Primer group, kit for detecting multiple mutations of human fetal beta-thalassemia and application thereof

Abstract

The present application relates to a primer set for detecting multiple mutations of human embryo beta-thalassemia, a kit and application thereof. The primer set comprises five primers for amplifying HBB a plurality of mutations in the gene region: HBB-F1, HBB-F2, HBB-F3, HBB-R1 and HBB-R2, the sequences of which are shown in SEQ ID NO: 1-5 respectively; and eight primers for amplifying HBB linkage SNP sites within 1Mb range of the upstream and downstream genes: L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F and L4-R, the sequences of which are shown in SEQ ID NO: 6-13 respectively. Based on the specific combination of long fragment PCR amplification and long fragment high-throughput sequencing, the present application can simultaneously detect multiple mutations of beta-thalassemia in multiple embryo samples with high specificity, accuracy and speed.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to a primer set, reagent kit, and their applications for detecting multiple mutations in human embryonic β-thalassemia using a long-read sequencing platform. Background Technology

[0002] Thalassemia is the most common single-gene inherited disease in the world. It is an autosomal recessive disorder, with high incidence rates in regions including southern China, Southeast Asia, the Mediterranean region, India, the Middle East, and Africa. Thalassemia is mainly divided into α-thalassemia and β-thalassemia, which are caused by the absence or insufficiency of β-globin synthesis, respectively. 1,2,3 .

[0003] β-thalassemia is a hereditary hemolytic anemia caused by insufficient Hb synthesis due to reduced or complete absence of β-globin peptide chain synthesis. 4,5 Severe β-thalassemia was first reported by pediatrician Cooley in 1925, hence it is also known as Cooley's anemia. The disease is inherited in an autosomal recessive manner, with the causative gene being HBB. The incidence is roughly equal in males and females. The clinical phenotypes of individuals with β-thalassemia and their dependence on blood transfusions can be categorized as follows: 6,7 (1) Carrier. Usually without clinical signs; (2) Patient. According to their condition and dependence on blood transfusion, it is further divided into intermediate β-thalassemia and severe β-thalassemia. In the absence of treatment, it can cause hepatosplenomegaly, distinctive facial features, osteoporosis and other symptoms. The β-globin gene cluster is located on chromosome 11 and contains 5 gene loci: 5'- HBE - HBG2 - HBG1 - HBD (δ)- HBB (β)-3'. Wherein HBB The gene encodes globin in adults, while the other four genes are expressed during embryonic development or at very low levels in adults. The vast majority of mutations leading to β-thalassemia are non-deletion mutations, with a minority being large deletions. 3 Based on the Ithanet, HbVar, LOVD, and LOVD-China thalassemia databases, more than 1,000 non-deletion mutations of the HBB gene have been identified worldwide.

[0004] Thalassemia is congenital, lifelong, and familial. Currently, there is no ideal treatment, and patients require lifelong treatment, enduring not only suffering but also incurring high costs. 3However, since genetic diseases usually occur through the reproductive behavior of gene carriers, assisted reproductive technologies can be used to block their transmission to offspring, reducing the number of people with genetic diseases. Preimplantation genetic testing (PGT) involves genetically testing embryos before implantation into the uterus to select normal or non-pathogenic embryos for transfer. PGT includes preimplantation genetic testing for aneuploidy (PGT-A), preimplantation genetic testing for chromosomal structural rearrangements (PGT-SR), and preimplantation genetic testing for monogenic / single-gene defects (PGT-M). PGT-M is for parents who have or carry known monogenic genetic diseases, such as thalassemia, and involves testing the embryo for monogenic genetic diseases before implantation. 7,8,9 .

[0005] Because PGT technology yields a small number of cells and has limited usable genetic material, whole genome amplification (WGA) is generally required. Currently, WGA methods are mainly divided into three categories: one is based on polymerase chain reaction (PCR) technology, such as primer extension preamplification PCR (PEP-PCR) and degenerate oligomer primer PCR (DOP-PCR); another is isothermal whole genome amplification reactions, such as multiple displacement amplification (MDA); and the third is multiple annealing and looping-based amplification cycles (MALBAC). Although many commercial kits are available, with varying key parameters, each method and kit has amplification biases, allele dropout (ADO), and coefficient of variation, among others. 10 This can lead to false negatives or false positives, resulting in diagnostic failure or even misdiagnosis.

[0006] To improve diagnostic accuracy, PGT-M requires simultaneous haplotype linkage analysis. Currently, there are three main linkage analysis methods: short tandem repeats (STRs), single-nucleotide polymorphism arrays (SNPs), and next-generation sequencing (NGS) technology. 7 However, STR technology is relatively cumbersome and costly, and STR and SNP microarrays can only analyze known sites, thus missing some unknown potential SNP sites that can be used for haplotype identification. NGS technology can only detect DNA fragments of about 300 bases, while the average spacing of SNP sites in the human genome is about 1000 bases. Therefore, NGS can only obtain at most one SNP site per DNA fragment on average, making it difficult to find consecutive SNPs and limiting the selection of SNP sites to construct haplotypes, resulting in a waste of SNP sites. Furthermore, all three methods require a proband, but some families do not have a proband or cannot obtain the proband's genetic material. Currently, methods that do not require a proband include third-generation whole-genome sequencing. 11 However, this method is expensive and has poor market accessibility and promotion in clinical applications.

