Primer group and application thereof in preparation of thalassemia detection kit

Abstract

The invention relates to the technical field of biology, in particular to a primer group and application thereof in preparation of a thalassemia detection kit. The primer group is designed and obtained on the basis of at least one of sequences 1)-13): 1) chr16: 219267-219290; (2) chr16: 228804 to 228829, and the molecular weight of chr16: (3) chr16: 215255 to 215276; (4) chr16: 235945 to 235971, and chr16: 235945 to And (5) chr16: 196488 to 196508, and the molecular weight of chr16: 196488 (6) chr < 11 >: 5276986 to 5277008; (7) chr11: 5257290-5257314, and (7) chr11: 5257290- (8) chr < 11 >: 5249915 to 5249939; (9) chr11: 5258978 to 5259000, and (9) chr11: 5258978 (10) chr11: 5221888-5221912, (10) chr11: (5221888- (11) chr11: 5189975-5189996, (11) chr11: (5189975- (12) chr11: 5152341-5152365, (12) chr11: ( (11, 13) chr11: 5133905-5133925, (11, 12, 13) chr11 Wherein the primer is at least partially complementary to the sequences 1)-13).

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically, relates to a primer set and its use in preparing a thalassemia detection kit. Background Art

[0002] Thalassemia (abbreviated as "thalassemia") is an autosomal recessive genetic disease, mainly caused by globin gene defects leading to reduced or missing globin chain synthesis. According to the type of globin chain affected, thalassemia is mainly divided into α-thalassemia and β-thalassemia. α-thalassemia is caused by the lack or reduction of α-globin gene synthesis, while β-thalassemia is caused by the reduction or loss of β-globin gene synthesis.

[0003] α-thalassemia is mainly caused by the loss or mutation of the α-globin gene, which leads to an imbalance in the ratio of globin chains, manifesting as symptoms such as chronic hemolytic anemia. Since there are two copies of the α-globin gene on each chromosome, gene loss or mutation can manifest as a variety of different inheritance patterns, leading to abnormalities in the number of genes, such as the formation of triplets or quadruplets. These complex structural variations are often difficult to identify through conventional detection methods. β-thalassemia is caused by the loss or mutation of the β-globin gene, leading to red blood cell damage, sclerosis and hemolysis. Point mutations, copy number variations (CNVs) and structural variations (SVs) of genes are all the main causes of thalassemia.

[0004] Existing thalassemia detection technologies have certain limitations. For example, methods such as reverse dot blot (RDB), multicolor melting curve analysis (MMCA), multiplex ligation-linked probe amplification (MLPA) and gap-PCR (Gap-PCR) can only detect common α-thalassemia, and can even detect HKαα / αα and HKαα / -α3.7, but cannot detect α-globin gene triplets and quadruplets; for example, multiplex ligation-linked probe amplification (MLPA) can only determine the copy number of deleted or repeated sequences, but cannot locate alleles; although high-throughput sequencing technology (NGS) performs well in the detection of point mutations, it is still unable to detect complex structural variations such as α-globin gene triplets, quadruplets, HKαα / αα, and cannot distinguish between homologous genes HBA1 / 2 and various variant cis-trans forms.

[0005] For this reason, the current thalassemia detection methods still need to be improved for complex variant types. Summary of the invention

[0006] The present application aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the present application proposes a detection method based on long-range PCR amplification technology. This method amplifies the α-globin gene and β-globin gene deletion regions related to thalassemia, and uses single-molecule library construction and sequencing technology to comprehensively detect copy number variations (CNVs), single nucleotide variations (SNVs) and structural variations (SVs) of thalassemia. On the basis of detecting common gene deletion types, it is possible to further detect 6 complex structural variations including αααanti3.7, αααanti4.2, HKαα, anti-HKαα, ααααanti3.7 and ααααanti4.2. In addition, it is also possible to accurately distinguish 1046 point mutations in homologous genes HBA1 and HBA2 and 1085 point mutations on the HBB gene, and to identify whether multiple mutation sites are located in cis or trans structures.

[0007] Specifically, this application provides the following technical solutions:

[0008] In the first aspect of the present application, the present application proposes a primer set. According to an embodiment of the present application, the aforementioned primer set includes: designed based on at least one of sequences 1)-13): 1) chr16:219267-219290; 2) chr16:228804-228829; 3) chr16:215255-215276; 4) chr16:235945-235971; 5) chr16:196488-196508; 6) chr11:5276986-5277008; 7) chr11:5257 290-5257314;8)chr11:5249915-5249939;9)chr11:5258978-5259000;10)chr11:5221888-5221912;11)chr11:5189975-5189996;12)chr11:5152341-5152365;13)chr11:5133905-5133925;Wherein, the aforementioned primers are at least partially complementary to the aforementioned sequences 1)-13). In some examples of the present application, the inventors have found through extensive research and experimental verification that the primer set designed based on the aforementioned sequences can accurately detect multiple complex variant types of thalassemia.

[0009] It should be noted that, in this article, each primer designed based on the above sequence can be completely complementary to sequence 1)-13), or partially complementary. Exemplarily, the length of sequence 1) is 23bp, and the primer involved in sequence 1) can be 23bp, or 17bp, 18bp, 19bp, 20bp, 21bp, 22bp or 23bp. The remaining sequence design methods are the same as above, and are limited to space reasons, so they will not be repeated here.

