Primer group and kit for detecting 11 types of thalassemia copy number variation

Abstract

The invention provides a primer group. The primer group comprises 16 primers as shown in SEQ ID NO: 1-16; wherein the upstream primer is a nucleoxamic acid sequence as shown in SEQ ID NO: 1-9, and the downstream primer is a nucleoxamic acid sequence as shown in SEQ ID NO: 10-16. When the primer group is used for PCR (Polymerase Chain Reaction) detection, 11 thalassemia copy number variation types can be detected by a single reaction hole.

Description

Primer sets and kits for detecting copy number variations in 11 thalassemia genes Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a primer set and a kit for detecting copy number variations of 11 types of thalassemia. Background Art

[0002] Thalassemia (abbreviated as "thalassemia") is a common autosomal single-gene genetic disease in the world, and the clinical manifestations of different genotypes may be different. Mild thalassemia can be a mild anemia with microcytic hypochromia, while intermediate and severe thalassemia can cause moderate to severe hemolytic anemia, and may also be accompanied by jaundice, hepatosplenomegaly, developmental delay and other manifestations. Children with severe thalassemia may even die before or after birth. Currently, it can only be cured by hematopoietic stem cell transplantation or gene editing, but matching is difficult and expensive, and it cannot be widely used clinically.

[0003] α-globin gene deletion or mutation can lead to the absence or reduction of α-globin chain synthesis, an imbalance in the ratio of α and β globin chains, and then chronic hemolytic anemia, clinically known as α-thalassemia. α-thalassemia is mainly divided into α 0 -Thalassemia and α + There are two types of thalassemia. β-thalassemia is caused by reduced or absent β-globin chain synthesis, resulting in an imbalance in the ratio of α to β-globin chains. Excess α-globin chains are deposited on the red blood cell membrane, causing oxidative damage and hardening of the red blood cells, leading to hemolysis. Currently, 934 β-gene mutations have been identified worldwide, and as of 2017, over 60 β-gene mutations have been identified in my country.

[0004] Thalassemia is a hereditary blood disorder with a high incidence in the Mediterranean, Southeast Asia, and the Pacific region. According to reports, Thailand has a population of 66.2 million, of which 30.0-40.0% carry the thalassemia gene; approximately 3.0-10.0% of the population in Indonesia carry the β-thalassemia gene, and 2.6-11.0% of the population carry the α-thalassemia gene; and 4.5% to 5.0% of the population in Malaysia carry the thalassemia gene. In Southeast Asia, common deletional thalassemias include -α 3.7 、-α 4.2 , 3.5kb del, HPFH-6, Asian Ind-inv-delGγ(Aγδβ)0, siriraj Gγ(Aγδβ), --THAI, --FIL, Hb Leppre, --SEA.

[0005] Currently, domestic test kits can only detect three or four common α-thalassemia deletion types in China, but lack the thalassemia copy number variants that are prevalent in Southeast Asian countries. Therefore, there is an urgent need to develop primers or test kits that can detect the thalassemia copy number variants that are prevalent in Southeast Asian countries.

[0006] Summary of the Invention

[0007] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0008] Gap polymerase chain reaction (Gap-PCR) technology uses specific primers to amplify specific regions of gene rearrangement or deletion, accurately detecting copy number variants (CNVs) of target genes. Gap-PCR offers advantages such as high sensitivity, high specificity, ease of use, low cost, and easy interpretation of results. It is widely used for genetic testing of copy number variants in thalassemia (abbreviated as "thalassemia").

[0009] The inventors provide a primer set and kit for detecting thalassemia copy number variation based on multiple Gap-PCR technology, which can achieve αα / αααanti 3.7 、αα / αααanti 4.2 , Asian Ind-inv-delGγ(Aγδβ)0, 3.5kb del, Siriraj Gγ(Aγδβ), HPFH-6, Hb Lepore, Taiwanese type, 619bp del, Thai 12.5kb del, Filipino del, etc. Detection of 11 types of thalassemia copy number variations. The detection range is mainly for the thalassemia copy number variation types that are highly prevalent in Southeast Asian countries (such as Thailand, Indonesia, India, Malaysia, Singapore, etc.). The present invention can be used independently to detect 11 types of thalassemia copy number variations, or in combination with other thalassemia gene detection technologies (such as PCR-RDB, ARMS-PCR, MLPA, NGS, etc.) to achieve more comprehensive detection of thalassemia genes.