[0007] Current methods based on STR, SNP arrays, NGS, and long-read whole-genome sequencing have the following limitations: 1. Dependence on the proband: However, some families do not have a proband, or cannot obtain the genetic material of the proband; 2. Low efficiency of linkage analysis: It can only use known sites or can only select a limited number of sites to construct haplotypes, resulting in a waste of linkage sites and requiring many linkage fragments for analysis; 3. High cost: Traditional methods are cumbersome, costly, and time-consuming, while third-generation whole genome sequencing is expensive. Summary of the Invention

[0008] The purpose of this invention is to address the current problems of human embryo β-thalassemia detection, which relies on probands, has low linkage analysis efficiency, and is cumbersome, costly, and time-consuming. This invention utilizes long-fragment PCR to simultaneously amplify β-thalassemia gene mutation-related fragments and... HBB Four linked fragments from linked SNP sites within a 1Mb range upstream and downstream of the gene, and the preparation of long-fragment sequencing libraries, enable comprehensive, accurate, efficient, and rapid detection of multiple samples. HBB The goal of detecting multiple gene mutations is to ensure the accuracy and sensitivity of embryo testing while avoiding the need for a proband and reducing the financial burden of testing for patients.

[0009] Therefore, this invention provides a method for detecting multiple mutations in human embryonic β-thalassemia based on multiplex long-fragment PCR amplification and long-fragment sequencing, without requiring a proband. Long-fragment PCR amplification is performed in separate reaction tubes for detection. HBB All point mutation fragments within the primer range of the gene locus and gap fragments for detecting large deletions, as well as HBB Linked fragments of linked SNP sites within a 1Mb range upstream and downstream of a gene; combined with read length determination features of long-fragment sequencing platforms, this invention enables accurate, rapid, and high-throughput detection of gene mutations in human embryonic β-thalassemia. The method described in this invention is simple to operate, and the long-fragment PCR and long-fragment libraries are reliable and highly reproducible, which is beneficial for the application of long-fragment sequencing technology in clinical testing.

[0010] Therefore, the present invention provides a primer set for simultaneous amplification HBB Multiple mutations in the gene region and linked single nucleotide polymorphisms (SNPs) within a 1Mb range upstream and downstream, among which those used for amplification HBB Five primers for various mutations in the gene region are as follows: HBB-F1, HBB-F2, HBB-F3, HBB-R1, and HBB-R2, with sequences shown in SEQ ID NO:1-5, respectively; among them, the primers used for amplification are... HBB The eight primers for the linked SNP sites within a 1Mb range upstream and downstream of the gene are as follows: L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F, and L4-R, and their sequences are shown in SEQ ID NO: 6-13.

[0011] The positions of the above 13 primers are as follows: Figure 1 As shown. The primer set can simultaneously amplify HBB The complete sequence within the primer range of the gene locus, including any type of mutant sequence within the primer range, as well as sequences of linked fragments within a 1Mb range upstream and downstream.

[0012] In some implementations, the primer set is used to simultaneously detect one or more of the following mutations: HBB Point mutations, structural variations, and linked SNP sites within a 1Mb range upstream and downstream of the gene locus primer range.

[0013] In this invention, the point mutations and structural variations comprise data from four thalassemia databases: Ithanet, HbVar, LOVD, and LOVD-China. HBB Gene mutation.

[0014] In some implementations, the structural variation includes large segment deletions.

[0015] In some implementations, the large fragment deletions include HBB-SEA_HPFH, HBB-Chinese, HBB-3.5kb_del, HBB-HPFH6, HBB-Beta21.9kb_del, HBB-Hb_Kenya, HBB-8.2k_del (2022), HBB-Kabylia_deletion_insertion, HBB-Czech_deletion, HBB-4.9kdel (2021), HBB-Indian_deletion, HBB-1.6kb_del, HBB-Hb_Lepore-Hollandia, HBB-Afghan_909bp_deletion, HBB-290bp_ deletion,HBB-538bp_deletion,HBD-2.9kb_del,HBB-49.3kb_0_Asian_del,HBB-Caucasian_HPFH,HBB-VCV000831400,HBB-VCV000639377,HBB-VCV000253972,HBB-6 19bp_deletion, HBB-1517bp_deletion, HBB-British, HBB-Hb_Lepore-Boston-Washington, HBB-Delta87_beta16, HBB-Hb_Lepore-Baltimore, and HBB-468bp_deletion.

[0016] In some embodiments, the amplified fragment size of the primer set is 1k to 20kb. In some embodiments, the amplified product size of the primer set is approximately 1k to 20kb.

[0017] In some implementations, degenerate base primers are used if the primer position contains an SNP.

[0018] In some implementations, the 5' ends of the primers in the primer set also contain DNA barcodes to distinguish different samples. The 5' end barcodes of the F and R primers may be the same or different, and those skilled in the art can choose as needed.

[0019] In some implementations, the DNA barcodes are different and have a length of 5-50 nt.

