCh37 version as the standard reference genome, the primer pairs for sex identification include 29 primer pairs, and the binding positions of the 29 primer pairs are shown as follows:

[0066]

[0067] In the second aspect of the present application, a kit for detecting thalassemia genes is provided, and the kit includes the primer combination product of the first aspect of the present application.

[0068] In some embodiments, the kit further includes PCR amplification reagents. Without limitation, the PCR amplification reagents include one or more of PCR buffer, DNA polymerase, and dNTPs.

[0069] In some embodiments, the kit further includes one or more of tag primers, DNA extraction reagents, and DNA purification reagents.

[0070] In the third aspect of the present application, a method for detecting thalassemia genes for non-diagnostic purposes is provided, including performing sequencing detection on the genomic DNA of a sample to be tested using the primer combination product of the first aspect of the present application or the kit of the second aspect of the present application.

[0071] In the fourth aspect of the present application, a library construction method for detecting thalassemia genes is provided, including the following steps:

[0072] S100: Extract the genomic DNA of the sample to be tested;

[0073] S200: Using the genomic DNA as a template, perform PCR amplification with the primer combination product of the first aspect of the present application or the kit of the second aspect of the present application to obtain a first-round amplification product;

[0074] S300: Using the first-round amplification product as a template, perform PCR amplification with the tag primer to obtain the second-round amplification product; and,

[0075] S400: Construct the library for detecting thalassemia genes based on the second-round amplification product.

[0076] In some embodiments, in step S100, the test sample is selected from one or more of a blood sample, a tissue sample, and an embryo sample.

[0077] In some embodiments, in step S100, it further includes performing whole-genome amplification using the genomic DNA of the embryo sample as a template.

[0078] In the fifth aspect of the present application, there is provided an application of the primer combination product of the first aspect of the present application in the preparation of a product for detecting thalassemia.

[0079] In some embodiments, thalassemia includes α-thalassemia and β-thalassemia.

[0080] Some examples are provided below.

[0081] The embodiments of the present application will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specified conditions in the following examples, the guidance given in the present application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturers, or referring to the experimental methods known in the art.

[0082] In the following embodiments, for the measurement parameters of the raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0083] In the following embodiments, the sequencing adapter and primer sequences include, but are not limited to, the adapter and primer sequences applicable to the MGI / BGI, Ion Torrent, and Illumina platforms. The library sequences of different platforms are different.

[0084] In the following embodiments, the library sequence for the BGI platform is as follows:

[0085] 5'Phos-CTCTCAGTACGTCAGCAGTT-Index2-CAACTCCTTGGCTCACAGAACGACATGGCTACGAT CCGACTT-Insert-;

[0086] AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA-Index1-CTGATAAGGTCGCCATGC-3'.

[0087] In the following examples, according to the different sequencing platforms, the library can be processed accordingly. For example, the library required for loading on the BGI platform is the nanoball (DNB). The prepared target gene library needs to be denatured into single-stranded DNA, and then the single-stranded DNA is ligated into single-stranded circular DNA through a ligation reaction. After preparing the nanoball library, it is sequenced on the BGI platform.

[0088] In the following examples, the information of the paired-end tag primers is as follows: UDIPrimer 1-96 (5 μM each, for MGI, plate) (purchased from AGTech, product number: M70132).

[0089] Example 1

[0090] 1. Six thalassemia family samples (74 samples) were recruited, mainly families carrying common thalassemia mutations (including -α 3.7 , -α 4.2 , -- SEA , α CS α, β CD17 , β IVS-Ⅰ-1 , β IVS-Ⅱ-654 , β CD41-42 mutations). Among them, there were 30 peripheral blood samples and 44 embryo samples, and the method was used to detect pathogenic variants.

[0091] 2. Template DNA preparation

[0092] 1) Genomic DNA extraction: Genomic DNA of 30 peripheral blood samples was extracted using nucleic acid extraction or purification reagents (Suzhou Beikang Medical Devices Co., Ltd., product number: Su Su Xie Bei 20210902), and the extracted DNA was subjected to quality inspection. 2) Whole-genome amplification: Whole-genome amplification of 44 embryo samples was performed using the REPLI-g Single Cell Kit, and the amplification products were subjected to quality inspection.

[0093] 3. First-round multiplex PCR amplification

[0094] 1) Using 849 pairs of multiplex primers designed in this application, prepare the reaction system on an ice box according to Table 1 below.

[0095] Table 1 Multiplex PCR reaction system

[0096] Component Volume (μL) Nuclease-free water 9-X DNA sample X Multiplex primer mix 5 Amplification enzyme mix 16 Total volume 30

[0097] Note: "X" represents the volume of the 40 ng DNA sample.

[0098] 2) After preparing the above system, vortex and mix well, then centrifuge briefly.

[0099] 3) Place the PCR tube into the gene amplifier and perform the reaction according to the program in Table 2 (hot lid at 105 °C).

