Primer set and kit for detecting 11 thalassemia copy number variations

Abstract

The application provides a primer set, which comprises 16 primers shown in SEQ ID NO: 1-16; wherein the upstream primer is a nucleic acid sequence shown in SEQ ID NO: 1-9, and the downstream primer is a nucleic acid sequence shown in SEQ ID NO: 10-16. When the primer set is used for PCR detection, 11 thalassemia copy number variation types can be detected in a single reaction well.

Description

Technical Field

[0001] This invention relates to the field of biological detection, specifically to a primer set and kit for detecting 11 copy number variations in thalassemia. Background Technology

[0002] Thalassemia is a common autosomal single-gene inherited disease worldwide, with different genotypes presenting with varying clinical manifestations. Mild thalassemia presents as microcytic hypochromic anemia, while intermediate and severe thalassemia can lead to moderate to severe hemolytic anemia, and may also be accompanied by jaundice, hepatosplenomegaly, and developmental delays. Children with severe thalassemia may even die before or after birth. Currently, it can only be cured through hematopoietic stem cell transplantation or gene editing, but matching is difficult and expensive, and these methods are not yet widely used in clinical practice.

[0003] Deletion or mutation of the α-globin gene can lead to a lack or reduction in the synthesis of α-globin chains, resulting in an imbalance in the ratio of α to β-globin chains, and consequently, chronic hemolytic anemia, clinically known as α-thalassemia. α-thalassemia is mainly divided into α... 0 -Thalassemia and alpha + There are two types of thalassemia. β-thalassemia is caused by a reduction or absence of β-globin chain synthesis, resulting in an imbalance in the ratio of α-globin chains to β-globin chains. Excess α-globin chains deposit on the erythrocyte membrane, leading to oxidative damage and hardening of erythrocytes, and ultimately causing hemolysis. Currently, 934 β-gene mutations have been discovered worldwide, and as of 2017, more than 60 β-gene mutations have been discovered in my country.

[0004] Thalassemia is a hereditary blood disorder with a high prevalence in the Mediterranean, Southeast Asia, and the Pacific region. In Southeast Asia, common deletion-type thalassemia includes -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, domestically produced kits can only detect 3-4 common α-thalassemia deletion types in China, but lack copy number variants of thalassemia that are prevalent in Southeast Asian countries. Therefore, there is an urgent need to develop primers or kits for detecting copy number variants of thalassemia that are prevalent in Southeast Asian countries. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0007] Gap polymerase chain reaction (Gap-PCR) is a technique that uses specially designed primers to amplify specific gene rearrangement or deletion regions, enabling accurate detection of copy number variants (CNVs) of target genes. Gap-PCR offers advantages such as high sensitivity, high specificity, ease of operation, low cost, and easy result interpretation, and is widely used for detecting copy number variant genes in thalassemia.

[0008] Based on multiplex gap-PCR technology, the inventors have provided a primer set and kit for detecting copy number variations in thalassemia, which can achieve αα / αααanti- PCR in a single tube. 3.7 ,αα / αααanti 4.2 This invention detects 11 copy number variants of thalassemia, including Asian Ind-inv-delGγ(Aγδβ)0, 3.5 kb del, Siriraj Gγ(Aγδβ), HPFH-6, Hb Lepore, Taiwan type, 619 bp del, Thai 12.5 kb del, and Filipino del. The detection scope primarily covers thalassemia copy number variants prevalent in Southeast Asian countries (such as Thailand, Indonesia, Malaysia, and Singapore). This invention can be used independently to detect these 11 thalassemia copy number variants, or in combination with other thalassemia gene detection technologies (such as PCR-RDB, ARMS-PCR, MLPA, and NGS) to achieve more comprehensive thalassemia gene detection.

[0009] Therefore, in a first aspect, the present invention provides a primer set. According to embodiments of the present invention, the primer set comprises 16 primers as shown in SEQ ID NO:1-16, wherein the upstream primer is a nucleotide sequence shown in SEQ ID NO:1-9, and the downstream primer is a nucleotide sequence shown in SEQ ID NO:10-16. Through continuous experimentation, the inventors have discovered that using the primer set of the present invention for PCR detection can achieve the detection of 11 copy number variants of thalassemia in a single reaction well, while also possessing advantages such as low template quality requirements, high specificity, good reproducibility, simple operation, low cost, and short cycle time.

