Primer set and use thereof in preparation of thalassemia detection kit

Abstract

The present invention relates to the technical field of biology, and specifically relates to a primer set and the use thereof in the preparation of a thalassemia detection kit. The primer set comprises primers designed on the basis of 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) chr11:5257290-5257314; 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 primers are at least partially complementary to sequences 1)-13).

Description

Primer set and use thereof in preparing a thalassemia detection kit TECHNICAL FIELD

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

[0002] Thalassemia (referred to as "thalassemia" for short) is an autosomal monogenic recessive genetic disease mainly caused by globin gene defects leading to reduced or absent synthesis of globin chains. According to the type of globin chain affected, thalassemia is mainly divided into alpha-thalassemia and beta-thalassemia. Alpha-thalassemia is caused by the synthesis deletion or reduction of alpha-globin genes, and beta-thalassemia is caused by the synthesis reduction or deletion of beta-globin genes.

[0003] Alpha-thalassemia is mainly caused by the deletion or mutation of alpha-globin genes, leading to an imbalance in the proportion of globin chains, and presenting symptoms such as chronic hemolytic anemia. Since there are two copies of alpha-globin genes on each chromosome, gene deletion or mutation can exhibit a variety of different inheritance patterns, leading to abnormalities in the number of genes, such as the formation of triplets or quadruplets, which are often difficult to identify by conventional detection methods. Beta-thalassemia is caused by the deletion or mutation of beta-globin genes, leading to red blood cell damage, hardening and hemolysis. Point mutations, copy number variations (CNV) and structural variations (SV) of genes are the main causes of thalassemia.

[0004] Existing thalassemia detection techniques have certain limitations. For example, reverse dot blot (RDB), multi-color melting curve analysis (MMCA), multiplex ligation-dependent probe amplification (MLPA) and gap-PCR can only detect common alpha-thalassemia, and even HKαα / αα and HKαα / -α3.7, but cannot detect alpha-globin gene triplets and quadruplets; multiplex ligation-dependent probe amplification (MLPA) can only determine the copy number of deletion or duplication sequences, and cannot locate alleles; although high-throughput sequencing technology (NGS) performs well in point mutation detection, it still cannot detect alpha-globin gene triplets, quadruplets, HKαα / αα and other complex structural variations, and cannot distinguish between homologous genes HBA1 / 2 and various variants in trans.

[0005] Therefore, the current thalassemia detection method still needs to be improved for complex variation types.

[0006] SUMMARY

[0007] The present application aims to at least partially solve one of the problems in the related art. To this end, the present application proposes a detection method based on long-range PCR amplification technology. The method amplifies the deletion region of the alpha-globin gene and beta-globin gene related to thalassemia, and uses single molecule library construction and sequencing technology to comprehensively detect the copy number variation (CNV), single nucleotide variation (SNV) and structural variation (SV) of thalassemia. In addition to detecting common gene deletion types, it can further detect 6 complex structural variations including αααanti3.7, αααanti4.2, HKαα, anti-HKαα, ααααanti3.7 and ααααanti4.2. In addition, it can accurately distinguish 1046 point mutations of homologous genes HBA1 and HBA2 and 1085 point mutations on the HBB gene, and can identify whether multiple mutation sites are in cis or trans structure.

[0008] Specifically, the present application provides the following technical solutions:

[0009] In the first aspect of the present application, a primer set is proposed. According to the embodiments of the present application, the aforementioned primer set comprises: 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) chr11:5257290-5257314; 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 primer is at least partially complementary to the aforementioned sequence 1)-13). In some examples of the present application, the inventors have found through a large number of research and experimental verifications that the primer set designed based on the aforementioned sequence can accurately detect various complex variation types of thalassemia.

[0010] It should be noted that in the present document, each primer based on the above sequence design and sequence 1)-13) can be fully complementary or partially complementary. For example, the length of sequence 1) is 23 bp, and the primer based on sequence 1) can be 23 bp, or 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp or 23 bp. The rest of the sequence design is as described above, and is not repeated here due to space constraints.

[0011] In some examples of the present application, the foregoing primer set can further comprise at least one of the following additional technical features:

[0012] In some examples of the present application, the foregoing primer set is selected from at least one of SEQ ID NO: 1-SEQ ID NO: 13. The foregoing 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 based on SEQ ID NO: 1-SEQ ID NO: 13, the related complex variations of thalassemia can be accurately detected. Those skilled in the art can understand that the foregoing SEQ ID NO: 1-SEQ ID NO: 13 is only one primer set based on sequences 1)-13), and primer sets obtained by increasing or decreasing the number of bases based on this primer set also fall within the scope of the present application.