[0010] Therefore, in the first aspect of the present invention, the present invention proposes a primer set. According to an embodiment of the present invention, the primer set includes 16 primers shown in SEQ ID NOs: 1 to 16, wherein the upstream primers are the nucleotide sequences shown in SEQ ID NOs: 1 to 9, and the downstream primers are the nucleotide sequences shown in SEQ ID NOs: 10 to 16. After continuous experiments, the inventors found that when the primer set of the present invention is used for PCR detection, a single reaction well can be used to detect 11 types of thalassemia copy number variation, while having the advantages of low template quality requirements, good specificity, good repeatability, simple operation, low cost, and short cycle time.

[0011] It should be noted that the nucleotide sequences shown in SEQ ID NOs: 1 and 3 in the upstream primers and the nucleotide sequence shown in SEQ ID NO: 10 in the downstream primer are common primers.

[0012] In a second aspect of the present invention, the present invention provides use of the primer set described in the first aspect in preparing a kit for detecting typing of thalassemia copy number variation.

[0013] In a third aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit includes the primer set described in the first aspect.

[0014] According to an embodiment of the present invention, the kit further comprises: DNase / RNase-free water, DNA polymerase, cresol red and a buffer.

[0015] According to an embodiment of the present invention, the concentrations of the 16 primers represented by SEQ ID NOs: 1 to 16 in the primer set are 90 to 110 μM, respectively.

[0016] According to a specific embodiment of the present invention, the concentrations of the 16 primers represented by SEQ ID NOs: 1 to 16 in the primer set are 100 μM respectively.

[0017] According to an embodiment of the present invention, the usage ratio of the 16 primers shown in SEQ ID NOs: 1 to 16 in the primer set is 1:1:1:1:1:1:1:1:1:1:1:0.2:0.13:0.8:0.7:1:1.

[0018] According to an embodiment of the present invention, the concentration of the DNA polymerase is 0.5-1.5 U / μL.

[0019] According to a specific embodiment of the present invention, the concentration of the DNA polymerase is 1 U / μL.

[0020] According to an embodiment of the present invention, the concentration of cresol red is 0.5-1.5 mg / mL.

[0021] According to a specific embodiment of the present invention, the concentration of cresol red is 1 mg / mL.

[0022] According to an embodiment of the present invention, Mg in the buffer solution is 2+ The concentration of ions is 1.5 to 2.5 mM.

[0023] According to a specific embodiment of the present invention, Mg in the buffer solution is 2+ The concentration of the ion is 2 mM.

[0024] According to an embodiment of the present invention, the concentration of each dNTP in the buffer is 380-420 μM.

[0025] According to a specific embodiment of the present invention, the concentration of each dNTP in the buffer is 400 μM.

[0026] It should be noted that "dNTPs" as used herein refer to deoxyribonucleoside triphosphates. N refers to a nitrogenous base, and the representative variable refers to one of A, T, G, C, and U. When N refers to A, T, G, C, and U, respectively, their concentrations are 380 to 420 μM, specifically 400 μM.

[0027] According to an embodiment of the present invention, the usage ratio of the primer set, the DNase / RNase-free water, the DNA polymerase, the cresol red and the buffer is (1-2): (2-4): (0.1-0.3): (0.5-1.5): (7-8).

[0028] According to a specific embodiment of the present invention, the usage ratio of the primer set, the DNase / RNase-free water, the DNA polymerase, the cresol red and the buffer is 1.4:2.9:0.2:1:7.5.

[0029] In a fourth aspect of the present invention, the present invention provides use of the kit described in the third aspect for preparing a product for detecting typing of thalassemia copy number variation.