[0010] It should be noted that the nucleotide sequences shown in SEQ ID NO:1 and 3 of the upstream primer and the nucleotide sequence shown in SEQ ID NO:10 of the downstream primer are common primers.

[0011] In a second aspect of the invention, the invention proposes the use of the primer set described in the first aspect in the preparation of a kit for detecting thalassemia copy number variants.

[0012] In a third aspect, the present invention provides a kit. According to an embodiment of the invention, the kit comprises the primer set described in the first aspect.

[0013] According to an embodiment of the present invention, the kit further comprises: DNase / RNase-free water, DNA polymerase, cresol red, and buffer solution.

[0014] According to an embodiment of the present invention, the concentrations of the 16 primers shown in SEQ ID NO:1~16 in the primer set are 90~110 μM, respectively.

[0015] According to a specific embodiment of the present invention, the concentrations of the 16 primers shown in SEQ ID NO:1~16 in the primer set are all 100 μM.

[0016] According to an embodiment of the present invention, the ratio of the 16 primers shown in SEQ ID NO:1~16 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.

[0017] According to an embodiment of the present invention, the concentration of the DNA polymerase is 0.5~1.5 U / μL.

[0018] According to a specific embodiment of the present invention, the concentration of the DNA polymerase is 1 U / μL.

[0019] According to an embodiment of the present invention, the concentration of cresol red is 0.5~1.5 mg / mL.

[0020] According to a specific embodiment of the present invention, the concentration of cresol red is 1 mg / mL.

[0021] According to an embodiment of the present invention, Mg in the buffer solution 2＋ The concentration of ions is 1.5~2.5mM.

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

[0023] According to an embodiment of the present invention, the concentration of dNTPs in the buffer solution is 380~420 μM.

[0024] According to a specific embodiment of the present invention, the concentration of each dNTP in the buffer solution is 400 μM.

[0025] It should be noted that the "dNTP" mentioned in this application refers to deoxyribonucleoside triphosphate. Here, N refers to a nitrogenous base, and the representative variable refers to one of A, T, G, C, U, etc. When N represents A, T, G, C, and U respectively, its concentration is 380~420μM, specifically 400μM.

[0026] According to an embodiment of the present invention, the ratio of the primer set, the DNase / RNase-free water, the DNA polymerase, the cresol red, and the buffer solution is (1~2):(2~4):(0.1~0.3):(0.5~1.5):(7~8).

[0027] According to a specific embodiment of the present invention, the ratio of the primer set, the DNase / RNase-free water, the DNA polymerase, the cresol red, and the buffer solution is 1.4:2.9:0.2:1:7.5.

[0028] In a fourth aspect of the invention, the present invention provides for the use of the kit described in the third aspect in the preparation of a product for detecting thalassemia copy number variants.

[0029] In a fifth aspect, the present invention provides a method for detecting thalassemia copy number variants. According to an embodiment of the invention, the method includes: detecting the nucleic acid sample to be tested using the primer set described in the first aspect or the kit described in the third aspect; and determining the thalassemia copy number variant type based on the detection results. Using the above method, it is possible to detect 11 thalassemia copy number variant types in a single reaction well, and it has advantages such as low template quality requirements, high specificity, good reproducibility, simple operation, low cost, and short cycle time.

[0030] According to an embodiment of the present invention, the nucleic acid sample to be tested is derived from blood.

[0031] It should be noted that the "method for detecting copy number variations in thalassemia" described in this invention can be a method for non-disease diagnosis purposes. For example, the method of this application can be used to detect in vitro blood to determine the specific type of gene copy number variation in the blood, so as to screen out blood with the same type of gene copy number variation.

[0032] According to an embodiment of the present invention, the detection includes performing PCR amplification on the nucleic acid sample to be tested to obtain PCR products; and performing electrophoresis and imaging on the PCR products to obtain an electrophoresis image.

[0033] According to an embodiment of the present invention, the ratio of the 16 primers shown in SEQ ID NO:1~16 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.