[0013] In a second aspect of the present application, the present application proposes the use of the foregoing primer set in the preparation of a thalassemia detection kit. In some examples of the present application, the kit prepared based on the foregoing primer set can be used for portable and accurate detection of thalassemia variation types.

[0014] Those skilled in the art can understand that the kit prepared based on the foregoing primer set can detect thalassemia variation types by means of conventional sequencing or PCR, and the sequencing or PCR is selected from routine laboratory operations, which will not be repeated here.

[0015] In some examples of the present application, the foregoing use can further comprise at least one of the following additional technical features:

[0016] In some examples of the present application, the foregoing thalassemia includes: alpha-thalassemia and beta-thalassemia; wherein the primer obtained based on at least one of sequences 1)-5) is used for the foregoing alpha-thalassemia detection; and the primer obtained based on at least one of sequences 6)-13) is used for the foregoing beta-thalassemia detection. The primer based on the foregoing sequence design can accurately detect various complex variation types of alpha-thalassemia and beta-thalassemia, such as special structure variations, etc.

[0017] In some examples of the present application, the primers designed based on sequences 1)-5) are shown as SEQ ID NO: 1-5; and the primers designed based on sequences 6)-13) are shown as SEQ ID NO: 6-13.

[0018] In a third aspect of the present application, a method for detecting thalassemia mutations is provided. According to an embodiment of the present application, the method comprises: performing PCR amplification on a nucleic acid sample to be tested using the primer set of the first aspect; performing library construction and sequencing on the PCR amplification product; and performing mutation analysis on the sequencing results. In some examples of the present application, the method is suitable for multiplex PCR amplification, and the optimization of the number of primers reduces the non-specific amplification of multiplex PCR, and the design of index sequences improves the sample throughput of single library construction, thereby further reducing the cost of single molecule library sequencing. The method has the advantages of low template quality requirement, good specificity, good repeatability, simple library construction operation, large single detection throughput, short cycle, etc.

[0019] In some examples of the present application, the method for detecting thalassemia mutations can further comprise at least one of the following additional technical features:

[0020] In some specific examples of the present application, the nucleic acid sample to be tested is selected from DNA, for example, DNA extracted from a human peripheral blood sample.

[0021] In some examples of the present application, in order to facilitate the differentiation of multiple samples, the 5' end of the primer set is connected with an index sequence. The design of the index sequence is selected from a conventional design scheme in a laboratory, which will not be described here.

[0022] In some examples of the present application, the thalassemia mutations include alpha-thalassemia mutations and beta-thalassemia mutations. The types of mutations include at least one of copy number variations (CNV), single nucleotide polymorphisms (SNP), and structural variations (SV). The primer set of the present application can be used to detect deletions, mutations, and structural variations in alpha-thalassemia mutations; and the primer set of the present application can be used to detect deletions and mutations in beta-thalassemia.

[0023] In some examples of the present application, the aforementioned alpha-thalassemia mutation includes at least one of the following: -SEA, -a3.7 I, -a6.9, -AW, -a6.3, -a3.7 II, -a2.8, -FIL, -a5.9, -a3.7 III, -a2.7, -MED-II, -a27.6, -14.9, -9.7, -a21.9, -a4.2, -aMAL3.5, -JS, -27.2, -11.1, -THAI, -a2.4, -20.5, -a7.9, -a10.3, -a5.3, -a0.8, aaaaanti3.7, HKaa, aaaaanti3.7, aaaaanti4.2, anti-HKaa, and aaaaanti4.2. Among them, -SEA, -a3.7 I, -a6.9, -AW, -a6.3, -a3.7 II, -a2.8, -FIL, -a5.9, -a3.7 III, -a2.7, -MED-II, -a27.6, -14.9, -9.7, -a21.9, -a4.2, -aMAL3.5, -JS, -27.2, -11.1, -THAI, -a2.4, -20.5, -a7.9, -a10.3, -a5.3, and -a0.8 belong to the alpha-globin gene deletion type; aaaaanti3.7, HKaa, aaaaanti3.7, aaaaanti4.2, anti-HKaa, and aaaaanti4.2 belong to the alpha-globin gene structural variation type.

[0024] In some examples of the present application, the aforementioned beta-thalassemia mutation includes at least one of the following: Chinese Gγ (Aγδβ)0, Yunnanese, -β21.9, Taiwanese deletion, SEA-HPFH, Filipino del, Siriraj Gγ (Aγδβ)0, HPFH-6, 78.9kb Gγ (Aγδβ)0 del, 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. The aforementioned mutation type belongs to the beta-globin gene deletion type.