[0010] In some examples of the present application, the aforementioned primer set may also include at least one of the following additional technical features:

[0011] In some examples of the present application, the aforementioned primer set is selected from at least one of the following: SEQ ID NO: 1-SEQ ID NO: 13. The aforementioned sequences SEQ ID NO: 1-SEQ ID NO: 13 (Table 3) are primer set sequences designed by the inventors based on sequences 1)-13), and complex variations related to thalassemia can be accurately detected based on SEQ ID NO: 1-SEQ ID NO: 13. It can be understood by those skilled in the art that the aforementioned SEQ ID NO: 1-SEQ ID NO: 13 is only a primer set involved in sequences 1)-13), and a primer set obtained by adding or reducing bases on the basis of this primer set also belongs to the protection scope of the present application.

[0012] In the second aspect of the present application, the present application proposes the use of the aforementioned primer set in preparing a thalassemia detection kit. In some examples of the present application, the kit prepared based on the aforementioned primer set can be used for portable and accurate detection of thalassemia variant types.

[0013] It can be understood by those skilled in the art that the kit prepared based on the aforementioned primer set can be used to detect the thalassemia variant type by conventional sequencing or PCR. Sequencing or PCR is selected from routine laboratory operations and will not be described in detail here.

[0014] In some examples of the present application, the aforementioned use may also include at least one of the following additional technical features:

[0015] In some examples of the present application, the aforementioned thalassemia includes: α-thalassemia and β-thalassemia; wherein the primers designed based on at least one of sequences 1)-5) are used for the aforementioned α-thalassemia detection; the primers designed based on at least one of sequences 6)-13) are used for the aforementioned β-thalassemia detection. The primers designed based on the aforementioned sequences can accurately detect a variety of complex variant types of α-thalassemia and β-thalassemia, such as special structural variants.

[0016] In some examples of the present application, the primers designed based on sequences 1)-5) are shown as SEQ ID NOs: 1-5; the primers designed based on sequences 6)-13) are shown as SEQ ID NOs: 6-13.

[0017] In the third aspect of the present application, the present application proposes a method for detecting thalassemia mutations. According to an embodiment of the present application, the aforementioned method includes: using the primer set of the first aspect to perform PCR amplification on the nucleic acid sample to be tested; performing library construction and sequencing on the PCR amplification product; and performing variation analysis on the sequencing results. In some examples of the present application, the aforementioned method is suitable for multi-tube PCR amplification, optimizing the number of primers to reduce non-specific amplification of multiple PCR, and improving the sample throughput of a single library construction through the design of the index sequence to further reduce the cost of single-molecule library construction and sequencing. This method has the advantages of low template quality requirements, good specificity, good repeatability, simple library construction operation, large single detection throughput, and short cycle.

[0018] In some examples of the present application, the aforementioned detection method may further include at least one of the following additional technical features:

[0019] In some specific examples of the present application, the aforementioned nucleic acid sample to be tested is selected from DNA, for example, DNA extracted from human peripheral blood samples.

[0020] In some examples of the present application, in order to facilitate the distinction of multiple samples, the 5' end of the aforementioned primer set is connected with an index sequence, wherein the design of the index sequence is selected from a conventional laboratory design scheme, which will not be described in detail here.

[0021] In some examples of the present application, the aforementioned thalassemia variants include: α-thalassemia variants and β-thalassemia variants. Among them, the types of variants include: at least one of copy number variation (CNV), single nucleotide polymorphism (SNP) and structural variation (SV). The primer set of the present application can be used to detect deletion type, mutation type and structural variation in α-thalassemia variants; the primer set of the present application can be used to detect deletion type and mutation type in β-thalassemia.

[0022] In some examples of this application, the aforementioned α-thalassemia variants include: --SEA, -α3.7Ⅰ, -α6.9, -AW, -α6.3, -α3.7II, -α2.8, - -FIL, -α5.9, -α3.7III, -α2.7, --MED-II, -α27.6, --14.9, --9.7, -α21.9, -α4.2, -αM At least one of AL3.5, --JS, --27.2, --11.1, --THAI, -α2.4, --20.5, -α7.9, -α10.3, -α5.3, -α0.8, ααanti3.7, HKαα, αααanti3.7, αααanti4.2, anti-HKαα and αααanti4.2. Among them, --SEA, -α3.7Ⅰ, -α6.9, -AW, -α6.3, -α3.7Ⅱ, -α2.8, --FIL, -α5.9, -α3.7Ⅲ, -α2. 7. --MED-II, -α27.6, --14.9, --9.7, -α21.9, -α4.2, -αMAL3.5, --JS, --27.2, --1 1.1, -THAI, -α2.4, -20.5, -α7.9, -α10.3, -α5.3 and -α0.8 belong to the α-globin gene deletion type; αααanti3.7, HKαα, αααanti3.7, αααanti4.2, anti-HKαα and ααααanti4.2 belong to the α-globin gene structural variant.

[0023] In some examples of the present application, the aforementioned β-thalassemia variants include: Chinese Gγ(Aγδβ)0, Yunnanese, -β21.9, 1357bp deletion, SEA-HPFH, Filipino del, Siriraj Gγ(Aγδβ)0, HPFH-6, 78.9kb Gγ(Aγδβ)0del, Caucasian HPFH, HPFH-7, HPFH-5, Thai del, Turkish, Asian Indian(Aγδβ)0, Hb Lepore, 3.5kb del, Croatian 1605bp del, British 1393bpdel, 619bp del and 105bp del. At least one of the aforementioned variant types belongs to the β-globin gene deletion type.