[0034] According to an embodiment of the present invention, the PCR amplification reaction system is as follows:

[0035]

[0036] According to an embodiment of the present invention, the concentrations of the 16 primers shown in SEQ ID NO:1~16 in the primer set are 90~110 μM, respectively.

[0037] According to a specific embodiment of the present invention, the concentrations of the 16 primers shown in SEQ ID NO:1~16 in the primer set are all 100 μM.

[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 2＋ The concentration of ions is 1.5~2.5 mM.

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

[0044] According to an embodiment of the present invention, the concentration of dNTPs in the buffer solution is 380~420 μM.

[0045] According to a specific embodiment of the present invention, the concentration of each dNTP in the buffer solution 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 as follows:

[0048]

[0049] 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 image.

[0050] According to an embodiment of the present invention, when bands appear in the electrophoresis pattern in the ranges of 1800~2000bp, 3500~3900bp, and 5000~5500bp, it is αα / αααanti 3.7 Fractal indication.

[0051] According to an embodiment of the present invention, when bands appear in the electrophoresis pattern in the ranges of 1500~2000 bp, 3500~3900 bp, and 5000~5500 bp, it is αα / αααanti 4.2 Fractal indication.

[0052] According to an embodiment of the present invention, when the electrophoresis pattern shows bands in the ranges of 2500~3000bp, 3500~3900bp, and 5000~5500bp, it indicates Asian-Indian typing.

[0053] According to an embodiment of the present invention, when bands appear in the electrophoresis pattern in the ranges of 1400~1700bp, 3500~3900bp, and 5000~5500bp, it indicates the Filipino del typing.

[0054] According to an embodiment of the present invention, when the electrophoresis pattern shows bands in the ranges of 1000~1500bp, 3500~3900bp, and 5000~5500bp, it indicates the Thai 12.5kb del typing.

[0055] According to an embodiment of the present invention, when bands appear in the electrophoresis pattern in the ranges of 700~1000 bp, 1700~2100 bp, 3500~3900 bp and 5000~5500 bp, it is an indication of Siriraj typing.

[0056] According to an embodiment of the present invention, when bands appear in the electrophoresis pattern in the ranges of 200~500 bp, 3500~3900 bp, and 5000~5500 bp, it is an indication of HPFH-6 typing.

[0057] According to an embodiment of the present invention, when bands appear in the electrophoresis pattern in the ranges of 4000~4300 bp, 3500~3900 bp, and 5000~5500 bp, it is an indication of Hb Lepore typing.

[0058] According to an embodiment of the present invention, when the electrophoresis pattern shows bands in the ranges of 100~300bp, 3500~3900bp, and 5000~5500bp, it indicates a 3.5kb del typing.

[0059] According to an embodiment of the present invention, when the electrophoresis pattern shows bands in the ranges of 2200~2500bp, 2800~3200, 3500~3900 bp, and 5000~5500 bp, it indicates the Taiwan type typing.

[0060] According to an embodiment of the present invention, when the electrophoresis pattern shows bands in the ranges of 2800~3200, 3500~3900 bp and 5000~5500 bp, it indicates a 619bp del typing.

[0061] Beneficial effects

[0062] This invention, based on multiplex Gap-PCR detection technology, enables the detection of 11 copy number variants of thalassemia in a single reaction well. Through testing, screening, and optimization, this invention completed the design and optimization of primer sequences, the testing and optimization of reaction procedures and systems, determined the optimal reaction system and procedure, and established standardized and executable result interpretation criteria. This technical solution has advantages such as low template quality requirements, high specificity, good reproducibility, simple operation, low cost, and short cycle time.

[0063] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0064] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0065] Figure 1 These are electrophoresis images of the amplification results of 8 whole blood samples according to an embodiment of the present invention;

[0066] Figure 2 This is an electrophoresis diagram of the verification amplification results of known thalassemia copy number variants according to Comparative Example 1 of the present invention;

[0067] Figure 3 This is an electrophoresis diagram of the verification amplification results of known thalassemia copy number variants according to Comparative Example 2 of the present invention;

[0068] Figure 4 This is an electrophoresis diagram of the verification amplification results of known thalassemia copy number variants according to Comparative Example 3 of the present invention. Detailed Implementation

[0069] 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 construed as limiting the present invention.

