Multiplex PCR primer and probe combination and kit

Abstract

The present invention discloses a multiplex PCR primer and probe combination and a kit. anti3.7 and ααα anti4.2 A multiplex PCR primer and probe combination was constructed that can simultaneously detect the 19 α gene variations, including α-thalassemia, using a two-tube reaction system and the same reaction conditions. Based on the composition, the present invention immobilizes the probe sequences in the composition on a solid phase carrier to form a gene chip. Combined with multiplex PCR and flow-through hybridization, it achieves visual detection of the 19 α gene variations and the genotypes of the tested samples. The detection has good specificity, high accuracy and sensitivity, and is simple to operate, time-saving, and easy to read results. It is conducive to the carrier screening of α gene variations and can provide an accurate reference for clinical diagnosis or genetic counseling of α-thalassemia.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection technology, and more specifically relates to a multiplex PCR primer and probe combination and a kit. Background Art

[0002] α-thalassemia (α-thalassemia) is a group of inherited chronic hemolytic anemias caused by variations (mutation, deletion, or recombination) in the α-globin gene (α gene), resulting in the absence or reduced synthesis of α-globin. It is one of the most common single-gene genetic diseases, with many people carrying α-gene mutations. The severity of α-thalassemia increases with the number of α-gene mutations, and the disease is clinically categorized as quiescent, mild, intermediate, and severe. Currently, there is no cost-effective treatment for α-thalassemia. Screening for α-gene mutations has become a crucial measure to control the birth of children with moderate and severe α-thalassemia, thereby reducing the incidence of α-thalassemia.

[0003] α gene mutation types include deletion type and non-deletion type (point mutation, recombination); deletion type mutation accounts for the majority. Common α gene deletion type mutations include: SEA 、-α 3.7 and -α 4.2 , common α gene point mutations include QS, CS and WS, accounting for about 95% to 96.3% of all α gene mutations. Although most existing α gene mutation or α-thalassemia genotype detection kits can detect the above common α gene mutations, with the further elucidation of the pathogenesis of α-thalassemia, some new α-thalassemia genotypes have been discovered and reported. The original α gene mutation or α-thalassemia genotype detection products can no longer meet the current detection needs and are prone to missed detection. For example, the fusion gene is an α gene mutation caused by homologous recombination of the α2 gene and the ψα1 gene, and its clinical phenotype is α + Thalassemia, it is of great significance to carry out screening for carriers of this mutation. anti3.7 and ααα anti4.2 α-triplet duplication (α-triplet duplication) is caused by mispairing and unequal exchange between homologous chromosomes in normal cells. Alone, α-triplet duplication does not lead to abnormal hematological parameters. However, when α-triplet duplication is combined with β-gene mutation, the increased number of alleles exacerbates the imbalance in the α-globin / β-chain ratio, potentially leading to more severe anemia.

[0004] In summary, there is an urgent need to establish a kit and method that can easily and quickly detect more than 99% of common α gene mutations, avoid missing α gene mutations that are subsequently discovered, and reduce the difficulty and cost of detection, so as to facilitate the screening and genetic diagnosis of people carrying α gene mutations and prevent the occurrence of α-thalassemia. Summary of the Invention

[0005] The present invention aims to solve the above-mentioned deficiencies in the prior art and provides a multiplex PCR primer and probe combination and a kit. The combination or kit can be used to detect fusion gene, ααα anti3.7 and ααα anti4.2 The 19 α gene variations, including α-thalassemia, are detected to detect the genotype of the tested samples, thereby providing a reference for the clinical diagnosis or genetic counseling of α-thalassemia.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The present invention targets 7 deletion types (-α 3.7 、-α 4.2 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 ), 9 mutant types (Init CD ATG>AG, CD30 -GAG, CD35 TCC>CCC, CD59 GGC>GAC, Poly A AATAAA>AATGAA, CD122 CAC>CAG, CD125 CTG>CCG, CD142 TAA>CAA, CD142 TAA>TAT) and 3 recombinant types (ααα anti3.7 、ααα anti4.2 , Fusion gene) α gene variation, and designed a multiplex PCR primer and probe composition that can simultaneously detect the above 19 α gene variations using a two-tube reaction system and the same reaction conditions. Based on the composition, the present invention immobilizes the probe sequences in the composition on a solid phase carrier to prepare a corresponding gene chip, and combines multiplex PCR and flow-through hybridization to achieve visual detection of the above 19 α gene variations. The operation is simple, time-saving, and the results are easy to read, which is conducive to the carrier screening of α gene variations. Therefore, the present invention requests protection for the multiplex PCR primer and probe composition.

[0008] Specifically, the composition of the present invention includes composition 1 and composition 2; composition 1 contains PCR amplification primers shown in SEQ ID NOs. 1 to 15 and probes shown in SEQ ID NOs. 22 to 29; composition 2 contains PCR amplification primers shown in SEQ ID NOs. 1 and 16 to 20 and probes shown in SEQ ID NO. 22 and SEQ ID NOs. 30 to 48.

