Primer compositions, methods and kits for detecting hba1 / 2 monogenic defects

Abstract

The application provides a primer composition, a method and a kit for detecting HBA1 / 2 single gene defect, comprising a first primer composition and a second primer composition designed according to SNP and STR sites screened in the HBA1 / 2 single gene region, the first primer composition is forward and reverse primers in SEQ ID NO: (2n-1) and SEQ ID NO: (2n), n is a positive integer from 1 to 467; the second primer composition is forward and reverse primers in SEQ ID NO: (2n-1) and SEQ ID NO: (2n), n is a positive integer from 468 to 496; the kit contains the primer composition, and the primer composition or the kit is used for detecting HBA1 / 2 single gene defect. The primer composition, the method and the kit can identify whether the HBA1 / 2 single gene of a preimplantation embryo is varied, and have the advantages of low cost and accurate diagnosis.

Description

Technical Field

[0001] This invention relates to the field of gene detection technology, specifically to a primer composition, method, and kit for detecting HBA1 / 2 single gene defects. Background Technology

[0002] Alpha-thalassemia is a prevalent single-gene inherited disease worldwide. It is a hereditary hemolytic anemia caused by insufficient hemoglobin (Hb) synthesis due to partial or complete inhibition of alpha-globin peptide chain synthesis. It is inherited in an autosomal recessive manner, and the pathogenic genes are HBA1 (OMIM141800) and HBA2 (OMIM141850). The incidence is equal in males and females. Currently, for couples carrying certain known genetic abnormalities, preimplantation genetic testing (PGT-M) is often used in reproductive medicine. By testing for single-gene diseases before embryo implantation, "disease-causing" embryos are excluded, and embryos without pathogenic mutations are selected for transfer to avoid the suffering of offspring from genetic diseases.

[0003] Cells used for PGT-M detection are typically few in number (3-10 cells). To ensure sufficient DNA for subsequent testing, whole-genome amplification of the cell sample is usually required. This introduces a disconnection phenomenon, where one allele is dominantly amplified while the other is suppressed. Therefore, PGT-M detection also requires detecting linkage marker sites upstream and downstream of the pathogenic gene. Linkage analysis is used to determine whether offspring embryos carry the risk chromosome inherited from their parents. For example, for PGT-M in α-thalassemia, multiplex PCR is used to target and capture SNP sites in the HBA1 / 2 single gene and its upstream and downstream adjacent ranges. After next-generation sequencing analysis, each SNP site is interpreted to construct a haplotype of the HBA1 / 2 single gene. Finally, it is assessed whether offspring embryos inherit the risk chromosome containing the pathogenic thalassemia gene mutation from the father and / or mother. The selection of SNP sites in the target gene should avoid sequences with high homology, high GC content, or polynucleotides. However, because the HBA1 / 2 single gene is located at the end of the chromosome, and some families cannot find enough effective sites on the telomere side of the HBA1 / 2 gene, the possibility of embryo recombination cannot be ruled out, which leads to the failure to construct haplotype and makes it impossible to determine whether the embryo is a qualified embryo.

[0004] Short tandem repeats (STRs) are polymorphic repetitive DNA sequences of 2-6 bp distributed throughout human chromosomes. Due to their high polymorphism, short repetitive sequences, ease of PCR amplification, and convenient detection, STRs have significant applications in genetic mapping, pathogenic gene localization, population genetics, and reproductive medicine. Currently, methods for constructing haplotypes using STRs are also used to analyze whether embryos carry pathogenic mutations, thereby determining whether the embryo is viable. However, constructing haplotypes using STRs often encounters problems such as: slippage severely affecting the accuracy of STR detection and analysis; STR haplotype construction primarily relies on capillary electrophoresis, resulting in low throughput and potential signal interference between different STR loci; and the limited number of publicly available STR loci for the HBA1 / 2 single gene makes it difficult to meet the needs of different clinical applications.

