Probe, primer pair, kit and detection method for beta-thalassemia gene detection

Abstract

The invention relates to a probe, a primer pair, a kit and a detection method for beta-thalassemia gene detection. The detection probes are selected from one or more of P1 to P6 of which the sequences are as shown in SEQ ID NO. 9 to SEQ ID NO. 14. An amplified fragment of the detection primer pair can be specifically combined with a detection probe. The detection primer pair is selected from one or more of a primer pair 1, a primer pair 2, a primer pair 3, a primer pair 4 and a primer pair 4, wherein the sequences of the primer pair 1, the primer pair 2, the primer pair 3 and the primer pair 4 are shown as SEQ ID NO.1 to SEQ ID NO.8. According to the invention, the detection of eight mutation sites in the beta-thalassemia gene can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and relates to a probe, a primer pair, a kit and a detection method for beta-thalassemia gene detection. Background Art

[0002] β-thalassemia refers to hereditary hemolytic anemia caused by insufficient synthesis of hemoglobin (Hb) due to reduced or complete absence of β-globin peptide chain synthesis. Severe β-thalassemia was first reported by American pediatrician Cooley in 1925, so it is also called Cooley anemia. The disease is inherited in an autosomal recessive manner, and the pathogenic gene is HBB. The incidence of males and females is roughly the same.

[0003] The high incidence areas of β-thalassemia mainly include the Mediterranean region, North Africa, the Sahara Desert, the Middle East, the Indian subcontinent and Southeast Asia including southern my country, with a mutation carrier rate of 2% to 30%. Epidemiological surveys show that the three provinces with the highest β-thalassemia carrier rates in my country are Guangxi (6.66%), Hainan (5.11%) and Guizhou (4.63%), and Guangdong, Yunnan, Hunan, Jiangxi and other places also have high rates.

[0004] β-thalassemia is caused by mutations in the β-globin gene, which are mainly point mutations, with a few being deletion-type. The β-globin gene cluster is located at 11p15.3 and includes two adult-expressed genes (β and δ). The gene arrangement order is 5'-δ-β-3'. β-thalassemia can be divided into three categories according to the degree of β-globin chain synthesis: (1) β 0 Thalassemia, β-globin chain cannot be synthesized at all, and the allele is called β 0 Thalassemia allele; (2) β + Thalassemia, reduced synthesis of β-globin chains, the allele in which it is located is called β + Thalassemia allele; (3) β ++ Thalassemia, also known as silent β-thalassemia, is a disease characterized by a slight reduction in the synthesis of β-globin chains.

[0005] The clinical phenotypes of β-thalassemia can be divided into four types: (1) Silent β-thalassemia, with a genotype of β ++ / β N , the hematological phenotype is normal or borderline, and can generally only be identified through molecular diagnosis; (2) mild β-thalassemia, the genotype is β 0 / β N or β + / β N , clinical manifestations are microcytic hypochromia and elevated HbA2 values; (3) intermediate β-thalassemia, genotype is β + / β+ or β + / β 0 The phenotypic variation range is large, ranging from mild anemia to moderate anemia, and moderate anemia often occurs in early childhood; (4) severe β-thalassemia, the genotype is β + / β 0 or β 0 / β 0 , usually accompanied by severe anemia, requiring regular blood transfusions to survive. Children are asymptomatic at birth and usually develop symptoms in infancy (3-12 months of age). If untreated, most children die before the age of 5. In addition, the molecular basis of the intermediate type is more complex. Heterozygotes with dominant β-thalassemia mutations (β D / β N ), homozygous for β-thalassemia mutation combined with α-thalassemia mutation (β 0 / β 0 ), or heterozygous for β-thalassemia mutations with α-globin triads (β 0 / β N or β + / β N ), will also show an intermediate phenotype.

[0006] At present, 129 β-globin gene point mutations (including abnormal hemoglobin) and 16 β-thalassemia deletion mutations have been found in the Chinese population. Among them, 8 mutations (HBB: c.126_129delCTTT, HBB: c.52A>T, HBB: c.316-197C>T, HBB: c.-78A>G, HBB: c.216_217insA, HBB: c.79G>A, HBB: c.92+1G>T, HBB: c.-79A>G) account for more than 95% of the total β-thalassemia mutations in Chinese people. For these high-frequency mutation sites, detection products are urgently needed to meet clinical needs.

