Multiplex PCR primer and probe composition and kit

Abstract

The invention discloses a multiplex PCR (Polymerase Chain Reaction) primer and probe composition and a kit. Aiming at 19 kinds of alpha gene variation including Fusion gene, alpha alpha alpha anti3.7 and alpha alpha alpha anti4.2, the invention constructs a multiplex PCR primer and probe composition capable of simultaneously detecting the 19 kinds of alpha gene variation by using the same reaction condition through a two-tube reaction system. Based on the composition, a probe sequence in the composition is fixed on a solid-phase carrier to prepare a gene chip, visual detection of 19 alpha gene variations and the genotype of a detected sample is realized by combining multiple PCR and diversion hybridization, the detection specificity is good, the accuracy and the sensitivity are high, the operation is simple, the consumed time is short, and the result is easy to read; and carrying and screening of alpha gene variation are facilitated, and accurate reference can be provided for clinical diagnosis or genetic counseling and the like of alpha-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 a deficiency 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] In view of the deficiencies in the above-mentioned existing technologies, the present invention provides a multiplex PCR primer and probe composition and a kit. By using the composition or the kit, 19 types of α gene mutations including Fusion gene, ααα anti3.7 and ααα anti4.2 can be detected, the genotype of the sample to be detected can be determined, and thus a reference can be provided for the clinical diagnosis or genetic counseling of α-thalassemia, etc.

[0006] The above object of the present invention is achieved by the following technical solutions: The present invention is directed to 7 deletion types (-α 3.7 , -α 4.2 , -- SEA , -- THAI , -- FIL , -- MED I , -α 20.5 ), 9 mutation types (Init CD ATG>A-G, 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) of α gene mutations, and designs a multiplex PCR primer and probe composition that can detect the above 19 types of α gene mutations simultaneously under the same reaction conditions through a two-tube reaction system. Based on the composition, the present invention fixes the probe sequences in the composition on a solid-phase carrier to make a corresponding gene chip, and realizes the visual detection of the above 19 types of α gene mutations by combining multiplex PCR and flow-through hybridization. Its operation is simple, time-consuming is short and the result is easy to read, which is beneficial to the screening of carriers of α gene mutations. Therefore, the present invention requests protection for the multiplex PCR primer and probe composition.

[0007] Specifically, the composition of the present invention includes Composition 1 and Composition 2; Composition 1 contains the PCR amplification primers shown in SEQ ID NO.1 to 15 and the probes shown in SEQ ID NO.22 to 29; Composition 2 contains the PCR amplification primers shown in SEQ ID NO.1 and SEQ ID NO.16 to 20 and the probes shown in SEQ ID NO.22 and SEQ ID NO.30 to 48.

[0008] The above 19 α gene mutations can be detected by using the composition of the present invention. Therefore, the present invention also claims the use of the multiplex PCR primer and probe composition in the preparation of a product for detecting one or more of the 19 α gene mutations.

[0009] Specifically, the 19 α gene mutations are -α 3.7 、-α 4.2 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 、InitCD ATG>A-G, 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.

[0010] 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; the primers shown in SEQ ID NO.3 and SEQ ID NO.2 and the probe shown in SEQ ID NO.27 are used to detect -α 20.5 mutation; the primers shown in SEQ ID NO.4 and SEQ ID NO.5 and the probe shown in SEQ ID NO.22 are used to detect -- SEA mutation; the primers shown in SEQ ID NO.6 and SEQ ID NO.7 and the probe shown in SEQ ID NO.25 are used to detect -- FIL mutation; the primers shown in SEQ ID NO.8 and SEQ ID NO.9 and the probe shown in SEQ ID NO.26 are used to detect -- THAI mutation; the primers shown in SEQ ID NO.10 and SEQ ID NO.11 and the probe shown in SEQ ID NO.23 are used to detect -- MED I mutation; the primers shown in SEQ ID NO.12 and SEQ ID NO.13 and the probe shown in SEQ ID NO.28 are used to detect ααα anti3.7 mutation; the primers shown in SEQ ID NO.14 and SEQ ID NO.15 and the 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.

[0011] 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.

[0012] 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.