[0005] Therefore, the above-mentioned methods for HBA1 / 2 single gene detection need to be improved to increase the accuracy of HBA1 / 2 single gene disease detection and ensure the accuracy of preimplantation embryo screening. Summary of the Invention

[0006] The purpose of this invention is to design a primer composition, method, and kit for detecting HBA1 / 2 single gene defects. By screening SNP and STR sites in the HBA1 / 2 single gene and preparing primer compositions based on the screened SNP and STR sites, the invention identifies whether there are variations / mutations in preimplantation embryos, thereby more efficiently and accurately screening qualified embryos for implantation and reducing the risk of disease.

[0007] The technical solution to achieve the purpose of the invention is as follows: In a first aspect, the present invention provides a primer composition for detecting HBA1 / 2 single gene defects, comprising: The first set of primer compositions was designed based on the SNP sites screened in the HBA1 / 2 single gene region. The first set of primer compositions consists of the forward and reverse primers corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 1 to 467. A second set of primer compositions was designed based on the STR sites screened in the HBA1 / 2 single gene region. The second set of primer compositions consists of the forward and reverse primers corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 468 to 496.

[0008] Furthermore, the range of SNP sites screened in the HBA1 / 2 single gene region is the HBA1 / 2 single gene and the DNA fragments within a 3Mb range upstream and downstream, and the first set of primer compositions includes: Primer composition one designed based on the SNP sites screened upstream of the HBA1 / 2 single gene, wherein the primer composition one is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 1 to 164; Primer composition two is designed based on the SNP sites selected by HBA1 / 2 single gene screening. The primer composition two is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 165 to 184. Primer composition three is designed based on the SNP sites screened downstream of the HBA1 / 2 single gene. Primer composition three is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 185 to 467.

[0009] Furthermore, the selection criteria for SNP sites include: (1) The minimum allele frequency of the SNP locus is greater than or equal to 0.2; (2) Remove SNP sites containing polynucleotides; (3) Remove SNP sites with GC content greater than 60% within a 200 bp range upstream and downstream; (4) Remove SNP sites that are homologous to each other in the human genome within 200 bp upstream and downstream.

[0010] Furthermore, the range of STR sites to be screened in the HBA1 / 2 single gene region is the HBA1 / 2 single gene and the DNA fragment within a 200Kb range upstream of it, and the second set of primer compositions includes: Primer composition four was designed based on the STR site selected upstream of the HBA1 / 2 single gene. The primer composition four is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 468 to 494. Primer composition five is designed based on the STR site of HBA1 / 2 single gene screening. Primer composition five is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 495 to 496.

[0011] Furthermore, the selection criteria for STR loci include: (1) Remove STR sites containing polynucleotides; (2) Retain STR sites with polymorphic information content (PIC) > 0.2; (3) Retain STR sites with repeating unit bases of 3, 4, 5, and 6; (4) Remove STR sites with a total length greater than 150 bp; (5) Remove STR sites with GC content greater than 60% within a 200 bp range upstream and downstream; (6) Remove STR sites that are homologous to each other in the human genome within 200 bp upstream and downstream.

[0012] In a second aspect, the present invention provides a kit for detecting HBA1 / 2 single gene defects, comprising the primer composition described in the first aspect, as well as a super reaction solution, an end repair reagent, a adapter ligation reagent, and an amplification reaction reagent.

[0013] Furthermore, the super reaction solution comprises 1.5× to 2.5× PCR buffer, 150 to 250 U / mL hot-start Taq enzyme, 5 to 15 U / mL UDG enzyme, 0.2 to 0.6 mM dNTP, 0.2 to 0.4 mM dUTP, 2 to 8 mM MgCl2, 0.5% to 2.5% BSA, and 0.5% to 2.5% mannitol.

[0014] Furthermore, the end-repair reagent comprises DNA fragment end-repair and Adenytion Buffer Mix, DNA fragment end-repair and Adenytion Enzyme Mix, and Nuclease-free Water.

[0015] Furthermore, the adapter ligation reagent comprises a ligase enzyme mixture and an adapter sequence barcode adapter.