[0009] The composition of the present invention can be used to detect the above 19 α gene variations. Therefore, the present invention also claims protection for the use of the multiplex PCR primer and probe composition in preparing a product for detecting one or more of the 19 α gene variations.

[0010] Specifically, the 19 α gene mutations are -α 3.7 、-α 4.2 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 , InitCD ATG>AG, CD30 -GAG, CD35 TCC>CCC, CD59 GGC>GAC, Poly A AATAAA>AATGAA, CD 122CAC>CAG, CD125 CTG>CCG, CD142 TAA>CAA, CD142 TAA>TAT, ααα anti3.7 、ααα anti4.2 and Fusiongene.

[0011] Specifically, in the composition, the primers shown in SEQ ID NO.1 and SEQ ID NO.2 and the probe shown in SEQ ID NO.24 are used to detect -α 3.7 mutation; primers shown in SEQ ID NO.3 and SEQ ID NO.2 and probe shown in SEQ ID NO.27 are used to detect -α 20.5 mutation; primers shown in SEQ ID NO.4 and SEQ ID NO.5 and probe shown in SEQ ID NO.22 are used to detect-- SEA mutation; primers shown in SEQ ID NO.6 and SEQ ID NO.7 and probe shown in SEQ ID NO.25 are used to detect -- FIL mutation; primers shown in SEQ ID NO.8 and SEQ ID NO.9 and probe shown in SEQ ID NO.26 are used to detect-- THAI mutation; primers shown in SEQ ID NO.10 and SEQ ID NO.11 and probe shown in SEQ ID NO.23 are used to detect -- MED I mutation; primers shown in SEQ ID NO.12 and SEQ ID NO.13 and probe shown in SEQ ID NO.28 are used to detect ααα anti3.7 mutation; primers shown in SEQ ID NO.14 and SEQ ID NO.15 and probe shown in SEQ ID NO.29 are used to detect ααα anti4.2mutation; primers shown in SEQ ID NO.1 and SEQ ID NO.16 and probes shown in SEQ ID NO.32 to 47 are used to detect the above 9 mutations; primers shown in SEQ ID NO.17 and SEQ ID NO.18 and probe shown in SEQ ID NO.31 are used to detect -α 4.2 mutation; the primers shown in SEQ ID NO.19 and SEQ ID NO.20 and the probe shown in SEQ ID NO.48 are used to detect Fusiongene mutation; the probe shown in SEQ ID NO.30 is the normal control probe corresponding to the deletion type and recombinant type α gene mutations.

[0012] The present invention achieves visual detection of the aforementioned 19 α gene mutations by immobilizing the probe sequences in the composition on a solid-phase carrier to form a corresponding gene chip. Specifically, the solid-phase carrier of the gene chip immobilizes probes with nucleotide sequences shown in SEQ ID NOs. 22 to 48 and a colorimetric control probe, the nucleotide sequence of which is shown in SEQ ID NO. 21.

[0013] Specifically, the 5' end of the probe having the nucleotide sequence as shown in SEQ ID NOs. 22 to 48 is amino-modified, and the color development system controls the 5' end of the probe to be bound to a label.

[0014] Specifically, the label is biotin.

[0015] Optionally, the solid phase carrier is nitrocellulose, cellulose acetate, glass sheet, silica gel wafer, nylon membrane, polypropylene membrane or micro-magnetic beads.

[0016] In a specific embodiment of the present invention, the solid phase carrier used is a nylon membrane.

[0017] Specifically, the concentration of the probe solution used in preparing the chip is 5-25 μM.

[0018] Preferably, the concentration of the probe solution is 20-25 μM.

[0019] More preferably, the concentration of the probe solution is 20 μM.

[0020] In a specific embodiment of the present invention, the gene chip includes a gene chip 1 and a gene chip 2; the gene chip 1 is used to detect -α 3.7 、-- SEA 、-- Thai 、-- Fil 、-- MED I 、-α 20.5 、ααα anti3.7 and αααanti4.2 variation; the gene chip 2 is used to detect the remaining variations among the 19 variations; the N and M marked on the gene chip refer to the normal control point and the mutation site, respectively; for example, Init N refers to the normal control point (wild type) of Init CD ATG>AG, and Init M refers to Init CD ATG>AG.

[0021] The present invention also provides a kit that, when combined with multiplex PCR and flow-through hybridization, can be used to visually detect the aforementioned 19 α gene variants. Specifically, the kit contains PCR amplification primers with nucleotide sequences as shown in SEQ ID NOs. 1-20 and the aforementioned gene chip; each of the PCR amplification primers has a label attached to its 5' end. Specifically, the label is biotin.

[0022] Specifically, the kit also contains reagents required for PCR amplification reaction and flow-through hybridization reaction.