[0020] In some implementations, the primer set is used for simultaneous amplification HBB At least one segment of the gene region and HBB Four segments of the linkage region within a 1Mb range upstream and downstream of the gene region.

[0021] In some embodiments, the primer set performs multiplex long-fragment PCR amplification in a single reaction tube. In some embodiments, the amplification product of the multiplex long-fragment PCR amplification contains... HBB At least one segment of the gene region and HBB Four segments within a 1Mb linkage region upstream and downstream of the gene region can be detected simultaneously, allowing for at least the following: HBB All point mutations within the primer range of the gene locus, and 30 large deletions (including HBB-SEA_HPFH, HBB-Chinese, HBB-3.5kb_del, HBB-HPFH6, HBB-Beta21.9kb_del, HBB-Hb_Kenya, HBB-8.2k_del (2022), HBB-Kabylia_deletion_insertion, HBB-Czech_deletion, HBB-4.9kdel (2021), HBB-Indian_deletion, HBB-1.6kb_del, HBB-Hb_Lepore-Hollandia, HBB-Afghan_909bp_deletion, HBB-290bp_deletion). The list includes the following SNPs: HBB-538bp_deletion, HBD-2.9kb_del, HBB-49.3kb_0_Asian_del, HBB-Caucasian_HPFH, HBB-VCV000831400, HBB-VCV000639377, HBB-VCV000253972, HBB-619bp_deletion, HBB-1517bp_deletion, HBB-British, HBB-Hb_Lepore-Boston-Washington, HBB-Delta87_beta16, HBB-Hb_Lepore-Baltimore, and HBB-468bp_deletion, as well as linked SNPs within a 1Mb range upstream and downstream. Point mutations and structural variations include those from four thalassemia databases (Ithanet, HbVar, LOVD, and LOVD-China). HBB Gene mutation.

[0022] In some implementations, the primer set is used for simultaneous direct detection HBB Gene regions and HBB The detection of haplotypes in linkage regions within 1Mb upstream and downstream of the gene region utilizes blastocyst and parental samples to infer haplotype linkage, and the detection is independent of the proband, thereby achieving proband-independent PGT-M detection.

[0023] On the other hand, the present invention provides the application of the primer set described herein in the preparation of a kit for detecting multiple mutations in human embryonic β-thalassemia.

[0024] In another aspect, the present invention provides a kit for detecting multiple mutations in human embryonic β-thalassemia, comprising the following reagents: (1) reagents for multiplex long fragment PCR amplification; (2) reagents for constructing long fragment sequencing libraries; wherein the reagents for multiplex long fragment PCR amplification contain the primer set as described in the present invention.

[0025] In some implementations, the kit is used for simultaneous direct detection HBB Gene regions and HBB The detection of haplotypes in linkage regions within 1Mb upstream and downstream of the gene region utilizes blastocyst and parental samples to infer haplotype linkage, and the detection is independent of the proband, thereby achieving proband-independent PGT-M detection.

[0026] In some implementations, the kit is used to simultaneously detect one or more of the following mutations: HBB Point mutations, structural variations, and linked SNP sites within a 1Mb range upstream and downstream of the gene locus primer range.

[0027] In some embodiments, the reagents for multiplex long fragment PCR amplification also include DNA polymerase and reaction buffer.

[0028] In one implementation, the long-fragment PCR amplification products obtained using the kit may or may not be purified before proceeding to the next reaction, as those skilled in the art may choose as needed.

[0029] In some embodiments, the primer set performs multiplex long-fragment PCR amplification in a single reaction tube. In some embodiments, the amplification product of the multiplex long-fragment PCR amplification contains... HBB At least one segment of the gene region and HBB Four segments within a 1Mb linkage region upstream and downstream of the gene region can be detected simultaneously, allowing for at least the following: HBBAll point mutations within the primer range of the gene locus, and 30 large deletions (including HBB-SEA_HPFH, HBB-Chinese, HBB-3.5kb_del, HBB-HPFH6, HBB-Beta21.9kb_del, HBB-Hb_Kenya, HBB-8.2k_del (2022), HBB-Kabylia_deletion_insertion, HBB-Czech_deletion, HBB-4.9kdel (2021), HBB-Indian_deletion, HBB-1.6kb_del, HBB-Hb_Lepore-Hollandia, HBB-Afghan_909bp_deletion, HBB-290bp_deletion). The list includes the following SNPs: HBB-538bp_deletion, HBD-2.9kb_del, HBB-49.3kb_0_Asian_del, HBB-Caucasian_HPFH, HBB-VCV000831400, HBB-VCV000639377, HBB-VCV000253972, HBB-619bp_deletion, HBB-1517bp_deletion, HBB-British, HBB-Hb_Lepore-Boston-Washington, HBB-Delta87_beta16, HBB-Hb_Lepore-Baltimore, and HBB-468bp_deletion, as well as linked SNPs within a 1Mb range upstream and downstream. Point mutations and structural variations include those from four thalassemia databases (Ithanet, HbVar, LOVD, and LOVD-China). HBB Gene mutation.

[0030] In some implementations, the reagents used to construct long-fragment sequencing libraries include adapters, ligases, DNA purification magnetic beads, reaction buffers, and exonucleases.