[0030] In the fifth aspect of the present invention, the present invention proposes a method for detecting the typing of thalassemia copy number variation. According to an embodiment of the present invention, the method comprises: using the primer set described in the first aspect or the kit described in the third aspect to detect the nucleic acid sample to be tested, and determining the typing of the thalassemia copy number variation based on the test results. The above method can realize the detection of 11 types of thalassemia copy number variation types in a single reaction well, and has the advantages of low template quality requirement, good specificity, good repeatability, simple operation, low cost, and short cycle.

[0031] According to an embodiment of the present invention, the nucleic acid sample to be tested is derived from blood.

[0032] It should be noted that the "method for detecting typing of thalassemia copy number variation" described in the present invention may be a method for non-disease diagnosis purposes, for example, using the method of the present application to detect blood in vitro to determine the specific type of gene copy number variation in the blood, so as to screen out blood with the same gene copy number variation type.

[0033] According to an embodiment of the present invention, the detection includes performing PCR amplification on the nucleic acid sample to be tested to obtain a PCR product; and performing electrophoresis and imaging on the PCR product to obtain an electrophoresis pattern.

[0034] According to an embodiment of the present invention, the usage ratio of the 16 primers shown in SEQ ID NOs: 1 to 16 in the primer set is 1:1:1:1:1:1:1:1:1:1:1:0.2:0.13:0.8:0.7:1:1.

[0035] According to an embodiment of the present invention, the PCR amplification reaction system is:

[0036] According to an embodiment of the present invention, the concentrations of the 16 primers represented by SEQ ID NOs: 1 to 16 in the primer set are 90 to 110 μM, respectively.

[0037] According to a specific embodiment of the present invention, the concentrations of the 16 primers represented by SEQ ID NOs: 1 to 16 in the primer set are 100 μM respectively.

[0038] According to an embodiment of the present invention, the concentration of the DNA polymerase is 0.5-1.5 U / μL.

[0039] According to a specific embodiment of the present invention, the concentration of the DNA polymerase is 1 U / μL.

[0040] According to an embodiment of the present invention, the concentration of cresol red is 0.5-1.5 mg / mL.

[0041] According to a specific embodiment of the present invention, the concentration of cresol red is 1 mg / mL.

[0042] According to an embodiment of the present invention, Mg in the buffer solution is 2+ The concentration of ions is 1.5 to 2.5 mM.

[0043] According to a specific embodiment of the present invention, Mg in the buffer solution is 2+ The concentration of the ion is 2 mM.

[0044] According to an embodiment of the present invention, the concentration of each dNTP in the buffer is 380-420 μM.

[0045] According to a specific embodiment of the present invention, the concentration of each dNTP in the buffer is 400 μM.

[0046] According to an embodiment of the present invention, the concentration of the nucleic acid sample to be tested is 10-100 ng / μL.

[0047] According to an embodiment of the present invention, the PCR amplification detection conditions are:

[0048] According to an embodiment of the present invention, the typing of the thalassemia copy number variation is determined based on the bands appearing in the electrophoresis diagram.

[0049] According to an embodiment of the present invention, when bands appear in the electrophoresis graph within the range of 1800-2000 bp, 3500-3900 bp and 5000-5500 bp, it is αα / αααanti 3.7 Instructions for classification.

[0050] According to an embodiment of the present invention, when bands appear in the electrophoresis graph within the range of 1500-2000 bp, 3500-3900 bp and 5000-5500 bp, it is αα / αααanti 4.2 Instructions for classification.

[0051] According to an embodiment of the present invention, the presence of bands in the ranges of 2500-3000 bp, 3500-3900 bp, and 5000-5500 bp in the electrophoresis graph is an indication of Asian-Indian typing.

[0052] According to an embodiment of the present invention, the presence of bands in the ranges of 1400-1700 bp, 3500-3900 bp, and 5000-5500 bp in the electrophoresis graph is an indication of Filipino delta typing.

[0053] According to an embodiment of the present invention, when bands appear in the ranges of 1000-1500 bp, 3500-3900 bp, and 5000-5500 bp on the electrophoresis graph, it indicates Thai 12.5 kb del typing.