[0016] Furthermore, the amplification reaction reagents include a PCR Enzyme Mix, a PCR Primer Mix, and Nuclease-free Water.

[0017] Thirdly, this invention provides a method for detecting HBA1 / 2 single-gene defects, comprising the following steps: Step 1: Obtain DNA samples from the proband, both parents, and the offspring embryo; Step 2: Use the primer composition described in the first aspect or the kit described in the second aspect to perform multiplex PCR amplification on the DNA samples of the proband and both parents, construct a DNA library and perform sequencing analysis; Step 3: Based on the sequencing analysis results, obtain the haplotypes of the proband and both parents; Step 4: Determine the HBA1 / 2 single-gene defect in the offspring embryos based on the haplotypes of the proband and both parents.

[0018] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention employs a method of screening SNP and STR sites in DNA fragments of the HBA1 / 2 single gene region according to rules and designing primer compositions. Multiplex PCR is performed on the samples using these primer compositions to construct libraries, followed by high-throughput sequencing analysis. By constructing haplotypes of mutated alleles in conjunction with pedigree information, the genotypes of offspring embryos are determined, thereby confirming the HBA1 / 2 gene defect detection results in the offspring embryos. The scheme designed in this invention is based on high-throughput sequencing technology, eliminating the need for cellular-level preliminary experiments. It allows for the analysis of a large number of samples simultaneously, with an average sequencing depth exceeding 100X, significantly improving detection efficiency and accuracy.

[0019] 2. Commonly used linkage loci include SNP loci and STR loci. SNP loci are frequently used in linkage analysis, but STR loci are rarely used in linkage analysis due to their length and susceptibility to slippage. Since the single gene HBA1 / 2 is only about 200 kb from the telomere side of chromosome 16, conventional methods that only detect SNP loci may not yield enough effective SNP loci in some families. Therefore, this application combines SNP and STR loci to form linkage loci for analysis, which can minimize misdiagnosis caused by ADO (Adaptive Dosing). Detailed Implementation

[0020] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0021] In this specific embodiment, reads refer to sequence fragments obtained through sequencing; single nucleotide polymorphism (SNP) mainly refers to DNA sequence polymorphism caused by variations in a single nucleotide at the genomic level; haplotype refers to a group of related single nucleotide polymorphisms located in a specific region of a chromosome that tend to be inherited as a whole by offspring, also known as haplotype or haplotype; sequencing depth, for example, in one embodiment, a sequencing depth of 1000x means that the specific PCR amplification product has been sequenced 1000 times; effective site refers to a certain SNP site or STR site in both partners of a couple, where one partner is homozygous and the other is heterozygous, then the site is called the heterozygous effective site.

[0022] It should be noted that the offspring embryos used in this invention are fertilized eggs obtained through in vitro fertilization. The cells extracted during the in vitro development to the cleavage stage or blastocyst stage are not directly applied to living human or animal bodies and do not belong to the diagnosis and treatment methods for diseases.

[0023] Meanwhile, the genomic reference sequences used in this specific embodiment can be from public databases, such as human genome sequences like hg19 and hg38 from the NCBI or UCSC databases.

[0024] Example 1: This embodiment focuses on the detection of alpha-thalassemia, a single-gene disorder. The alpha-thalassemia gene is located on chromosome 16, relatively close to the telomeres. Mutations at certain sites in the HBA1 / 2 gene on chromosome 16 can increase the risk of disease in the individual or their offspring. Based on the severity of symptoms, it can be divided into Hb H disease and Hb Bart's hydrops fetalis syndrome. Hb H disease, also known as intermediate alpha-thalassemia, often presents with moderate hemolysis. These individuals are asymptomatic at birth, but gradually develop anemia, hepatosplenomegaly, jaundice, etc., during infancy. Some individuals may experience skeletal deformities, resulting in distinctive facial features such as maxillary hyperplasia and a prominent forehead. Infections or the use of oxidizing drugs can induce acute hemolysis or even a hemolytic crisis. Most patients develop normally, with no significant impact on daily life, and do not require blood transfusions. A small number of individuals with severe anemia may have hypersplenism, which can be improved after splenectomy. Common complications include gallstones and lower extremity ulcers. Elderly patients may experience iron overload due to chronic hemolysis. HbBart's hydrops fetalis syndrome, also known as severe alpha-thalassemia, affects fetuses with severe anemia and hypoxia, resulting in generalized edema, jaundice, significant hepatosplenomegaly, and developmental delays. Pregnant women carrying such fetuses may experience preeclampsia, premature birth, and abnormal bleeding. Due to the large placenta, serious complications may occur during delivery.