[0007] In the process of detecting mutations in the eight common β-globin genes, traditional β-thalassemia detection products require one pair of primers to match two probes for each detection site, without considering the optimized design of primers and probes, resulting in high consumption of primers and probes and a complex reaction system. The experimental process is relatively cumbersome (with many steps), the detection cycle is long, the results must be manually interpreted, the product tube must be opened for analysis, and the risk of contamination is high. Summary of the invention

[0008] Based on this, the purpose of the embodiments of the present application includes providing a probe, a primer pair, a kit and a detection method for β-thalassemia gene detection.

[0009] In the first aspect of the present application, a β-thalassemia gene detection probe is provided, wherein the detection probe is selected from one or more of the following detection probes P1 to P6:

[0010] The sequence of the detection probe P1 is shown in SEQ ID NO.9;

[0011] The sequence of the detection probe P2 is shown in SEQ ID NO.10;

[0012] The sequence of the detection probe P3 is shown in SEQ ID NO.11;

[0013] The sequence of the detection probe P4 is shown in SEQ ID NO.12;

[0014] The sequence of the detection probe P5 is shown in SEQ ID NO.13; and,

[0015] The sequence of the detection probe P6 is shown in SEQ ID NO.14.

[0016] In some embodiments of the present application, the detection probe is a self-quenching fluorescent probe, and the 5′ end and 3′ end of the self-quenching fluorescent probe are labeled with a fluorescent group and a quenching group, respectively.

[0017] In the second aspect of the present application, a β-thalassemia gene detection primer pair is provided, and the amplified fragment of the detection primer pair can be specifically bound by the detection probe described in the first aspect.

[0018] In some embodiments of the present application, the detection primer pair is selected from one or more pairs of the following primer pairs 1 to 4:

[0019] Primer pair 1, comprising: a forward primer F1 having a sequence as shown in SEQ ID NO.1, and a reverse primer R1 having a sequence as shown in SEQ ID NO.2;

[0020] Primer pair 2, comprising: a forward primer F2 having a sequence as shown in SEQ ID NO.3, and a reverse primer R2 having a sequence as shown in SEQ ID NO.4;

[0021] Primer pair 3, comprising: a forward primer F3 having a sequence as shown in SEQ ID NO.5, a reverse primer R3 having a sequence as shown in SEQ ID NO.6; and,

[0022] Primer pair 4 includes: a forward primer F4 whose sequence is shown as SEQ ID NO.7, and a reverse primer R4 whose sequence is shown as SEQ ID NO.8.

[0023] In a third aspect of the present application, a β-thalassemia gene detection kit is provided, the kit comprising:

[0024] The detection probe described in the first aspect; or / and,

[0025] The detection primer pair described in the second aspect.

[0026] In some embodiments of the present application, the detection kit further comprises PCR buffer, Mg 2+ , one or more of dNTP and TaqDNA polymerase, and also includes dUTP and UNG enzyme.

[0027] In some embodiments of the present application, the detection kit includes a PCR reaction solution and an enzyme reagent;

[0028] The PCR reaction solution comprises the forward primer F1, the reverse primer R1, the forward primer F2, the reverse primer R2, the forward primer F3, the reverse primer R3, the forward primer F4, the reverse primer R4, the detection probe P1, the detection probe P2, the detection probe P3, the detection probe P4, the detection probe P5, the detection probe P6, and the Mg 2+ , dNTPs, dUTPs and PCR buffer;

[0029] The enzyme reagent comprises Taq DNA polymerase and UNG enzyme.

