A kit for non-invasive prenatal detection of paternal alpha-thalassemia large fragment deletion based on capillary electrophoresis SNP analysis and application

Abstract

The application discloses a kit for noninvasive prenatal detection of paternal alpha-thalassemia large fragment deletion based on linkage SNP analysis and capillary electrophoresis and application, and belongs to the technical field of molecular diagnosis and medical genetics. The SNP molecular marker contains at least three of 30 specific SNP sites which are in strong linkage disequilibrium with normal chromosome chains and are located in the range of large fragment deletion of chromosome 16. The detection method is as follows: extracting maternal plasma free DNA and both peripheral blood genomic DNA, using 60 pairs of primers with nucleotide sequences shown in SEQ ID NO. 1-87 for multiplex PCR amplification, performing capillary electrophoresis typing, constructing a father normal chromosome haplotype, and judging whether the fetus inherits the paternal deletion chain by analyzing the fetal SNP allele proportion in the plasma. The application breaks through the bottleneck of maternal homozygous deletion background interference, and provides a reliable means for clinical noninvasive prenatal diagnosis of paternal alpha-thalassemia large fragment deletion.

Description

Technical Field

[0001] This invention relates to the fields of molecular diagnostics and medical genetics, and in particular to a kit and its application for non-invasive prenatal detection of large fragment deletions in paternal α-thalassemia using capillary electrophoresis based on linkage SNP analysis. Background Technology

[0002] Alpha-thalassemia (α-thalassemia) is a common single-gene genetic disorder caused by the deletion or mutation of the α-globin gene cluster on the short arm of human chromosome 16, resulting in insufficient synthesis of α-globin chains. In southern China and Southeast Asia, the Southeast Asian deletion type (α-thalassemia) is more prevalent. -- SEA Deletion type is the most common pathogenic deletion, with a carrier rate as high as 5-10% in the population. If one spouse carries the SEA deletion, and the other carries another type of alpha-thalassemia defect (such as deletion type), it can lead to a high incidence of disease. -α 3.7 , -α 4.2 If the mutation is either paternal or non-deletion type, the fetus has a 25% risk of inheriting combined alpha-thalassemia. Fetuses inheriting paternal SEA deletion and other maternal deletions may present with hemoglobin H disease (HbH disease), with clinical severity ranging from mild to severe anemia, some requiring lifelong blood transfusions, significantly impacting quality of life. Therefore, paternal... -- SEA Accurate prenatal assessment of whether a missing allele has been passed on to the fetus is of vital clinical value.

[0003] Currently, the gold standard for diagnosis relies on invasive procedures such as chorionic villus sampling and amniocentesis to obtain fetal samples. While these methods are accurate, they carry a clear risk of miscarriage and complications (approximately 0.1-0.5%), often placing a significant physical and psychological burden on the pregnant woman's family. Non-invasive prenatal testing technology based on cell-free fetal DNA from maternal peripheral blood has become a routine method for screening for chromosomal aneuploidy, and its success has greatly promoted its application in the detection of single-gene genetic diseases.

[0004] However, developing a universally applicable and accurate non-invasive technology for detecting large paternal deletions faces a series of complex and challenging problems that vary depending on the mother's genotype: The most complex scenario: The mother is a heterozygous for a large homologous deletion. In this case, approximately 50% of the deleted DNA is present in the maternal plasma, creating extremely high homology background noise. Any method that directly targets the deletion breakpoint or region (such as digital PCR) cannot effectively distinguish whether the detected "deletion signal" originates from the mother or the fetus. The maternal "background noise" completely drowns out the weak fetal signal, making it impossible to determine the paternal origin. --SEA Whether the missing allele is inherited is the biggest technical bottleneck faced by existing non-invasive methods.