[0025] This embodiment provides a primer composition for detecting HBA1 / 2 single gene defects, including a first set of primer compositions and a second set of primer compositions. The first set of primer compositions is designed based on SNP sites selected from the HBA1 / 2 single gene region. The selection principles for these SNP sites are general selection principles, including: (1) The minimum allele frequency of the SNP locus is greater than or equal to 0.2; (2) Remove SNP sites containing polynucleotides; (3) Remove SNP sites with GC content greater than 60% within a 200 bp range upstream and downstream; (4) Remove SNP sites that are homologous to each other in the human genome within 200 bp upstream and downstream.

[0026] After screening, a total of 467 SNP sites were obtained. Primers were designed for each SNP site to obtain the first set of primer compositions. The first set of primer compositions consisted of the forward and reverse primers corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 1 to 467. Furthermore, the range of SNP sites to be screened in the HBA1 / 2 single gene region is the HBA1 / 2 single gene and the DNA fragments within a 3Mb range upstream and downstream, and the first set of primer compositions includes primer composition one, primer composition two, and primer composition three.

[0027] Primer composition one is designed based on the SNP sites screened upstream of the HBA1 / 2 single gene. Primer composition one is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 1 to 164. Primer composition two is designed based on the SNP sites of HBA1 / 2 single gene screening. Primer composition two is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 165 to 184. Primer composition three is designed based on the SNP sites screened downstream of the HBA1 / 2 single gene. Primer composition three is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 185 to 467.

[0028] The second set of primers was designed based on STR sites selected from the HBA1 / 2 single gene region. The selection principles for STR sites include: (1) Remove STR sites containing polynucleotides; (2) Retain STR loci with polymorphic information content (PIC) > 0.2. PIC reflects the polymorphism of the target sequence. When the PIC value of the STR locus is higher, it indicates that the heterozygosity of the STR locus is higher and the genetic polymorphism is better.

[0029] The PIC value was calculated as follows: peripheral blood was collected from 200 unrelated individuals, genomic DNA was extracted, and 29 STR loci associated with the HBA1 / 2 single gene were amplified and screened using fluorescent PCR. The amplified products were subjected to capillary electrophoresis using an ABI3500Dx genetic analyzer, and the results were analyzed using Genemaper 4.1 software. Finally, the allele frequency distribution and PIC of the 29 HBA-STR loci were calculated using Microsoft Excel software. The results showed that the PIC values ​​of all 29 loci were greater than 0.5, indicating that these 29 STR loci possess high genetic polymorphism and have high application value in genetic diagnosis. (3) Retain STR sites with repeating units of 3, 4, 5, and 6 bases. Since STR sites with repeating units of 2 bases are prone to slippage, STR sites with repeating units of 2 bases are removed. (4) Remove STR sites with a total length greater than 150 bp; (5) Remove STR sites with GC content greater than 60% within a 200 bp range upstream and downstream; (6) Remove STR sites that are homologous to each other in the human genome within 200 bp upstream and downstream.

[0030] After screening, a total of 29 STR loci were obtained. Primers were designed for each STR locus to obtain a second set of primer compositions. The second set of primer compositions consisted of the forward and reverse primers corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 468 to 496. Furthermore, the range of STR sites to be screened in the HBA1 / 2 single gene region is the HBA1 / 2 single gene and the DNA fragment within 200Kb upstream of it, and the second set of primer compositions includes primer composition four and primer composition five.

