Method and kit for detecting alpha and beta globin gene copy number and tiny variation and application thereof

Abstract

The invention discloses a method and a kit for detecting copy number and tiny variation of an alpha-globin gene and a beta-globin gene and application of the method and the kit. An alpha-globin gene and a beta-globin gene are amplified at the same time in a single-tube PCR reaction, and as many as 61 deletion or repetition types such as normal types, 8 heterozygous deletion types, 36 homozygous deletion types, 2 repetition types, 14 composite heterozygous deletion / repetition types and the like can be detected at a time. Meanwhile, three types of non-deletion type alpha-thalassemia and 18 types of non-deletion type beta-thalassemia can be respectively detected, and normal, heterozygous and homozygous mutations can be distinguished. According to the method, deletion, repetition and tiny variation detection of alpha and beta-thalassemia can be achieved only through one-time PCR, the process is simple, standardization is easy to achieve, meanwhile, the detection time is greatly shortened, and integrity detection is provided for thalassemia.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for detecting the copy number and minor variation of alpha and beta globin genes, a kit and application thereof. Background Art

[0002] Thalassemia (abbreviated as thalassemia) is a monogenic hereditary hemolytic disease caused by a mutation (point mutation or deletion, etc.) in one or more globin genes, which leads to a reduction in the synthesis of globin peptide chains or a complete inability to synthesize them, resulting in an imbalance in the number of peptide chains. Clinically, α and β-thalassemia are the most common. Thalassemia has obvious racial characteristics and regional distribution differences, and is mainly distributed in the Mediterranean coast, Southeast Asia and a few African regions. In southern China, thalassemia is one of the most common and most harmful genetic diseases. Among them, the carrier rate of thalassemia in Guangxi can reach more than 20%.

[0003] α-thalassemia can be divided into deletion type and non-deletion type, of which the deletion type accounts for the majority. ＋ -Thalassemia (missing 1 α gene, -α / ) and α 0 - Thalassemia (missing 2 α genes, - / ). α common in India, Southeast Asia and Africa ＋ -Thalassemia is -α 3.7 , -α 4.2 , α 0 -Thalassemia is- SEA ; The Mediterranean region is dominated by -- MED Type α 0 -Thalassemia is the most common. There are 3 types of non-deletion α-thalassemia: α WS α (α2: c.369C>G), α QS α (α2: c.427T>C), α CS α (α2:c.377T>C) plus 3 deletion types of α-thalassemia: -α 3.7 , -α 4.2 and-- SEA , accounting for 98% of the total number of patients in China. Generally, 3 α-gene dysfunctions manifest as intermediate α-thalassemia (HbH disease), and 4 α-gene dysfunctions manifest as severe α-thalassemia (Hb Bart's). However, since the expression level of α2 is higher than that of α1, at a ratio of about 2:1, some patients with homozygous non-deletion α-thalassemia or non-deletion compound deletion α-thalassemia may also manifest as intermediate α-thalassemia. There are also a small number of case reports that non-deletion compound homozygous deletion α-thalassemia may also manifest as severe α-thalassemia. In addition, HS-40 is the main regulatory sequence of the α-globin gene. Its deletion will reduce the expression of the α-globin gene. If combined with α ＋ - Thalassemia or α 0-Thalassemia may result in α-thalassemia intermedia or major.

[0004] The genetic variation of β-thalassemia is mainly point mutation or small deletion of β gene, and a few are large deletion. In China, 8 mutations (β: c.124_127delTTCT, β: c.52A>T β: c.316-197C>T, β: c.-78A>G, β: c.216_217insA, β: c.79G>A, β: c.92+1G>T, β: c.-79A>G) account for more than 95% of the total β-thalassemia mutations. The genotype β++ / βN belongs to the static β-thalassemia; the genotype β0 / βN or β+ / βN belongs to the mild β-thalassemia; the genotype β+ / β+ or β+ / β0 belongs to the intermediate β-thalassemia; the genotype β+ / β0 or β0 / β0 belongs to the severe β-thalassemia. The phenotype of intermediate β-thalassemia varies greatly, with patients having mild to moderate anemia, and severe β-thalassemia patients having severe anemia, requiring regular blood transfusions throughout their lives to survive. At present, it has been found that the clinical phenotype of β-thalassemia is related to the balance of the α / β-globin peptide chain ratio. For example, the combination of β-thalassemia heterozygotes with α-globin gene triplets or tetrads can aggravate the imbalance of the α / β-globin peptide chain ratio, causing the individual's clinical phenotype to worsen from asymptomatic to intermediate thalassemia, or making the clinical phenotype of intermediate or severe β-thalassemia patients more severe; and the combination of α-thalassemia mutations can make the α / β-globin peptide chain ratio of β-thalassemia patients tend to balance, thereby alleviating the anemia phenotype.