[0013] Specifically, the label is biotin.

[0014] Optionally, the solid phase carrier is nitrocellulose, cellulose acetate, glass sheet, silica gel wafer, nylon membrane, polypropylene membrane or micro-magnetic beads.

[0015] In a specific embodiment of the present invention, the solid phase carrier used is a nylon membrane.

[0016] Specifically, the concentration of the probe solution used in preparing the chip is 5-25 μM.

[0017] Preferably, the concentration of the probe solution is 20-25 μM.

[0018] More preferably, the concentration of the probe solution is 20 μM.

[0019] 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 Mutation; the gene chip 2 is used to detect the remaining mutations among 19 mutations; N and M marked on the gene chip respectively refer to the normal control point and the mutation site; for example, Init N refers to the normal control point (wild type) of Init CD ATG>A-G, and Init M refers to Init CD ATG>A-G.

[0020] The present invention also provides a kit. By using the kit and combining multiplex PCR and flow-through hybridization, visual detection of the above 19 α-gene mutations can be carried out. Specifically, the kit contains PCR amplification primers with nucleotide sequences shown in SEQ ID NO.1 to 20 and the above gene chip; markers are bound to the 5' ends of the PCR amplification primers. Specifically, the marker is biotin.

[0021] Specifically, the kit also contains reagents required for PCR amplification reaction and flow-through hybridization reaction.

[0022] When detecting using the kit of the present invention, since composition 1 mainly detects deletion-type and recombinant mutations and the fragments are very long, negative samples cannot be normally amplified by GAP-PCR, so the chromogenic system control probe on gene chip 1 corresponding to composition 1 is used as a monitor. The primers shown in SEQ ID NO.1 and SEQ ID NO.16 in composition 2 can amplify normal fragments and can be used as internal standard primers.

[0023] The present invention also claims the application of the kit in the preparation of a product for detecting one or more of the 19 α-gene mutations.

[0024] Specifically, the 19 α-gene mutations are -α 3.7 、-α 4.2 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 、InitCD ATG>A-G, 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.

[0025] The application of the multiplex PCR primer and probe composition or kit of the present invention in the preparation of an α-thalassemia diagnosis product should also be within the protection scope of the present invention.

[0026] The present invention also provides a method for detecting the 19 α-gene mutations by using the kit, comprising the following steps: S1. Extract genomic DNA of the sample to be tested; S2. Using the obtained genomic DNA as a template, perform multiplex PCR reactions with the primers shown in Group 1 and Group 2 respectively; the reaction procedure is: 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 finally extension at 72°C for 5 min; in the multiplex PCR reaction system, the final concentration of the PCR amplification primers is 0.1 - 0.5 μM; the final concentration of Mg 2+ is 1.0 - 3.0 mM; the amount of Taq enzyme used is 2.5 U; the amount of sample genomic DNA used is 80 - 140 ng.

[0027] S3. Use a gene chip for detection and result interpretation.

[0028] Preferably, in the multiplex PCR reaction system, the final concentration of the PCR amplification primers is 0.3 - 0.4 μM; the final concentration of Mg 2+ is 1.5 - 2.5 mM; the amount of sample genomic DNA used is 100 - 120 ng.

[0029] More preferably, in the multiplex PCR reaction system, the final concentration of the PCR amplification primers is 0.3 μM; the final concentration of Mg 2+ is 1.5 - 2.0 mM; the amount of sample genomic DNA used is 100 ng.

[0030] The present invention has the following beneficial effects: For 19 α-gene mutations including Fusion gene, ααα anti3.7 and ααα anti4.2 the present invention constructs a multiplex PCR primer and probe composition that can detect the 19 α-gene mutations simultaneously under the same reaction conditions through a two-tube reaction system. Based on the composition, the present invention makes a gene chip by immobilizing the probe sequences in the composition on a solid-phase carrier, and realizes the visual detection of the 19 α-gene mutations and the genotypes of the samples to be tested by combining multiplex PCR and flow-through hybridization. It has good detection specificity, high accuracy and sensitivity, simple operation, short time consumption, easy-to-read results, is conducive to the screening of carriers of α-gene mutations, and can provide accurate references for clinical diagnosis or genetic counseling of α-thalassemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of the gene chip prepared by the present invention.