[0031] Primer composition four is designed based on the STR site selected upstream of the HBA1 / 2 single gene. Primer composition four is the forward and reverse primers corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 468 to 494. Primer composition five is designed based on the STR site for HBA1 / 2 single gene screening. Primer composition five is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 495 to 496.

[0032] In this embodiment, since the HBA1 / 2 gene is located near the telomere of chromosome 16, and its coding region covers the range of 222846-223709 and 226679-227520 on chromosome 16 (taking hg19 as an example), when using conventional methods to detect only the SNP site of the HBA1 / 2 gene (i.e., using only the first set of primers (i.e., primers designed through SNPs), some families may not be able to find enough effective sites, resulting in a high ADO value. This cannot rule out the possibility of embryo recombination, leading to failure in haplotype construction and making it impossible to determine whether the embryo is a qualified embryo or causing a risk of misjudgment. Therefore, the first set of primers and the second set of primers must be used simultaneously.

[0033] Example 2: This embodiment provides a kit for detecting HBA1 / 2 single gene defects, which contains the primer composition in Example 1, as well as a super reaction solution, an end repair reagent, a linker ligation reagent, and an amplification reaction reagent.

[0034] The super reaction solution includes 1.5× to 2.5× PCR buffer, 150 to 250 U / mL hot-start Taq enzyme, 5 to 15 U / mL UDG enzyme, 0.2 to 0.6 mM dNTP, 0.2 to 0.4 mM dUTP, 2 to 8 mM MgCl2, 0.5% to 2.5% BSA, and 0.5% to 2.5% mannitol.

[0035] The end-repair reagent comprises DNA fragment end-repair and Adenytion Buffer Mix, DNA fragment end-repair and Adenytion Enzyme Mix, and Nuclease-free Water.

[0036] The adapter ligation reagent comprises a ligase enzyme mixture (Ligase Enzyme Mix) and an adapter sequence (Barcode Adapter).

[0037] The amplification reaction reagents include a PCR Enzyme Mix, a PCR Primer Mix, and Nuclease-free Water.

[0038] Example 3: This embodiment provides a method for detecting HBA1 / 2 single gene defects, including the following steps: Step 1: Obtain DNA samples from the proband and both parents.

[0039] DNA samples from both parents can be selected from one or more of the following: peripheral blood genomic DNA, semen DNA, oral mucosal cell DNA, and whole-genome amplification products of cells.

[0040] Preferably, each DNA sample contains more than 500 ng of DNA.

[0041] Step 2: Using the primer composition in Example 1 or the kit in Example 2, perform multiplex PCR amplification on the DNA samples of the proband and both parents to construct a DNA library and perform sequencing analysis.

[0042] When the DNA sample to be tested comes from multiple test samples, a different tag sequence (barcode) can be added to each sample to distinguish the samples during the sequencing process, so as to achieve simultaneous sequencing and sequencing analysis of multiple samples.

[0043] During sequencing: the average sequencing depth of the target region > 100X, the sequencing depth of the target region > 30X, and the proportion of sites > 50%. The average sequencing depth is calculated as follows: For example, a panel size of 3M produces 3G of data. For libraries with a panel size greater than 1M, the capture efficiency is often around 60%, meaning that 60% of the sequencing data is the desired data. Therefore, the data in the target region is 3G * 60% = 1.8G. A rough calculation of the sequencing depth is 1.8G / 3M = 600X. Therefore, for a library with a panel size of 3M and a data output of 3G, the sequencing depth is approximately 600X. If the average sequencing depth is low, it may directly lead to the inability to detect truly existing low-abundance mutations. In bioinformatics analysis, it is generally considered that a mutation is considered credible only when there are 5 independent positive reads. Therefore, if it is necessary to detect mutations as low as 1%, 5 / (0.01) = 500 total reads are required. Sequencing depth refers to the ratio of the total number of bases obtained from sequencing to the size of the genome being tested. It can be understood as the average number of times each base in the genome is sequenced. A sequencing depth greater than 20X for a certain site indicates that the site has been sequenced more than 20 times, and the results are more reliable.