[0005] At present, the detection technologies for thalassemia include: GAP-PCR (GAP-PCR), which uses a pair of primers corresponding to a mutation type, and detects gene fragment deletion mutations based on the presence or absence of amplified products and fragment length through PCR amplification and agarose gel electrophoresis; PCR-reverse dot hybridization (PCR-RDB) technology is to fix a series of specific oligonucleotides of known mutation sites on the membrane, and simultaneously analyze multiple point mutations that may exist in a specimen in one hybridization reaction. It is a commonly used molecular diagnostic technology for detecting thalassemia point mutations; PCR-flow-through hybridization method forces all hybridization liquids to flow through low-density gene chips, allowing all single-stranded DNA to fully contact the probes, and hybridize with the corresponding complementary single-stranded probes fixed in the micropores to detect gene mutations. The basic principle of multiple ligation-dependent probe amplification (MLPA) technology is that the probe hybridizes with the target sequence DNA, through ligation and PCR amplification, and then the amplified product is subjected to capillary electrophoresis fragment analysis on a first-generation sequencer to detect fragment deletions or duplications. The above detection methods are cumbersome to operate and take a long time to detect. The types of thalassemia gene mutations are complex. In addition to multiple deletion types, complex genotypes such as compound α-triplets, HS-40 deletions or non-deletion α-thalassemia need to be considered. Therefore, it is urgent to develop new detection methods that can detect the copy number of multiple deletion and duplication α-thalassemia, and simultaneously detect non-deletion α and β-thalassemia, so as to achieve high coverage, rapid, economical and accurate diagnosis to meet multi-level clinical needs. Summary of the invention

[0006] The object of the present invention is to provide a method, a kit and application thereof for detecting the copy number and minor variation of α-globin gene and β-globin gene.

[0007] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme: An amplification primer for detecting the copy number and minor variation of α-globin gene and β-globin gene, which can simultaneously amplify α-globin gene, β-globin gene, internal reference gene and sex chromosome gene when performing single-tube or split-tube amplification reaction; the amplification primer comprises: Thirty-eight amplification primers were designed within, upstream, or downstream of the ζ2, Ψζ1, Ψα2, Ψα1, α2, α1, and θ1 gene regions of the α-globin gene cluster; Known minor variants in the α2 gene WS α, α QS α and α CS α / Design 6 amplification primers; Known minor variants in the β-globin gene -32 , β -30 , β -29 , β -28 , β Cap+1 , βIntM , β CD14-15 , β CD17 , β CD26 , β CD27 / 28 , β IVS- I -1(G>T) , β IVS-I-1(G>A) , β IVS-I-5 , β CD71-72 , β CD43 , β CD41-42 , β CD31 and β IVS-II-654 Design 38 amplification primers; Design 8 amplification primers for the internal reference gene; Four amplification primers were designed for sex chromosome genes.

[0008] The internal reference gene is one or more of GAPDH, ACTB, CFTR, HPRT, RPP30 or RPP40; The sex chromosome gene is one or more of AMEL, ZFX, ZFY, TAF9 or SRY.

[0009] In the PCR amplification reaction, the amplification primer contains at least one fluorescent group; the fluorescent group is selected from any one of FAM, VIC, HEX, JOE, TMR, NED, PET, and ROX.

[0010] The nucleotide sequences of primers in the ζ2, Ψζ1, Ψα2, Ψα1, α2, α1 and θ1 gene regions of the α globin gene cluster are shown in SEQ ID NOs. 1-38; the nucleotide sequences of primers with known minor variations on the α2 gene are shown in SEQ ID NOs. 39-44; the known minor variations of the β gene include SEQ ID NOs. 45-82; the nucleotide sequences of primers for the internal reference genes are shown in SEQ ID NOs. 83-90; and the nucleotide sequences of primers for amplifying sex chromosome genes are shown in SEQ ID NOs. 91-94.

[0011] A kit for detecting the copy number and minor variation of α and β globin genes, the kit comprising the above-mentioned amplification primers; the kit also comprises an amplification reagent, an α / β normal control, an α / β positive control, and an α / β blank control.