[0031] In some implementations, the long fragment sequencing is selected from single-molecule real-time sequencing (SMRT) and nanopore technology platforms.

[0032] In a preferred embodiment, long-fragment sequencing is performed using SMRT-based sequencing from Pacific Biosciences (PacBio) or Nanopore sequencing from ONT.

[0033] In one specific implementation, the SMRT library connector can be connected using either a flat-end connection or a TA connection.

[0034] In one specific implementation, the universal blunt-ended adapter sequence for SMRT is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3' (SEQ ID NO: 14), which is annealed to form a blunt-ended stem-loop adapter aptamer. Different DNA sequences (barcodes) of 5-50 nt can be added to the stem to form different barcode-bearing adapter aptamers. SMRT libraries with different barcodes can be mixed together for sequencing.

[0035] In one specific implementation, the universal SMRT adapter sequence is 5'-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 15), which is annealed to form a blunt-ended stem-loop adapter aptamer. Different DNA sequences (barcodes) of 5-50 nt can be added to the stem to form different barcode-bearing adapter aptamers. SMRT libraries with different barcodes can be mixed together for sequencing.

[0036] In one implementation, the SMRT connector may or may not have a barcode. Preferably, the SMRT connector has a barcode designed by PacBio or a custom-designed barcode, which can be selected by those skilled in the art as needed.

[0037] In a preferred embodiment, the SMRT library is matched with the PacBio sequencing platform.

[0038] In a preferred embodiment, the reagents used to construct long-fragment Nanopore libraries include end-repair enzymes, adapters, ligases, DNA purification magnetic beads, 80% ethanol, and reaction buffer.

[0039] In one implementation, the Nanopore library connector can be connected using either a flat-end connector or a TA connector.

[0040] In one implementation, the Nanopore connector may or may not have a barcode. Preferably, the Nanopore connector has a barcode designed by ONT or a custom-designed barcode, which can be selected by those skilled in the art as needed.

[0041] In a preferred embodiment, the Nanopore library is matched with the ONT sequencing platform.

[0042] On another aspect, the present invention provides a system for detecting multiple mutations in human embryonic β-thalassemia, comprising the following modules: (1) a collection module for obtaining subject samples; (2) an amplification module for performing multiplex long-fragment PCR amplification on the samples; (3) a library construction module for constructing long-fragment sequencing libraries; (4) a sequencing module for sequencing; and (5) an analysis module for analyzing mutation types and linkage relationships between different fragments; wherein the multiplex long-fragment PCR amplification in the amplification module is performed using primer sets as described in the present invention or kits as described in the present invention; wherein the multiplex long-fragment PCR amplification in the amplification module is performed in a single reaction system; wherein the multiple mutations in β-thalassemia include HBB Point mutations, structural variations, and linked SNP sites within a 1Mb range upstream and downstream of the gene locus primer range.

[0043] In some implementations, the subject sample includes paternal and maternal samples and / or blastocyst samples other than the proband.

[0044] On another aspect, the present invention provides a system for detecting haplotype linkage in parent samples and / or blastocyst samples other than the proband, comprising: (1) a collection module for acquiring parent samples and / or blastocyst samples other than the proband; (2) an amplification module for performing multiplex long-fragment PCR amplification on the parent samples and blastocyst samples respectively; (3) a library construction module for constructing a long-fragment sequencing library; (4) a sequencing module for long-fragment sequencing; and (5) an analysis module for constructing haplotype linkage based on the parent samples and blastocyst samples. HBB Mutations in gene regions and HBB Haplotype linkage detection was performed on SNP sites within a 1Mb range upstream and downstream of the gene region; wherein the multiplex long fragment PCR amplification in the amplification module was performed using the primer set or kit described in this invention; wherein the amplification product of the multiplex long fragment PCR amplification in the amplification module contained HBB At least one segment of the gene region and HBB Four fragments within a 1Mb range upstream and downstream of the gene region are linked; the multiplex long fragment PCR amplification in the amplification module is performed in a single reaction system; the system performs multiplex long fragment PCR amplification only on parent and blastocyst samples and is independent of the proband.

[0045] The detection, construction, and analysis of haplotype linkage involve obtaining parental samples and blastocyst samples other than the proband (which are then subjected to whole-genome amplification to obtain WGA products), and performing multiplex long-fragment PCR amplification on the parental samples and blastocyst samples other than the proband using the primer set or kit described in this invention, thereby obtaining individual parental samples and blastocyst samples other than the proband.HBB At least one segment of the gene region and HBB Four fragments within a 1Mb range upstream and downstream of the HBB gene region are identified. SNV calling is performed on mutation sites within the primer range and within a 1Mb range upstream and downstream of the HBB gene locus in samples from parents other than the proband, and each fragment is genotyped. The haplotype information of all fragments within the 1Mb range upstream and downstream of the HBB gene region in the samples from parents other than the proband and blastocyst samples is summarized to obtain the linkage relationship between the linked fragments and the HBB gene region in the samples from parents other than the proband and blastocyst samples, thus constructing the two haplotype information of the proband. Finally, the haplotype information of the HBB gene region in the blastocyst sample to be tested can be obtained from the haplotype information of the linked fragments within a 1Mb range upstream and downstream and their linkage relationship with the HBB gene region. Therefore, using the primer set (kit) and method (system) of this invention, PGT-M detection can be truly achieved without relying on the proband.