[0054] According to an embodiment of the present invention, when bands appear in the electrophoresis graph within the ranges of 700-1000 bp, 1700-2100 bp, 3500-3900 bp, and 5000-5500 bp, it is an indication of Siriraj typing.

[0055] According to an embodiment of the present invention, the presence of bands in the ranges of 200-500 bp, 3500-3900 bp, and 5000-5500 bp in the electrophoresis graph is an indication of HPFH-6 typing.

[0056] According to an embodiment of the present invention, the presence of bands in the ranges of 4000-4300 bp, 3500-3900 bp, and 5000-5500 bp in the electrophoresis graph is an indication of Hb Lepore typing.

[0057] According to an embodiment of the present invention, when bands appear in the ranges of 100-300 bp, 3500-3900 bp and 5000-5500 bp on the electrophoresis graph, it is an indication of 3.5 kb del typing.

[0058] According to an embodiment of the present invention, when bands appear in the electrophoresis graph within the ranges of 2200-2500 bp, 2800-3200, 3500-3900 bp, and 5000-5500 bp, it is an indication of Taiwan type typing.

[0059] According to an embodiment of the present invention, when bands appear in the ranges of 2800-3200 bp, 3500-3900 bp, and 5000-5500 bp on the electrophoretogram, it is an indication of 619 bp del typing. Beneficial effects

[0060] This invention, based on multiplex Gap-PCR detection technology, achieves the goal of detecting 11 thalassemia copy number variant types in a single reaction well. Through testing, screening, and optimization, the invention completes the design and optimization of primer sequences, tests and optimizes the reaction procedures and reaction systems, determines the optimal reaction system and reaction procedures, and establishes standardized and executable result interpretation criteria. This technical solution has the advantages of low template quality requirements, high specificity, good reproducibility, simple operation, low cost, and short cycle time.

[0061] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0063] FIG1 is an electrophoresis diagram of amplification results of 8 whole blood samples according to an embodiment of the present invention;

[0064] FIG2 is an electrophoresis diagram of the verification amplification results of known thalassemia copy number variation types according to Comparative Example 1 of the present invention;

[0065] FIG3 is an electrophoresis diagram of the verification amplification results of known thalassemia copy number variation types according to Comparative Example 2 of the present invention;

[0066] FIG4 is an electrophoresis diagram of the verification amplification results of known thalassemia copy number variation types according to Comparative Example 3 of the present invention. DETAILED DESCRIPTION

[0067] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0068] Definition and Description

[0069] Unless otherwise specified or clearly contradicted by context, the articles "a," "an," and "the" as used herein are intended to include "at least one" or "one or more." Thus, as used herein, these articles refer to one or more than one (i.e., at least one) of the objects. For example, "a component" refers to one or more components, i.e., more than one component may be contemplated for use or use in implementing the described embodiments.

[0070] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0071] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0072] The present invention provides a primer set and its use, a kit and its use, and a method for detecting thalassemia copy number variation typing, which will be described in detail below.

[0073] Primer set

[0074] The present invention designs detection primers for the copy number variation regions of 11 thalassemia genes, a total of 16 detection primers are designed, multiplex Gap-PCR amplification is performed in a single well, the amplified products are detected by agarose gel electrophoresis, and the copy number variation type is interpreted by the electrophoresis bands.

[0075] In one aspect of the present invention, a primer set is provided, comprising 16 primers shown in SEQ ID NOs: 1 to 16, as specifically shown in Table 1. According to an embodiment of the present invention, the primer set comprises an upstream primer and a downstream primer: the upstream primer has a nucleotide sequence shown in SEQ ID NOs: 1 to 9, and the downstream primer has a nucleotide sequence shown in SEQ ID NOs: 10 to 16.

[0076] Reagent test kit

[0077] In another aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit contains the primer set described above, as well as DNase / RNase-free water, DNA polymerase, cresol red and a buffer. According to an embodiment of the present invention, the concentrations of the 16 primers are respectively 100 μM, the usage ratios of the 16 primers in the primer set are 1:1:1:1:1:1:1:1:1:1:0.2:0.13:0.8:0.7:1:1, the concentration of the DNA polymerase is 1 U / μL, the concentration of the cresol red is 1 mg / mL, and the Mg content in the buffer is 0.1%. 2+ The concentration of ions was 2 mM, and the concentration of each dNTP in the buffer was 400 μM.