[0025] In some examples of the present application, in the PCR amplification processing system, the concentration of the aforementioned primer set, the aforementioned 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). The detection of various complex mutations of alpha-thalassemia and beta-thalassemia can be achieved within the aforementioned concentration range.

[0026] In some examples of the present application, in the PCR amplification processing system, the concentration of the aforementioned 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 aforementioned primer set is 0.4 μM.

[0027] 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 20 ng / μL-100 ng / μL, optionally 20 ng / μL, 30 ng / μL, 40 ng / μL, 50 ng / μL, 60 ng / μL, 70 ng / μL, 80 ng / μL, 90 ng / μL, or 100 ng / μL.

[0028] In some examples of the present application, in the PCR amplification processing system, the concentration of the aforementioned DNA polymerase is 0.8 U / μL-1.2 U / μL, optionally 0.8 U / μL, 0.9 U / μL, 1.0 U / μL, 1.1 U / μL, or 1.2 U / μL. In some preferred examples of the present application, the concentration of the aforementioned DNA polymerase is 1.0 U / μL.

[0029] In some examples of the present application, based on the above amplification system, the reaction procedure of PCR amplification processing is shown in Table 1:

[0030] Table 1

[0031] It can be understood by those skilled in the art that simple adjustments to temperature, time, or cycles 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 processing is shown in Table 2:

[0033] Table 2

[0034] In some examples of the present application, after the PCR amplification processing, before the library construction processing, further comprising: pooling processing. By mixing multiple PCR amplification products, simultaneous processing in subsequent library construction and sequencing is achieved, sequencing efficiency is improved, and sequencing cost is reduced.

[0035] In some examples of the present application, after the above-mentioned pooling processing, further comprising a purification processing. The sample quality is improved through the purification processing, ensuring efficient library construction.

[0036] In some examples of the present application, further comprising quality control of the purification processing product after the purification processing. The quality and concentration of the DNA sample for library construction are ensured to meet the requirements through quality control. The quality control sample that does not meet the quality control requirements needs to be reprocessed by PCR amplification.

[0037] In some examples of the present application, the aforementioned library construction processing is selected from single molecule sequencing library construction. Without PCR amplification, bias is reduced, long read sequencing is supported, and various types of thalassemia variants can be accurately detected. The method has low sample quantity requirements, is suitable for research on precious or limited samples, and reduces errors in the processing process, improving the quality and accuracy of sequencing data.

[0038] In some examples of the present application, further comprising quality control processing of the above-mentioned single molecule sequencing library, ensuring the quality of the single molecule sequencing library, avoiding unnecessary errors and data problems in the sequencing process, and improving the reliability and accuracy of the sequencing results.

[0039] In some specific examples of the present application, the aforementioned single molecule sequencing library construction comprises: performing end repair processing and A addition processing on the PCR amplification processing product; and performing purification processing on the A addition processing product to obtain a single molecule sequencing library.

[0040] In some examples of the present application, the variant analysis is realized through an automatic bioinformatics analysis process matched with the sequencing platform (such as Huada Gene), and different sequencing platforms correspond to different variant analysis processes. Those skilled in the art can understand that, based on the primer set of the present application for single molecule library construction and sequencing, combined with a conventional variant analysis process, the variant types of thalassemia can be obtained.

[0041] In a third aspect of the present application, a kit is provided. According to an embodiment of the present application, the aforementioned kit comprises the primer set of the first aspect. In some examples of the present application, based on the aforementioned kit, accurate detection of various variant types (including special structure variants) of thalassemia can be realized.

[0042] In some examples of the present application, the aforementioned kit can further comprise at least one of the following additional technical features:

[0043] In some examples of the present application, the aforementioned kit further comprises at least one of the following: a DNA polymerase and a buffer, nuclease-free water, a library construction reagent, and a sequencing reagent.

[0044] In some examples of the present application, the aforementioned 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.

[0045] It can be understood by those skilled in the art that the aforementioned kit can also include some commonly used reagents and instructions, etc.

[0046] In a fourth aspect of the present application, a nucleic acid sequencing method is provided. According to an embodiment of the present application, the sequencing method comprises: performing PCR amplification on a nucleic acid sample to be tested using the primer set of the first aspect; performing sequencing library construction on the PCR amplification product to obtain a sequencing library; performing sequencing processing on the sequencing library using a single molecule sequencing platform to obtain sequencing data; and performing variant analysis on the sequencing data. Without PCR amplification, bias is reduced, long read sequencing is supported, and various types of thalassemia variants can be accurately detected. In some examples of the present application, the aforementioned method is suitable for multi-tube PCR amplification, the optimization of primer quantity reduces non-specific amplification of multiplex PCR, and the design of index sequences improves the sample throughput of single library construction, further reducing the cost of single molecule library construction sequencing. This method has the advantages of low template quality requirement, good specificity, good repeatability, simple library construction operation, large single detection throughput, short cycle, etc.