[0100] Table 2 Multiplex PCR reaction program

[0101]

[0102] 4. Purification of the first-round PCR product

[0103] 1) Prepare 80% ethanol in advance and keep it at room temperature for later use. Try to use freshly prepared 80% ethanol for magnetic bead purification. 2) Take out the AMPure XP magnetic beads from the 4 °C refrigerator in advance, mix well and equilibrate at room temperature for 30 minutes; vortex and mix the AMPure XP magnetic beads that have been equilibrated to room temperature for later use. 3) Take out the PCR tube after the reaction in the previous step and centrifuge briefly. Add 0.9 times the volume of AMPure XP magnetic beads (27 μL) to each PCR tube, vortex and mix well, centrifuge briefly, and let stand at room temperature for 5 minutes. 4) Place the PCR tube on the magnetic stand for 3 minutes until the solution becomes clear. 5) Keep the PCR tube on the magnetic stand, carefully discard the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, let stand for 30 seconds, and discard the supernatant. 6) Repeat the previous step once. 7) Cover the tube cap, centrifuge briefly, place the PCR tube on the magnetic stand, and carefully use a 10 μL pipette to discard the residual liquid at the bottom, taking care not to aspirate the magnetic beads. 8) Keep the PCR tube on the magnetic stand and let stand at room temperature for 3 - 5 minutes to dry the magnetic beads and completely volatilize the residual ethanol. 9) Remove the PCR tube from the magnetic stand, add 18 μL of nuclease-free water to the tube, vortex and mix well, centrifuge briefly, and let stand at room temperature for 5 minutes. 10) Place the PCR tube on the magnetic stand for 2 minutes until the solution becomes clear. 11) Use a pipette to aspirate 15.5 μL of the supernatant and transfer it to a new PCR tube.

[0104] 5. Second-round adapter PCR amplification

[0105] 1) Prepare the reaction system on an ice box according to Table 3 below.

[0106] Table 3 Second-round PCR reaction system

[0107] Component Volume (μL) Previous purification product 15.5 Dual-tag primer 2 Amplification enzyme mix 12.5 Total volume 30

[0108] 2) After preparing the above system, vortex and mix well, then centrifuge briefly.

[0109] 3) Place the PCR tube into the gene amplifier and perform the reaction according to the program in Table 4 (hot lid at 105 °C).

[0110] Table 4 Second-round PCR reaction procedure

[0111]

[0112] 6. Purification of the second-round PCR products

[0113] 1) Take out the PCR tubes after the previous reaction and centrifuge briefly. Add 0.9 times the volume of AMPure XP magnetic beads (27 μL) to each PCR tube, vortex thoroughly, centrifuge briefly, and let stand at room temperature for 5 minutes. 2) Place the PCR tubes on the magnetic stand for 3 minutes until the solution is clear. 3) Keep the PCR tubes on the magnetic stand and carefully discard the supernatant. Add 180 μL of 80% ethanol solution to the PCR tubes, let stand for 30 seconds, and discard the supernatant. 4) Repeat the previous step once. 5) Cover the tube caps, centrifuge briefly, place the PCR tubes on the magnetic stand, and carefully use a 10-μL pipette to discard the residual liquid at the bottom, taking care not to aspirate the magnetic beads. 6) Keep the PCR tubes on the magnetic stand and let stand at room temperature for 3 - 5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely. 7) Remove the PCR tubes from the magnetic stand, add 24 μL of nuclease-free water to the tubes, vortex thoroughly, centrifuge briefly, and let stand at room temperature for 5 minutes. 8) Place the PCR tubes on the magnetic stand for 2 minutes until the solution is clear. 9) Transfer 22 μL of the supernatant to a new centrifuge tube.

[0114] 7. Quantification: Use a Qubit fluorometer for concentration determination.

[0115] 8. Sequencing on the machine

[0116] For high-throughput sequencing, use the BGI DA500 gene sequencer with a sequencing read length of PE150 and perform according to the standard operating procedures of the sequencer.

[0117] 9. Data analysis

[0118] Adopt bioinformatics to analyze the sequencing results. The detection results are shown in Tables 5 - 10 as follows:

[0119] Table 5 Basic quality control analysis results of sequencing data

[0120]

[0121]

[0122] Note: F0 represents the male partner, M0 represents the female partner, S1 represents the offspring, F1 represents the father of the female partner, M1 represents the mother of the female partner, F2 represents the father of the male partner, M2 represents the mother of the male partner, and P01 represents embryo No. 1.

[0123] Table 6 Detection results of family members and embryos

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[0125]

[0126] Table 7 Statistics of Valid SNP Loci

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[0128]

[0129] Table 8 Gender Identification Results

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[0131]

[0132]

[0133] Table 9 Paternity Test Results of Pedigree 2

[0134] SamPair CPI Judgment result M0_S1 2.09E+11 Parent-child relationship M0_P01 7.58E+09 Parent-child relationship M0_P02 6.59E+11 Parent-child relationship M0_P03 9.37E+11 Parent-child relationship M0_P04 1.30E+11 Parent-child relationship M0_P05 7.59E+10 Parent-child relationship M0_P06 1.12E+12 Parent-child relationship M0_P07 6.25E+10 Parent-child relationship M0_P08 7.73E+10 Parent-child relationship F0_S1 6.54E+13 Parent-child relationship F0_P01 8.04E+14 Parent-child relationship F0_P02 6.70E+13 Parent-child relationship F0_P03 8.46E+08 Parent-child relationship F0_P04 7.33E+12 Parent-child relationship F0_P05 3.00E+13 Parent-child relationship F0_P06 2.95E+13 Parent-child relationship F0_P07 2.40E+13 Parent-child relationship F0_P08 5.66E+13 Parent-child relationship

[0135] Note: If the Cumulative Paternity Index (CPI) > 10000, it is determined as a paternity relationship.