[0012] The amplification reagent comprises an α / β reaction solution, an α / β primer mixture and a hot start polymerase; the α / β reaction solution comprises 10X Taq Buffer, MgCl2, dATP, dTTP, dGTP, dCTP and betaine; the α / β normal control is human genomic DNA of normal type of α or β-thalassemia; the α / β positive control is human genomic DNA of positive type of α or β-thalassemia; the blank control is a Tris buffer with a pH of 8.5; and the α / β primer mixture is obtained by mixing the above primers.

[0013] A method for detecting α and β globin gene copy numbers and minor variations for non-diagnostic purposes, comprising the following steps: (1) Add the DNA sample to be tested, α / β normal control, α / β positive control, and α / β blank control to the amplification reagent respectively; (2) subjecting the mixture obtained in step (1) to a PCR reaction, and after the reaction is completed, analyzing the PCR amplification product by fluorescent labeling capillary electrophoresis; (3) Capillary electrophoresis analysis separates amplification products with different fluorescence and fragment lengths and generates detection peak area information; (4) Copy number interpretation: S1: The 19 detection peak areas of each sample in the ζ2, Ψζ1, Ψα2, Ψα1, α2, α1 and θ1 gene regions, upstream or downstream sites, and the 18 detection peak areas of the normal type of minor mutations in the β-globin gene cluster are divided by the sum of the detection peak areas of the internal reference genes, and then the sample to be tested is divided by the corresponding value of each site of the α / β normal control, which is the detection peak ratio of each site. The corresponding copy number result is obtained by corresponding the value of the detection peak ratio to the corresponding copy number result, and the α-globin gene cluster is 0, 1, 2 or 3 copies; S2: The following genotypes can be determined by the copy number combination of 19 loci in the α-globin gene cluster: αα / αα, -α 3.7 / αα、-α 4.2 / αα,-- SEA / αα,-- THAI / αα,-- FIL / αα,-- MEDI / αα,-- MEDII / αα、-α 20.5 / αα、-α 3.7 / -α 3.7 , -α 4.2 / -α 4.2 、-- SEA / -- SEA 、-- THAI / -- THAI 、-- FIL / --FIL 、-- MEDI / -- MEDI 、-- MEDII / -- MEDII 、-α 20.5 / -α 20. 5、-α 3.7 / -α 4.2 、-α 3.7 / -- SEA 、-α 3.7 / -- THAI 、-α 3.7 / -- FIL 、-α 3.7 / -- MEDI 、-α 3.7 / -- MEDII 、-α 3.7 / -α 20.5 、-α 4.2 / -- SEA 、-α 4.2 / -- THAI 、-α 4.2 / -- FIL 、-α 4.2 / -- MEDI 、-α 4.2 / -- MEDII 、-α 4.2 / -α 20.5 、-- SEA / -- THAI 、-- SEA / -- FIL 、-- SEA / -- MEDI 、-- SEA / -- MEDII 、-- SEA / -α 20.5 、-- THAI / -- FIL 、-- THAI / -- MEDI 、-- THAI / -- MEDII 、-- THAI / -α 20.5 、-- FIL / -- MEDI 、-- FIL / -- MEDII 、-- FIL / -α 20.5 、-- MEDI / - MEDII 、-- MEDI / -α 20.5 、-- MEDII / -α 20.5、ααα anti3.7 / αα、ααα anti4.2 / αα、-α 4.2 / ααα anti3.7 、-- SEA / ααα anti3.7 、-- THAI / ααα anti3.7 、-- FIL / ααα anti3.7 、-- MEDI / ααα anti3.7 、-- MEDII / ααα anti3.7 , -α 20.5 / ααα anti3.7 , -α 3.7 / ααα anti4.2 、-- SEA / ααα anti4.2 、-- THAI / ααα anti4.2 、-- FIL / ααα anti4.2 、-- MEDI / ααα anti4.2 、-- MEDII / ααα anti4.2 , -α 20.5 / ααα anti4.2 , del HS-40 / αα, del HS-40 / del HS-40; S3: The β-globin gene was judged as normal type, heterozygous deletion type or homozygous deletion type by the copy number results of 18 loci in the β-globin gene cluster; (5) Interpretation of minor variations: Minor variations are known in the α2 gene and β-globin gene. The normal type, heterozygous mutant type, or homozygous mutant type can be interpreted based on the presence or absence of the detection peak.

[0014] Furthermore, the PCR reaction procedure in step (2) is: 95°C, 5 min; 95°C 30 sec, 66°C 40 sec, 72°C 40 sec, 28 cycles; 72°C 45 min; stored at 4°C.

[0015] Application of the above primers or the above kit in nucleic acid detection products for α- and β-thalassemia related genes.