[0005] Widespread and technically demanding scenarios: Mothers carrying other types of alpha-thalassemia defects (such as...) -α 3.7 , -α 4.2 (Deletion or point mutation). In this case, although direct interference from maternal isotypes does not exist, the technical challenges are not eliminated and are more complex. First, the detection target—the paternal isotype... -- SEA A deletion is itself a "deletion" event. Given the low proportion (typically 5%-15%) and highly fragmented nature of fetal cell-free DNA, reliably confirming the "absence" of a specific large segment on a haploidentical genome within a large amount of normal maternal DNA results in an extremely low signal-to-noise ratio, demanding extremely high sensitivity and specificity, and easily leading to false negatives. Secondly, clinical needs extend beyond simply determining "presence"; type differentiation is also required. For example, it's necessary to clearly distinguish whether the fetus inherited a paternal SEA deletion, or a paternal normal allele, and possibly also maternal inheritance. -α 3.7 Missing. This requires the detection method to have excellent allele-specific resolution capabilities.

[0006] Currently, there are two main approaches to non-invasive prenatal testing for paternal pathogenic alleles, but in the face of... -- SEA Significant limitations exist when the content is missing: 1. The paternal-specific point mutation detection pathway is not applicable: For genetic diseases caused by point mutations, the specific mutation site carried by the father can be directly detected. However, SEA deletion is a large segment of genomic deletion, and there is no "paternal-specific point mutation" that can be directly targeted. Therefore, this technical pathway is completely unsuitable.

[0007] 2. The high-throughput sequencing-based family haplotype construction and relative haplotype dosimetry pathway is costly and complex: Represented by technologies such as cSMART, this pathway first requires high-throughput sequencing of core family members to construct an accurate haplotype map. The complete protocol relies on subsequent relative haplotype dosimetry analysis to infer the fetal genetic status. This process has high sample requirements (usually requiring samples from parents and probands), cumbersome testing procedures (involving library construction, deep sequencing, etc.), and extremely complex bioinformatics analysis (such as RHDO analysis), making standardization and automation difficult. More importantly, this technology is cumbersome, time-consuming, and has limited throughput, with a single test costing thousands of yuan. It is completely unsuitable for the general requirements of clinical prenatal screening for economy, timeliness, and accessibility, making it difficult to widely adopt in public health or as a first-line screening technology.

[0008] In conclusion, there is an urgent need in clinical practice for a universally applicable, economical, and highly specific non-invasive method to determine whether a fetus has inherited paternal genetic traits. -- SEA This invention aims to overcome the aforementioned multiple technical barriers and provide an innovative solution to meet the needs of high-risk (both parents) genotype detection technologies that lack alleles, are not limited by the mother's specific genotype, and can effectively identify genotypes. -- SEA From missing) to broader risks (one side is) -- SEA Prenatal diagnostic needs of families with missing features (or other types of defects). Summary of the Invention

[0009] The purpose of this invention is to provide a kit and its application for non-invasive prenatal detection of large fragment deletions in paternal α-thalassemia based on linked SNP analysis using capillary electrophoresis, thereby addressing the problems existing in the prior art. This invention does not directly detect... -- SEA The absence itself, but through detection and non- -- SEA Using tightly linked allele SNP sites on the deletion strand as "molecular tags," capillary electrophoresis is used to perform SNP typing on cell-free DNA from parents and blood plasma to construct the father's haplotype, thereby tracing carriers. -- SEA This strategy can accurately and non-invasively determine whether a fetus has inherited a missing paternal chromosome from its father. -- SEA The absence of alleles effectively eliminates background interference caused by the mother being a homologous carrier.

[0010] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of reagents for detecting SNP molecular markers in any of the following: (1) Application in the preparation of non-invasive prenatal testing products for large fragment deletions in paternal α-thalassemia; (2) Application in the preparation of products for assessing the risk of a fetus having severe α-thalassemia or hemoglobin H disease before birth; (3) Application in the preparation of products for diagnosing fetal severe α-thalassemia or hemoglobin H disease; The SNP molecular markers include SNP sites that are strongly linked to the normal chromosome chain in disequilibrium. The SNP sites include at least three of the 30 sites shown in the table below, and the site information is shown in Table 1. The SNP site is located within the deletion range of the large segment deletion type on human chromosome 16.

[0011] Preferably, capillary electrophoresis is used to detect the genotype of the SNP locus in order to identify the fetal paternal thalassemia large fragment deletion type.