[0032] Figure 2 For the detection results of the 20 validation samples and the blank control using the kit of the present invention.

[0033] Figure 3 For the detection results of test samples 1 to 15 using the kit of the present invention.

[0034] Figure 4 For the detection results of test samples 16 to 30 using the kit of the present invention.

[0035] Figure 5 For the detection results of test samples 31 to 45 using the kit of the present invention.

[0036] Figure 6 For the detection results of test samples 46 to 60 using the kit of the present invention.

[0037] Figure 7 For the detection results of test samples 61 to 75 using the kit of the present invention.

[0038] Figure 8 For the detection results of test samples 76 to 90 using the kit of the present invention.

[0039] Figure 9 For the detection results of test samples 91 to 105 using the kit of the present invention.

[0040] Figure 10 For the detection results of test samples 106 to 120 using the kit of the present invention. Detailed implementation manners

[0041] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0042] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0043] Example 1 Obtaining of multiplex PCR primer and probe combinations 1. Based on materials such as the "Clinical Genetic Counseling Expert Consensus on α-Thalassemia Gene Detection", the present invention sorted out the currently relatively common α gene variations, including 7 deletion types, 9 mutation types, and 3 recombinant α gene variations. The variation sites, mutation types, and other information of the α gene variations are shown in Table 1; in the table, α + Thalassemia refers to the deletion or functional defect (such as point mutation) of a single α-globin gene, resulting in partial reduction of the expression of this gene, but not complete loss of function; α0 Alpha thalassemia refers to the deletion or complete inactivation of both alpha-globin genes on the same chromosome, resulting in the inability of this chromosome to produce any alpha chains.

[0044] Table 1 Common alpha gene mutations

[0045] For the 19 alpha gene mutations shown in Table 1, the present invention first designed multiple individual primers / probes separately and carried out screening in a singleplex system. On the basis of the obtained primer pairs and probes, cross-reaction tests were conducted to exclude non-specific amplification and binding between primers / probes, as well as to exclude mutual inhibition. After repeated testing and optimization, a set of multiplex PCR primers and probe compositions that can simultaneously detect the above 19 alpha-thalassemia gene mutations through a two-tube reaction system was finally obtained.

[0046] The multiplex PCR primers and probe compositions include Composition 1 and Composition 2; among them, Composition 1 detects 8 alpha-thalassemia gene mutations, namely: -α 3.7 、-α 4.2 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 、ααα anti3.7 、ααα anti4.2 , including Primer 1 to Primer 15 and Probe 1 to Probe 9; Composition 2 detects 11 alpha-thalassemia gene mutations, namely -α 4.2 、Init CD ATG>A-G、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, Primer 16 to Primer 20, Probe 1 and Probe 10 to Probe 28. The detection targets and nucleotide sequences of the primers 1 to 20 and probes 1 to 28 are shown in Table 2 and Table 3 respectively.

[0047] Table 2 Nucleotide sequences of primers in the multiplex PCR primer and probe compositions

[0048] Note: The target point mutations described in the table refer to the 9 mutant α-thalassemia genes, namely Init CD ATG>A-G, 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; the 5' ends of the primers are all labeled with biotin.

[0049] Table 3 Nucleotide Sequences of Probes in the Multiplex PCR Primer and Probe Composition

[0050] Note: Probe 10 (NP) is the normal control probe corresponding to the deleted and recombinant α-thalassemia genes, and probe 1 (Bio) is the color development system control probe.

[0051] Example 2 Gene Chip and Detection Kit Based on the multiplex PCR primers and probes described in Example 1, the present invention developed a kit for detecting the above 19 α-thalassemia gene mutations, including the multiplex PCR primers and a gene chip prepared using the probes. Among them, the gene chip includes a fixed carrier and probes fixed on the fixed carrier, namely the probe sequences shown in SEQ ID NO.21-48; the 5' ends of the probes were subjected to amination treatment (amino group Amino), and the color development system control probe was labeled with biotin.