[0078] According to an embodiment of the present invention, the usage ratio of the components in the kit, that is, the usage ratio of the primer set, the DNase / RNase-free water, the DNA polymerase, the cresol red and the buffer is (1-2): (2-4): (0.1-0.3): (0.5-1.5): (7-8).

[0079] Therefore, by using the kit of the present invention, the specific typing of thalassemia copy number variation can be accurately and effectively detected.

[0080] use

[0081] In another aspect of the present invention, the present invention provides use of the aforementioned primer set in preparing a kit for detecting typing of thalassemia copy number variation.

[0082] In another aspect of the present invention, the present invention provides use of the aforementioned kit for preparing a product for detecting typing of thalassemia copy number variation.

[0083] Methods for detecting copy number variations in thalassemia

[0084] In another aspect, the present invention provides a method for detecting the typing of thalassemia copy number variation. According to an embodiment of the present invention, the method comprises: testing the nucleic acid sample to be tested using the primer set or kit described above, and determining the typing of the thalassemia copy number variation based on the test results; wherein the nucleic acid sample to be tested is derived from blood, and the testing comprises performing PCR amplification on the nucleic acid sample to obtain a PCR product, and performing electrophoresis and imaging of the PCR product to obtain an electropherogram.

[0085] According to an embodiment of the present invention, the PCR amplification reaction system is:

[0086] The usage ratio of the 16 primers shown in SEQ ID NO: 1 to SEQ ID NO: 16 in the primer set is 1: 1: 1: 1: 1: 1: 1: 1: 0.2: 0.13: 0.8: 0.7: 1: 1; the concentrations of the 16 primers shown in SEQ ID NO: 1 to 16 in the primer set are 100 μM respectively; the concentration of the DNA polymerase is 1 U / μL; the concentration of the cresol red is 1 mg / mL; the Mg content in the buffer is 100 μM. 2+ The concentration of the ion is 2 mM.; the concentration of each dNTP in the buffer is 400 μM; and the concentration of the nucleic acid sample to be tested is 10-100 ng / μL.

[0087] According to an embodiment of the present invention, the PCR amplification detection conditions are:

[0088] According to an embodiment of the present invention, the typing of the thalassemia copy number variation is determined based on the bands appearing in the electrophoresis diagram.

[0089] When bands appear in the electrophoresis graph within the ranges of 1800-2000 bp, 3500-3900 bp, and 5000-5500 bp, it is αα / αααanti 3.7 Instructions for classification.

[0090] When the electrophoresis graph shows bands in the range of 1500-2000 bp, 3500-3900 bp and 5000-5500 bp, it is αα / αααanti 4.2 Instructions for classification.

[0091] When bands appear in the ranges of 2500-3000 bp, 3500-3900 bp, and 5000-5500 bp in the electrophoretogram, it is an indication of Asian-Indian typing.

[0092] When bands appear in the ranges of 1400-1700 bp, 3500-3900 bp, and 5000-5500 bp in the electrophoretogram, it is an indication of Filipino delta typing.

[0093] When bands appear in the ranges of 1000-1500 bp, 3500-3900 bp, and 5000-5500 bp on the electrophoretogram, it indicates Thai 12.5 kb del typing.

[0094] When bands appear in the ranges of 700-1000 bp, 1700-2100 bp, 3500-3900 bp and 5000-5500 bp in the electrophoresis graph, it is an indication of Siriraj typing.

[0095] When bands appear in the ranges of 200-500 bp, 3500-3900 bp, and 5000-5500 bp on the electrophoresis graph, it is an indication of HPFH-6 typing.

[0096] When bands appear in the ranges of 4000-4300 bp, 3500-3900 bp and 5000-5500 bp in the electrophoretogram, it is an indication of Hb Lepore typing.