[0016] The beneficial effects of the present invention are: (1) Combine multiple-stage PCR amplification reactions in the same tube to solve the quantitative and qualitative problems of multiple PCR amplification at one time. Use multiple different types of primers in a single-tube amplification reaction to simultaneously amplify the α-globin gene cluster and the β-globin gene cluster, and detect deletions, duplications, and minor variations at one time. The entire process only takes 2.5 hours, which can greatly shorten the detection time to meet clinical needs and provide complete detection for thalassemia.

[0017] (2) Design 19 sites within or outside the upstream and downstream regions of the ζ2, Ψζ1, Ψα2, Ψα1, α2, α1, and θ1 gene regions of the α-globin gene cluster, such as Figure 1 , copy number detection sites are set in the deletion range and outside the two sides of the deletion type --MEDII, --THAI, --FIL, -α20.5, --MEDI, --SEA, -α4.2, -α3.7, and each heterozygous or homozygous deletion can be detected through the combination of copy numbers. In addition to detecting deletion types, the present invention can also detect duplication types, such as ααα anti3.7 / αα、ααα anti4.2 / αα, the specific deletion and duplication copy number combinations at 19 loci are shown in Figure 2 Compared with GAP-PCR, which can only be designed for known deletion types, the present invention can detect up to 61 deletion or duplication types, including normal type, 8 heterozygous deletion types, 36 homozygous deletion types, 2 duplication types, 14 compound heterozygous deletion / duplication types, etc., in addition to single-tube amplification, because 19 loci are scattered in the α-globin gene cluster. The copy number combinations of 61 types at 19 loci are shown in Figure 2. Figure 2 At the same time, if a copy number combination that does not match the above types appears, it may indicate the presence of other deletion types.

[0018] (3) In addition to the deletion type, the present invention can also detect the duplication type. The α-globin gene triplets are the result of unequal exchange of the homologous fragments X, Y, and Z boxes of the α-globin gene cluster on chromosome 16 during meiosis. Generally, different allelic exchanges can produce two common α-globin gene triplets, namely ααα anti3.7 and ααα anti4.2, the α-globin gene trio alone will not cause clinical symptoms or significant hematological changes. However, studies have found that carrying both the α-globin gene trio and heterozygous β-thalassemia will increase the imbalance between α-globin and β-globin, change the hematological characteristics of the carrier, and convert the clinical phenotype from β-thalassemia heterozygotes to intermediate or severe β-thalassemia. Therefore, for individuals who carry both the α-globin gene trio and β-thalassemia heterozygotes, it is recommended to strengthen clinical attention after the onset of anemia symptoms. The trio is a type that cannot be detected by the commonly used thalassemia detection technology GAP-PCR. The special copy number detection technology of the present invention on the α-globin gene cluster can accurately detect ααα anti3.7 and ααα anti4.2 , providing accurate assessment of thalassemia inheritance and disease severity.

[0019] (4) The present invention can detect the copy number of HS-40, the regulatory sequence of the α-globin gene. HS-40 deficiency can lead to a decrease in the expression of α-globin and aggravate the symptoms of α-thalassemia. Therefore, the detection of HS-40 deficiency is helpful for the accurate diagnosis of α-thalassemia and genetic risk assessment.

[0020] (5) β-thalassemia is mainly caused by point mutations of the β gene, supplemented by large deletions. In addition to detecting 18 minor mutations in β-thalassemia, the present invention sets wild-type and mutant primers for each mutation, thereby distinguishing between heterozygous and homozygous mutations. At the same time, large deletions can be further calculated through the wild-type detection peaks of 18 sites. Compared with other technologies, point mutations are mostly detected by reverse dot hybridization or first-generation sequencing, and large deletions are mostly detected by GAP-PCR. The present invention can detect 18 minor mutations at one time and indicate whether there is a large deletion, such as the Chinese type Gγ+(Aγδβ)0 deletion and the Southeast Asian type HPFH deletion.

[0021] (6) In order to avoid the deviation caused by the difference in amplification efficiency between PCR wells and reduce the amplification preference caused by the difference in the length of the target gene fragment, the present invention sets four internal control genes with different fragment lengths as internal controls, incorporates them into the calculation of the detection peak ratio, and standardizes the amplification efficiency for effective monitoring of the sample extraction and amplification process, and improves the accuracy of copy number detection of the α-globin gene and the β-globin gene, and distinguishes between the deletion type and the duplication type.