[0012] Preferably, the detection method includes the following steps: S1: Collect peripheral blood samples from pregnant women and simultaneously collect peripheral blood samples from their spouses as paternal controls; S2: Extract cell-free DNA from the plasma of pregnant women and extract genomic DNA from peripheral blood samples of pregnant women and their partners; S3: Using 60 pairs of primers with nucleotide sequences as shown in SEQ ID NO.1-87, amplify the cell-free DNA from the pregnant woman's plasma, the pregnant woman's peripheral blood DNA, and the partner's peripheral blood DNA obtained in step S2, respectively; S4: Analyze the amplification products of step S3 using capillary electrophoresis to obtain allelic genotyping data for each SNP locus. S5: Based on the allelic genotyping data of each SNP locus, construct the normal chromosome haplotype of the spouse; S6: By analyzing the proportion of SNP alleles in fetal samples from pregnant women's cell-free plasma DNA and comparing it with the haplotypes of the pregnant woman and her spouse, it can be determined whether the fetus has inherited a haplotype from the father's normal chromosome, and further determine whether it has inherited the paternal α-large fragment deletion type.

[0013] Preferably, the method of determination is as follows: if an allele signal from the father is detected in the pregnant woman's plasma DNA on a linked SNP allele unique to the spouse, then the fetus is determined to have inherited the normal paternal chromosome chain; otherwise, the fetus is determined to have inherited the large deletion chain from the father.

[0014] Preferably, the pregnant woman's blood sample is any one of the following types of samples: (1) -- SEA Large-segment missing heterozygotes; (2) Carry -α 3.7 , -α 4.2 Deletion or non-deletion point mutation of α-globin; (3) The α-globin genotype is completely normal.

[0015] The present invention also provides a kit for non-invasive prenatal detection of large fragment deletions in paternal α-thalassemia based on linkage SNP analysis by capillary electrophoresis, comprising: reagents for detecting the genotype of the SNP locus.

[0016] Preferably, the reagent comprises 60 pairs of primers with nucleotide sequences as shown in SEQ ID NO.1-87.

[0017] Preferably, in the primer set, the 5' end of each upstream primer is labeled with FAM, NED, and VIC fluorescent groups, respectively, and the same fluorescent label corresponds to amplification fragments of different lengths to achieve synchronous differentiation of multiple sites.

[0018] Preferably, the kit further includes 2×PCR amplification premix, betaine solution, dimethyl sulfoxide, plasma free DNA and genomic DNA extraction reagents, GeneScan-600 LIZ molecular weight internal standard and highly deionized formamide.

[0019] This invention also provides the use of the kit in any of the following: (1) Application in the preparation of non-invasive prenatal testing products for large fragment deletions in paternal α-thalassemia; (2) Application in the preparation of products for assessing the risk of a fetus having severe α-thalassemia or hemoglobin H disease before birth; (3) Application in the preparation of products for diagnosing fetal severe α-thalassemia or hemoglobin H disease.

[0020] The present invention discloses the following technical effects: (1) Overcoming maternal homologous background interference and achieving paternal allele specific tracking. This invention does not directly detect large fragment deletions, but indirectly tracks the paternal chromosome inheritance status by detecting SNP sites closely linked to non-deleted strands as "molecular tags". This effectively avoids the background noise of about 50% of deletion DNA generated when the mother is also a deletion carrier, and completely solves the fundamental problem that traditional quantitative methods cannot distinguish the source of the signal.

[0021] (2) It has a wide range of applications and is not limited by the mother's thalassemia genotype. Regardless of the mother's thalassemia genotype... -- SEAMissing carrier, or - α 3.7 , -α 4.2 This invention is applicable to other deletion or point mutation carriers and has strong clinical applicability.

[0022] (3) High accuracy. Capillary electrophoresis technology has high resolution and good repeatability, and can accurately quantify the alleles of multiple SNPs, effectively eliminate non-specific interference, ensure reliable interpretation results, and help identify weak signals from the fetus.

[0023] (4) Non-invasive and safe. Only peripheral blood of the pregnant woman needs to be collected, which completely avoids the risk of miscarriage and infection caused by invasive procedures and significantly reduces the burden on the pregnant woman's family.