[0024] In some examples of the present application, in the PCR amplification processing system, the concentration ratio of the aforementioned primer set, the aforementioned nucleic acid sample to be tested, and the DNA polymerase is (0.3μM-0.6μM): (20ng / μL-100ng / μL): (0.8U / μL-1.2U / μL). Within the aforementioned concentration range, the detection of multiple complex variations of α-thalassemia and β-thalassemia can be achieved.

[0025] In some examples of the present application, in the PCR amplification treatment system, the concentration of the primer set is 0.3 μM-0.6 μM, optionally 0.3 μM, 0.4 μM, 0.5 μM or 0.6 μM. In some preferred examples of the present application, the concentration of the primer set is 0.4 μM.

[0026] In some examples of the present application, in the PCR amplification processing system, the concentration of the aforementioned nucleic acid sample to be tested is 20ng / μL-100ng / μL, optionally, 20ng / μL, 30ng / μL, 40ng / μL, 50ng / μL, 60ng / μL, 70ng / μL, 80ng / μL, 90ng / μL or 100ng / μL.

[0027] In some examples of the present application, in the PCR amplification processing system, the aforementioned DNA polymerase concentration is 0.8U / μL-1.2U / μL, optionally 0.8U / μL, 0.9U / μL, 1.0U / μL, 1.1U / μL or 1.2U / μL. In some preferred examples of the present application, the aforementioned DNA polymerase concentration is 1.0U / μL.

[0028] In some examples of the present application, based on the above amplification system, the reaction procedure of PCR amplification process is shown in Table 1:

[0029] Table 1

[0030]

[0031] It will be understood by those skilled in the art that simple adjustments of temperature, time or cycle based on the above reaction procedure are also within the scope of protection of the present application.

[0032] In some preferred examples of the present application, the reaction procedure of the aforementioned PCR amplification process is shown in Table 2:

[0033] Table 2

[0034]

[0035]

[0036] In some examples of the present application, after the PCR amplification process and before the library construction process, a pooling process is further included, which mixes multiple PCR amplification products so that they can be processed simultaneously in the subsequent library construction and sequencing, thereby improving the sequencing efficiency and reducing the sequencing cost.

[0037] In some examples of the present application, after the above-mentioned pooling process, a purification process is further included to improve the sample quality and ensure efficient library construction.

[0038] In some examples of the present application, the purification process further includes quality control of the purified product. The quality control ensures that the quality and concentration of the DNA sample used for library construction meet the requirements. The quality control samples that do not meet the quality control requirements need to be re-PCR amplified.

[0039] In some examples of this application, the aforementioned library construction process is selected from single-molecule sequencing library construction. No PCR amplification is required, bias is reduced, and long-read sequencing is supported, and various types of thalassemia variants can be accurately detected. This method has low sample size requirements and is suitable for the study of precious or limited samples. It also reduces errors in the processing process and improves the quality and accuracy of sequencing data.

[0040] In some examples of the present application, the single-molecule sequencing library is further subjected to quality control processing to ensure the quality of the single-molecule sequencing library, avoid unnecessary errors and data problems during the sequencing process, and improve the reliability and accuracy of the sequencing results.

[0041] In some specific examples of the present application, the aforementioned single-molecule sequencing library construction includes: performing end repair and A addition treatment on the PCR amplification product; and purifying the A addition product to obtain the single-molecule sequencing library.

[0042] In some examples of the present application, variation analysis is achieved through an automated bioinformatics analysis process that is compatible with a sequencing platform (such as BGI). Different sequencing platforms have corresponding variation analysis processes. Those skilled in the art will appreciate that by performing single-molecule library construction and sequencing based on the primer set of the present application, combined with conventional variation analysis processes, the variation type of thalassemia can be obtained.

[0043] In the third aspect of the present application, the present application proposes a kit. According to an embodiment of the present application, the aforementioned kit includes: the aforementioned primer set of the first aspect. In some examples of the present application, based on the aforementioned kit, accurate detection of multiple variant types (including special structural variants) of thalassemia can be achieved.

[0044] In some examples of the present application, the aforementioned kit may also include at least one of the following additional technical features:

[0045] In some examples of the present application, the aforementioned kit further includes: at least one of: DNA polymerase and buffer, nuclease-free water, library construction reagents and sequencing reagents.

[0046] In some examples of the present application, the aforementioned library construction reagents include: at least one of: end repair enzyme, end repair buffer, polyA, ligase, ligation buffer, elution buffer and purification magnetic beads.

[0047] Those skilled in the art will appreciate that the above-mentioned kit may also include some commonly used reagents and instructions and the like.

[0048] In the fourth aspect of the present application, the present application proposes a nucleic acid sequencing method. According to an embodiment of the present application, the sequencing method includes: using the primer set of the first aspect to perform PCR amplification on the nucleic acid sample to be tested; constructing a sequencing library for the PCR amplification product to obtain a sequencing library; using a single-molecule sequencing platform to perform sequencing on the sequencing library to obtain sequencing data; performing variation analysis on the sequencing data. No PCR amplification is required, deviations are reduced, and long-read sequencing is supported, and various types of thalassemia mutations can be accurately detected. In some examples of the present application, the aforementioned method is suitable for multi-tube PCR amplification, and the optimization of the number of primers reduces the non-specific amplification of multiple PCR, and the design of the index sequence increases the sample throughput of a single library construction to further reduce the cost of single-molecule library construction sequencing. The method has the advantages of low template quality requirements, good specificity, good repeatability, simple library construction operation, large single detection throughput, and short cycle.