[0022] (7) Combined detection of α- and β-thalassemia is of great significance for assessing the severity of the disease. Through semi-quantitative detection of the α-thalassemia gene and qualitative detection of non-deletion α- and β-thalassemia, the balance of α / β-globin can be known, the severity of α- and β-thalassemia can be assessed, and it can be used as a prognosis assessment for clinical drug treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Schematic diagram for the design of the α-globin gene cluster and β-globin gene cluster loci.

[0024] Figure 2 The copy numbers of 61 types of α-thalassemia deletion and duplication types at 19 detection sites are shown.

[0025] Figure 3 This is the test result diagram of the αα / αα normal male sample.

[0026] Figure 4 -α 3.7 / αα-deficient α-thalassemia carrier male sample test results.

[0027] Figure 5 for-- SEA / αα-deficient α-thalassemia carrier female sample test results.

[0028] Figure 6 is α WS Figure 2. Test results of female samples of α / αα non-deletional α-thalassemia carriers.

[0029] Figure 7 β CD41-42 (c.126_129delCTTT) / β N Figure 2. Detection results of samples from female non-deletional β-thalassemia carriers. DETAILED DESCRIPTION

[0030] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and the best embodiments.

[0031] Example 1 Primer Design According to the NCBI database, the α and β-globin gene clusters were searched, and specific primers were designed in the ζ2, Ψζ1, Ψα2, Ψα1, α2, α1 and θ1 gene regions and upstream and downstream, β gene and four internal control genes of the α-globin gene cluster. The specific sequences are shown in Table 1. The preparation of α / β reaction solution and α / β primer mixture is shown in Tables 2 and 3 below. The prepared α / β reaction solution and α / β primer mixture were stored at -20±5℃ for future use.

[0032] Table 1 Amplification composition information Table 1-1

[0033] Table 1-2

[0034] Table 1-3

[0035] Table 1-4

[0036] Table 2 Allocation of α / β reaction solution components

[0037] Table 3 Allocation of α / β primer mixture composition Table 3-1

[0038] Table 3-2

[0039] Example 2 PCR amplification and result analysis 1. Sample processing: The nucleic acid extraction kit produced by Xiamen Bioxun Biotechnology Co., Ltd. (centrifugal column method, catalog number 04020001, registration number: Fujian Xiaxiebei 20200355) was used to extract human whole blood genomic DNA as the test sample for subsequent PCR reactions. The DNA concentration was 5ng / uL~60ng / uL, OD 260 nm / OD 280 The ratio of nm is between 1.6-2.0.

[0040] 2. Preparation of amplification reagents: (1) Take out the α / β reaction solution and α / β primer mixture and thaw them at room temperature. Mix by inverting the tubes and centrifuge briefly in a microcentrifuge to allow all the liquid to settle to the bottom of the tube.

[0041] (2) Preparation of amplification reagents: Prepare amplification reagents according to Table 4.

[0042] Table 4 Amplification reagent preparation table

[0043] 3. Sample addition: Take 21 μL of the above amplification reagent, add 4 μL of the sample to be tested, α / β normal control, α / β positive control, and α / β blank control to the corresponding PCR reaction tube containing the amplification reagent, cover the PCR reaction tube cap, and centrifuge briefly. The α / β normal control is the human genomic DNA of normal type of α or β-thalassemia; the α / β positive control is the human genomic DNA of positive type of α or β-thalassemia; the blank control is Tris buffer at pH 8.5.

[0044] 4. PCR amplification and capillary electrophoresis analysis: (1) Set the PCR reaction program as shown in Table 5, and the reaction volume is set to 25 μL; Table 5 PCR amplification program

[0045] (2) Analysis of PCR amplification products by fluorescent labeling capillary electrophoresis: Use ABI3130, ABI3730, ABI3500Dx, or ABI SeqStudio Genetic Analyzer capillary genetic analyzer for detection. Take 2 μL of amplification product and 10 μL of 1% GeneScan 500 LIZ Size Standard (prepared with 10 μL GeneScan 500 LIZ Size Standard and 990 μL Hi-Di Formamide, i.e. 1 vol% GeneScan 500 LIZ Size Standard), denature at 95°C for 3 minutes, immediately put on ice for 2 minutes, and then detect on the analyzer.

[0046] 5. Software analysis: After the reaction program is completed, the PCR amplification product is analyzed for fragment size using GeneMapper software. The Analysis Method, Panel, and Size Standard files required for analysis can be downloaded from the website. For specific information on data import, analysis parameter settings, and result analysis, please refer to the GeneMapper User Manual.