[0023] When using the kit described herein for detection, since Composition 1 primarily detects deletional and recombinant variants, resulting in very long fragments, GAP-PCR cannot amplify negative samples normally. Therefore, the colorimetric control probe on Gene Chip 1 corresponding to Composition 1 is used as a monitoring tool. The primers represented by SEQ ID NO. 1 and SEQ ID NO. 16 in Composition 2 can amplify normal fragments and serve as internal standard primers.

[0024] The present invention also claims protection for the use of the kit in preparing a product for detecting one or more of the 19 α gene variations.

[0025] Specifically, the 19 α gene mutations are -α 3.7 、-α 4.2 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 , InitCD ATG>AG, CD30 -GAG, CD35 TCC>CCC, CD59 GGC>GAC, Poly A AATAAA>AATGAA, CD 122CAC>CAG, CD125 CTG>CCG, CD142 TAA>CAA, CD142 TAA>TAT, ααα anti3.7 、ααα anti4.2 and Fusiongene.

[0026] The use of the multiplex PCR primer and probe composition or kit of the present invention in the preparation of α-thalassemia diagnostic products should also be within the scope of protection of the present invention.

[0027] The present invention also provides a method for detecting the 19 α gene mutations using the kit, comprising the following steps:

[0028] S1. Extract genomic DNA from the sample to be tested;

[0029] S2. Using the obtained genomic DNA as a template, multiplex PCR reactions were performed using the primers shown in Group 1 and Group 2, respectively. The reaction procedure was as follows: 35 cycles of hot start at 95°C for 15 min, denaturation at 98°C for 40 sec, annealing at 62°C for 1 min, and extension at 72°C for 2 min, with a final extension step at 72°C for 5 min. In the multiplex PCR reaction system, the final concentration of the PCR amplification primers was 0.1–0.5 μM. Mg 2+ The final concentration of Taq was 1.0-3.0 mM; the amount of Taq enzyme used was 2.5 U; and the amount of sample genomic DNA used was 80-140 ng.

[0030] S3. Use gene chips to perform detection and interpret the results.

[0031] Preferably, in the multiplex PCR reaction system, the final concentration of the PCR amplification primers is 0.3-0.4 μM; Mg 2+ The final concentration is 1.5-2.5 mM; the amount of sample genomic DNA is 100-120 ng.

[0032] Further preferably, in the multiplex PCR reaction system, the final concentration of the PCR amplification primer is 0.3 μM; Mg 2+ The final concentration was 1.5-2.0 mM; the amount of sample genomic DNA was 100 ng.

[0033] The present invention has the following beneficial effects:

[0034] Targeting Fusion gene, ααα anti3.7 and ααα anti4.2 The present invention has constructed a multiplex PCR primer and probe composition that can simultaneously detect the 19 α gene variations, including 19 α gene variations, using a two-tube reaction system and the same reaction conditions. Based on the composition, the present invention fixes the probe sequence in the composition on a solid phase carrier to prepare a gene chip, and combines multiplex PCR and flow-through hybridization to achieve visual detection of the 19 α gene variations and the genotype of the tested samples. The detection has good specificity, high accuracy and sensitivity, simple operation, short time consumption, and easy-to-read results, which is conducive to the carrier screening of α gene variations and can provide an accurate reference for the clinical diagnosis or genetic counseling of α-thalassemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1Schematic diagram of the gene chip prepared by the present invention.

[0036] Figure 2 These are the test results of the 20 validation samples and blank controls using the kit of the present invention.

[0037] Figure 3 These are the test results of test samples 1 to 15 using the kit of the present invention.

[0038] Figure 4 These are the test results of test samples 16 to 30 using the kit of the present invention.

[0039] Figure 5 These are the test results of test samples 31 to 45 using the kit of the present invention.

[0040] Figure 6 These are the test results of test samples 46 to 60 using the kit of the present invention.

[0041] Figure 7 These are the test results of test samples 61 to 75 using the kit of the present invention.

[0042] Figure 8 These are the test results of test samples 76 to 90 using the kit of the present invention.

[0043] Figure 9 These are the test results of test samples 91 to 105 using the kit of the present invention.

[0044] Figure 10 These are the test results of test samples 106 to 120 using the kit of the present invention. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] Unless otherwise specified, all reagents and materials used in the following examples were commercially available.

[0047] Example 1 Obtaining multiplex PCR primer and probe combinations

[0048] 1. Based on the "Consensus of Clinical Genetic Counseling Experts on Genetic Testing for α-thalassemia" and other materials, this paper summarizes the relatively common α gene variations, including 7 deletion types, 9 mutation types, and 3 recombinant α gene variations. The information on the mutation sites and mutation types of the α gene variations is shown in Table 1; +Thalassemia refers to the deletion or functional defect (e.g., point mutation) of a single α-globin gene, resulting in a partial reduction in the expression of the gene but not a complete loss of function; 0 Thalassemia occurs when both alpha-globin genes on the same chromosome are missing or completely inactivated, resulting in the inability of that chromosome to produce any alpha chains.