[0052] 1. Preparation of Gene Chip (1) Arrangement of Probes The fixed carrier used to prepare the gene chip in this example is a nylon membrane (2 pieces); among them, membrane 1 (hereinafter referred to as gene chip 1, Thal a8MY) is divided into 9 small grids, and probes 1 and 10-28 are sequentially fixed in each small grid. The specific positions of each probe are shown in Table 4 (represented by the detection target corresponding to the probe); Table 4

[0053] Membrane 2 (hereinafter referred to as gene chip 2, Thal a11MY) is divided into 20 small grids, and probes 1-9 are sequentially fixed in each small grid. The specific positions of each probe are shown in Table 5 (represented by the detection target corresponding to the probe); Table 5

[0054] (2) Treatment of Nylon Membrane The nylon membrane was treated. First, it was immersed in a 0.1 M HCl solution for 30 s. Then, the membrane with the residual solution removed was immersed in a 20% EDAC solution for 15 min. Finally, it was placed in a membrane washing tray and rinsed with 200 mL of purified water for 10 s. This step was repeated 4 times, and then it was placed on absorbent paper to remove the excess residual liquid. The membrane was transferred to a drying oven at a temperature of 20°C and a humidity of 45% and dried for 12 hours. The dried nylon membranes were separated with Kimwipes paper, transferred into a sealed plastic bag, and stored at 4°C for later use.

[0055] (3)Spotting The synthesized probe (the probe shown in Table 3) was dissolved with a probe diluent (a mixture of 0.5 M Na2CO3 and 0.5 M NaHCO3 in a volume ratio of 1:99, pH = 8.4). Then, the prepared probe solution was spotted onto the above-mentioned nylon membrane through a micropipetting device (the distribution positions of the probes on the membrane are shown in Table 4 and Table 5 respectively), 0.4 μL per drop. After spotting, the membrane was placed at room temperature for 15 min for reaction. Then, the membrane was transferred to a 0.1 M NaOH solution and immersed for 10 min, and then placed in a membrane washing tray and rinsed with 200 mL of purified water for 10 s. This step was repeated 4 times, and then it was placed on absorbent paper to remove the excess residual liquid. The washed membrane was transferred to a drying oven at a temperature of 20°C and a humidity of 45% and dried for 12 hours to obtain a gene chip.

[0056] The schematic diagram of the gene chip prepared by the present invention is as Figure 1 shown, and the sorting of the detected mutation sites is marked on the figure.

[0057] 2. Detection kit In addition, the present invention also provides a kit for detecting the above-mentioned 19 α-thalassemia gene mutations. The kit contains primers 1-20 shown in Table 2 (the primers are divided into two groups, group 1 contains primers 1-15, and group 2 contains primer 1 and primers 16-20), the prepared gene chips (Thal a8MY and Thal a11MY), reagents required for PCR reaction (PCR buffer, dNTPs, MgCl2, Taq enzyme), and sterilized water; the 5' ends of all primer pairs in the kit are labeled with biotin.

[0058] 3. Method for using the kit Based on the above-mentioned kit, the present invention also provides a method for detecting the above-mentioned 19 α-thalassemia gene mutations, including the following steps: (1)Extract genomic DNA from the sample to be tested; (2) Using the obtained genomic DNA as a template, perform multiplex PCR reactions with the primers shown in Group 1 and Group 2 respectively. The reaction systems are shown in Table 6 and Table 7 respectively. The reaction program (the same for both) is: 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 finally extension at 72°C for 5 min; Among them, the PCR amplification reaction system for the primers in Group 1 is shown in Table 6: The reagents used for amplification are HotStarTaq DNA Polymerase enzyme with the product number 203203 purchased from Qiagen, its supporting 10×PCR Buffer, Q-solution (gene amplification auxiliary reagent), and 25 mM MgCl2.

[0059] Table 6

[0060] The PCR amplification reaction system for the primers in Group 2 is shown in Table 7.

[0061] Table 7

[0062] Note: The 5' ends of the primers are all labeled with biotin.

[0063] The present invention can amplify multiple α-thalassemia gene mutations under the same conditions, reducing the consumption of PCR reagents and the demand for PCR instruments, reducing the operation steps, and lowering the cost.