[0070] Definitions and Explanations

[0071] Unless otherwise stated or there is a clear conflict in the context, the articles “a,” “an,” and “described” as used herein are intended to include “at least one” or “one or more.” Therefore, these articles as used herein refer to articles for one or more (i.e., at least one) objects. For example, “a component” refers to one or more components, meaning that more than one component may be considered for use or adoption in the implementation of the described embodiments.

[0072] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0073] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0074] This invention proposes a primer set and its uses, a kit and its uses, and a method for detecting copy number variations in thalassemia, which will be described in detail below.

[0075] Primer set

[0076] This invention designs detection primers for copy number variation regions of 11 thalassemia genes, with a total of 16 detection primers designed. Multiplex Gap-PCR amplification is performed in a single well, and the amplification products are detected by agarose gel electrophoresis. The copy number variation type is determined by the electrophoretic bands.

[0077] In one aspect of the invention, a primer set is provided, comprising 16 primers as shown in SEQ ID NO: 1-16, as detailed in Table 1. According to an embodiment of the invention, the primer set includes an upstream primer and a downstream primer: the upstream primer has the nucleotide sequences shown in SEQ ID NO: 1-9, and the downstream primer has the nucleotide sequences shown in SEQ ID NO: 10-16.

[0078] Reagent test kit

[0079] In another aspect, the present invention provides a kit. According to an embodiment of the invention, the kit contains the aforementioned primer set, as well as DNase / RNase-free water, DNA polymerase, cresol red, and buffer. According to an embodiment of the invention, the concentration of each of the 16 primers is 100 μM, the ratio of the 16 primers 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, the concentration of the DNA polymerase is 1 U / μL, the concentration of the cresol red is 1 mg / mL, and the buffer contains Mg... 2＋ The concentration of the ions is 2 mM, and the concentration of each dNTP in the buffer solution is 400 μM.

[0080] According to an embodiment of the present invention, the ratio of the components in the kit, namely the ratio of the primer set, the DNase / RNase-free water, the DNA polymerase, the cresol red, and the buffer solution, is (1~2):(2~4):(0.1~0.3):(0.5~1.5):(7~8).

[0081] Therefore, the kit of the present invention can accurately and effectively detect the specific subtype of copy number variation in thalassemia.

[0082] use

[0083] In another aspect of the invention, the invention proposes the use of the aforementioned primer set in the preparation of a kit for detecting thalassemia copy number variations.

[0084] In another aspect of the invention, the present invention provides for the use of the aforementioned kit in the preparation of a product for detecting thalassemia copy number variants.

[0085] Methods for Detecting Copy Number Variations in Thalassemia

[0086] In another aspect, the present invention provides a method for genotyping copy number variations in thalassemia. According to an embodiment of the present invention, the method includes: detecting the nucleic acid sample to be tested using the primer set or kit described above; and determining the genotype of the thalassemia copy number variation based on the detection results; wherein the nucleic acid sample to be tested is derived from blood; and the detection includes performing PCR amplification on the nucleic acid sample to be tested to obtain PCR products, and performing electrophoresis and imaging on the PCR products to obtain an electrophoresis image.

[0087] According to an embodiment of the present invention, the PCR amplification reaction system is as follows:

[0088]

[0089] The ratio of the 16 primers shown in SEQ ID NO:1 to SEQ ID NO:16 in the primer set is 1:1:1:1:1:1:1:1:1:1:0.2:0.13:0.8:0.7:1:1; the concentration of each of the 16 primers shown in SEQ ID NO:1 to 16 in the primer set is 100 μM; the concentration of the DNA polymerase is 1 U / μL; the concentration of the cresol red is 1 mg / mL; and the amount of Mg in the buffer solution is... 2＋ The concentration of the ions is 2 mM; the concentration of each dNTP in the buffer solution is 400 μM; and the concentration of the nucleic acid sample to be tested is 10-100 ng / μL.

[0090] According to an embodiment of the present invention, the PCR amplification detection conditions are as follows:

[0091]

[0092] 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 image.

[0093] When the electrophoresis pattern shows bands in the ranges of 1800~2000bp, 3500~3900bp, and 5000~5500bp, it indicates αα / αααanti 3.7 Fractal indication.