[0049] Table 1 Common α gene mutations

[0050]

[0051] For the 19 α-thalassemia gene variants shown in Table 1, the present invention first designed multiple separate primers / probes and performed single-plex screening. Based on the resulting primer pairs and probes, cross-reaction testing was performed to eliminate nonspecific amplification and binding between the primers / probes, as well as mutual inhibition. After repeated testing and optimization, a set of multiplex PCR primers and probe compositions was finally obtained that can simultaneously detect the above 19 α-thalassemia gene variants using a two-tube reaction system.

[0052] The multiplex PCR primer and probe composition includes composition 1 and composition 2; wherein, composition 1 detects eight α-thalassemia gene variations, namely: -α 3.7 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 、ααα anti3.7 、ααα anti4.2 , including primers 1 to 15 and probes 1 to 9; Composition 2 detects 11 α-thalassemia gene mutations, namely -α 4.2 , Init CD ATG>AG, CD30 -GAG, CD35 TCC>CCC, CD59 GGC>GAC, Poly A AATAAA>AATGAA, CD 122 CAC>CAG, CD125 CTG>CCG, CD142 TAA>CAA, CD142 TAA>TAT, Fusion gene, including primer 1, primers 16 to 20, probe 1, and probes 10 to 28. The detection targets of the primers 1 to 20 and probes 1 to 28 and their nucleotide sequences are shown in Tables 2 and 3, respectively.

[0053] Table 2 Nucleotide sequences of primers in multiplex PCR primer and probe combinations

[0054]

[0055] Note: The target point mutations mentioned in the table refer to the nine mutant α-thalassemia genes, namely, Init CD ATG>AG, CD30 -GAG, CD35 TCC>CCC, CD59 GGC>GAC, Poly A AATAAA>AATGAA, CD 122 CAC>CAG, CD125 CTG>CCG, CD142 TAA>CAA, and CD142 TAA>TAT; the 5' ends of the primers are all labeled with biotin.

[0056] Table 3 Nucleotide sequences of probes in multiplex PCR primer and probe combinations

[0057]

[0058] Note: Probe 10 (NP) is the normal control probe corresponding to the deletion and recombinant α-thalassemia genes, and probe 1 (Bio) is the color development system control probe.

[0059] Example 2 Gene Chip and Detection Kit

[0060] Based on the multiplex PCR primers and probes described in Example 1, the present invention has developed a kit for detecting the aforementioned 19 α-thalassemia gene mutations, comprising the multiplex PCR primers and a gene chip prepared using the probes. The gene chip comprises a fixed support and probes fixed to the fixed support, namely, the probe sequences shown in SEQ ID NOs. 21 to 48; the 5′ end of the probes is amino-treated (amino group), and the color development system control probes are labeled with biotin.

[0061] 1. Preparation of gene chips

[0062] (1) Probe arrangement

[0063] The immobilization carrier used in preparing the gene chip in this example was a nylon membrane (2 sheets). Membrane 1 (hereinafter referred to as gene chip 1, Thal a8MY) was divided into 9 small cells, each of which contained probes 1 to 9. The specific positions of the probes are shown in Table 4 (indicated by the corresponding detection targets).

[0064] Table 4

[0065]

[0066] Membrane 2 (hereinafter referred to as gene chip 2, Thal a11MY) is divided into 20 small grids, each of which is fixed with probe 1 and probes 10 to 28 in sequence. The specific positions of the probes are shown in Table 5 (indicated by the detection target corresponding to the probe);

[0067] Table 5

[0068]

[0069] (2) Treatment of nylon membrane

[0070] To treat the nylon membrane, first soak it in 0.1 M HCl solution for 30 seconds. Then, after removing the residual solution, soak the membrane in 20% EDAC solution for 15 minutes. Finally, place it in a membrane washing tray and rinse it with 200 mL of purified water for 10 seconds. Repeat this step four times and place it on absorbent paper to remove excess residual liquid. Transfer the membrane to a drying oven at a temperature of 20°C and a humidity of 45% and dry it for 12 hours. Separate the dried nylon membrane with Kimwipes paper, transfer it to a sealed film bag, and store it at 4°C until used.

[0071] (3) Sample

[0072] The synthesized probes (probes shown in Table 3) were dissolved in probe diluent (a mixture of 0.5 M Na2CO3 and 0.5 M NaHCO3 in a volume ratio of 1:99, pH = 8.4). The prepared probe solutions were then spotted on the nylon membrane using a micropipette (the distribution of the probes on the membranes is shown in Tables 4 and 5, respectively), with 0.4 μL per drop. After spotting, the membranes were placed at room temperature for 15 minutes for reaction. The membranes were then transferred to a 0.1 M NaOH solution and soaked for 10 minutes. Subsequently, the membranes were placed in a membrane washing tray and rinsed with 200 mL of purified water for 10 seconds. This step was repeated four times, and the membranes were placed on absorbent paper to remove excess liquid. The washed membranes were then placed in a drying oven at 20°C and 45% humidity for 12 hours to produce gene chips.

[0073] The schematic diagram of the gene chip prepared by the present invention is as follows Figure 1 As shown, the ranking of the detected variant sites is marked on the figure.