[0030] In some embodiments of the present application, the PCR reaction solution comprises 0.1 μM to 0.2 μM forward primer F1, 1 μM to 2 μM reverse primer R1, 0.1 μM to 0.2 μM forward primer F2, 1 μM to 2 μM reverse primer R2, 0.1 μM to 0.2 μM forward primer F3, 1 μM to 2 μM reverse primer R3, 0.1 μM to 0.2 μM forward primer F4, 1 μM to 2 μM reverse primer R4, 0.2 μM to 0.3 μM detection probe P1, 0.2 μM to 0.3 μM detection probe P2, 0.2 μM to 0.3 μM detection probe P3, 0.3 μM to 0.4 μM detection probe P4, 0.3 μM to 0.4 μM detection probe P5, 0.2 μM to 0.3 μM detection probe P6, 2 mM to 3 mM Mg 2+ , 0.2mM~0.3mM dNTP, 0.2mM~0.3mM dUTP and PCR buffer, and the fluorescent groups of each detection probe are different; or / and,

[0031] The enzyme reagent comprises 3U / μL to 8U / μL Taq DNA polymerase and 1U / μL to 2U / μL UNG enzyme.

[0032] In some embodiments of the present application, the concentration ratio of the forward primer and the reverse primer paired therewith in the PCR reaction solution is 1:(8-12).

[0033] In some embodiments of the present application, the detection kit further comprises one or more of a β-thalassemia standard control and a β-thalassemia negative control;

[0034] The β-thalassemia standard control is a wild-type plasmid containing the β-globin gene or normal human genomic DNA;

[0035] The β-thalassemia negative control is 1×TE buffer solution or water.

[0036] In a fourth aspect of the present application, a method for detecting a β-globin gene is provided, the method comprising the following steps:

[0037] Providing the kit described in the third aspect;

[0038] The PCR reaction solution and the enzyme reagent are mixed and centrifuged to prepare a PCR reaction premix;

[0039] Putting the DNA of the sample to be tested into the PCR reaction premix, and performing PCR amplification to obtain the amplified product of the sample to be tested;

[0040] Based on the probe melting curve method, the amplified product of the test sample is detected and compared with the β-thalassemia standard control, and the corresponding Tm values ​​of the test sample and the β-thalassemia standard control are obtained, which are recorded as Tm1 and Tm2, ​​respectively. The mutation status of the β-globin gene in the test sample is determined based on the change of Tm1 relative to Tm2.

[0041] In some embodiments of the present application, judging the mutation of the β-globin gene in the sample to be tested according to the change of Tm1 relative to Tm2 includes:

[0042] The difference between Tm2 and Tm1 is recorded as ΔTm, and judging whether the β-globin gene of the sample to be tested contains a mutation according to the ΔTm, and / or determining the genotype of the mutation, optionally includes:

[0043] Under the condition of ΔTm=5.03±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β IVS-II-654 ;

[0044] Under the condition of ΔTm=5.51±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-71-72 ;

[0045] Under the condition of ΔTm=11.05±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-41-42 ;

[0046] Under the condition of ΔTm=9.59±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β IVS-I-1 ;

[0047] Under the condition of ΔTm=7.74±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-26 ;

[0048] Under the condition of ΔTm=4.68±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-17 ;

[0049] Under the condition of ΔTm=5.18±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β -28 ;

[0050] Under the condition of ΔTm=3.75±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β -29 .

[0051] The present application can detect multiple sites by sharing one pair of primers and matching two probes, which greatly saves the consumption of primers and probes, has simple detection steps and a short detection cycle, does not require a hybridization instrument during the detection process, and only requires an extraction instrument and a PCR instrument to realize the detection process, and the instrument can automatically interpret the test results, and the product is analyzed in a closed tube, with a low risk of contamination. In addition, the present application can simultaneously detect non-deletion mutations and point mutations in the synthesis of β-globin peptide chains.