[0047] 6. Calculation of detection peak ratio: The detection peak areas of the copy number detection sites of the test sample and the α / β normal control and the internal reference gene are divided by the total area of ​​the internal reference gene, and then the value corresponding to each site of the test sample divided by the normal control is the detection peak ratio (R) of each site.

[0048] 7. Interpretation of results: (1) The peak ratio (R) of the internal reference gene detection falls between 1.80 and 2.20, and the peak ratio of the copy number detection site detection is used to interpret the copy number according to Table 6: Table 6

[0049] (2) The copy number results of 19 loci of the α-globin gene cluster were based on Figure 2 The type of test is determined. Figure 5 As shown; (3) If the copy number of the wild-type β-globin gene detection peak is 0 copies, it may indicate the Chinese type Gγ+(Aγδβ)0 homozygous deletion and the Southeast Asian type HPFH homozygous deletion. If it is 1 copy, it may indicate the Chinese type Gγ+(Aγδβ)0 heterozygous deletion and the Southeast Asian type HPFH heterozygous deletion. (4) Interpretation of minor variations: When the mutant type of the minor variation site has a detection peak, it is judged as a positive variation; when there is no detection peak, it is judged as a negative variation.

[0050] Example 3 Reagent Performance Verification 1. Evaluation of the diagnostic accuracy of the test kit 16 accuracy reference samples (including 6 deletion type, 1 α-triplet, 2 non-deletion type α-thalassemia reference samples and 7 non-deletion type β-thalassemia reference samples) were tested. The samples were shown in Table 7. High, medium and low concentrations were tested respectively. The sample concentrations were 60 ng / uL, 25ng / uL and 15ng / uL, respectively. Each concentration was tested in triplicate. Three batches of reagents were tested. The copy numbers of all detection sites and detection peaks met the requirements.

[0051] Table 7 Accuracy references Deletion type α-thalassemia and non-deletion type α and β-thalassemia

[0052] 2. Repeatability Four repeatability references were tested (Table 8), each concentration was tested ten times, three batches of reagents were tested, the copy number of the detection site and the detection peak judgment were correct, and the test results were all consistent.

[0053] Table 8 Repeatability references Deletion α-thalassemia and non-deletion α- and β-thalassemia

[0054] 4. Detection Limit 16 detection limit reference samples were tested, as shown in the table below. The sample concentrations were diluted to 20ng / µL and 15ng / µL, respectively. Each test was repeated 20 times. Three batches of reagents were tested, and the number of test results met the requirements.

[0055] Table 9 Detection limit reference substances Deletion type α-thalassemia and non-deletion type α and β-thalassemia

[0056] Example 4 1. 150 EDTA-anticoagulated whole blood samples were collected. The SALSA® MLPA® Probemix P140 developed by MRC-Holland of the Netherlands was used as the reference method for deletion α-thalassemia and α-triple to verify the consistency of the results.

[0057] 2. Collect EDTA anticoagulated whole blood samples and use the nucleic acid extraction kit produced by Xiamen Bioxun Biotechnology Co., Ltd. (centrifugal column method, product number 04020001, registration number: Fujian Xiaxie Bei 20200355) to extract human genomic DNA. Use a micro-spectrophotometer to detect the concentration and purity of DNA. The concentration of 150 samples is 15ng / uL-50ng / uL, and the OD 260nm / OD 280nmThe ratio is between 1.6-2.0.

[0058] 3. According to the steps of Example 2, DNA was added and PCR amplification reaction was performed.

[0059] 4. According to the steps of Example 2, the results were analyzed. The results are shown in the following table, which all met the requirements for the copy number of the α-globin gene detection site. Among them, 6 samples were tested from α-thalassemia patients, 97 samples were tested from α-thalassemia carriers, and 47 samples were tested from α-thalassemia normal individuals, for a total of 150 samples (Table 10).

[0060] Table 10 Test results of 150 samples Table 10-1

[0061] Table 10-2

[0062] Table 10-3

[0063] Table 10-4

[0064] Table 10-5

[0065] *MLPA: Multiplex Ligand Probe Amplification, which serves as a positive control group in the present disclosure.

[0066] According to Table 10 and Figures 1 to 5 ( Figure 3 For sample number 104, Figure 4 For sample number 13, Figure 5 The results of sample number 7 show that the present invention can accurately determine the number of copies of the α-globin gene in human individuals. The method can not only identify α-thalassemia patients, carriers and normal individuals, but also further distinguish the gender of the samples.

[0067] Example 5 1. For non-deletion α- and β-thalassemia samples, 21 EDTA-anticoagulated whole blood samples were collected, and first-generation sequencing was used as a reference method to verify the consistency of the results.