[0074] 2. Detection kit

[0075] In addition, the present invention also provides a kit for detecting the above-mentioned 19 α-thalassemia gene mutations, which contains primers 1 to 20 shown in Table 2 (the primers are divided into two groups, group 1 contains primers 1 to 15, and group 2 contains primer 1 and primers 16 to 20), the prepared gene chip (Thal a8MY and Thal a11MY), reagents required for PCR reaction (PCR buffer, dNTPs, MgCl2, Taq enzyme) and sterile water; the 5' end of all primer pairs in the kit is labeled with biotin.

[0076] 3. How to use the kit

[0077] Based on the kit, the present invention also provides a method for detecting the 19 α-thalassemia gene mutations, comprising the following steps:

[0078] (1) Extract genomic DNA from the sample to be tested;

[0079] (2) Using the obtained genomic DNA as a template, multiplex PCR reactions were performed using the primers shown in Group 1 and Group 2, respectively. The reaction systems are shown in Tables 6 and 7, respectively. The reaction procedures (all the same) were as follows: hot start at 95°C for 15 min, denaturation at 98°C for 40 sec, annealing at 62°C for 1 min, extension at 72°C for 2 min, 35 cycles, and a final extension step at 72°C for 5 min.

[0080] Among them, the PCR amplification reaction system of the primers contained in Group 1 is shown in Table 6: the reagents used for amplification are HotStarTaq DNA Polymerase enzyme with catalog number 203203 purchased from Qiagen and its matching 10× PCR Buffer, Q-solution (gene amplification auxiliary reagent) and 25 mM MgCl2.

[0081] Table 6

[0082]

[0083] The PCR amplification reaction system of the primers contained in Group 2 is shown in Table 7.

[0084] Table 7

[0085]

[0086] Note: The 5' end of the primers are labeled with biotin.

[0087] The present invention can amplify multiple α-thalassemia gene mutations under the same conditions, reducing the amount of PCR reagents and the demand for PCR instruments, reducing the number of operating steps, and lowering costs.

[0088] (3) Using gene chips for testing and interpretation of results

[0089] The obtained amplified product was denatured at 95°C for 5 min, quickly transferred to an ice box, placed for 2 min, and then added to 0.8 mL of hybridization solution (2×SSC / 0.1% SDS) pre-warmed to 44°C. After mixing, it was placed in the reaction well of the hybridizer and applied to the prepared gene chip. It was hybridized at 44°C for 30 min, washed four times with solution WB1 (0.5×SSC / 0.1% SDS, warmed at 44°C), and 0.5 mL of blocking solution (0.25% skim milk powder, 0.05% thimerosal) was added. It was blocked at 25°C for 5 min, dried, and added with 0.5 mL of enzyme labeling solution (AP enzyme labeled with streptavidin dissolved in TBS). The enzyme was labeled for 5 minutes, washed four times with 0.8 mL of solution A (TBS, 0.1% Tween20 and 0.05% sodium azide), and 0.5 mL of solution A was added. mL of color development solution (NBT / BCIP), develop color for 5 minutes in the dark, and finally rinse three times with hybridization solution (2×SSC / 0.1%SDS), dry, analyze the color development, and interpret the results.

[0090] The method for interpreting gene chip results is:

[0091] ① Blue-purple spots of similar color appear at the mutation detection probe and the corresponding negative control probe of the gene chip. The corresponding sites are heterozygous for α-thalassemia gene mutation, α-thalassemia gene deletion, or α-thalassemia gene recombination;

[0092] ② Blue-purple spots appear at the mutation detection probe of the gene chip, while no blue-purple spots appear at the corresponding negative control probe. The corresponding sites are α-thalassemia gene mutations, α-thalassemia gene deletions, or α-thalassemia gene recombination homozygotes;

[0093] ③ If the gene chip only shows blue-purple spots at the Bio site and the negative control probe, no α-thalassemia gene mutation, α-thalassemia gene deletion, or α-thalassemia gene recombination was detected;

[0094] ④ No blue-purple spots appear at the Bio site of the gene chip, indicating the test is invalid;

[0095] ⑤ No blue-purple spots appeared at all mutation detection probes and negative control probes in the gene chip, indicating that the test was invalid;

[0096] ⑥ If no blue-purple spots appear at a mutation detection probe and the corresponding negative control probe on the gene chip, while the test results at other probes are normal, a new mutation type may have occurred at this site and further sequencing analysis is required.

[0097] Example 3 Test kit detection effect test

[0098] In order to test whether the kit described in Example 2 of the present invention can accurately detect the 19 α-thalassemia gene mutations, the present invention used 20 human peripheral venous blood samples with known genotypes (the sample numbers and their corresponding genotypes (carried mutations) are shown in Table 8) as validation samples, and used the DNA of the validation samples as templates to test the detection effect of the kit. Sterile water was added during the test as validation sample 21 (blank control).