[0046] On another front, the present invention provides a system for detecting human embryonic β-thalassemia using only parental samples, comprising the following modules: (1) a collection module for obtaining parental samples and blastocyst samples; (2) an amplification module for performing multiplex long-fragment PCR amplification on the parental samples and blastocyst samples respectively; (3) a library construction module for constructing a long-fragment sequencing library; (4) a sequencing module for long-fragment sequencing; and (5) an analysis module for embryonic pathogenicity analysis, based on the parental samples and blastocyst samples. HBB Mutations in gene regions and HBB Haplotype linkage was constructed at SNP sites within a 1Mb range upstream and downstream of the gene region, and embryo pathogenicity was analyzed. The multiplex long-fragment PCR amplification in the amplification module was performed using the primer set described in this invention or the kit described in this invention. The amplification products of the multiplex long-fragment PCR amplification in the amplification module contained... HBB At least one segment of the gene region and HBB Four fragments within a 1Mb range upstream and downstream of the gene region are linked; the multiplex long fragment PCR amplification in the amplification module is performed in a single reaction system; the system performs multiplex long fragment PCR amplification only on parent and blastocyst samples and is independent of the proband.

[0047] In some embodiments, the sequencing module in the system described in this invention utilizes a technology platform selected from single-molecule real-time sequencing (SMRT) and nanopore technology.

[0048] In some embodiments, the amplification module in the system described in this invention is also used to perform whole-genome amplification on blastocyst samples.

[0049] In some implementations, the parental samples are derived from blood, oral swabs, hair, urine, saliva, feces, synovial fluid, cerebrospinal fluid, ascites, pleural effusion, bile, or pancreatic fluid.

[0050] Based on a specific combination of long-fragment PCR amplification and long-fragment high-throughput sequencing, this invention enables the simultaneous detection of multiple mutations in β-thalassemia in multiple embryonic samples with high specificity, accuracy, and speed.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0052] Definition In this invention, the term " HBB A gene is a set of genes that encode β-globin in humans and other mammals. The HBB gene, located on chromosome 11, encodes adult β-globin. Mutations or abnormal expression of the HBB gene can lead to a range of diseases, such as β-thalassemia, which is usually associated with the deletion or non-functionality of the β chain gene.

[0053] In this invention, the term "multiple mutations in human embryonic β-thalassemia" refers to... HBB The gene loci include point mutations, structural variations, and SNPs linked within a 1 Mb range upstream and downstream of the primers used in this invention. The point mutations and structural variations comprise data from four thalassemia databases: Ithanet, HbVar, LOVD, and LOVD-China. HBBGene mutations. These structural variations include large deletions, such as those in HBB-SEA_HPFH, HBB-Chinese, HBB-3.5kb_del, HBB-HPFH6, HBB-Beta21.9kb_del, HBB-Hb_Kenya, HBB-8.2kb_del (2022), HBB-Kabylia_deletion_insertion, HBB-Czech_deletion, HBB-4.9kdel (2021), HBB-Indian_deletion, HBB-1.6kb_del, HBB-Hb_Lepore-Hollandia, HBB-Afghan_909bp_deletion, and HBB-290bp_. deletion,HBB-538bp_deletion,HBD-2.9kb_del,HBB-49.3kb_0_Asian_del,HBB-Caucasian_HPFH,HBB-VCV000831400,HBB-VCV000639377,HBB-VCV000253972,HBB-6 19bp_deletion, HBB-1517bp_deletion, HBB-British, HBB-Hb_Lepore-Boston-Washington, HBB-Delta87_beta16, HBB-Hb_Lepore-Baltimore, and HBB-468bp_deletion.

[0054] In this invention, the term "linkage" refers to the phenomenon that two or more loci tend to be passed on together to offspring during inheritance. This phenomenon typically occurs between genes that are physically close to each other on chromosomes.

[0055] In this invention, the terms "SNP", "single nucleotide polymorphism" or "SNP site" and "single nucleotide polymorphism site" are used interchangeably. They refer to DNA sequence polymorphism caused by a single amino acid variation at the genomic level, including single base conversion, transversion, insertion and deletion.

[0056] In this invention, the term "structural variation" refers to a structural variation in a nucleic acid sequence involving changes in at least two adjacent bases, preferably at least four bases, and can refer to, for example, deletions, insertions, substitutions, sequence duplications, or translocations of multiple nucleotides. According to some embodiments, the structural variation affects a sequence length of at least about 50 bases, preferably at least about 100 bases, and more preferably at least about 1 kb (= 1000 bases). According to some embodiments, the structural variation affects a sequence length of up to 300 Mb (megabases = 1,000,000 bases), for example, up to 30 Mb, for example, up to 3 Mb.