[0024] (5) Simple operation, short cycle and economical cost. No library construction, deep sequencing or complex bioinformatics analysis is required. It can be completed by multiplex PCR and conventional capillary electrophoresis. It is efficient and low cost, and is suitable for large-scale screening and promotion.

[0025] (6) The interpretation is intuitive and easy to standardize. Qualitative interpretation based on capillary electrophoresis peak diagrams is logically simple and clear, and it is easy to achieve automated analysis and standardized reporting.

[0026] (7) Highly practical. It is particularly suitable for prenatal screening and diagnosis in scenarios where both parents are SEA deletion carriers. It can clearly distinguish whether the fetus is normal, heterozygous, or severe, and has significant clinical guiding value. Attached Figure Description

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

[0028] Figure 1 A schematic diagram of capillary electrophoresis at 30 SNP sites; Figure 2 Capillary electrophoresis results of SNP sites in the father's (002F) blood sample; Figure 3 Capillary electrophoresis results of SNP sites in the mother's (002M) blood sample; Figure 4 Capillary electrophoresis results of SNP sites in the father's (003F) blood sample; Figure 5 Capillary electrophoresis results of SNP sites in the mother's (003M) blood sample; Figure 6 Capillary electrophoresis results of SNP sites in the father's (025F) blood sample; Figure 7 Capillary electrophoresis results of SNP sites in the mother's (025M) blood sample; Figure 8 Capillary electrophoresis results of SNP sites in the father's (049F) blood sample; Figure 9 Capillary electrophoresis results of SNP sites in the mother's (049M) blood sample; Figure 10 Capillary electrophoresis results of SNP sites in maternal plasma samples (fetal 002C); Figure 11 Capillary electrophoresis results of SNP sites in maternal plasma samples (fetal 003C); Figure 12 Capillary electrophoresis results of SNP sites in maternal plasma samples (fetal O25C); Figure 13 This is the result of capillary electrophoresis detection of SNP sites in a pregnant woman's plasma sample (fetal 049C). Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] This invention discloses a non-invasive prenatal method and a dedicated kit for detecting whether a fetus has inherited paternal alpha-thalassemia deletion, including the following deletion types: -α 3.7 ，-α 4.2 ，-- SEA ，-- THAI ，-- FIL This invention provides a universal, non-invasive detection method for paternal thalassemia with large deletions. The method involves collecting peripheral blood from pregnant women to obtain cell-free plasma DNA; performing multiplex PCR amplification on a set of single nucleotide polymorphisms (SNPs) specifically located within the large deletion region and strongly linked to the normal strand; using capillary electrophoresis to perform high-precision fragment analysis and allele typing of the amplified products; and specifically identifying and determining whether alleles covering the large deletion region on the paternal chromosome have been passed on to the fetus by analyzing the SNP haplotype linkage between the fetus and parents. This invention is not limited by the specific thalassemia genotype of the mother (regardless of the specific genotype). - - SEA Missing carrier, or -α 3.7 , -α 4.2 By limiting the number of individuals with other types of deletions or mutations (such as carriers of other types), this invention can effectively eliminate interference from maternal DNA background, enabling non-invasive, accurate, and economical identification of whether a fetus has inherited a large deletion type of paternal thalassemia, thereby assessing the risk of the fetus having severe alpha-thalassemia or hemoglobin H disease (HbH disease). This invention solves the problem of specific haplotype tracking in complex family backgrounds using traditional non-invasive methods.

[0035] The following specific examples further illustrate the method for non-invasive prenatal testing to determine whether a fetus has inherited paternal alpha-thalassemia deficiency.

[0036] Example 1: Both parents are - - SEA Non-invasive testing of fetuses with heterozygous deletion 1. SNP site selection and primer design Peripheral blood samples from 400 healthy individuals were analyzed using nanopore third-generation sequencing. SEA Sequencing of the deleted regions yielded 30 highly heterozygous SNP sites, as shown in Table 1. Primers for each SNP were designed using Primer5 software (Table 2), with the 5' end of the upstream primer labeled with FAM fluorescence. All amplicon lengths were designed between 30 and 135 bp to ensure clear separation in capillary electrophoresis. Results are shown in [Table 1]. Figure 1 .