[0064] (3) Use a gene chip for detection and result interpretation Denature the obtained amplification product at 95°C for 5 min, quickly transfer it to an ice box, place it for 2 min, then add it to 0.8 mL of hybridization solution (2×SSC / 0.1% SDS) pre-warmed to 44°C. After mixing, place it in the reaction wells of a hybridization instrument, apply it to the prepared gene chip, hybridize at 44°C for 30 min, wash 4 times with solution WB1 (0.5×SSC / 0.1% SDS, pre-warmed at 44°C), add 0.5 mL of blocking solution (0.25% skim milk powder, 0.05% thimerosal), block at 25°C for 5 min, drain, then add 0.5 mL of enzyme-labeled solution (AP enzyme labeled with streptavidin dissolved in TBS), enzyme-label for 5 min, wash 4 times with 0.8 mL of solution A (TBS, 0.1% Tween20 and 0.05% sodium azide), add 0.5 mL of chromogenic solution (NBT / BCIP), develop color in the dark for 5 min, finally rinse 3 times with hybridization solution (2×SSC / 0.1% SDS), air dry, analyze the color development situation, and interpret the results.

[0065] The result interpretation method of the gene chip is as follows: ① If blue-purple spots with similar colors appear at the mutation detection probe of the gene chip and the corresponding negative control probe, the corresponding site is a heterozygote of α-thalassemia gene mutation, α-thalassemia gene deletion, or α-thalassemia gene recombination; ② If a blue-purple spot appears at the mutation detection probe of the gene chip, but no blue-purple spot appears at the corresponding negative control probe, the corresponding site is a homozygote of β-thalassemia gene mutation, α-thalassemia gene deletion, or α-thalassemia gene recombination; ③ If blue-purple spots only appear at the Bio site and the negative control probe of the gene chip, then no α-thalassemia gene mutation, α-thalassemia gene deletion, or α-thalassemia gene recombination is detected; ④ If no blue-purple spot appears at the Bio site of the gene chip, the detection is invalid; ⑤ If no blue-purple spot appears at all the mutation detection probes and the negative control probes of the gene chip, the detection is invalid; ⑥ If no blue-purple spot appears at a certain mutation detection probe and the corresponding negative control probe of the gene chip, while the detection results of other probes are normal, a new mutation type may occur at this site, and further sequencing analysis is required.

[0066] Example 3 Testing the Detection Effect of the Kit To test whether the kit described in Example 2 of the present invention can accurately detect the 19 α-thalassemia gene variations, 20 human peripheral venous blood samples with known genotypes (the sample numbers and their corresponding genotypes (carried variations) are shown in Table 8) of the present invention were used as verification samples. Using the DNA of the verification samples as a template, the detection effect of the kit was tested, and sterile water was added as verification sample 21 (blank control) during the testing process.

[0067] The detection results of using the kit of the present invention to detect the 20 verification samples and the blank control are as Figure 2 shown in

[0068] Table 8 Variations Carried by the Verification Samples and the Kit Detection Results

[0069] Note: The negative sample is a sample in which none of the 7 deletion-type α-thalassemia genes, 3 recombination-type α-thalassemia genes, and 9 mutation-type α-thalassemia genes have mutations.

[0070] From Figure 2It can be seen that in the color development results of the blank control, only the Bio point shows a blue-purple spot; when the kit shown in Example 2 is used to detect the verification samples, the test results are consistent with the actual genotypes of the samples, indicating that the kit shown in Example 2 can be used to detect α-thalassemia gene mutations, deletions and recombinations.

[0071] Example 4 Performance Test of the Kit 1. Accuracy and Specificity Test Using 120 human peripheral venous blood samples with known genotypes as test samples (Test Samples 1 - 120), the DNA concentrations of the test samples were all controlled at 20 - 40 ng / μL, and the accuracy and specificity of the kit prepared in Example 2 were detected as follows: The results of the 120 test samples were judged 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 Bori gene amplifier, and flow-through hybridization was performed on a Kip medical nucleic acid molecule hybridization instrument HBHM - 3001S.