[0094] When the electrophoresis pattern shows bands in the ranges of 1500~2000 bp, 3500~3900 bp, and 5000~5500 bp, it indicates αα / αααanti 4.2 Fractal indication.

[0095] When the electrophoresis pattern shows bands in the ranges of 2500~3000bp, 3500~3900bp, and 5000~5500bp, it indicates Asian-Indian typing.

[0096] When the electrophoresis pattern shows bands in the ranges of 1400~1700bp, 3500~3900bp, and 5000~5500bp, it indicates Filipino del typing.

[0097] When the electrophoresis pattern shows bands in the ranges of 1000~1500bp, 3500~3900bp, and 5000~5500bp, it indicates the Thai 12.5kb del typing.

[0098] When the electrophoresis pattern shows bands in the ranges of 700~1000 bp, 1700~2100 bp, 3500~3900 bp, and 5000~5500 bp, it indicates Siriraj typing.

[0099] When the electrophoresis pattern shows bands in the ranges of 200~500 bp, 3500~3900 bp, and 5000~5500 bp, it indicates HPFH-6 typing.

[0100] When the electrophoresis pattern shows bands in the ranges of 4000~4300 bp, 3500~3900 bp, and 5000~5500 bp, it indicates Hb Lepore typing.

[0101] When the electrophoresis pattern shows bands in the ranges of 100~300bp, 3500~3900bp, and 5000~5500bp, it indicates a 3.5kb del typing.

[0102] When the electrophoresis pattern shows bands in the ranges of 2200~2500bp, 2800~3200, 3500~3900 bp, and 5000~5500 bp, it indicates that the Taiwan type has been identified.

[0103] When the electrophoresis pattern shows bands in the ranges of 2800~3200, 3500~3900 bp, and 5000~5500 bp, it indicates a 619bp del typing.

[0104] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0105] Example 1

[0106] Based on the copy number variation types of thalassemia genes, the inventors determined the two ends of the variation intervals and used SnapGene software to design primers outside the variation regions to amplify the genes in the variation intervals. Detection primers were designed for 11 copy number variation regions of thalassemia genes, for a total of 16 detection primers, as shown in Table 1.

[0107] Table 1

[0108]

[0109] Example 2

[0110] 1) DNA extraction: DNA was extracted from human peripheral blood samples using a magnetic bead extraction kit (brand: MAGEN, catalog number: MD5111-02).

[0111] 2) Prepare the Gap-PCR primer mix according to Table 2.

[0112] Table 2

[0113]

[0114] 3) Configure the individual reaction wells for Gap-PCR according to Table 3.

[0115] Table 3

[0116]

[0117] DNase / RNase-free water (brand: BGI, catalog number: FQD-48A), cresol red (brand: Merck, catalog number: 114472-25G), primers (brand: Sangon Biotech), DNA polymerase, buffer (brand: Akerui, catalog number: AG12204).

[0118] 4) Set the Gap-PCR reaction program according to Table 4. This invention is applicable to the commonly used 96-well PCR instrument in China: ETC821M PCR instrument.

[0119] Table 4

[0120]

[0121] 5) PCR Product Electrophoresis: Weigh a certain amount of agarose and add the corresponding 1×TAE buffer (brand: Sangon Biotech, catalog number: B540023-0001) to prepare a 0.8% agarose gel. Mix the agarose powder and 1×TAE in a microwave-safe conical flask. Heat in a microwave oven until the agarose is completely dissolved (but do not overcook the solution, as the buffer will evaporate, thus changing the final agarose ratio in the gel). Then remove the flask, shake well, and cool to 50-60°C. Add 0.005% nucleic acid dye (brand: Sangon Biotech, catalog number: A616697-0500) to the agarose gel, stir well, and prepare a 0.8% agarose gel. Immediately pour the agarose gel solution into a gel tray 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 number: 3423A) and PCR products to the wells of an agarose gel. Electrophoresis conditions: 135V, electrophoresis time 1 hour (the speed may vary slightly depending on the electrophoresis apparatus). Stop electrophoresis when the indicator band is about 1.5 cm from the bottom of the gel.

[0122] 6) Result interpretation: After the reaction is completed, the PCR products are subjected to electrophoresis, gel imaging, and analysis.