[0099] The test results of the 20 validation samples and blank controls using the kit of the present invention are as follows: Figure 2 and shown in Table 8.

[0100] Table 8 Variants carried by validation samples and kit detection results

[0101]

[0102] Note: Negative samples are samples in which none of the 7 deletion-type α-thalassemia genes, 3 recombinant α-thalassemia genes, and 9 mutant α-thalassemia genes are mutated.

[0103] Depend on Figure 2 It can be seen that only blue-purple spots appear in the color development results of the blank control; the verification sample was tested using the kit shown in Example 2, and the test results were consistent with the actual genotype of the sample, indicating that the kit shown in Example 2 can be used to detect α-thalassemia gene mutation, deletion and recombination.

[0104] Example 4 Test kit detection performance test

[0105] 1. Accuracy and specificity testing

[0106] 120 human peripheral venous blood samples with known genotypes were used as test samples (test samples 1 to 120). The DNA concentration of the test samples was controlled at 20 to 40 ng / μL. The accuracy and specificity of the kit prepared in Example 2 were tested as follows:

[0107] 120 test samples were respectively subjected to result judgment using the kit shown in Example 2 in combination with its usage method to obtain the test results of the test samples; PCR amplification was performed on a Biori gene amplifier, and flow-through hybridization was performed on a Hybribio medical nucleic acid molecular hybridizer HBHM-3001S.

[0108] Control group: Mutation samples were detected by Sanger sequencing, and deletion and recombinant samples were detected by agarose gel electrophoresis.

[0109] 2. Detection sensitivity test

[0110] Nineteen heterozygous DNA samples with known genotypes were used as sensitivity test samples (sensitivity test sample 1 to sensitivity test sample 19). Two DNA concentrations were set for each sensitivity test sample for testing; the two DNA concentrations were 2 ng / μL and 105 ng / μL, respectively.

[0111] The sensitivity test samples were respectively judged using the kit shown in Example 2 in combination with its usage method to obtain the test results of the sensitivity test samples; each sensitivity test sample was tested 20 times, repeated 3 times and the test results of each time were recorded; "+" represents the clarity of the test results, and the more "+"s, the clearer the test results, which is manifested as a darker color development. Among them, the color development of the "+++" test results is the same as that of the Figure 2 same.

[0112] 3. Experimental results

[0113] (1) The test results of the kit’s accuracy and specificity are shown in Table 9. The color development results of test samples 1 to 15 are shown in the figure below. Figure 3 As shown, the color development results of test samples 16 to 30 are shown in the figure. Figure 4 As shown in the figure, the color development results of test samples 31 to 45 are as follows Figure 5 As shown, the color development results of test samples 46 to 60 are shown in the figure. Figure 6 As shown in the figure, the color development results of test samples 61 to 75 are as follows Figure 7 As shown in the figure, the color development results of test samples 76 to 90 are as follows Figure 8 As shown, the color development results of test samples 91 to 105 are shown in the figure Figure 9 As shown, the color development results of test samples 106 to 120 are shown in FIG. Figure 10 shown.

[0114] Table 9 Accuracy and specificity test results of the kit

[0115]

[0116] Combine Figures 3 to 10As shown in Table 9, when the kit of the present invention was used to detect thalassemia gene mutations in 120 test samples, the thalassemia gene mutation detection results were consistent with the reagent gene mutation results for each test sample, with an accuracy rate of 100%. In addition, when the kit of Example 2 was used to detect β-thalassemia and non-deletion β-thalassemia genotype-positive samples (test samples 100-105) and negative samples, the test results were all negative, with a negative consistency rate of 100% and a specificity of 100%, indicating that the kit of Example 2 can only specifically detect α-thalassemia gene mutations or deletions.

[0117] The detection sensitivity test results of the kit are shown in Table 10.

[0118] Table 10 Detection sensitivity test results of the kit

[0119]

[0120] The sensitivity test results show that when the kit shown in Example 2 was used to detect thalassemia gene mutations in 20 test samples, the test was repeated 20 times in two DNA concentration ranges of 2 ng / μL and 105 ng / μL, and the hybridization results presented were clearly visible and the signals were strong; therefore, the lower limit of detection of the kit shown in Example 3 is 2 ng / μL (10 ng / reaction), and the upper limit of detection is 105 ng / μL.

[0121] Example 5 Kit Optimization

[0122] 1. Optimization of probe concentration on gene chip

[0123] Referring to the gene chip preparation method described in Example 2, corresponding gene chips were prepared in this example using probe solutions of varying concentrations. The 20 validation samples described in Example 3 were used as test samples, and the detection performance of the gene chips prepared using probe solutions of varying concentrations was tested using the test method described in Example 2. The probe concentrations tested in this example were 5, 10, 15, 20, and 25 μM, respectively. The test results are shown in Table 11.

[0124] Table 11 Optimization test results of probe concentration on gene chip

[0125]

[0126] The results shown in Table 11 show that when the probe concentration is 5 to 25 μM, the detection results are consistent and non-specific. Among them, when the probe concentration is 20 to 25 μM, the detection results are relatively clear and non-specific, and the detection results are consistent with the known genotype of the sample. When preparing the gene chip, the probe concentration of the probe liquid should be 20 μM.