[0049] In some examples of the present application, the aforementioned nucleic acid sequencing method may further include at least one of the following additional technical features:

[0050] In some specific examples of the present application, the aforementioned nucleic acid sample to be tested is selected from DNA, for example, DNA extracted from human peripheral blood samples.

[0051] In some examples of the present application, in order to facilitate the distinction of multiple samples, the 5' end of the aforementioned primer set is connected with an index sequence, wherein the design of the index sequence is selected from a conventional laboratory design scheme, which will not be described in detail here.

[0052] In some examples of the present application, the aforementioned sequencing library is selected from a single-molecule sequencing library. Correspondingly, the construction of the sequencing library is also selected from the single-molecule sequencing library construction process, including: performing end repair and A addition treatment on the PCR amplification product; and purifying the A addition product and performing sequencing adapter connection treatment. It is understandable that the aforementioned end repair and A addition treatment are performed in an end repair and A addition reaction system. This step is a conventional step for library construction, and the specific operation scheme can be performed based on the instructions of the commercially available kit, which will not be repeated here.

[0053] The reaction procedures of the aforementioned end repair and A treatment include: 20°C, 8min-15min; 65°C, 8min-15min. In a preferred example of the present application, the reaction procedures of the aforementioned end repair and A treatment are selected from 20°C, 10min; 65°C, 10min.

[0054] Wherein, the aforementioned purification process is achieved by purification magnetic beads.

[0055] Wherein, the ligation process is carried out in the presence of DNA ligase. In some examples of the present application, the aforementioned DNA ligase is selected from T4 ligase.

[0056] Single-molecule sequencing library construction does not require PCR amplification, reduces bias, supports long-read sequencing, and can accurately detect various types of thalassemia variants. This method has low sample size requirements and is suitable for the study of precious or limited samples. It also reduces errors in the processing process and improves the quality and accuracy of sequencing data.

[0057] In some examples of the present application, the single-molecule sequencing library is further subjected to quality control processing to ensure the quality of the single-molecule sequencing library, avoid unnecessary errors and data problems during the sequencing process, and improve the reliability and accuracy of the sequencing results.

[0058] In some examples of the present application, the aforementioned thalassemia variation includes: α-thalassemia variation and / or β-thalassemia variation. Wherein, the type of variation includes: at least one of copy number variation (CNV), single nucleotide polymorphism (SNP) and structural variation (SV). The primer set of the present application can be used to detect deletion type, mutation type and structural variation in α-thalassemia variation; the primer set of the present application can be used to detect deletion type and mutation type in β-thalassemia.

[0059] In some examples of this application, the aforementioned α-thalassemia variants include: --SEA, -α3.7I, -α6.9, -AW, -α6.3, -α3.7II, -α2.8, - -FIL, -α5.9, -α3.7III, -α2.7, --MED-II, -α27.6, --14.9, --9.7, -α21.9, -α4.2, -αM At least one of AL3.5, --JS, --27.2, --11.1, --THAI, -α2.4, --20.5, -α7.9, -α10.3, -α5.3, -α0.8, ααanti3.7, HKαα, αααanti3.7, αααanti4.2, anti-HKαα, and αααanti4.2. Among them, --SEA, -α3.7Ⅰ, -α6.9, -AW, -α6.3, -α3.7Ⅱ, -α2.8, --FIL, -α5.9, -α3.7Ⅲ, -α2. 7. --MED-II, -α27.6, --14.9, --9.7, -α21.9, -α4.2, -αMAL3.5, --JS, --27.2, --1 1.1, --THAI, -α2.4, --20.5, -α7.9, -α10.3, -α5.3 and -α0.8 belong to the α-globin gene deletion type; αααanti3.7, HKαα, ααααanti3.7, αααanti4.2, anti-HKαα and ααααanti4.2 belong to the α-globin gene structural variant.

[0060] In some examples of the present application, the aforementioned β-thalassemia variants include: Chinese Gγ(Aγδβ)0, Yunnanese, -β21.9, 1357bp deletion, SEA-HPFH, Filipino del, Siriraj Gγ(Aγδβ)0, HPFH-6, 78.9kb Gγ(Aγδβ)0del, Caucasian HPFH, HPFH-7, HPFH-5, Thai del, Turkish, Asian Indian(Aγδβ)0, Hb Lepore, 3.5kb del, Croatian 1605bp del, British 1393bpdel, 619bp del and 105bp del. At least one of the aforementioned variant types belongs to the β-globin gene deletion type.

[0061] In some examples of the present application, variation analysis is achieved through an automated bioinformatics analysis process that is compatible with a sequencing platform (such as BGI). Different sequencing platforms have corresponding variation analysis processes. Those skilled in the art will appreciate that by performing single-molecule library construction and sequencing based on the primer set of the present application, combined with conventional variation analysis processes, the variation type of thalassemia can be obtained.