[0123] The absence of a 3712bp band and a copy number variation band within the detection range indicates experimental failure. DNA needs to be extracted again for PCR testing.

[0124] There was a 3712bp band and a copy number variation band within the detection range. Based on the correspondence between the electrophoretic bands and the genotype (see Table 5), the corresponding thalassemia copy number variation type (homozygous deletion type) was determined.

[0125] No 3712bp band was found, but there were copy number variation bands within the detection range. Based on the correspondence between electrophoretic bands and genotypes (see Table 5), the corresponding thalassemia copy number variation type (heterozygous deletion type) was determined.

[0126] (Homozygous heterozygosity is used to determine the deletion type of thalassemia. Alleles on homologous chromosomes are deleted. The deletion of one allele is heterozygous deletion, and the deletion of two alleles at the same time is homozygous deletion.)

[0127] Table 5

[0128]

[0129] Example 3

[0130] Eight whole blood samples with known blood types were used for testing. The information on the known blood types of the samples is shown in Table 6.

[0131] Table 6

[0132]

[0133] 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 sizes of the electrophoretic bands in the eight examples are shown in Table 7, and the electrophoresis results are shown in the figure below. Figure 1 As shown, the experimental results indicate that the types detected in all 8 samples were consistent with the known types.

[0134] Table 7

[0135]

[0136] Comparative Example 1

[0137] 1) DNA extraction: DNA was extracted from human peripheral blood samples using a magnetic bead extraction kit (brand: MAGEN, catalog number: MD5111-02).

[0138] 2) Configure the Gap-PCR primer mix according to Table 2 in Example 2.

[0139] 3) Configure individual reaction wells for Gap-PCR according to Table 8.

[0140] Table 8

[0141]

[0142] 4) Set the Gap-PCR reaction program according to Table 9. This invention is applicable to the commonly used 96-well PCR instrument in China: ETC821M PCR instrument.

[0143] Table 9

[0144]

[0145] The remaining steps are the same as in Example 2. The Gap-PCR detection results are shown in the figure below. Figure 2 As shown, when the amount of ApexHF HsDNA polymerase in the PCR system is 0.5 μL and the amount of primer is 2 μL, multiple non-specific bands appear in the electrophoresis image of the PCR product.

[0146] Comparative Example 2

[0147] 1) DNA extraction: DNA was extracted from human peripheral blood samples using a magnetic bead extraction kit (brand: MAGEN, catalog number: MD5111-02).

[0148] 2) Configure the Gap-PCR primer mix according to Table 2 in Example 2.

[0149] 3) Configure a single reaction well for Gap-PCR according to Table 10.

[0150] Table 10

[0151]

[0152] 4) Set the Gap-PCR reaction program according to Table 11. This invention is applicable to the commonly used 96-well PCR instrument in China: ETC821M PCR instrument.

[0153] Table 11

[0154]

[0155] The remaining steps are the same as in Example 2. The Gap-PCR detection results are shown in the figure below. Figure 3 As shown, when the reaction buffer and reaction enzyme in the PCR system are changed, the PCR reaction cannot effectively amplify specific bands.

[0156] Comparative Example 3

[0157] 1) DNA extraction: DNA was extracted from human peripheral blood samples using a magnetic bead extraction kit (brand: MAGEN, catalog number: MD5111-02).

[0158] 2) Configure the Gap-PCR primer mix according to Table 2 in Example 2.

[0159] 3) Configure individual reaction wells for Gap-PCR according to Table 12.

[0160] Table 12

[0161]

[0162] 4) Set the Gap-PCR reaction program according to Table 13. This invention is applicable to the commonly used 96-well PCR instrument in China: ETC821M PCR instrument.

[0163] Table 13

[0164]

[0165] The remaining steps are the same as in Example 2. The Gap-PCR detection results are shown in the figure below. Figure 4 As shown, when the reaction program is extended for 5 minutes, the results show that nonspecific bands appear in the reaction.

[0166] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0167] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A primer set, characterized in that, Includes the 16 primers shown in SEQ ID NO:1~16; The upstream primer is the nucleotide sequence shown in SEQ ID NO:1~9, and the downstream primer is the nucleotide sequence shown in SEQ ID NO:10~16.