[0068] 2. Collect EDTA anticoagulated whole blood samples and use the nucleic acid extraction kit produced by Xiamen Bioxun Biotechnology Co., Ltd. (centrifugal column method, product number 04020001, registration number: Fujian Xiaxiebei 20200355) to extract human genomic DNA. Use a micro-spectrometer to detect the concentration and purity of DNA. The concentration of 21 samples was 15ng / uL-60ng / uL, OD 260nm / OD 280nm The ratio is between 1.6-2.0.

[0069] 3. According to the steps of Example 2, DNA was added and PCR amplification was performed. 4. According to the steps of Example 2, the results were analyzed. The results are shown in Table 11. WS α / αα(α2:c.369C>G)detected 2 samples, α QS α / αα(α2:c.377T>C)detected 1 sample, α CS α / αα (α2:c.427T>C) detected 1 sample, β-thalassemia patients β IVS-II-654 / β IVS-II-654 (β:c.316–197C>T) detected 1 sample, β-thalassemia carrier β CD41-42 / β N (β:c.126_129delCTTT) tested 6 samples, β IVS-II-654 / β N (β:c.316–197C>T) tested 7 samples, β CD26 / β N (β:c.79G>A) 3 samples were tested, and a total of 21 samples were tested, all of which were consistent with the results of first-generation sequencing. The experimental results showed that the present invention can identify α-thalassemia carriers and β-thalassemia patients.

[0070] Table 11 Test results of 21 samples

[0071] According to Table 11 and Figure 6 (Sample No. 1) and Figure 7 From the results of (sample number 6), it can be seen that the present invention can accurately measure the known minor variations of the α2 gene and β gene of human individuals, and further identify thalassemia patients, thalassemia carriers and normal individuals.

[0072] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. An amplification primer for detecting the copy number and minor variation of α-globin gene and β-globin gene, characterized in that: When performing a single-tube or split-tube amplification reaction, the α-globin gene, the β-globin gene, the internal reference gene and the sex chromosome gene can be amplified simultaneously; the amplification primers include: Thirty-eight amplification primers were designed within, upstream, or downstream of the ζ2, Ψζ1, Ψα2, Ψα1, α2, α1, and θ1 gene regions of the α-globin gene cluster; Known minor variants in the α2 gene WS α, α QS α and α CS α / Design 6 amplification primers; Known minor variants in the β-globin gene -32 , β -30 , β -29 , β -28 , β Cap+1 , β IntM , β CD14-15 , β CD17 , β CD26 , β CD27 / 28 , β IVS- I -1(G>T) , β IVS-I-1(G>A) , β IVS-I-5 , β CD71-72 , β CD43 , β CD41-42 , β CD31 and β IVS-II-654 Design 38 amplification primers; Design 8 amplification primers for the internal reference gene; Four amplification primers were designed for sex chromosome genes.

2. The amplification primer according to claim 1, characterized in that: The internal reference gene is one or more of GAPDH, ACTB, CFTR, HPRT, RPP30 or RPP40; The sex chromosome gene is one or more of AMEL, ZFX, ZFY, TAF9 or SRY.

3. The amplification primer according to claim 1, characterized in that: In the PCR amplification reaction, the primer contains at least one fluorescent group; the fluorescent group is selected from any one of FAM, VIC, HEX, JOE, TMR, NED, PET, and ROX.

4. The amplification primer according to claim 1, characterized in that: The nucleotide sequences of primers in the ζ2, Ψζ1, Ψα2, Ψα1, α2, α1 and θ1 gene regions of the α globin gene cluster are shown in SEQ ID NOs. 1-38; the nucleotide sequences of primers with known minor variations on the α2 gene are shown in SEQ ID NOs. 39-44; the primers with known minor variations on the β gene include SEQ ID NOs. 45-82; the nucleotide sequences of primers for internal reference genes are shown in SEQ ID NOs. 83-90; The nucleotide sequences of primers used to amplify sex chromosome genes are shown in SEQ ID NOs. 91-94.

5. A kit for detecting the copy number and minor variation of α and β globin genes, characterized in that: The kit comprises the amplification primers according to any one of claims 1 to 4; the kit also comprises an amplification reagent, an α / β normal control, an α / β positive control, and an α / β blank control.

6. The detection kit according to claim 5, characterized in that: The amplification reagent comprises an α / β reaction solution, an α / β primer mixture and a hot start polymerase; the α / β reaction solution comprises 10X Taq Buffer, MgCl2, dATP, dTTP, dGTP, dCTP and betaine; the α / β normal control is human genomic DNA of normal type for α or β-thalassemia; the α / β positive control is human genomic DNA of positive type for α or β-thalassemia; the blank control is a Tris buffer with a pH of 8.5; the α / β primer mixture is obtained by mixing the primers described in claim 4.