[0062] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0064] Figure 1 A schematic diagram of thalassemia variant type detection provided in an embodiment of the present application;

[0065] Figure 2 A schematic diagram of the design positions of α-globin gene primers provided in the examples of the present application;

[0066] Figure 3 A schematic diagram of the design positions of the β-globin gene primers provided in the examples of the present application;

[0067] Figure 4 A schematic diagram of the electrophoresis results of the Long PCR extension of different deletion samples provided in the embodiments of the present application;

[0068] FIG5 is a schematic diagram of cyclone SEQ sequencing results of different deletion samples and mutation samples provided in the embodiments of the present application; wherein A is αα / -α 3.7 Sample result diagram, B is Chinese (Aγδβ)0 / β N Schematic diagram of sample results, C is αα / ααα anti3.7 Schematic diagram of sample results, D is a schematic diagram of the results of α-globin gene point mutation Hb Constant Spring, E is a schematic diagram of the results of β-globin gene point mutation Codons 41 / 42(-TTCT)beta0, F is a schematic diagram of the results of β-globin gene point mutation Codon43(G>T)beta0 and Codons 41 / 42(-TTCT)beta0 trans mutation samples, and G is a schematic diagram of the results of HKαα / αα samples. DETAILED DESCRIPTION

[0069] The embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, but cannot be understood as limiting the present application.

[0070] In this application, unless otherwise specified, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of this application, "plurality" means at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0071] In this article, unless otherwise specified, the term "single-molecule library preparation" is a technical method that directly uses a single DNA molecule to construct a sequencing library without PCR amplification. It minimizes sequence deviations and errors by directly connecting the original DNA molecule to the sequencing adapter without amplification steps, retaining the true information of the genome, and is suitable for long-read sequencing and accurate detection of variations in the genome.

[0072] In this article, unless otherwise specified, the term "sequencing library" refers to a collection of processed DNA or RNA samples before genome or transcriptome sequencing. The sample usually contains DNA fragments or cDNA fragments to which adapters have been connected so that they can be identified and read during the sequencing process.

[0073] In this article, unless otherwise specified, there is no particular limitation on the sequencing platform, which can be any common third-generation sequencing platform. In this article, the sequencing platform that can be used to perform the above sequencing method can be selected from but not limited to PacBio's SMRT sequencing platform, Oxford Nanopore sequencing platform, and BGI's Cyclone SEQ sequencing platform.

[0074] The sequences involved in this application are shown in Table 3.

[0075] Table 3

[0076]

[0077]

[0078] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0079] Embodiment 1:

[0080] This example is intended to exemplify the effectiveness and feasibility of the complex variation detection method for thalassemia. Figure 1 , the specific steps are as follows:

[0081] 1) DNA extraction

[0082] Peripheral blood samples from clinical subjects with thalassemia were selected and DNA was extracted using a magnetic bead extraction kit. The gDNA was stored at -20°C.

[0083] 2) Long PCR amplification system

[0084] Take out 48 clinical sample DNA, ApexHF Hs DNA polymerase, ApexHF Hs CL buffer, and PrimerMix (index-primer) from -20°C, vortex and mix after thawing, and prepare the reaction system (α-thalassemia deletion and mutation) of a single PCR reaction well according to Table 4;

[0085] Table 4 Single PCR reaction system (tube 1)

[0086] Reagents Volume (μL) Enzyme-free water (DNase / RNase-free water) 8.25 Buffer (ApexHF Hs CL buffer) 12.5 Primer Mix, 5 μM 2 DNA polymerase (ApexHF Hs DNA polymerase) 0.25 Template DNA 2

[0087] Note: Primer design positions are as follows Figure 2 As shown, the specific sequences are shown in SEQ ID NO: 1-5.

[0088] Prepare the reaction system (β-thalassemia deletion and mutation) for a single PCR reaction well according to Table 5;

[0089] Table 5 Single PCR reaction system (tube 2)

[0090] Reagents Volume (μL) DNase / RNase-free water 8 ApexHF Hs CL buffer 12.5 Primer Mix (5 μM) 2 ApexHF Hs DNA polymerase 0.5 Template DNA 2

[0091] Note: Primer design positions are as follows Figure 3 As shown, the specific sequences are shown in SEQ ID NO:6-13.

[0092] The Long PCR reaction program was set according to Table 6, and the reaction was carried out on a 96-well PCR instrument for about 5.5 h.

[0093] Table 6 Long PCR reaction program

[0094]

[0095] 3) PCR product pooling and purification

[0096] Pool 48 PCR products from tube 1 into 1.5 mL centrifuge tubes at an equal volume of 15 μL to obtain 720 μL of PCR product mixture 1; pool 48 PCR products from tube 2 into 1.5 mL centrifuge tubes at an equal volume of 15 μL to obtain 720 μL of PCR product mixture 2;

[0097] Add 0.5 times the volume of purified magnetic beads to the PCR mixture (the beads were allowed to stand at room temperature in advance and vortexed to mix evenly), and incubate at room temperature for 10 minutes to allow the DNA to bind to the magnetic beads;

[0098] Place the 1.5 mL EP tube on a magnetic stand and carefully remove the supernatant after the solution becomes clear (approximately 5 minutes).