[0047] In some examples of the present application, the aforementioned nucleic acid sequencing method can also include at least one of the following additional technical features:

[0048] In some specific examples of the present application, the aforementioned nucleic acid sample to be tested is selected from DNA, for example, DNA extracted based on a human peripheral blood sample.

[0049] In some examples of the present application, in order to facilitate the differentiation of multiple samples, the 5' end of the aforementioned primer set is connected with an index sequence. The design of the index sequence is selected from a conventional design scheme in a laboratory, which will not be described here.

[0050] 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 a single molecule sequencing library construction process, which includes: performing end repair and A addition processing on the PCR amplification product; and performing purification processing and sequencing adapter ligation processing on the A addition processing product. It can be understood that the aforementioned end repair and A addition processing is performed in an end repair and A addition processing reaction system. This step is a conventional library construction step, and the specific operation scheme can be operated based on the instructions of a commercially available kit, which will not be described here.

[0051] The reaction procedure of the end repair and A-tailing includes 20℃, 8-15 min; 65℃, 8-15 min. In some examples of the present application, the reaction procedure of the end repair and A-tailing is selected from 20℃, 10 min; 65℃, 10 min.

[0052] The purification is achieved by using a purification magnetic bead.

[0053] The ligation is performed in the presence of a DNA ligase. In some examples of the present application, the DNA ligase is selected from T4 ligase.

[0054] The single molecule sequencing library construction does not require PCR amplification, reduces bias, supports long read sequencing, and can accurately detect various types of thalassemia mutations. The method has low sample size requirement, is suitable for the study of precious or limited samples, reduces errors in the processing process, and improves the quality and accuracy of sequencing data.

[0055] In some examples of the present application, further comprising quality control processing of the single molecule sequencing library to ensure the quality of the single molecule sequencing library, avoid unnecessary errors and data problems in the sequencing process, and improve the reliability and accuracy of the sequencing results.

[0056] In some examples of the present application, the thalassemia mutation includes an α-thalassemia mutation and / or a β-thalassemia mutation. The type of mutation includes at least one of a copy number variation (CNV), a single nucleotide polymorphism (SNP), and a structural variation (SV). The primer set of the present application can be used to detect deletions, mutations, and structural variations in α-thalassemia mutations; the primer set of the present application can be used to detect deletions and mutations in β-thalassemia.

[0057] In some examples of the present application, the aforementioned alpha-thalassemia mutation includes at least one of the following: -SEA, -a3.7 I, -a6.9, -AW, -a6.3, -a3.7 II, -a2.8, -FIL, -a5.9, -a3.7 III, -a2.7, -MED-II, -a27.6, -14.9, -9.7, -a21.9, -a4.2, -aMAL3.5, -JS, -27.2, -11.1, -THAI, -a2.4, -20.5, -a7.9, -a10.3, -a5.3, -a0.8, aaaaanti3.7, HKa a, aaaaanti3.7, aaaaanti4.2, anti-HKa a, and aaaaanti4.2. Among them, -SEA, -a3.7 I, -a6.9, -AW, -a6.3, -a3.7 II, -a2.8, -FIL, -a5.9, -a3.7 III, -a2.7, -MED-II, -a27.6, -14.9, -9.7, -a21.9, -a4.2, -aMAL3.5, -JS, -27.2, -11.1, -THAI, -a2.4, -20.5, -a7.9, -a10.3, -a5.3, and -a0.8 belong to the alpha-globin gene deletion type; aaaaanti3.7, HKa a, aaaaanti3.7, aaaaanti4.2, anti-HKa a, and aaaaanti4.2 belong to the alpha-globin gene structural variation type.

[0058] In some examples of the present application, the aforementioned beta-thalassemia mutation includes at least one of the following: Chinese Gγ (Aγδβ)0, Yunnanese, -β21.9, Taiwanese deletion, SEA-HPFH, Filipino del, Siriraj Gγ (Aγδβ)0, HPFH-6, 78.9kb Gγ (Aγδβ)0 del, 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. The aforementioned mutation type belongs to the beta-globin gene deletion type.

[0059] In some examples of the present application, the variant analysis is realized through the automatic bioinformatics analysis process matched with the sequencing platform (such as Huada Gene), and the variant analysis processes corresponding to different sequencing platforms are different. Those skilled in the art can understand that, based on the primer set of the present application for single molecule library construction and sequencing, combined with the conventional variant analysis process, the variant type of thalassemia can be obtained.