[0097] When bands appear in the ranges of 100-300 bp, 3500-3900 bp, and 5000-5500 bp on the electrophoretogram, it is an indication of 3.5 kb del typing.

[0098] When bands appear in the ranges of 2200-2500 bp, 2800-3200, 3500-3900 bp and 5000-5500 bp in the electrophoretogram, it is an indication of Taiwan type typing.

[0099] When bands appear in the ranges of 2800-3200, 3500-3900 bp, and 5000-5500 bp on the electrophoretogram, it is an indication of 619 bp del typing.

[0100] The following embodiments of the present invention are described in detail. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this field or the product specifications are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be obtained commercially.

[0101] Example 1

[0102] Based on the thalassemia gene copy number variation type, the inventors determined the sites at both ends of the variation interval, used SnapGene software to design primers outside the variation region for amplifying the variation interval gene, and designed detection primers for 11 thalassemia gene copy number variation regions, respectively. A total of 16 detection primers were designed, as shown in Table 1.

[0103] Table 1

[0104] Example 2

[0105] 1) DNA extraction: DNA was extracted from human peripheral blood samples using a magnetic bead extraction kit (brand: MAGEN, product number: MD5111-02).

[0106] 2) Prepare the Gap-PCR primer mix according to Table 2.

[0107] Table 2

[0108] 3) Configure a single reaction well for Gap-PCR according to Table 3.

[0109] Table 3

[0110] DNase / RNase-free water (Brand: BGI, Catalog No.: FQD-48A), Cresol Red (Brand: Merck, Catalog No.: 114472-25G), Primers (Brand: Sangon), DNA polymerase and buffer (Brand: Acryl, Catalog No.: AG12204).

[0111] 4) The Gap-PCR reaction program was set according to Table 4. The present invention is applicable to a common domestic 96-well PCR instrument: ETC821M PCR instrument.

[0112] Table 4

[0113] 5) Electrophoresis of PCR Products: Weigh a certain amount of agarose and add the corresponding 1× TAE buffer (Brand: Sangon, Catalog No.: B540023-0001) to prepare a 0.8% agarose gel. Mix the agarose powder and 1× TAE in a microwaveable Erlenmeyer flask. Heat in a microwave until the agarose is completely dissolved (but do not overcook the solution, as the buffer will evaporate, changing the final ratio of agarose in the gel). Then remove the flask, shake well, and cool to 50-60°C. Add 0.005% nucleic acid dye (Brand: Sangon, Catalog No.: A616697-0500) to the agarose gel and stir well to prepare a 0.8% agarose gel. Pour the agarose gel solution into the gel tray promptly using a suitable comb. Allow the agarose solution to cool until the agarose gel is completely solidified. Add 5 μL of DNA Marker (Brand: Takara, Catalog No.: 3423A) and the PCR product to the wells of the agarose gel. Electrophoresis conditions: voltage 135 V, electrophoresis time 1 hour (speeds vary slightly between different electrophoresis instruments). Stop electrophoresis when the indicator band is approximately 1.5 cm from the bottom of the gel.

[0114] 6) Result interpretation: After the reaction is completed, the PCR products are subjected to electrophoresis, gel imaging, and analysis.

[0115] There is no 3712bp band and no copy number variation band within the detection range. The experiment is considered a failure and the DNA needs to be re-extracted and PCR detection is required;

[0116] There is a 3712 bp band and a copy number variation band within the detection range. According to the correspondence between the electrophoresis band and the genotype (see Table 5), the corresponding thalassemia copy number variation type (homozygous deletion type) is determined.

[0117] There is no 3712bp band, but there is a copy number variation band within the detection range. According to the correspondence between the electrophoresis band and the genotype (see Table 5), the corresponding thalassemia copy number variation type (heterozygous deletion type) is determined to be detected.

[0118] (Pure heterozygosity is used to determine the thalassemia deletion type. For the alleles of the homologous chromosomes, if one allele is missing, it is a heterozygous deletion, and if both alleles are missing, it is a pure and homozygous deletion)

[0119] Table 5

[0120] Example 3

[0121] Eight whole blood samples of known types were used for testing. The known type information of the samples is shown in Table 6:

[0122] Table 6

[0123] Eight whole blood samples were tested according to the method described in Example 2. After the reaction, the PCR products were subjected to electrophoresis, gel imaging, and analysis. The electrophoresis band sizes for the eight samples are shown in Table 7, and the electrophoresis results are shown in Figure 1. The experimental results indicate that the types detected in all eight samples were consistent with the known types.