[0072] Control group: Mutant samples were detected by Sanger sequencing, and deletion and recombinant samples were detected by agarose gel electrophoresis.

[0073] 2. Detection Sensitivity Test Using the DNA of 19 heterozygous samples with known genotypes as sensitivity test samples (Sensitivity Test Samples 1 - 19), each sensitivity test sample was detected at two DNA concentrations; the two DNA concentrations were 2 ng / μL and 105 ng / μL respectively.

[0074] The results of the sensitivity test samples were 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 results of each test were recorded; “+” was used to represent the clarity of the test results, and the more “+” there were, the clearer the test results were, manifested as a deeper color development degree. Among them, the color development degree of the “+++” test results was the same as Figure 2 the same.

[0075] 3. Experimental Results (1) The accuracy and specificity test results of the kit are shown in Table 9, and the color development result diagrams of Test Samples 1 - 15 are as Figure 3 shown, the color development result diagrams of Test Samples 16 - 30 are as Figure 4 shown, the color development result diagrams of Test Samples 31 - 45 are as Figure 5 shown, the color development result diagrams of Test Samples 46 - 60 are as Figure 6As shown, the color development result diagrams of test samples 61 to 75 are as Figure 7 shown, and the color development result diagrams of test samples 76 to 90 are as Figure 8 shown, and the color development result diagrams of test samples 91 to 105 are as Figure 9 shown, and the color development result diagrams of test samples 106 to 120 are as Figure 10 shown.

[0076] Table 9 Accuracy and Specificity Test Results of the Kit

[0077] Combined Figures 3 to 10 with the results shown in Table 9, it can be seen that when using the kit of the present invention to detect thalassemia gene mutations in 120 test samples, the thalassemia gene mutation detection results are consistent with the reagent gene mutation results of each test sample, and the accuracy rate is 100%. In addition, when using the kit shown in Example 2 to detect β-thalassemia and non-deletion β-thalassemia genotype positive samples (test samples 100 to 105) and negative samples, the detection results are all negative, the negative coincidence rate is 100%, and the specificity is 100%, indicating that the kit shown in Example 2 can only specifically detect α-thalassemia gene mutations or deletions.

[0078] The detection sensitivity test results of the kit are shown in Table 10.

[0079] Table 10 Detection Sensitivity Test Results of the Kit

[0080] From the sensitivity test results, it can be seen that when using the kit shown in Example 2 to detect thalassemia gene mutations in 20 test samples, within the two ranges of DNA concentrations set at 2 ng / μL and 105 ng / μL DNA concentration, each was repeatedly detected 20 times, and the presented hybridization results showed clear color development and strong signals; therefore, the detection lower limit of the kit shown in Example 3 is 2 ng / μL (10 ng / reaction), and the detection upper limit is 105 ng / μL.

[0081] Example 5 Kit Optimization 1. Optimization of Probe Concentration on the Gene Chip Referring to the method for preparing a gene chip described in Example 2, in this example, gene chips corresponding to different concentrations of probe solutions were prepared. Using the 20 validation samples described in Example 3 as test samples, the detection effects of the gene chips prepared with different concentrations of probe solutions were tested in combination with the test method described in Example 2. The probe concentrations tested in this example were 5, 10, 15, 20, and 25 μM respectively, and the test results are shown in Table 11.

[0082] Table 11 Optimization test results of probe concentrations on the gene chip

[0083] As can be seen from the results shown in Table 11, when the probe concentration was 5 - 25 μM, the detection results could all be shown and there was no non-specificity; among them, when the probe concentration was 20 - 25 μM, the detection results were relatively the clearest and there was no non-specificity, and the detection results were all the same as the known genotypes of the samples. When preparing the gene chip, the probe concentration of the probe solution should be 20 μM.

[0084] 2. Optimization of the reaction system (1) Optimization of primer concentration Using the 20 validation samples described in Example 3 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, and were denoted as experimental groups 1 - 5 in sequence. The results are shown in Table 12.