[0044] Step 3: Based on the sequencing analysis results, obtain the haplotypes of the proband and both parents.

[0045] Step 4: Determine the HBA1 / 2 single-gene defect in the offspring embryos based on the haplotypes of the proband and both parents.

[0046] This example uses the detection of sea locus defects in the HBA1 / 2 single gene as an example to specifically illustrate the method for detecting HBA1 / 2 single gene defects. In this example, a group of carriers in both spouses with defects at the sea locus were selected. Both carriers of the HBA gene (hg19, NM_000558.5, chr16: 215400-234700) showed loss of sea heterozygosity. The detection process is as follows: First, peripheral blood samples from both parents were processed to extract DNA; the proband's cell sample underwent WGA amplification; offspring embryo samples were obtained through in vitro fertilization and amplified to obtain DNA samples from both parents, the proband, and the offspring embryos. The DNA samples from the offspring embryos were taken from fertilized eggs obtained through in vitro fertilization, specifically cells extracted when the fertilized eggs developed to the cleavage or blastocyst stage, such as trophoblast cells at the blastocyst stage.

[0047] Secondly, using the kit of Example 2 designed with primer compositions from Example 1, and the kit designed with only the first set of primer compositions from Example 1, DNA samples were constructed according to the Illumina standard library construction procedure, and sequenced using Miseq.

[0048] Then, based on the sequencing analysis results, the haplotypes of the proband and both parents are obtained, and the risk chromosomes of both parents are analyzed and determined. If the patient's family has entered an assisted reproductive cycle and there are offspring embryos (i.e., obtained through in vitro fertilization), the risk chromosomes of both parents can be used to further determine whether each embryo sample carries a risk chromosome from the father or the mother.

[0049] The test results of the proband and both parents using the two kits were analyzed in detail. Trimmomatic software was used to remove adapter sequences from the raw data generated by the Illumina sequencer, and BWA software was used to align to the human hg19 reference genome. Finally, haplotype SNP coverage and genotype were analyzed, and lobSTR was used to analyze STR coverage and genotype. The haplotypes of both parents and the proband obtained using the kit in Example 2 are shown in the table below: 1. When using a kit prepared with only the first primer combination for testing, the results show that both parents have more than three valid SNP sites downstream of the HBA1 / 2 gene, meeting the recommended testing criteria of the "Expert Consensus on Preimplantation Genetic Testing of Embryos with Single-Gene Diseases". Specifically, upstream of the HBA1 / 2 gene, the female parent had more than three valid SNP sites, but the male parent had only one. Based on these results, it cannot be determined whether homologous recombination occurred in the offspring embryo in the aforementioned region, and therefore it is impossible to determine whether the offspring carry the pathogenic mutation.

[0050] 2. When the kit prepared using the first primer composition and the second primer composition (i.e., the kit in Example 2) is used, according to the detection results, and as shown in the table above, both parents were found to have more than 3 valid SNP sites downstream of HBA1 / 2. Specifically, upstream of the HBA1 / 2 gene, the female parent was found to have more than 3 valid SNP sites, and the male parent was found to have one valid SNP site and one valid STR site, which meets the recommended detection standards of the "Expert Consensus on Preimplantation Genetic Testing for Single Gene Disease Embryos".

[0051] Based on the table above, it can be determined that chromosome M0, which is the same as the female parent, is a risk chromosome; chromosome M1, which is a normal chromosome, is the same as the male parent; chromosome F0, which is a risk chromosome; and chromosome F1, which is a normal chromosome. The haplotype results of both parents and the proband are shown in the table below: In the table above, F0 represents the risk chromosome of the male parent, F1 represents the normal chromosome of the male parent, M0 represents the risk chromosome of the female parent, and M1 represents the normal chromosome of the female parent.