[0136] Table 10 Full Sibship Test Results of Pedigree 2

[0137]

[0138]

[0139] Note: If the Cumulative Full Sibship Index (CFSI) > 10000, it is determined as a full sibship relationship.

[0140] The above results show that:

[0141] Results of data quality control analysis: A multiplex amplicon library of 74 thalassemia samples to be tested (S001 - S074) was constructed and sequenced by high-throughput sequencing on the MGI platform. After sequencing, the sequencing data was analyzed, and the data quality control met the analysis requirements, as shown in Table 5.

[0142] Direct detection results: The direct detection results of 73 samples were consistent with the known variant information, and the detection results of 1 embryo sample were inconsistent, as shown in Table 6. First-generation verification was performed on the 1 embryo sample with inconsistent detection results, and it was found that the first-generation verification results were consistent with the direct detection results. Due to the particularity of embryo single-cell level amplification, it was speculated that ADO (allele dropout) might occur at the IVS-II-654 locus of embryo P01 in pedigree 4. The first-generation verification results are as Figure 5 shown.

[0143] Haplotype genotyping results: The genotyping results of 44 embryos were all consistent with the known variant information, as shown in Table 6. In addition, the number of effective SNP sites all met the genotyping requirements, as shown in Table 7.

[0144] Sex identification results: The sex identification results of 74 samples were all consistent with the known sex, as shown in Table 8.

[0145] Kinship identification results: The kinship identification results of 6 families were all correct. Taking Family 2 as an example, the identification results are shown in Tables 9 and 10.

[0146] The method provided by the embodiment of the present application only requires one experiment to integrally and efficiently detect CNVs, SNVs, and indels occurring in common thalassemia-related pathogenic genes. It can directly detect variants and indirectly analyze the variant information of embryos through haplotype genotyping results. The results of the two can be mutually verified, thereby improving the accuracy of the results and reducing verification work. In addition, the method provided by the embodiment of the present application can also perform accurate sex identification and kinship identification, and has the advantages of comprehensive, efficient, and accurate detection, saving detection costs. It can be used in directions such as pre-implantation thalassemia detection of embryos, preventing the birth of thalassemic children, reducing their birth defects, and is conducive to popularization and application in clinical practice.

[0147] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0148] The above-described embodiments only represent several implementation manners of the present application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description and drawings can be used to explain the scope of the claims.

Claims

1. A primer combination product, characterized in that: The primer combination product includes primer pairs for detecting thalassemia gene mutations, using the GRCh37 version of the human nuclear genome as a standard reference genome, and the primer pairs include 670 primer pairs, and the binding positions of the 670 primer pairs are respectively as follows:

2. The primer combination product according to claim 1, characterized in that: The primer combination product also includes primer pairs for kinship identification, using the GRCh37 version of the human nuclear genome as a standard reference genome, and the primer pairs for kinship identification include 150 primer pairs, and the binding positions of the 150 primer pairs are respectively as follows:

3. The primer combination product according to claim 1 or 2, characterized in that: The primer combination product also includes primer pairs for sex identification, using the GRCh37 version of the human nuclear genome as a standard reference genome, and the primer pairs for sex identification include 29 primer pairs, and the binding positions of the 29 primer pairs are respectively as follows:

4. A kit for detecting thalassemia gene, characterized in that: The kit comprises the primer combination product according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also includes a PCR amplification reagent; Optionally, the PCR amplification reagent includes one or more of a PCR buffer, a DNA polymerase and dNTPs.

6. The kit according to claim 4 or 5, characterized in that The kit also includes one or more of a label primer, a DNA extraction reagent and a DNA purification reagent.

7. A method for constructing a library for detecting thalassemia genes, characterized in that: The following steps are involved: Extracting genomic DNA from the sample to be tested; Using the genomic DNA as a template, performing PCR amplification using the primer combination product according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 6 to obtain a first-round amplification product; Using the first round amplification product as a template, using the label primer to perform PCR amplification to obtain the second round amplification product; and, The library for detecting thalassemia genes is constructed based on the second round of amplification products.

8. The library construction method according to claim 7, characterized in that, The sample to be tested is selected from one or more of a blood sample, a tissue sample and an embryo sample.

9. The library construction method according to claim 8, characterized in that, The step of extracting genomic DNA from the sample to be tested also includes: performing whole genome amplification using the genomic DNA from the embryo sample as a template.

10. Use of the primer combination product according to any one of claims 1 to 3 in the preparation of a product for detecting thalassemia.

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