[0085] Table 12 Detection results of experimental groups 1 - 5

[0086] As can be seen from the results shown in Table 12, when the concentrations of primers 4 and 5 in multiplex PCR reaction system A are 0.1 μM, the detection result for (SEA, sample 3) is the clearest and there is no non-specificity; when the concentrations of primers 14 and 15 are 0.2 μM, the detection result for (anti4.2, sample 8) is the clearest and there is no non-specificity. When the concentrations of primers 1, 6-9, 12, and 13 are 0.3 μM, the detection results for (3.7, FIL, THAI, anti3.7, samples 1, 4, 5, 7) are the clearest and there is no non-specificity. When the concentrations of primers 10 and 11 are 0.4 μM, the detection result for (MED, sample 6) is the clearest and there is no non-specificity. When the concentration of primer 3 is 0.5 μM and the concentration of primer 2 is 0.8 μM, the detection result for (20.5, sample 2) is the clearest and there is no non-specificity. When the concentrations of primer 17 and primer 18 in multiplex system B are 0.5 μM, the detection result for the deleted α-thalassemia gene (4.2, sample 18) is the clearest and there is no non-specificity. When the concentrations of other primers are 0.3 μM, the detection results for (samples 9 - sample 17, sample 19) are the clearest and there is no non-specificity, and the detection results are all the same as the known genotypes of the samples. When the primers in multiplex PCR reaction system A and multiplex PCR system B are not within the above ranges, the clarity and specificity of the combined detection of multiple mutation types of α-thalassemia genes will decrease to a certain extent.

[0087] (2)Mg 2+ Optimization of ion concentration Using the 20 validation samples described in Example 3 as test samples, referring to the reaction system described in Example 2, adjust the concentration of Mg 2+ in the multiplex PCR reaction system to 0.5, 1.0, 1.5, 2.0, 2.5, 3.0 mM, denoted as experimental groups 6 - 10 in sequence, and the results are shown in Table 13.

[0088] Table 13 Detection results of experimental groups 6 - 11

[0089] As can be seen from the results shown in Table 13, when the concentration of Mg 2+ in multiplex PCR reaction system A is 2 mM, the detection results for 6 deleted and 2 recombinant α-thalassemia genes (samples 1 - sample 8) are clear and there is no non-specificity; when the concentration of Mg 2+When the concentration is 1.5 mM, the detection results for 9 mutant types, 1 deletion type, and 1 recombinant type of α-thalassemia genes (Samples 9 - 19) are clear and there is no non-specificity. Therefore, the added volume of MgCl2 in multiplex PCR reaction system A is 4 μL, and the added volume of MgCl2 in multiplex PCR reaction system B is 3 μL.

[0090] (3) Optimization of Taq enzyme addition amount Using the 20 validation samples described in Example 3 as test samples, referring to the reaction system described in Example 2, adjust the added volume of Taq enzyme in the multiplex PCR reaction system to 1.5, 2.0, 2.5, 3.0, 3.5 U, denoted as experimental groups 11 - 15 in sequence. The results are shown in Table 14.

[0091] Table 14 Detection results of experimental groups 12 - 16

[0092] As can be seen from the results shown in Table 14, when the added volume of Taq enzyme in multiplex PCR system A and multiplex PCR system B is 0.5 μL (Taq addition amount is 2.5 U), the detection results for 20 samples are clear and there is no non-specificity, and the detection results are all the same as the known genotypes of the samples; while when the added volume of Taq enzyme in multiplex PCR system A and multiplex PCR system B is 0.3 μL (Taq addition amount is 1.5 U), the detection results for 20 samples are relatively unclear and the color development degree is lighter; when the added volume of Taq in multiplex PCR system A and multiplex PCR system B is 0.4 μL, 0.6 μL, and 0.7 μL (Taq addition amounts are 2.0 U, 3.0 U, and 3.5 U), among the detection results of 20 samples, there are individual samples with relatively unclear detection results and non-specificity.

[0093] (4) Optimization of the added amount of the sample to be tested Using the 20 validation samples described in Example 3 as test samples, referring to the reaction system described in Example 2, adjust the added volume of the sample to be tested in the multiplex PCR reaction system to 2, 3, 4, 5, 6, 7 μL (sample concentration is 20 ng / μL), denoted as experimental groups 17 - 22 in sequence. The results are shown in Table 15.