[0060] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0061] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0062] Fig. 1 is a schematic diagram of thalassemia variant type detection provided by the embodiments of the present application;

[0063] Fig. 2 is a schematic diagram of the design position of the a-globin gene primer provided by the embodiments of the present application;

[0064] Fig. 3 is a schematic diagram of the design position of the β-globin gene primer provided by the embodiments of the present application;

[0065] Fig. 4 is a schematic diagram of the electrophoresis results of Long PCR extension of different deletion samples provided by the embodiments of the present application;

[0066] Fig. 5 is a schematic diagram of the cyclone SEQ sequencing results of different deletion samples and mutation samples provided by the embodiments of the present application; wherein, A is aα / -α 3.7 sample result schematic diagram, B is Chinese (Aγδβ)0 / β N sample result schematic diagram, C is αα / ααα anti3.7 sample result schematic diagram, D is a schematic diagram of the point mutation Hb Constant Spring of the a-globin gene, E is a schematic diagram of the point mutation Codons 41 / 42 (-TTCT) beta0 of the β-globin gene, F is a schematic diagram of the trans-mutation sample of the point mutation Codon 43 (G > T) beta0 and the point mutation Codons 41 / 42 (-TTCT) beta0 of the β-globin gene, and G is a schematic diagram of the HKαα / αα sample. DETAILED DESCRIPTION

[0067] 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 by referring to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0068] In the present application, unless otherwise specified, the terms "first", "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, for example, two, three, etc., unless otherwise specifically limited.

[0069] In the present application, unless otherwise specified, the term "single molecule library preparation" refers to a technical method for constructing a sequencing library directly using a single DNA molecule without PCR amplification. It directly connects the original DNA molecule to the sequencing adapter without amplification step, thereby minimizing sequence bias and errors, preserving the true information of the genome, and being suitable for long read sequencing and accurate detection of genomic variations.

[0070] In the present application, 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 that have been connected to adapters so that they can be recognized and read during sequencing.

[0071] In the present application, unless otherwise specified, the sequencing platform is not particularly limited and can be any common third-generation sequencing platform. In the present application, the sequencing platform that can be used to perform the above-mentioned sequencing method can be selected from, but not limited to, the SMRT sequencing platform of PacBio, the Oxford Nanopore sequencing platform, and the Cyclone SEQ sequencing platform of Huada Gene.

[0072] The sequences involved in the present application are shown in Table 3.

[0073] Table 3

[0074] The scheme of the present application will be explained below in combination with examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be obtained by purchase.

[0075] Example 1:

[0076] This example aims to exemplarily verify the effectiveness and feasibility of the complex thalassemia variant detection method. Referring to FIG. 1, the specific steps are as follows:

[0077] 1) DNA extraction

[0078] Peripheral blood samples of thalassemia clinical testers were selected, and DNA was extracted by magnetic bead method. gDNA was stored at -20°C.

[0079] 2) Long PCR amplification system

[0080] The DNA of 48 clinical samples, ApexHF Hs DNA polymerase, ApexHF Hs CL buffer, and Primer Mix(index-primer) were taken out from -20°C, vortexed and mixed after melting, and the reaction system of individual PCR reaction wells was prepared according to Table 4(α-thalassemia deletion and mutation);

[0081] Table 4 Single PCR reaction system(tube 1) Note: The primer design position is shown in Figure 2, and the specific sequence is SEQ ID NO: 1-5.

[0082] The reaction system of individual PCR reaction wells was prepared according to Table 5(β-thalassemia deletion and mutation);

[0083] Table 5 Single PCR reaction system(tube 2) Note: The primer design position is shown in Figure 3, and the specific sequence is SEQ ID NO: 6-13.

[0084] The Long PCR reaction program was set according to Table 6, and the reaction was performed on a 96-well PCR instrument for about 5.5h.

[0085] Table 6 Long PCR reaction program

[0086] 3) PCR product pooling and purification

[0087] The PCR products of 48 tubes 1 were pooled into 1.5mL centrifuge tubes at a volume of 15μL, and a total of 720μL of PCR product mixture 1 was obtained; the PCR products of 48 tubes 2 were pooled into 1.5mL centrifuge tubes at a volume of 15μL, and a total of 720μL of PCR product mixture 2 was obtained;

[0088] 0.5 times the volume of purification magnetic beads were added to the PCR mixture(magnetic beads were pre-incubated at room temperature and vortexed and mixed uniformly), and incubated at room temperature for 10min to allow the DNA to bind to the magnetic beads;

[0089] Place 1.5 mL EP tubes on a magnetic stand, and after the solution is clear (about 5 min), carefully remove the supernatant;