[0057] In this invention, the "sample" or "subject sample" can be a parent sample and / or a blastocyst sample. The parent sample can be derived from blood, oral swabs, hair, urine, saliva, feces, synovial fluid, cerebrospinal fluid, ascites, pleural fluid, bile, or pancreatic fluid, etc. The blastocyst sample can be blastocyst trophoblast cells. It is known that PGT technology first requires obtaining the genetic material of the embryo, commonly using blastocyst trophoblast cells. Specifically, for example, on day 5-6 after in vitro fertilization, when the embryo has developed to the blastocyst stage, it has approximately 100-150 cells; 5-10 trophoblast cells are obtained for genetic testing.

[0058] In this invention, "embryo" or "embryo sample" refers to a blastocyst sample. In some specific embodiments, "embryo" or "embryo sample" is blastocyst trophoblast cells.

[0059] In this invention, the term "haplotype" refers to a combination of alleles at a series of closely linked loci on a chromosome in an individual. In genetics, haplotypes are often used to describe a group of alleles at a specific locus that are genetically transmitted as a whole.

[0060] In this invention, the term "proband" refers to a patient diagnosed as carrying a disease-causing gene and exhibiting symptoms of the disease, and is an organism with a genetic relationship to the subject.

[0061] In this invention, Ithanet, HbVar, LOVD, and LOVD-China are four databases focused on thalassemia, providing genetic information and related data about the disease. Ithanet is an international database supported by the World Health Organization (WHO) to collect and share information on genetic variations in thalassemia. It is a multi-ethnic, multi-national project containing extensive genetic data, helping researchers understand the genetic background of thalassemia in different populations. The HbVar database is a public, professional database focusing on hemoglobin variations, including those related to thalassemia. It provides detailed variation information, including gene sequence changes, related phenotypes, and literature citations, making it an important resource for research on hemoglobinopathies. LOVD is an open variation database developed by Leiden University Medical Center in the Netherlands. It allows researchers to submit and share data on genetic variations. LOVD contains multiple sub-databases covering various genetic diseases, including thalassemia. LOVD-China is a branch of LOVD focusing on genetic variations in the Chinese population. It provides information on the variations of thalassemia and other genetic diseases in the Chinese population, which helps to understand the genetic characteristics and disease risks of the Chinese population.

[0062] The superior technical effects of this invention are mainly in the following aspects: (1) Independent of prior witnesses. This invention can directly and simultaneously detect... HBB Haplotypes of gene regions and long linkage fragments, combined with parental genotypes, are used for linkage analysis, avoiding the need for a proband.

[0063] (2) Detection of multiple mutation types with a single kit. This invention can simultaneously detect multiple mutations, including SNVs, Indels, and structural variations, in a single reaction primer system.

[0064] (3) High sensitivity and low cost. This invention can efficiently utilize SNP sites for genotyping, greatly improving the ease of embryo genotyping, greatly improving sensitivity and accuracy, preventing misdiagnosis caused by amplified allele deactivation, and greatly reducing the time and labor costs of testing.

[0065] (4) High-throughput detection. Long-fragment sequencing can achieve 384 barcode adapters, and even more barcode adapters can be designed as needed. Alternatively, a dual-barcode system using primer-band barcodes and adapter-band barcodes can be used to achieve even more barcode combinations. The high-throughput characteristics of long-fragment sequencing platforms enable high-throughput sample detection.

[0066] (5) High accuracy. PacBio's SMRT dumbbell-shaped library can be interpreted multiple times during sequencing, and the base accuracy of the sequencing results after correction is greater than 99%. Moreover, SMRT sequencing errors are random, and the base accuracy is greater than 99.9% after correction by sequencing depth. Therefore, it can accurately interpret gene mutations within the primer detection range.

[0067] (6) Flexible detection time. The Nanopore platform can generate data within minutes and can start data analysis within minutes or hours depending on the actual data volume requirements. When the detection timeliness requirement is high, the Nanopore platform has a time advantage. Attached Figure Description

[0068] Figure 1 This is a schematic diagram of primer design for multiplex-long fragment PCR.

[0069] Figure 2 This is a gel electrophoresis image of DNA amplified from samples using the multiplex-long fragment PCR method described in Example 1.

[0070] Figure 3 This is a schematic diagram of IGV results and linkage relationships for the parents and some representative embryos of family 1.

[0071] Figure 4 This is a schematic diagram of IGV results and linkage relationships for the parents and embryos in family 8. Detailed Implementation

[0072] Exemplary embodiments of the present invention will now be described in detail, which should not be considered as limitations on the present invention, but rather as a more detailed description of certain aspects, features, and implementations of the present invention.

[0073] 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 within the scope of this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range. While any methods and materials similar to or equivalent to those described herein may be used in practice or experimentation with this disclosure, preferred materials and methods are described below.

[0074] The sequence used in this invention Table 1 Primer sequences

[0075] Example 1: Amplification using the multiplex-long fragment PCR method of the present invention HBBGene fragments and linkage analysis fragments Prepare the reaction system according to Table 2 below to amplify the DNA samples of the embryo (i.e., blastocyst sample) and parents: Table 2:

[0076] Pre-amplification was performed on a PCR instrument under the conditions shown in Table 3 below: Table 3:

[0077] After amplification, 5 μL of each sample was taken and detected on a 1% DNA gel. The results are as follows. Figure 2 As shown, using different samples as templates, HBB Both genes and linked fragments can be effectively amplified.