[0099] Keep the 1.5mL EP tube on the magnetic stand all the time, add 1000μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 seconds, and carefully remove the supernatant; repeat the previous rinsing step for a total of two rinses;

[0100] Keep the 1.5mL EP tube on the magnetic rack at all times, open the cover and dry the magnetic beads at room temperature for about 10 minutes;

[0101] Remove 1.5mL EP from the magnetic rack, add 50μL nuclease-free water, mix thoroughly with a pipette, and let stand at room temperature for 2 minutes. Let stand on the magnetic rack for 5 minutes until the solution is clear, then carefully pipette the supernatant into a new 1.5mL EP tube;

[0102] The purified product DNA was quantified using Qubit 2.0 and diluted to 50 ng / μL for later use.

[0103] 2.4.4 Single-molecule library construction

[0104] Take 500 ng of the purified products from PCR tube 1 and PCR tube 2 in a 0.2 mL PCR tube, add nuclease-free water to make the volume uniform to 44 μL, record it as tube A; similarly, take 500 ng of the purified products from PCR tube 1 and PCR tube 2 in a 0.2 mL PCR tube, add nuclease-free water to make the volume uniform to 44 μL, record it as tube B;

[0105] According to the proportions in Table 7, prepare the required amount of end repair plus "A" reaction solution in a centrifuge tube of appropriate specifications. This step needs to be prepared on ice. Vortex the prepared end repair plus "A" reaction solution 3 times, each time for 3 seconds, and centrifuge it instantly to collect the reaction solution at the bottom of the tube;

[0106] Table 7 End repair plus "A" reaction system

[0107] Reagents 2 reaction volumes (μL) End Repair Buffer 1 (EA Buffer 1) 12 EA Enzyme 1 6 End Repair Buffer 2 (EA Buffer 2) 6 Nuclease-free water 8 Template DNA 32

[0108] Pipette 16 μL of the prepared end repair reaction plus "A" reaction solution into tube A and tube B (0.2 mL PCR tube), gently pipette several times with a flared pipette tip until mixed, and centrifuge briefly to collect the reaction solution at the bottom of the tube;

[0109] Tube A and tube B (0.2 mL PCR tube) were placed in a PCR instrument and reacted according to the conditions in Table 8. After the reaction was completed, the reaction solution was collected to the bottom of the tube by instant centrifugation;

[0110] Table 8 Reaction procedure

[0111] temperature time Hot cover (105℃) ON 20℃ 10min 65℃ 10min 4℃ Hold

[0112] Take out the purified magnetic beads and place them at room temperature. Vortex and mix thoroughly before use.

[0113] Pipette 60 μL of purified magnetic beads into two new 1.5 mL EP tubes (labeled tube A and tube B), use a flared pipette tip to gently pipette all the samples from tube A and tube B (0.2 mL PCR tube) containing unrepaired products into the two tubes containing magnetic beads, and flick the tube wall to mix;

[0114] Tube A and tube B (1.5 mL EP tube) were fixed on a rotary mixer, rotated at a slow speed, and incubated at room temperature for 10 min;

[0115] Remove tube A and tube B (1.5 mL EP tube), centrifuge briefly, and place on a magnetic rack. Let stand for 2-5 minutes until the liquid is clear. Use a pipette to carefully pipette tube A and tube B (1.5 mL EP tube) onto the magnetic rack. Add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and tube walls. Carefully pipette the supernatant and discard it.

[0116] Repeat the previous step;

[0117] Remove tube A and tube B (1.5 mL EP tube) from the magnetic rack and centrifuge them instantly. After separation on the magnetic rack, use a small-range pipette to absorb the remaining liquid at the bottom of the tube;

[0118] Keep tube A and tube B (1.5 mL EP tube) on the magnetic rack, open the cap of the centrifuge tube, and dry at room temperature until the surface of the magnetic beads is not reflective or cracked;

[0119] Remove tube A and tube B (1.5mL EP tube) from the magnetic rack, add 62μL nuclease-free water to elute DNA, and flick the tube wall to mix. Centrifuge for 3 seconds and collect the liquid in the tube to the bottom of the tube. Incubate at room temperature for 10 minutes;

[0120] Centrifuge tube A and tube B (1.5 mL EP tube) for a short time and place them on a magnetic stand. Let them stand for 5 minutes until the liquid is clear. Transfer 60 μL of the supernatant to new tubes A and B (1.5 mL EP tube). The remaining sample can be used for concentration determination. It is recommended to use the Qubit dsDNA HS Assay Kit to determine the concentration of the purified end-repair plus "A" product.

[0121] Set the thermostatic metal bath to 25°C for preheating;

[0122] Take out the adapter and ligase, flick the tube wall to mix, centrifuge instantly, and place on ice for later use (for the specially designed adapter, do not use vortexing or pipetting to mix; the ligation buffer (4XLigation buffer) is relatively viscous, so please vortex and mix thoroughly).

[0123] Take out the ligation buffer (4X Ligation buffer) and thaw it at room temperature, shake and mix, centrifuge it instantly, and place it on ice for later use. Prepare the ligation reaction solution according to Table 9;

[0124] Table 9 Ligation reaction system

[0125]

[0126]

[0127] Add 2.5 μL of adapter to the unrepaired product tube A and tube B (1.5 mL EP tube), tap the tube wall to mix, and then centrifuge instantly;

[0128] Use a pipette to slowly pipette 37.5 μL of the prepared ligation reaction solution into tube A and tube B (1.5 mL EP tube), flick the tube wall until fully mixed, and centrifuge instantly to collect the reaction solution at the bottom of the tube (the ligation reaction solution is relatively viscous, so please slowly pipette and drain the liquid, and ensure that it is fully mixed. Insufficient mixing will reduce the efficiency of library construction);

[0129] Tube A and tube B (1.5 mL EP tube) were placed in a preheated thermostatic metal bath (25° C.) for ligation reaction with a reaction time of 30 min.