[0090] Keep the 1.5 mL EP tube on the magnetic stand at all times, add 1000 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature for 30 sec, and carefully remove the supernatant; repeat the last rinse step once, for a total of two rinses;

[0091] Keep the 1.5 mL EP tube on the magnetic stand at all times, and dry the magnetic beads at room temperature for about 10 min with the lid open;

[0092] Remove the 1.5 mL EP from the magnetic stand, add 50 μL of nuclease-free water, mix well using a pipette, and let stand at room temperature for 2 min. After the solution is clear on the magnetic stand for 5 min, carefully pipette the supernatant into a new 1.5 mL EP tube;

[0093] Quantify the purified product DNA using Qubit 2.0 and dilute to 50 ng / μL for later use.

[0094] 2.4.4 Single molecule library construction

[0095] Take 500 ng of PCR tube 1 and PCR tube 2 purified products into a 0.2 mL PCR tube, add nuclease-free water to make the volume uniform to 44 μL, and mark it as tube A; similarly, take 500 ng of PCR tube 1 and PCR tube 2 purified products into a 0.2 mL PCR tube, and add nuclease-free water to make the volume uniform to 44 μL, and mark it as tube B;

[0096] According to the proportions in Table 7, prepare the required amount of end repair + "A" reaction solution in a centrifuge tube of the appropriate size. This step needs to be prepared on ice, and the prepared end repair + "A" reaction solution is vortexed for 3 times, 3 s each time, and the reaction solution is collected at the bottom of the tube by instantaneous centrifugation;

[0097] Table 7 End repair + "A" reaction system

[0098] Do not pipette 16 μL of prepared end repair + "A" reaction solution into tube A and tube B (0.2 mL PCR tube), and gently blow with a dilator several times until mixed, and collect the reaction solution at the bottom of the tube by instantaneous centrifugation;

[0099] Place tube A and tube B (0.2 mL PCR tube) in a PCR instrument, and perform the reaction according to the conditions in Table 8. After the reaction is completed, collect the reaction solution at the bottom of the tube by instantaneous centrifugation;

[0100] Table 8 Reaction program

[0101] Take out the purified magnetic beads and place them at room temperature. Shake well before use.

[0102] Take 60 μL of purified magnetic beads and add them to two new 1.5 mL EP tubes (label tube A and tube B). Gently pipette the entire sample from the end repair product tubes A and B (0.2 mL PCR tubes) into the two tubes containing the magnetic beads. Mix by flicking the tube wall with your hand.

[0103] Fix tube A and tube B (1.5 mL EP tubes) on the rotating mixer and rotate slowly. Incubate at room temperature for 10 min.

[0104] Take tube A and tube B (1.5 mL EP tubes) off the magnetic stand and place them on the magnetic stand after centrifugation. Let stand for 2-5 min until the liquid is clear. Carefully pipette the supernatant and discard it. Keep tube A and tube B (1.5 mL EP tubes) on the magnetic stand. Add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads and the tube wall. Carefully pipette the supernatant and discard it.

[0105] Repeat the previous step.

[0106] Take tube A and tube B (1.5 mL EP tubes) off the magnetic stand and centrifuge them immediately. After separation on the magnetic stand, use a small-volume pipette to pipette the remaining liquid from the bottom of the tube.

[0107] Keep tube A and tube B (1.5 mL EP tubes) on the magnetic stand. Open the centrifuge tube cap and dry at room temperature until the magnetic beads are dry and there is no reflection or cracking on the surface.

[0108] Take tube A and tube B (1.5 mL EP tubes) off the magnetic stand and add 62 μL of nuclease-free water to elute the DNA. Mix by flicking the tube wall with your hand. Centrifuge for 3 seconds to collect the liquid in the tube. Incubate at room temperature for 10 min.

[0109] Place tube A and tube B (1.5 mL EP tubes) on the magnetic stand after centrifugation. Let stand for 5 min until the liquid is clear. Transfer 60 μL of the supernatant to new tube A and tube B (1.5 mL EP tubes). The remaining sample can be used to determine the concentration. It is recommended to use the Qubit dsDNA HS Assay Kit to determine the concentration of the purified end repair + A product.

[0110] Set the constant temperature metal bath to 25°C and preheat it.

[0111] Take out the adapters and DNA ligase. Mix well by flicking the tube wall and centrifuge immediately. Place on ice for later use (Special design adapters, do not use vortex shaking and pipette blowing to mix; Ligation buffer (4X) is thick, please shake well before use).