[0078] Example 2: Construction of PacBio sequencing libraries using the long-fragment PCR method involved in this invention. Step 1: Long fragment PCR amplification Prepare the reaction system according to Table 4 below, and amplify the DNA samples from embryonic and parental samples. HBB Gene fragments and linkage analysis fragments.

[0079] Table 4:

[0080] Pre-amplification was performed on a PCR instrument under the conditions shown in Table 5 below: Table 5:

[0081] After amplification, place the amplification product in a centrifuge and centrifuge at 10,000 rpm for 20 min. After centrifugation, allow it to stand horizontally, and take 4 μL of the supernatant to be added to a new tube.

[0082] Step 2: Constructing PacBio sequencing libraries Prepare the reaction system according to Table 6 below: Table 6:

[0083] The PCR reaction was performed under the following conditions: 37℃ for 20 min; 25℃ for 15 min; 65℃ for 10 min. After the reaction was complete, 0.5 μL of Exonuclease III (NEB, Cat#M0206L) and 0.5 μL of Exonuclease VII (NEB, Cat#M0379L) were added, and the reaction was continued at 37℃ for 1 hour. The DNA was purified twice using 0.6x Ampure PB magnetic beads (PacBio, Cat#100-265-900) according to the manufacturer's instructions, and finally eluted with 10 μL of Elution Buffer. The resulting DNA eluate was the target DNA PacBio sequencing library. The DNA concentration was determined using Qubit dsDNA HS reagent (ThermoFisher, Cat#Q32851) on a Qubit 3 Fluoromter (ThermoFisher, Cat#Q33216). When there are multiple PacBio sequencing libraries, equal amounts of the libraries can be mixed together to prepare a mixed library.

[0084] Step 3: PacBio sequencing and analysis Based on the total and molar concentrations of the library, an appropriate volume of library was reacted with the binding reagent (PacBio, Cat#101-820-200) and primers (PacBio, Cat#100-970-100) to prepare the final, sequence-ready library. Sequencing results from representative families are shown below. Figure 3 and Figure 4 As shown, where Figure 3 This is a schematic diagram of IGV detection results and linkage relationships for the parents and some representative embryos in family 1. Figure 4 This is a schematic diagram of IGV results and linkage for the parents and embryos in family 8.

[0085] Example 3: HBB Detection and verification of gene mutations Forty-one embryo samples (i.e., blastocyst samples) from eight families and their parents were collected. Following Example 2, the haplotype and genotype of the parents and embryos were simultaneously detected using the method (and kit) of this invention. NGS + Sanger assay was used as a control. The results obtained using this invention were compared with the control results, as shown in Table 7. All sample results were completely consistent.

[0086] Table 7

[0087] Therefore, the results detected using the method of this invention, compared with the NGS+Sanger method, show that both specificity and sensitivity reach 100%.

[0088] It should be noted that although the above-described implementation examples have demonstrated a series of features of the present invention, researchers and those skilled in the art can utilize the ideas of the present invention to adjust and modify the reaction reagents, reaction conditions, etc., involved in multiplex + long fragment PCR reactions and long fragment sequencing library construction according to specific needs. Therefore, those skilled in the art can make several simple substitutions without departing from the concept and principles of the present invention, and these should all be included within the scope of protection of the present invention.

[0089] References [1] Modell B, Darlison M. Bull World Health Organ. Globalepidemiology of haemoglobin disorders and derived service indicators. BullWorld Health Organ. 2008, 86(6):480-7. Doi:10.2471 / blt.06.036673. [2] Asia. Ann Hum Biol. 2005, 32(2):123-30. Doi: 10.1080 / 03014460500075084. [3] Xu Xiangmin. Operational Guidelines for the Prevention and Control of Thalassemia [M]. Beijing: People's Military Medical Publishing House, 2011.

[0090] [4] Taher AT,Weatherall DJ,Cappellini MD.Thalassaemia[J].Lancet,2018,391(10116):155-167.DOI:10.1016 / s0140-6736(17)31822-6. [5] Thein SL. Molecular basis of β thalassemia and potentialtherapeutic targets. Blood Cells Mol Dis. 2018, 70:54-65. Doi: 10.1016 / j.bcmd.2017.06.001. [6] Shang X, Xu X. Update in the genetics of thalassemia: Whatclinicians need to know. Best Pract Res Clin Obstet Gynaecol. 2017, 39:3-15.Doi: 10.1016 / j.bpobgyn.2016.10.012. [7] ESHRE PGT-M Working Group, Carvalho F, Moutou C, Dimitriadou E,Dreesen J, Giménez C, Goossens V, Kakourou G, Vermeulen N, Zuccarello D, DeRycke M. ESHRE PGT Consortium good practice recommendations for the detectionof monogenic disorders. Human Reproduction Open. 2020, 1–18. Doi:10.1093 / hropen / hoaa018. [8] De Rycke M, Berckmoes V. Preimplantation Genetic Testing forMonogenic Disorders. Genes. 2020, 11(8): 871. Doi:10.3390 / genes11080871. [9] ESHRE PGT-SR / PGT-A Working Group, Coonen E, Rubio C, ChristopikouD, Dimitriadou E, Gontar J, Goossens V, Maurer M, Spinella F, Vermeulen N, DeRycke M. ESHRE PGT Consortium good practice recommendations for the detectionof structural and numerical chromosomal aberrations. Human Reproduction Open.2020, 1-20. Doi: 10.1093 / hropen / hoaa017.