[0130] After the reaction is completed, centrifuge instantly and collect the reaction solution at the bottom of the tube;

[0131] Pipette 40 μL of purified magnetic beads into tube A and tube B (1.5 mL EP tube) respectively, and flick the tube wall to mix (take out the purified magnetic beads 30 minutes in advance and place them at room temperature, and shake them thoroughly before use);

[0132] Place tube A and tube B (1.5 mL EP tube) on a rotary mixer, rotate slowly, and incubate at room temperature for 10 min;

[0133] Centrifuge tube A and tube B (1.5 mL EP tube) for a short time and place them on a magnetic stand. Let them stand for 2-5 minutes until the liquid becomes clear. Use a pipette to carefully remove the supernatant and discard it.

[0134] Keep tube A and tube B (1.5mL EP tube) on the magnetic rack, add 150μL Wash Buffer 1, remove tube A and tube B (1.5mL EP tube) from the magnetic rack, flick the tube wall to mix the magnetic beads, centrifuge instantly, put them back on the magnetic rack, let stand for 2-5min until the liquid is clear, carefully aspirate the supernatant and discard;

[0135] Repeat the previous step;

[0136] Remove tube A and tube B (1.5 mL EP tube) from the magnetic rack and centrifuge them instantly. After separation on the magnetic rack, use a small-range pipette to absorb the remaining liquid at the bottom of the tube;

[0137] Remove tube A and tube B (1.5 mL EP tube) from the magnetic stand, add 22 μL elution buffer, and incubate at 37°C for 10-20 min. Centrifuge for 3 seconds and collect the liquid in the tube to the bottom of the tube;

[0138] Centrifuge tube A and tube B (1.5mL EP tube) for a short time and place them on a magnetic stand. Let them stand for 2-5 minutes until the liquid is clear. Transfer 20μL of the supernatant in tube A and tube B (1.5mL EP tube) to a new 1.5mL EP tube. The remaining sample can be used for concentration determination. Use the Qubit dsDNA HS Assay Kit to determine the concentration of the purified ligation product and use it as a standard sequencing library.

[0139] Based on the BGI Cyclone SEQ single-molecule sequencing platform, single-molecule sequencing was performed using the Cyclone WT sequencing chip and WT sequencing kit. A single chip sequenced a standard library and produced 10G of data.

[0140] 5) Single molecule information analysis of thalassemia

[0141] The thalassemia single-molecule bioinformatics analysis platform (BGI) was used to perform index splitting and primer splitting analysis of samples (reads were aligned to the thalassemia variation region), and the sample type (whether there is a variation, including CNV, SNV and SV) was automatically output.

[0142] 6) Test results

[0143] The electrophoresis results of Long PCR extension samples with different deletions are as follows Figure 4 As shown in Figure 2, the cyclone SEQ sequencing results of different deletion samples and mutation samples are as follows: Figure 5A -E. Among them, 48 clinical samples for thalassemia screening were tested, with a sensitivity of 100% for positive samples and a specificity of 100% for negative samples. Three cases of α-globin gene deletion αα / -α3.7 type, one case of β-globin gene deletion Chinese (Aγδβ) 0 type, one case of complex structural variation αα / αααanti3.7 type, and one case of complex structural variation HKαα / αα type were successfully detected. One case of α-globin gene point mutation Hb Constant Spring heterozygote, one case of β-globin gene point mutation Codons 41 / 42 (-TTCT) beta0 heterozygote, and one case of β-globin gene point mutation Codon 43 (G>T) beta0 and Codons 41 / 42 (-TTCT) beta0 compound heterozygote were detected, and the trans structural mutation was determined. The specific results are shown in Table 10.

[0144] Table 10 Test results

[0145]

[0146] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0147] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A primer set, characterized in that: include: Based on at least one of the sequences 1)-13), the following is designed: 1)chr16:219267-219290; 2)chr16:228804-228829; 3)chr16:215255-215276; 4)chr16:23 5945-235971; 5) chr16: 196488-196508; 6) chr11: 5276986-5277008; 7) chr11: 5257290-525 7314;8)chr11:5249915-5249939;9)chr11:5258978-5259000;10)chr11:5221888-522191 2;11)chr11:5189975-5189996;12)chr11:5152341-5152365;13)chr11:5133905-5133925; Wherein, the primer is at least partially complementary to the sequences 1)-13).

2. The primer set according to claim 1, characterized in that The primer set is selected from at least one of the following: SEQ ID NO:1-SEQ ID NO:

13.

3. Use of the primer set according to claim 1 or 2 in preparing a thalassemia detection kit.

4. The use according to claim 3, characterized in that The thalassemia includes: α-thalassemia and β-thalassemia; in, Primers designed based on at least one of sequences 1)-5) are used for the detection of α-thalassemia; The primers designed based on at least one of sequences 6)-13) are used for the detection of β-thalassemia.

5. The use according to claim 4, characterized in that The primers designed based on sequences 1)-5) are shown in SEQ ID NOs: 1-5; The primers designed based on sequences 6)-13) are shown in SEQ ID NOs: 6-13.