[0112] Thaw out the ligation buffer (4X Ligation buffer) room temperature, shake well after mixing, and centrifuge immediately. Prepare the ligation reaction solution according to Table 9;

[0113] Table 9 Ligation reaction system

[0114] Add 2.5 μL of adaptor to B (1.5 mL EP tube) respectively, shake well after mixing, and centrifuge immediately;

[0115] Slowly add 37.5 μL of prepared ligation reaction solution to tube A and tube B (1.5 mL EP tube) respectively with a pipette, shake the tube wall until fully mixed, and centrifuge immediately to collect the reaction solution at the bottom of the tube (the ligation reaction solution is sticky, please slowly suck and discharge, and make sure it is fully mixed, otherwise the library construction efficiency will be reduced);

[0116] Place tube A and tube B (1.5 mL EP tube) in a preheated constant temperature metal bath (25°C) for ligation reaction, and the reaction time is 30 min. After the reaction is completed, centrifuge immediately to collect the reaction solution at the bottom of the tube;

[0117] Add 40 μL of purified magnetic beads to tube A and tube B (1.5 mL EP tube) respectively, and shake the tube wall with your hand (take out the purified magnetic beads 30 min in advance and place them at room temperature, and mix well before use);

[0118] Fix tube A and tube B (1.5 mL EP tube) on the rotary shaker, rotate slowly, and incubate at room temperature for 10 min;

[0119] Place tube A and tube B (1.5 mL EP tube) on the magnetic stand after centrifugation, and stand for 2-5 min until the liquid is clear, and carefully suck the supernatant with a pipette and discard it;

[0120] Keep tube A and tube B (1.5 mL EP tube) on the magnetic stand, add 150 μL of Wash Buffer 1, take tube A and tube B (1.5 mL EP tube) off the magnetic stand, shake the magnetic beads well after mixing, and centrifuge immediately, and place them back on the magnetic stand. Stand for 2-5 min until the liquid is clear, and carefully suck the supernatant and discard it;

[0121] Repeat the previous step;

[0122] After taking tube A and tube B (1.5 mL EP tube) off the magnetic stand, centrifuge immediately, and separate on the magnetic stand, and use a small volume pipette to suck the remaining liquid at the bottom of the tube;

[0123] Remove tube A and tube B (1.5 mL EP tube) from the magnetic stand, add 22 μL elution buffer (Elution Buffer), incubate at 37°C for 10-20 min. Centrifuge for 3 seconds, collect the liquid in the tube to the bottom of the tube;

[0124] Place tube A and tube B (1.5 mL EP tube) on the magnetic stand after centrifugation, stand for 2-5 min until the liquid is clear, transfer 20 μL supernatant in tube A and tube B (1.5 mL EP tube) to a new 1.5 mL EP tube. The remaining sample can be used to determine the concentration, and the purified ligation product is determined by Qubit dsDNA HS Assay Kit and used as a standard sequencing library;

[0125] Based on the Huada Cyclone SEQ single molecule sequencing platform, use Cyclone WT sequencing chip and WT sequencing kit for single molecule sequencing, one standard library is sequenced per chip and 10G data is output.

[0126] 5) Thalassemia single molecule information analysis

[0127] Use the thalassemia single molecule bioinformatics analysis platform (Huada Gene) to analyze the index split and primer split samples (reads aligned to thalassemia variant regions), and automatically output sample types (whether there are variants, including CNV, SNV and SV).

[0128] 6) Detection results

[0129] The electrophoresis results of Long PCR extension of different deletion samples are shown in Figure 4, and the cyclone SEQ sequencing results of different deletion samples and mutant samples are shown in Figures 5A-E. Among them, 48 thalassemia screening clinical samples were detected, and the positive sample sensitivity reached 100%, and the negative sample specificity was 100%. 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 / ααα anti 3.7 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 structure mutation was determined. The specific results are shown in Table 10.

[0130] Table 10 Detection results

[0131] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Also, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, the person skilled in the art can combine and combine the different embodiments or examples described in the present specification and the features of the different embodiments or examples, without contradiction.

[0132] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and the person skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A primer set, characterized by, Comprising: The primer is designed based on at least one of sequences 1)-13) and obtained: 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) chr11:5257290-5257314; 8) chr11:5249915-5249939; 9) chr11:5258978-5259000; 10) chr11:5221888-5221912; 11) chr11:5189975-5189996; 12) chr11:5152341-5152365; 13) chr11:5133905-5133925; 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 of claim 1 or 2 in the preparation of a thalassemia detection kit.