[10] Zhang Ningyuan, Huang Guoning, Fan Liqing, Feng Yun, Shen Huan, Liu Ping, Lu Wenhong, Zhang Yunshan, Wang Xiuxia, Zhang Songying, Huang Xuefeng, Wu Qiongfang, Quan Song, Zhou Canquan, Zhou Congrong, Shi Juanzi, Sun Yingpu, Sun Haixiang. Laboratory technical guidelines for preimplantation genetic diagnosis and screening. Journal of Reproductive Medicine, 2018, 29(9). Doi:10.3969 / j.issn.1004-3845.2018.09.001.

[0091]

[11] Huang L, Ma F, Chapman A, Lu S, Xie

Claims

1. A primer set for simultaneous amplification HBB Multiple mutations in the gene region and linked single nucleotide polymorphisms (SNPs) within a 1 Mb range upstream and downstream, Among them, the amplification HBB The following are five primers for multiple mutations in the gene region: HBB-F1, HBB-F2, HBB-F3, HBB-R1 and HBB-R2, the sequences of which are shown in SEQ ID NO: 1-5 respectively; Among them, the amplification HBB The following are the eight primers for linked SNP sites within a 1Mb range upstream and downstream of the gene: L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F, and L4-R, respectively, are shown in SEQ ID NO: 6-13.

2. The primer set according to claim 1, wherein the primer set is used to simultaneously detect one or more of the following mutations: HBB Point mutations, structural variations, and linked SNP sites within a 1Mb range upstream and downstream of the gene locus primer range.

3. The primer set according to claim 2, wherein the structural variation comprises a large segment deletion.

4. The primer set according to claim 3, wherein the large fragment deletion includes HBB-SEA_HPFH, HBB-Chinese, HBB-3.5kb_del, HBB-HPFH6, HBB-Beta21.9kb_del, HBB-Hb_Kenya, HBB-8.2k_del (2022), HBB-Kabylia_deletion_insertion, HBB-Czech_deletion, HBB-4.9kdel (2021), HBB-Indian_deletion, HBB-1.6kb_del, HBB-Hb_Lepore-Hollandia, HBB-Afghan_909bp_deletion, HBB-2 90bp_deletion, HBB-538bp_deletion, HBD-2.9kb_del, HBB-49.3kb_0_Asian_del, HBB-Caucasian_HPFH, HBB-VCV000831400, HBB-VCV000639377, HBB-VCV000253972, HB B-619bp_deletion, HBB-1517bp_deletion, HBB-British, HBB-Hb_Lepore-Boston-Washington, HBB-Delta87_beta16, HBB-Hb_Lepore-Baltimore, and HBB-468bp_deletion.

5. The primer set according to claim 1, wherein the primer set is used for simultaneous amplification HBB At least one segment of the gene region and HBB Four segments of the linkage region within a 1Mb range upstream and downstream of the gene region.

6. The primer set according to claim 1, wherein the amplified fragment size of the primer set is from 1k to 20kb.

7. The primer set according to claim 1, wherein the primer set is used for simultaneous direct detection HBB Gene regions and HBB The haplotype of the linked region within 1 Mb upstream and downstream of the gene region is detected by using blastocyst samples and parental samples to infer haplotype linkage, and the detection does not depend on the proband.

8. The primer set according to claim 1, wherein the 5' end of the primer further comprises a DNA barcode.

9. The primer set according to claim 8, wherein the DNA barcodes are distinct and have a length of 5-50 nt.

10. Use of the primer set according to any one of claims 1-9 in the preparation of a kit for detecting multiple mutations in human embryonic β-thalassemia.

11. A kit for detecting multiple mutations in human embryonic β-thalassemia, comprising the following reagents: (1) Reagents for multiplex long fragment PCR amplification; (2) Reagents used to construct long-fragment sequencing libraries; The reagents used for multiplex long fragment PCR amplification comprise the primer set as described in any one of claims 1-9.

12. The kit according to claim 11, wherein the kit is used for simultaneous direct detection HBB Gene regions and HBB The haplotype of the linked region within 1 Mb upstream and downstream of the gene region is detected by using blastocyst samples and parental samples to infer haplotype linkage, and the detection does not depend on the proband.

13. The kit of claim 11, wherein the kit is used to simultaneously detect one or more of the following mutations: HBB Point mutations, structural variations, and linked SNP sites within a 1Mb range upstream and downstream of the gene locus primer range.

14. The kit of claim 11, wherein the reagent for multiplex long fragment PCR amplification further comprises DNA polymerase and reaction buffer.

15. The kit of claim 11, wherein the reagents for constructing long-fragment sequencing libraries include adapters, ligases, DNA purification beads, reaction buffers, and exonucleases.

16. The kit according to claim 11, wherein the long fragment sequencing is based on a platform of single-molecule real-time sequencing (SMRT) technology or nanopore technology.

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