[0094] Table 15 Detection results of experimental groups 17 - 22

[0095] As can be seen from the results shown in Table 15, the added volume of the test samples in multiplex PCR system A and multiplex PCR system B is 5 μL. The detection results of 20 samples are clear and there is no non-specificity, and the detection results are all the same as the known genotypes of the samples. However, increasing or decreasing the added volume of the test samples will reduce the clarity and specificity of the detection results.

[0096] (5)Optimization of the multiplex PCR amplification program Taking the 20 validation samples described in Example 3 as test samples, after preparing the reaction system according to Example 2, the following different reaction programs were used for amplification respectively, and the results are shown in Table 16.

[0097] The multiplex PCR amplification program 1 is: 95°C, 15 min; 98°C, 35 s, 60°C, 1 min, 72°C, 2 min, 30 cycles; 72°C, 5 min.

[0098] The multiplex PCR amplification program 2 is: 95°C, 15 min; 98°C, 40 s, 62°C, 1 min, 72°C, 2 min, 35 cycles; 72°C, 5 min.

[0099] The multiplex PCR amplification program 3 is: 95°C, 15 min; 97°C, 35 s, 60°C, 50 s, 72°C, 150 s, 40 cycles; 72°C, 7 min.

[0100] The multiplex PCR amplification program 4 is: 95°C, 15 min; 98°C, 40 s, 64°C, 50 s, 72°C, 150 s, 35 cycles; 72°C, 5 min.

[0101] Table 16 Detection results of multiplex PCR amplification programs 1 to 4

[0102] As can be seen from the results shown in Table 16, only when the multiplex PCR reaction systems A and B in the kit shown in Example 2 are combined with the multiplex PCR amplification program 2, the detection results of 20 samples are clear and there is no non-specificity, and the detection results are all the same as the known genotypes of the samples. When combined with the multiplex PCR amplification programs 1, 3, and 4, the clarity and specificity of the sample detection results are reduced.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A multiplex PCR primer and probe composition, characterized in that, It includes Composition 1 and Composition 2; Composition 1 includes PCR amplification primers with nucleotide sequences shown in SEQ ID NOs. 1 to 15 and probes with nucleotide sequences shown in SEQ ID NOs. 22 to 29; Composition 2 includes PCR amplification primers with nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NOs. 16 to 20 and probes with nucleotide sequences shown in SEQ ID NO. 22 and SEQ ID NOs. 30 to 48.

2. Use of the multiplex PCR primer and probe composition according to claim 1 in the preparation of a product for detecting one or more of 19 α gene variations, characterized in that, The 19 α gene mutations are -α 3.7 、-α 4.2 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 、Init CD ATG>A-G, 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 shown in SEQ ID NOs. 22 to 48 and a color development system control probe are fixed on the solid phase carrier of the chip, and the nucleotide sequence of the color development system control probe is shown in SEQ ID NO.

21.

4. The gene chip according to claim 3, wherein, The 5'-end of the probe with nucleotide sequence shown in SEQ ID NOs. 22 to 48 has an amino modification, and a label is bound to the 5'-end of the color development system control probe.

5. The gene chip according to claim 4, wherein The solid phase carrier is nitrocellulose, cellulose acetate, glass slide, silica wafer, nylon membrane, polypropylene membrane or microscale magnetic beads.

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

7. The gene chip according to claim 6, wherein 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 shown in SEQ ID NOs. 1 to 20 and the gene chip according to any one of claims 3 to 7; labels are bound to the 5'-ends of the PCR amplification primers.

9. The kit according to claim 8, wherein 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 the preparation of a product for detecting one or more of 19 α gene variations, characterized in that The 19 α gene mutations are -α 3.7 、-α 4.2 、-- SEA 、-- THAI 、-- FIL 、-- MED I 、-α 20.5 、Init CD ATG>A-G, CD30 -GAG, CD35 TCC>CCC, CD59 GGC>GAC, Poly A AATAAA>AATGAA, CD122 CAC>CAG, CD125 CTG>CCG, CD142 TAA>CAA, CD142 TAA>TAT, ααα anti3.7 、ααα anti4.2 and Fusion gene.

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