[0037] Table 1 Information on 30 loci Note: The physical location of the above sites is referenced from the Hg38 version of the genome.

[0038] Table 2 Primers 2. Sample processing and DNA extraction Five mL of EDTA-anticoagulated blood was collected from each of pregnant women at or above 12 weeks of gestation and their partners. The blood samples from the pregnant women were centrifuged in two steps to separate plasma. Cell-free DNA from the plasma and genomic DNA from the blood samples of the pregnant women and their partners were extracted using nucleic acid extraction or purification reagents (catalog number: A004) from Guangzhou Darui Biotechnology Co., Ltd.

[0039] 3. Multiplex PCR amplification Prepare a 25 μL PCR reaction mixture containing: 20 ng DNA, 12.5 μL 2×2× KeyPo SE MasterMix (Nanjing Novizan Biotechnology Co., Ltd., catalog number: PK512-C4), 2 pmol of each primer, 0.75 μL L betaine solution (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: B0300), and 0.25 μL dimethyl sulfoxide (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: D9170). The reaction program was: 95℃ for 10 min; 35 cycles (95℃ for 30 s, 68℃ for 30 s, 72℃ for 30 s); 72℃ for 10 min.

[0040] 4. Capillary electrophoresis and typing 1 μL of PCR product was mixed with 0.5 μL of GeneScan-600 LIZ internal standard and 9.5 μL of highly deionized (Hi-Di) formamide. Capillary electrophoresis was performed using an ABI 3500 series genetic analyzer. Data were analyzed using GeneMapper software to determine the genotype of each sample at each SNP locus based on fragment size and peak height.

[0041] 5. Data Analysis and Result Interpretation Step A: Constructing parental haplotypes. The following are the SNP genotypes of the parents (4 pairs: 002F and 002M; 003F and 003M; 025F and 025M; 049F and 049M) analyzed using the method of this invention. As shown in Table 3. The S11, S2, S3, S5, S6, S7, and S8 loci are located at -- SEA Outside the deleted region, it is therefore marked as a dibase, "- / -", and the remaining sites are located in - - SEA The missing area is therefore marked with "-", see Figures 2-9 .

[0042] Table 3. SNP genotypes of 4 pairs of parents Note: Bold text indicates the father and mother. - SEA The bases that are different at SNP sites within the missing region.

[0043] Step B: Fetal allele detection. Analyze the electrophoresis peaks of the maternal plasma DNA. Observe whether a small "A" peak (i.e., the paternal-specific allele peak of 002F) appears, in addition to the maternal peak (e.g., the maternal peak of HBA1-S4 in sample 002M is G). If a small peak appears, it indicates that the fetus has inherited the paternal non-deleted strand, because these SNP sites are located in the deletion region, and the detected SNP sites can only be located on the non-deleted strand. The bolded SNP sites in Table 4 represent the corresponding SNP sites on the paternal non-deleted strand, see Table 4. Figures 10-13 (The following explanation uses the fetal testing (002C, 003C, 025C, 049C) of the above four sets of parents as examples.)

[0044] Table 4. Fetal allele information Step C: Comprehensive Interpretation. If, when testing the pregnant woman's plasma sample, consistent paternally specific allele signals from the fetus are detected at multiple SNP loci, and their relative proportions match the fetal DNA concentration (usually 5%-25%), then it is determined that the fetus has inherited paternally non-specific alleles. -SEA The missing chromosome is the chromosome that was inherited from the father.

[0045] If no paternal-specific allele signal is detected, the fetus is determined to have inherited paternal genes. -- SEA Missing chain.

[0046] According to this interpretation principle, 002C and 003C inherited the paternal non-hereditary genes. - SEA The missing strands, 025C and 049C, inherited from the father. - SEA Missing chain.

[0047] Simultaneous GAP-PCR and Sanger sequencing verification were performed on amniotic fluid DNA from samples 002C, 003C, 025C, and 049C. The GAP-PCR results showed that 002C was - - SEA The heterozygous type is missing; 003C is normal; 025C is - - SEA The homozygous form is missing; 049C is - - SEA Missing heterozygous type, 025C is - - SEA The homozygous deletion without amplicon can be used for Sanger sequencing. The Sanger sequencing results of other samples are shown in Table 5.