[0127] 2. Optimization of reaction system

[0128] (1) Optimization of primer concentration

[0129] The 20 validation samples described in Example 3 were used as test samples. Referring to the reaction system described in Example 2, the final concentrations of each primer in the multiplex PCR reaction system were adjusted to 0.1, 0.2, 0.3, 0.4, and 0.5 μM, respectively. These were designated as experimental groups 1 to 5, and the results are shown in Table 12.

[0130] Table 12 Test results of experimental groups 1 to 5

[0131]

[0132] The results shown in Table 12 show that when the concentration of primers 4 and 5 in the multiplex PCR reaction system A was 0.1 μM, the detection result for (SEA, sample 3) was the clearest and there was no nonspecificity; when the concentration of primers 14 and 15 was 0.2 μM, the detection result for (anti4.2, sample 8) was the clearest and there was no nonspecificity; when the concentration of primers 1, 6 to 9, 12, and 13 was 0.3 μM, the detection result for (3.7, FIL, THAI, anti3.7, samples 1, 4, 5, and 7) was the clearest and there was no nonspecificity; when the concentration of primers 10 and 11 was 0.4 μM, the detection result for (MED, sample 6) was the clearest and there was no nonspecificity; when the concentration of primer 3 was 0.5 μM and the concentration of primer 2 was 0.8 μM, the detection result for (20.5, sample 2) was the clearest and there was no nonspecificity; when the concentration of primers 17 and 18 in the multiplex system B was 0.5 μM, the detection result for primer 17 and primer 18 was 0.5 μM. At a concentration of 0.1 μM, the detection results for the deletion-type α-thalassemia gene (4.2, sample 18) were clearest and free of nonspecificity. At a concentration of 0.3 μM, the detection results for the other samples (samples 9 to 17, and sample 19) were clearest and free of nonspecificity, and the test results were consistent with the known genotypes of the samples. When the primers in Multiplex PCR Systems A and B are outside the above ranges, the clarity and specificity of the results for the combined detection of multiple variants in the α-thalassemia gene are somewhat reduced.

[0133] (2) Mg 2+ Optimization of ion concentration

[0134] The 20 validation samples described in Example 3 were used as test samples. Referring to the reaction system described in Example 2, the Mg content in the multiplex PCR reaction system was adjusted. 2+ The concentrations were 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM, which were respectively recorded as experimental groups 6 to 10. The results are shown in Table 13.

[0135] Table 13 Test results of experimental groups 6 to 11

[0136]

[0137] As shown in Table 13, the Mg content in the multiplex PCR reaction system A was 2+ At a concentration of 2 mM, the detection results for 6 deletion-type and 2 recombinant α-thalassemia genes (samples 1 to 8) were clear and non-specific; Mg in multiplex PCR reaction system B 2+ At a concentration of 1.5 mM, the detection results for nine mutant, one deletion, and one recombinant α-thalassemia gene (samples 9 to 19) were clear and nonspecific. Therefore, the volume of MgCl2 added to multiplex PCR reaction system A was 4 μL, and the volume of MgCl2 added to multiplex PCR reaction system B was 3 μL.

[0138] (3) Optimization of Taq enzyme addition amount

[0139] The 20 validation samples described in Example 3 were used as test samples. Referring to the reaction system described in Example 2, the amount of Taq enzyme added in the multiplex PCR reaction system was adjusted to 1.5, 2.0, 2.5, 3.0, and 3.5 U, which were respectively recorded as experimental groups 11 to 15. The results are shown in Table 14.

[0140] Table 14 Test results of experimental groups 12 to 16

[0141]

[0142] The results shown in Table 14 show that when the added volume of Taq enzyme in Multiplex PCR System A and Multiplex PCR System B was 0.5 μL (Taq added amount was 2.5 U), the detection results for the 20 samples were clear and non-specific, and the detection results were consistent with the known genotypes of the samples. However, when the added volume of Taq enzyme in Multiplex PCR System A and Multiplex PCR System B was 0.3 μL (Taq added amount was 1.5 U), the detection results for the 20 samples were relatively unclear, and the color development was relatively shallow. When the added volumes of Taq enzyme in Multiplex PCR System A and Multiplex PCR System B were 0.4 μL, 0.6 μL, and 0.7 μL (Taq added amounts were 2.0 U, 3.0 U, and 3.5 U), the detection results of some of the 20 samples were relatively unclear and non-specific.

[0143] (4) Optimization of the amount of sample to be tested

[0144] The 20 validation samples described in Example 3 were used as test samples. Referring to the reaction system described in Example 2, the added volumes of the test samples in the multiplex PCR reaction system were adjusted to 2, 3, 4, 5, 6, and 7 μL (sample concentration was 20 ng / μL). These were designated as experimental groups 17 to 22, respectively. The results are shown in Table 15.