4. Use according to claim 3, characterized in that, The thalassemia includes: alpha-thalassemia and beta-thalassemia; The primer designed based on at least one of sequences 1)-5) is used for the detection of the alpha-thalassemia; The primer designed based on at least one of sequences 6)-13) is used for the detection of the beta-thalassemia. The primer designed based on sequences 1)-5) is shown as SEQ ID NO: 1-5; 5. Use according to claim 4, characterized in that, The primer designed based on sequences 6)-13) is shown as SEQ ID NO: 6-13. Comprising:

6. A method for detecting a thalassemia mutation, characterized by, The primer set of claim 1 or 2 is used for PCR amplification of the nucleic acid sample to be tested; The product of the PCR amplification is subjected to library construction and sequencing; The sequencing results are subjected to variation analysis. The thalassemia variation includes: alpha-thalassemia variation and / or beta-thalassemia variation; 7. The method of claim 6, wherein, Optionally, the type of variation includes at least one of copy number variation, single nucleotide polymorphism, and structural variation. ​ 8. The method of claim 7, wherein, The alpha-thalassemia mutation includes at least one of SEA, -a3.7 I, -a6.9, -AW, -a6.3, -a3.7 II, -a2.8, --FIL, -a5.9, -a3.7 III, -a2.7, --MED-II, -a27.6, --14.9, --9.7, -a21.9, -a4.2, -aMAL3.5, --JS, --27.2, --11.1, --THAI, -a2.4, --20.5, -a7.9, -a10.3, -a5.3, -a0.8, aaaa anti3.7, HK a a, aaaa anti3.7, aaaa anti4.2, anti-HK a a and aaaa anti4.

2.

9. The method of claim 7, wherein, The beta-thalassemia mutation includes at least one of Chinese G gamma (A gamma delta beta) 0, Yunnanese, -beta21.9, Taiwanese deletion, SEA-HPFH, Filipino del, Siriraj G gamma (A gamma delta beta) 0, HPFH-6, 78.9kb G gamma (A gamma delta beta) 0 del, Caucasian HPFH, HPFH-7, HPFH-5, Thai del, Turkish, Asian Indian (A gamma delta beta) 0, Hb Lepore, 3.5kb del, Croatian 1605bp del, British 1393bp del, 619bp del and 105bp del.

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

11. The method of claim 10, wherein, The reaction procedure of the PCR amplification processing is shown in Table 1: Table 1 12. The method of claim 11, wherein, The reaction procedure of the PCR amplification processing is shown in Table 2: Table 2 13. The method of claim 6, wherein, After the PCR amplification processing, before the library construction processing, further comprising: pooling processing; Optionally, after the pooling processing, further comprising: purification processing.

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

15. A kit comprising, Comprise: The primer set of claim 1 or 2. The primer set of claim 1 or 2.

16. The kit of claim 15, wherein The kit further comprises at least one of a DNA polymerase and a buffer, nuclease-free water, library building reagents, and sequencing reagents.

17. The kit of claim 16, wherein The library building reagents comprise at least one of a end repair enzyme, an end repair buffer, a poly A, a ligase, a ligation buffer, an elution buffer, and a purification magnetic bead.

18. A method of sequencing a nucleic acid, characterized by, The kit further comprises at least one of a DNA polymerase and a buffer, nuclease-free water, library building reagents, and sequencing reagents. The kit further comprises at least one of a DNA polymerase and a buffer, nuclease-free water, library building reagents, and sequencing reagents. The kit further comprises at least one of a DNA polymerase and a buffer, nuclease-free water, library building reagents, and sequencing reagents. The kit further comprises at least one of a DNA polymerase and a buffer, nuclease-free water, library building reagents, and sequencing reagents. The variant comprises a thalassemia variant, the thalassemia variant is selected from an alpha-thalassemia variant and / or a beta-thalassemia variant; 19. The method of claim 18, wherein, Optionally, the type of the variant comprises at least one of a copy number variant, a single nucleotide polymorphism, and a structural variant; Optionally, the alpha-thalassemia variant comprises at least one of --SEA, -α3.7 I, -α6.9, -AW, -α6.3, -α3.7 II, -α2.8, --FIL, -α5.9, -α3.7 III, -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, αααanti3.7, HKαα, ααααanti3.7, αααanti4.2, anti-HKαα, and ααααanti4.2; Optionally, the beta-thalassemia variant comprises at least one of Chinese Gγ(Aγδβ)0, Yunnanese, -β21.9, Taiwanese deletion, SEA-HPFH, Filipino del, Siriraj Gγ(Aγδβ)0, HPFH-6, 78.9kb Gγ(Aγδβ)0 del, 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. The variant analysis is achieved by an automated bioinformatics analysis pipeline of the sequencing platform.

20. The method of claim 18 or 19, wherein, The kit further comprises at least one of a DNA polymerase and a buffer, nuclease-free water, library building reagents, and sequencing reagents.

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