[0124] Table 7

[0125] Comparative Example 1

[0126] 1) DNA extraction: DNA was extracted from human peripheral blood samples using a magnetic bead extraction kit (brand: MAGEN, product number: MD5111-02).

[0127] 2) Prepare the Gap-PCR primer mix according to Table 2 in Example 2.

[0128] 3) Configure a single reaction well for Gap-PCR according to Table 8.

[0129] Table 8

[0130] 4) The Gap-PCR reaction program was set according to Table 9. The present invention is applicable to a common domestic 96-well PCR instrument: ETC821M PCR instrument.

[0131] Table 9

[0132] The remaining steps were the same as those in Example 2. The Gap-PCR test results are shown in FIG2 . When the amount of ApexHF Hs DNA polymerase used in the PCR system was 0.5 μL and the amount of primers used was 2 μL, multiple nonspecific bands appeared in the electrophoresis graph of the PCR product.

[0133] Comparative Example 2

[0134] 1) DNA extraction: DNA was extracted from human peripheral blood samples using a magnetic bead extraction kit (brand: MAGEN, product number: MD5111-02).

[0135] 2) Prepare the Gap-PCR primer mix according to Table 2 in Example 2.

[0136] 3) Configure a single reaction well for Gap-PCR according to Table 10.

[0137] Table 10

[0138] 4) The Gap-PCR reaction program was set according to Table 11. The present invention is applicable to a common domestic 96-well PCR instrument: ETC821M PCR instrument.

[0139] Table 11

[0140] The remaining steps were the same as those in Example 2. The Gap-PCR test results are shown in FIG3 . When the reaction buffer and the reaction enzyme in the PCR system were replaced, the PCR reaction could not effectively amplify the specific band.

[0141] Comparative Example 3

[0142] 1) DNA extraction: DNA was extracted from human peripheral blood samples using a magnetic bead extraction kit (brand: MAGEN, product number: MD5111-02).

[0143] 2) Prepare the Gap-PCR primer mix according to Table 2 in Example 2.

[0144] 3) Configure a single reaction well for Gap-PCR according to Table 12.

[0145] Table 12

[0146] 4) Set the Gap-PCR reaction program according to Table 13. The present invention is applicable to a common domestic 96-well PCR instrument: ETC821M PCR instrument.

[0147] Table 13

[0148] The remaining steps were the same as those in Example 2. The Gap-PCR detection results are shown in FIG4 . When the extension time of the reaction program was 5 min, it can be seen from the results that non-specific bands appeared in the reaction.

[0149] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean 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 can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0150] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A primer set, characterized in that: comprising 16 primers shown in SEQ ID NOs: 1 to 16; The upstream primers are nucleotide sequences shown in SEQ ID NOs: 1 to 9, and the downstream primers are nucleotide sequences shown in SEQ ID NOs: 10 to 16.

2. Use of the primer set according to claim 1 in preparing a kit for detecting typing of copy number variation in thalassemia.

3. A kit, characterized in that: Comprising the primer set of claim 1.

4. The kit according to claim 3, characterized in that Further including: DNase / RNase-free water, DNA polymerase, cresol red, and buffer; Optionally, the concentrations of the 16 primers shown in SEQ ID NOs: 1 to 16 in the primer set are 90 to 110 μM, respectively; Optionally, the usage ratio of the 16 primers shown in SEQ ID NOs: 1 to 16 in the primer set is 1:1:1:1:1:1:1:1:1:1:0.2:0.13:0.8:0.7:1:1; Optionally, the concentration of the DNA polymerase is 0.5 to 1.5 U / μL; Optionally, the concentration of cresol red is 0.5 to 1.5 mg / mL; Optionally, the buffer contains Mg 2+ The concentration of ions is 1.5 to 2.5 mM; Optionally, the concentration of each dNTP in the buffer is 380-420 μM; Optionally, the usage ratio of the primer set, the DNase / RNase-free water, the DNA polymerase, the cresol red and the buffer is (1-2): (2-4): (0.1-0.3): (0.5-1.5): (7-8).