2. Use of the primer set of claim 1 in the preparation of a kit for detecting copy number variations in thalassemia.

3. A reagent kit, characterized in that, Includes the primer set as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, Further includes: DNase / RNase-free water, DNA polymerase, cresol red, and buffer solution.

5. The reagent kit according to claim 4, characterized in that, The concentrations of the 16 primers shown in SEQ ID NO:1~16 in the primer set are 90~110μM, respectively.

6. The reagent kit according to claim 4, characterized in that, The concentration of the DNA polymerase is 0.5~1.5 U / μL.

7. The reagent kit according to claim 4, characterized in that, The concentration of cresol red is 0.5~1.5 mg / mL.

8. The reagent kit according to claim 4, characterized in that, Mg in the buffer solution 2＋ The concentration of ions is 1.5~2.5 mM.

9. The reagent kit according to claim 4, characterized in that, The concentration of dNTPs in the buffer solution is 380~420 μM.

10. The kit according to claim 4, characterized in that, The ratio of the primer set, the DNase / RNase-free water, the DNA polymerase, the cresol red, and the buffer solution is (1~2):(2~4):(0.1~0.3):(0.5~1.5):(7~8).

11. The kit according to any one of claims 3-10 is used in the preparation of a product for detecting thalassemia copy number variants.

12. A method for typing thalassemia copy number variations for non-disease diagnostic purposes, characterized in that, include: The nucleic acid sample to be tested was amplified by PCR to obtain the PCR product; The PCR products were subjected to electrophoresis and imaging to obtain an electrophoresis image. Based on the bands appearing in the electrophoresis image, the thalassemia copy number variation was determined. in: The reaction system for the PCR amplification is as follows: The detection conditions for the PCR amplification are as follows: 。 13. The method according to claim 12, characterized in that, The nucleic acid sample to be tested was derived from blood.

14. The method according to claim 12, characterized in that, The ratio of the 16 primers shown in SEQ ID NO:1~16 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.

15. The method according to claim 12, characterized in that, The concentrations of the 16 primers shown in SEQ ID NO:1~16 in the primer set are 90~110μM, respectively.

16. The method according to claim 12, characterized in that, The concentration of the nucleic acid sample to be tested is 10-100 ng / μL.

17. The method according to claim 12, characterized in that, When the electrophoresis pattern shows bands in the ranges of 1800~2000bp, 3500~3900bp, and 5000~5500bp, it indicates αα / αααanti 3.7 Fractal indication; When the electrophoresis pattern shows bands in the ranges of 1500~2000 bp, 3500~3900 bp, and 5000~5500 bp, it indicates αα / αααanti 4.2 Fractal indication; When the electrophoresis pattern shows bands in the ranges of 2500~3000bp, 3500~3900bp, and 5000~5500bp, it indicates the Asian-Indian typing. When bands appear in the electrophoresis pattern within the ranges of 1400~1700bp, 3500~3900bp, and 5000~5500bp, it indicates the Filipino del typing. When the electrophoresis pattern shows bands in the ranges of 1000~1500bp, 3500~3900bp, and 5000~5500bp, it indicates the Thai 12.5kb del typing. When bands appear in the electrophoresis pattern in the ranges of 700~1000 bp, 1700~2100 bp, 3500~3900 bp, and 5000~5500 bp, it indicates the Siriraj typing. When bands appear in the electrophoresis pattern within the ranges of 200~500 bp, 3500~3900 bp, and 5000~5500 bp, it indicates HPFH-6 typing. When bands appear in the electrophoresis pattern within the ranges of 4000~4300 bp, 3500~3900 bp, and 5000~5500 bp, it indicates Hb Lepore typing; When the electrophoresis pattern shows bands in the ranges of 100~300bp, 3500~3900bp, and 5000~5500bp, it indicates a 3.5kb del typing. When bands appear in the electrophoresis pattern within the ranges of 2200~2500bp, 2800~3200, 3500~3900 bp, and 5000~5500 bp, it indicates the Taiwan type typing. When the electrophoresis pattern shows bands in the ranges of 2800~3200, 3500~3900 bp, and 5000~5500 bp, it indicates a 619bp del typing.

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