[0052] The detection methods for other sites in the HBA1 / 2 single gene region are the same as those for the sea site in the above example, and will not be illustrated here.

[0053] Based on the haplotype construction results of the proband and both parents, it is possible to accurately determine whether the offspring embryo sample carries the risk chromosome of the female or male parent.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0055] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A primer composition for detecting HBA1 / 2 single gene defects, characterized in that, include: The first set of primer compositions was designed based on the SNP sites screened in the HBA1 / 2 single gene region. The first set of primer compositions consists of the forward and reverse primers corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 1 to 467. A second set of primer compositions was designed based on the STR sites screened in the HBA1 / 2 single gene region. The second set of primer compositions consists of the forward and reverse primers corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 469 to 496.

2. The primer composition for detecting HBA1 / 2 single gene defects according to claim 1, characterized in that, The range of SNP sites to be screened in the HBA1 / 2 single gene region is the HBA1 / 2 single gene and the DNA fragments within a 3Mb range upstream and downstream of it, and the first set of primer compositions consists of primer composition one, primer composition two, and primer composition three. composition: Among them, primer composition one is designed based on the SNP site screened upstream of the HBA1 / 2 single gene. Primer composition one is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 1 to 164. Primer composition two was designed based on the SNP sites selected by HBA1 / 2 single gene screening. The primer composition two is the forward and reverse primers corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 165 to 184. Primer composition three was designed based on the SNP sites screened downstream of the HBA1 / 2 single gene. Primer composition three is the forward and reverse primers corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 185 to 467.

3. The primer composition for detecting HBA1 / 2 single gene defects according to claim 2, characterized in that, The selection criteria for SNP sites include: (1) The minimum allele frequency of the SNP locus is greater than or equal to 0.2; (2) Remove SNP sites containing polynucleotides; (3) Remove SNP sites with GC content greater than 60% within a 200 bp range upstream and downstream; (4) Remove SNP sites that are homologous to each other in the human genome within 200 bp upstream and downstream.

4. The primer composition for detecting HBA1 / 2 single gene defects according to claim 1, characterized in that, The range of STR sites to be screened in the HBA1 / 2 single gene region is the HBA1 / 2 single gene and the DNA fragment within a 200Kb range upstream of it, and the second set of primer compositions consists of primer composition four and primer composition five. composition: Among them, primer composition four was designed based on the STR site screened upstream of the HBA1 / 2 single gene. Primer composition four is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 469 to 494. Primer composition five was designed based on the STR site of HBA1 / 2 single gene screening. Primer composition five is the forward primer and reverse primer corresponding to SEQ ID NO: (2n-1) and SEQ ID NO: (2n), where n is a positive integer from 495 to 496.

5. The primer composition for detecting HBA1 / 2 single gene defects according to claim 4, characterized in that, The selection criteria for STR loci include: (1) Remove STR sites containing polynucleotides; (2) Retain STR sites with polymorphic information content (PIC) > 0.2; (3) Retain STR sites with repeating unit bases of 3, 4, 5, and 6; (4) Remove STR sites with a total length greater than 150 bp; (5) Remove STR sites with GC content greater than 60% within a 200 bp range upstream and downstream; (6) Remove STR sites that are homologous to each other in the human genome within 200 bp upstream and downstream.

6. A kit for detecting HBA1 / 2 single gene defects, characterized in that, It includes the primer composition according to any one of claims 1 to 5, as well as the super reaction solution, end repair reagent, adapter ligation reagent, and amplification reaction reagent.

7. The reagent kit according to claim 6, characterized in that: The super reaction solution comprises 1.5× to 2.5× PCR buffer, 150 to 250 U / mL hot-start Taq enzyme, 5 to 15 U / mL UDG enzyme, 0.2 to 0.6 mM dNTP, 0.2 to 0.4 mM dUTP, 2 to 8 mM MgCl2, 0.5% to 2.5% BSA, and 0.5% to 2.5% mannitol.

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