5. Use of the kit according to claim 3 or 4 in preparing a product for detecting typing of thalassemia copy number variation.

6. A method for detecting thalassemia copy number variation typing, characterized in that, include: Using the primer set of claim 1 or the kit of claim 3 or 4 to detect the nucleic acid sample to be tested, and determining the typing of the thalassemia copy number variation based on the test result; Optionally, the nucleic acid sample to be tested is derived from blood.

7. The method according to claim 6, characterized in that The detection includes performing PCR amplification on the nucleic acid sample to be tested to obtain a PCR product; The PCR product is subjected to electrophoresis and imaging to obtain an electrophoresis diagram.

8. The method according to claim 6, characterized in that The usage ratio of the 16 primers shown in SEQ ID NOs: 1 to 16 in the primer set is 1:1:1:1:1:1:1:1:1:1:0.2:0.13:0.8:0.7:1:

1.

9. The method according to claim 7, characterized in that: The PCR amplification reaction system is: Optionally, the concentrations of the 16 primers shown in SEQ ID NOs: 1 to 16 in the primer set are 90 to 110 μM, respectively; Optionally, the concentration of the DNA polymerase is 0.5 to 1.5 U / μL; Optionally, the concentration of cresol red is 0.5 to 1.5 mg / mL; Optionally, the buffer contains Mg 2+ The concentration of ions is 1.5 to 2.5 mM; Optionally, the concentration of dNTPs in the buffer is 380-420 μM each; Optionally, the concentration of the nucleic acid sample to be tested is 10-100 ng / μL; Optionally, the PCR amplification detection conditions are:

10. The method according to claim 7, characterized in that The typing of the thalassemia copy number variation is determined based on the bands appearing in the electrophoresis diagram; Optionally, when the electrophoresis graph shows bands in the range of 1800-2000 bp, 3500-3900 bp and 5000-5500 bp, it is αα / αααanti 3.7 Instructions for typing; Optionally, when the electrophoresis graph shows bands in the range of 1500-2000 bp, 3500-3900 bp and 5000-5500 bp, it is αα / αααanti 4.2 Instructions for typing; Optionally, the presence of bands in the electrophoresis graph within the range of 2500-3000 bp, 3500-3900 bp, and 5000-5500 bp is indicative of Asian-Indian typing; Optionally, the electrophoretogram has stripes in the range of 1400-1700 bp, 3500-3900 bp and 5000-5500 bp. When it is with, it is an indication of Filipino del classification; Optionally, when bands appear in the electrophoresis graph within the range of 1000-1500 bp, 3500-3900 bp, and 5000-5500 bp, it is an indication of Thai 12.5 kb del typing; Optionally, when bands appear in the electrophoresis graph within the range of 700-1000 bp, 1700-2100 bp, 3500-3900 bp, and 5000-5500 bp, it is an indication of Siriraj typing; Optionally, the presence of bands in the electrophoresis graph within the range of 200 to 500 bp, 3500 to 3900 bp, and 5000 to 5500 bp is indicative of HPFH-6 typing; Optionally, when bands appear in the electrophoresis graph within the range of 4000-4300 bp, 3500-3900 bp, and 5000-5500 bp, it is an indication of Hb Lepore typing; Optionally, when bands appear in the electrophoresis graph within the range of 100-300 bp, 3500-3900 bp, and 5000-5500 bp, it is an indication of 3.5 kb del typing; Optionally, when the electrophoresis graph shows bands in the range of 2200-2500 bp, 2800-3200, 3500-3900 bp, and 5000-5500 bp, it is an indication of Taiwan type typing; Optionally, when bands appear in the electrophoresis graph within the ranges of 2800-3200, 3500-3900 bp and 5000-5500 bp, it is an indication of 619 bp del typing.