7. A method for detecting the copy number and minor variations of α and β globin genes for non-diagnostic purposes, characterized in that: The following steps are involved: (1) Add the DNA sample to be tested, α / β normal control, α / β positive control, and α / β blank control to the amplification reagent respectively; (2) subjecting the mixture obtained in step (1) to a PCR reaction, and after the reaction is completed, analyzing the PCR amplification product by fluorescent labeling capillary electrophoresis; (3) Capillary electrophoresis analysis separates amplification products with different fluorescence and fragment lengths and generates detection peak area information; (4) Copy number interpretation: S1: The 19 detection peak areas of each sample in the ζ2, Ψζ1, Ψα2, Ψα1, α2, α1 and θ1 gene regions, upstream or downstream sites, and the 18 detection peak areas of the normal type of minor mutations in the β-globin gene cluster are divided by the sum of the detection peak areas of the internal reference genes, and then the sample to be tested is divided by the corresponding value of each site of the α / β normal control, which is the detection peak ratio of each site. The corresponding copy number result is obtained by corresponding the value of the detection peak ratio to the corresponding copy number result, and the α-globin gene cluster is 0, 1, 2 or 3 copies; S2: The following genotypes can be determined by the copy number combinations at 19 loci within the α-globin gene cluster: αα / αα, -α 3.7 / αα, -α 4.2 / αα, -- SEA / αα, -- THAI / αα, -- FIL / αα, -- MEDI / αα, -- MEDII / αα, -α 20.5 / αα, -α 3.7 / -α 3.7 , -α 4.2 / -α 4.2 , -- SEA / -- SEA , -- THAI / -- THAI , -- FIL / -- FIL , -- MEDI / -- MEDI , -- MEDII / -- MEDII , -α 20.5 / -α 20. 5, -α 3.7 / -α 4.2 , -α 3.7 / -- SEA , -α 3.7 / -- THAI , -α 3.7 / -- FIL , -α 3.7 / -- MEDI , -α 3.7 / -- MEDII , -α 3.7 / -α 20.5 , -α 4.2 / -- SEA , -α 4.2 / -- THAI , -α 4.2 / -- FIL , -α 4.2 / -- MEDI , -α 4.2 / -- MEDII , -α 4.2 / -α 20.5 , -- SEA / -- THAI , -- SEA / -- FIL , -- SEA / -- MEDI , -- SEA / -- MEDII , -- SEA / -a 20.5 、-- THAI / -- FIL 、-- THAI / -- MEDI 、-- THAI / -- MEDII 、-- THAI / -a 20.5 、-- FIL / -- MEDI 、-- FIL / -- MEDII 、-- FIL / -a 20.5 、-- MEDI / - MEDII 、-- MEDI / -a 20.5 、-- MEDII / -a 20.5 、aaaa anti3.7 / aa、aaa anti4.2 / aa、-a 4.2 / aaaa anti3.7 、-- SEA / aaaa anti3.7 、-- THAI / aaaa anti3.7 、-- FIL / aaaa anti3.7 、-- MEDI / aaaa anti3.7 、-- MEDII / aaaa anti3.7 ,-a 20.5 / aaaa anti3.7 ,-a 3.7 / aaaa anti4.2 、-- SEA / aaaa anti4.2 、-- THAI / aaaa anti4.2 、-- FIL / aaaa anti4.2 、-- MEDI / aaaa anti4.2 、-- MEDII / aaaa anti4.2 ,-a 20.5 / aaaa anti4.2 、del HS-40 / aa、del HS-40 / del HS-40; S3: The β-globin gene was judged as normal type, heterozygous deletion type or homozygous deletion type by the copy number results of 18 loci within the β-globin gene cluster; Interpretation of minor variations: Minor variations are known in the α2 gene and the β-globin gene. The normal type, heterozygous mutant type or homozygous mutant type are interpreted based on the presence or absence of the detection peak.

8. The method according to claim 7, characterized in that: The PCR reaction procedure in step (2) is: 95°C, 5 min; 95°C 30 sec, 66°C 40 sec, 72°C 40 sec, 28 cycles; 72°C 45 min; stored at 4°C.

9. Use of the primers according to any one of claims 1 to 4 or the kit according to any one of claims 5 to 6 in products for nucleic acid detection of α- and β-thalassemia-related genes.

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