6. A method for detecting thalassemia mutation, characterized in that: include: Using the primer set described in claim 1 or 2 to perform PCR amplification on the nucleic acid sample to be tested; Perform library construction and sequencing on the PCR amplification products; Perform variation analysis on the sequencing results.

7. The method according to claim 6, characterized in that The thalassemia variants include: α-thalassemia variants and / or β-thalassemia variants; Optionally, the type of variation includes at least one of copy number variation, single nucleotide polymorphism and structural variation.

8. The method according to claim 7, characterized in that The α-thalassemia variants include: --SEA, -α3.7I, -α6.9, -AW, -α6.3, -α3.7II, -α2.8, --FIL, -α5 .9, -α3.7Ⅲ, -α2.7, --MED-II, -α27.6, --14.9, --9.7, -α21.9, -α4.2, -αMAL3.5 , --JS, --27.2, --11.1, --THAI, -α2.4, --20.5, -α7.9, -α10.3, -α5.3, -α0.8, at least one of αααanti3.7, HKαα, ααααanti3.7, αααanti4.2, anti-HKαα and αααanti4.

2.

9. The method according to claim 7, characterized in that: The β-thalassemia variations include: Chinese Gγ(Aγδβ)0, Yunnanese, -β21.9, 1357bp deletion, SEA-HPFH, Filipino del, Siriraj Gγ(Aγδβ)0, HPFH-6, 78.9kb Gγ(Aγδβ)0del, Caucasian HPFH, HPFH-7, HPFH-5, Thai del, Turkish, Asian Indian(Aγδβ)0, Hb Lepore, 3.5kb del, Croatian 1605bp del, British 1393bp del, 619bp del and 105bp del. At least one of them.

10. The method according to claim 8 or 9, characterized in that: In the PCR amplification processing system, the concentration ratio of the primer set, the nucleic acid sample to be tested, and the DNA polymerase is (0.3 μM-0.6 μM): (20 ng / μL-100 ng / μL): (0.8 U / μL-1.2 U / μL); Optionally, in the PCR amplification treatment system, the concentration of the primer set is 0.3 μM-0.6 μM; Optionally, in the PCR amplification processing system, the concentration of the nucleic acid sample to be tested is 20 ng / μL-100 ng / μL; Optionally, in the PCR amplification processing system, the DNA polymerase concentration is 0.8U / μL-1.2U / μL.

11. The method according to claim 10, characterized in that The reaction procedure of the PCR amplification process is shown in Table 1: Table 1 12. The method according to claim 11, characterized in that The reaction procedure of the PCR amplification process is shown in Table 2: Table 2 13. The method according to claim 6, characterized in that After PCR amplification and before library construction, the process further includes: pooling; Optionally, after the pooling process, the method further comprises: a purification process.

14. The method according to claim 6 or 13, characterized in that: The library construction process is selected from single molecule sequencing library construction; Optionally, the single molecule sequencing library construction comprises: The PCR amplification products were subjected to end repair and A addition treatment; The A-addition product is purified to obtain a single-molecule sequencing library.

15. A kit, characterized in that: include: The primer set according to claim 1 or 2.

16. The kit according to claim 15, characterized in that The kit further comprises: at least one of DNA polymerase and buffer, nuclease-free water, library construction reagents and sequencing reagents.

17. The kit according to claim 16, characterized in that The library construction reagents include: at least one of: end repair enzyme, end repair buffer, poly A, ligase, ligation buffer, elution buffer and purification magnetic beads.

18. A nucleic acid sequencing method, characterized in that: include: Using the primer set described in claim 1 or 2 to perform PCR amplification on the nucleic acid sample to be tested; Constructing a sequencing library for the PCR amplification product to obtain a sequencing library; Using a single-molecule sequencing platform to perform sequencing processing on the sequencing library to obtain sequencing data; The sequencing data are subjected to variation analysis.

19. The method according to claim 18, characterized in that The variation includes: a thalassemia variation, wherein the thalassemia variation is selected from an α-thalassemia variation and / or a β-thalassemia variation; Optionally, the type of variation includes at least one of copy number variation, single nucleotide polymorphism and structural variation; Optionally, the α-thalassemia variants include: --SEA, -α3.7I, -α6.9, -AW, -α6.3, -α3.7II, -α2.8, --FIL, -α5.9, -α3.7III, -α2.7, --MED-II, -α27.6, --14.9, --9.7, -α21.9, -α4.2, -αMAL3 .5, --JS, --27.2, --11.1, --THAI, -α2.4, --20.5, -α7.9, -α10.3, -α5.3, -α0.8, at least one of αααanti3.7, HKαα, ααααanti3.7, αααanti4.2, anti-HKαα and ααααanti4.2; Optionally, the β-thalassemia variation includes at least one of: Chinese Gγ(Aγδβ)0, Yunnanese, -β21.9, 1357bp deletion, SEA-HPFH, Filipino del, Siriraj Gγ(Aγδβ)0, HPFH-6, 78.9kb Gγ(Aγδβ)0del, Caucasian HPFH, HPFH-7, HPFH-5, Thai del, Turkish, Asian Indian(Aγδβ)0, Hb Lepore, 3.5kb del, Croatian 1605bp del, British 1393bp del, 619bp del and 105bpdel.

20. The method according to claim 18 or 19, characterized in that The variation analysis is achieved through the automated bioinformatics analysis process of the sequencing platform.

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