[0048] Table 5 Comparison results of the method of this invention with the gold standard amniotic fluid detection The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of reagents for detecting SNP molecular markers in any of the following: (1) Application in the preparation of non-invasive prenatal testing products for large fragment deletions in paternal α-thalassemia; (2) Application in the preparation of products for assessing the risk of a fetus having severe α-thalassemia or hemoglobin H disease before birth; (3) Application in the preparation of products for diagnosing fetal severe α-thalassemia or hemoglobin H disease; The SNP molecular markers include SNP sites that are in strong linkage disequilibrium with the normal chromosome chain. These SNP sites include at least three of the 30 sites shown in the table below, and the site information is as follows: The SNP site is located within the deletion range of the large segment deletion type on human chromosome 16.

2. The application as described in claim 1, characterized in that, The genotype of the SNP loci was detected by capillary electrophoresis to identify fetal paternal thalassemia with large fragment deletions.

3. The application as described in claim 2, characterized in that, The detection method includes the following steps: S1: Collect peripheral blood samples from pregnant women and simultaneously collect peripheral blood samples from their spouses as paternal controls; S2: Extract cell-free DNA from the plasma of pregnant women and extract genomic DNA from peripheral blood samples of pregnant women and their partners; S3: Using 60 pairs of primers with nucleotide sequences as shown in SEQ ID NO.1-87, amplify the cell-free DNA from the pregnant woman's plasma, the pregnant woman's peripheral blood DNA, and the partner's peripheral blood DNA obtained in step S2, respectively; S4: Analyze the amplification products of step S3 using capillary electrophoresis to obtain allelic genotyping data for each SNP locus. S5: Based on the allelic genotyping data of each SNP locus, construct the normal chromosome haplotype of the spouse; S6: By analyzing the proportion of SNP alleles in fetal samples from pregnant women's cell-free plasma DNA and comparing it with the haplotypes of the pregnant woman and her spouse, it can be determined whether the fetus has inherited a haplotype from the father's normal chromosome, and further determine whether it has inherited the paternal α-large fragment deletion type.

4. The application as described in claim 3, characterized in that, The method for determination is as follows: if an allele signal from the father is detected in the pregnant woman's plasma DNA on a linked SNP allele unique to the spouse, then the fetus is determined to have inherited the normal chromosome chain from the father. Conversely, if the fetus does not inherit a large missing segment from the father, it is determined that the fetus has inherited the missing segment from the father.

5. The application as described in claim 1, characterized in that, The pregnant woman's blood sample can be any of the following types of samples: (1) -- SEA Large-segment missing heterozygotes; (2) Carry -α 3.7 , -α 4.2 Deletion or non-deletion point mutation of α-globin; (3) The α-globin genotype is completely normal.

6. A kit for non-invasive prenatal detection of large fragment deletions in paternal α-thalassemia using capillary electrophoresis based on linkage SNP analysis, characterized in that... include: A reagent for detecting the genotype of the SNP site described in claim 1.

7. The kit according to claim 6, characterized in that, The reagent comprises 60 pairs of primers with nucleotide sequences as shown in SEQ ID NO.1-87.

8. The kit according to claim 7, characterized in that, In the primer set, the 5' end of each upstream primer is labeled with FAM, NED, and VIC fluorescent groups, respectively. The same fluorescent label corresponds to amplification fragments of different lengths to achieve synchronous differentiation of multiple sites.

9. The reagent kit as described in claim 6, characterized in that, The kit also includes 2×PCR amplification premix, betaine solution, dimethyl sulfoxide, plasma free DNA and genomic DNA extraction reagents, GeneScan-600 LIZ molecular weight internal standard and highly deionized formamide.

10. The use of the kit according to any one of claims 6-9 in any of the following: (1) Application in the preparation of non-invasive prenatal testing products for large fragment deletions in paternal α-thalassemia; (2) Application in the preparation of products for assessing the risk of a fetus having severe α-thalassemia or hemoglobin H disease before birth; (3) Application in the preparation of products for diagnosing fetal severe α-thalassemia or hemoglobin H disease.

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