[0145] Table 15 Test results of experimental groups 17 to 22

[0146]

[0147] The results shown in Table 15 show that when the added volume of the test samples in Multiplex PCR System A and Multiplex PCR System B was 5 μL, the test results for all 20 samples were clear and nonspecific, and the test results were consistent with the known genotypes of the samples. However, increasing or decreasing the added volume of the test samples would reduce the clarity and specificity of the test results.

[0148] (5) Optimization of multiplex PCR amplification program

[0149] The 20 validation samples described in Example 3 were used as test samples. After the reaction system was prepared according to Example 2, amplification was performed using the following different reaction procedures. The results are shown in Table 16.

[0150] The multiplex PCR amplification program 1 was as follows: 95°C, 15 min; 98°C, 35 s, 60°C, 1 min, 72°C, 2 min, 30 cycles; 72°C, 5 min.

[0151] The multiplex PCR amplification program 2 was as follows: 95°C, 15 min; 98°C, 40 s, 62°C, 1 min, 72°C, 2 min, 35 cycles; 72°C, 5 min.

[0152] Multiplex PCR amplification program 3 was as follows: 95°C, 15 min; 97°C, 35 s, 60°C, 50 s, 72°C, 150 s, 40 cycles; 72°C, 7 min.

[0153] Multiplex PCR amplification program 4 was as follows: 95°C, 15 min; 98°C, 40 s, 64°C, 50 s, 72°C, 150 s, 35 cycles; 72°C, 5 min.

[0154] Table 16 Detection results of multiplex PCR amplification procedures 1 to 4

[0155]

[0156] The results shown in Table 16 indicate that the multiplex PCR reaction system A and multiplex PCR reaction system B in the kit of Example 2 produced clear and nonspecific detection results for the 20 samples only when combined with multiplex PCR amplification procedure 2, and the detection results were consistent with the known genotypes of the samples. When combined with multiplex PCR amplification procedures 1, 3, and 4, the clarity and specificity of the detection results of the samples decreased.

[0157] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A multiplex PCR primer and probe combination, characterized in that: It includes composition 1 and composition 2; the composition 1 includes PCR amplification primers with nucleotide sequences as shown in SEQ ID NOs. 1 to 15 and probes with nucleotide sequences as shown in SEQ ID NOs. 21 to 29; the composition 2 includes PCR amplification primers with nucleotide sequences as shown in SEQ ID NOs. 1 and 16 to 20 and probes with nucleotide sequences as shown in SEQ ID NO. 21 and SEQ ID NOs. 30 to 48.

2. Use of the multiplex PCR primer and probe combination according to claim 1 in the preparation of a product for detecting 19 α-globin gene mutations, characterized in that: The 19 α-globin gene mutations are -α 3.7 、-α 4.2 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 , Init CD ATG>AG, CD30 -GAG, CD35 TCC>CCC, CD59 GGC>GAC, Poly AAATAAA>AATGAA, CD 122 CAC>CAG, CD125 CTG>CCG, CD142 TAA>CAA, CD142 TAA>TAT, ααα anti3.7 、ααα anti4.2 and Fusion gene.

3. A gene chip, characterized in that: Probes with nucleotide sequences as shown in SEQ ID NOs. 21 to 48 are fixed on the solid phase carrier of the chip; wherein the probe with nucleotide sequence as shown in SEQ ID NO. 21 is a control probe of the color development system.

4. The gene chip according to claim 3, characterized in that The 5' end of the probe with the nucleotide sequence shown in SEQ ID NOs. 22 to 48 is amino-modified, and the color development system controls the 5' end of the probe to be bound to a marker.

5. The gene chip according to claim 4, characterized in that The solid phase carrier is nitrocellulose, cellulose acetate, glass sheet, silica gel wafer, nylon membrane, polypropylene membrane or micro-magnetic beads.

6. The gene chip according to any one of claims 3 to 5, characterized in that The concentration of the probe solution used in preparing the chip is 5-25 μM.

7. The gene chip according to claim 6, characterized in that The concentration of the probe solution is 20-25 μM.

8. A kit, characterized in that The kit contains PCR amplification primers with nucleotide sequences as shown in SEQ ID NOs. 1 to 20 and the gene chip according to any one of claims 3 to 7; the 5' ends of the PCR amplification primers are all combined with markers.

9. The kit according to claim 8, characterized in that The kit also contains reagents required for PCR amplification reaction and flow-through hybridization.

10. Use of the kit according to claim 8 or 9 in preparing a product for detecting 19 α-globin gene mutations, characterized in that: The 19 α-globin gene mutations are -α 3.7 、-α 4.2 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 , Init CD ATG>AG, CD30 -GAG, CD35 TCC>CCC, CD59 GGC>GAC, Poly A AATAAA>AATGAA, CD 122 CAC>CAG, CD125 CTG>CCG, CD142 TAA>CAA, CD142 TAA>TAT, ααα anti3.7 、ααα anti4.2 and Fusion gene.

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