[0052] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0054] Figure 1 This is the experimental result diagram of the wild-type sample at the β site;

[0055] Figure 2 This is the experimental result diagram of IVS-II-654 (C>T) heterozygous sample;

[0056] Figure 3 This is the experimental result diagram of CD71 / 72 (+A) heterozygous sample;

[0057] Figure 4 This is the experimental result diagram of CD41 / 42 (-TTCT) heterozygous sample;

[0058] Figure 5 This is the experimental result diagram of IVS-I-1 (G>T) heterozygous sample;

[0059] Figure 6 This is a graph showing the experimental results of CD26 (G>A) heterozygous samples;

[0060] Figure 7 This is the experimental result diagram of CD17 (A>T) heterozygous sample;

[0061] Figure 8 This is the experimental result diagram of -28 (A>G) heterozygous sample;

[0062] Fig. 9 This is the experimental result diagram of -29 (A>G) heterozygous sample;

[0063] Fig.10 This is the result of the primer probe screening experiment for wild-type samples at the β site;

[0064] Fig.11 This is the result of the primer probe screening experiment for wild-type samples at the β site;

[0065] Fig.12 This is the result of the primer probe screening experiment for wild-type samples at the β site;

[0066] Fig.13 This is the result of the primer probe screening and reaction system adjustment experiment for IVS-II-654 (C>T) heterozygous sample;

[0067] Fig.14 This is a diagram showing the results of the primer probe screening and reaction system adjustment experiment for -28 (A>G) heterozygous samples;

[0068] Fig.15 This is a graph showing the results of the CD71 / 72 (+A) heterozygous sample reaction system optimization experiment;

[0069] Fig.16 This is a graph showing the results of the CD41 / 42 (-TTCT) heterozygous sample reaction system optimization experiment;

[0070] Fig.17 The results of the experiment for optimizing the reaction system of -28 (A>G) heterozygous sample;

[0071] Fig.18The results of the experiment for optimizing the reaction system of the heterozygous sample of IVS-II-654 (C>T);

[0072] Fig.19 Diagram of the design mechanism of primer pairs and probes for this application. DETAILED DESCRIPTION

[0073] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing implementation modes and embodiments and are not intended to limit the present invention.

[0075] the term

[0076] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0077] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0078] In the present invention, "plurality", "multiple", "multiple times", "multiple" and the like, unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0079] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0080] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0081] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0082] In the present invention, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0083] In the present invention, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0084] In the present invention, in the "first aspect", "second aspect", "third aspect", "fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0085] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0086] In the present invention, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum value and the maximum value) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0087] The temperature parameters in the present invention, if not specifically limited, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the precision range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0088] In the present invention, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0089] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0090] In view of the shortcomings of the existing technology such as complicated operation, long time consumption, high detection cost and easy contamination, a simple, fast, highly sensitive, reliable and low-cost β-thalassemia gene detection method based on probe melting curve analysis technology is provided.

[0091] The method of the present application uses a multicolor melting curve analysis technique (Multicolor Melting Curve Analysis, MMCA), which is based on the probe method and the melting curve analysis method, combined with multicolor fluorescence detection, and can simultaneously detect multiple targets (i.e., sites) in a reaction. The probe uses a double-labeled, self-quenched TaqMan probe, and the detection principle is: when the TaqMan probe is free in the solution and is in an irregular curling state, the fluorescent group labeled on the probe is relatively close to the quenching group, and the fluorescence signal is weak due to fluorescence resonance energy transfer (fluorescence resonance energy transfer, FRET). When the target sequence exists in the reaction system, the probe and the target sequence are complementary and paired to form an extended state, and the fluorescent group and the quenching group are relatively far away, emitting a strong fluorescence signal. When the amplification is completed, the product amount reaches the highest peak and the fluorescence signal is also the strongest. At this time, the amplified product is heated again for detection, and the probe hybridized on the product gradually dissociates from the target sequence, forming an irregular curling state, and the fluorescence signal gradually weakens. The temperature and fluorescence signal change curve graph is drawn, and the melting curve analysis can be performed.

[0092] The first aspect of the present application

[0093] The present application provides a β-thalassemia gene detection probe, wherein the detection probe is selected from one or more of the following detection probes P1 to P6:

[0094] The sequence of the detection probe P1 is shown in SEQ ID NO.9;

[0095] The sequence of the detection probe P2 is shown in SEQ ID NO.10;

[0096] The sequence of the detection probe P3 is shown in SEQ ID NO.11;

[0097] The sequence of the detection probe P4 is shown in SEQ ID NO.12;

[0098] The sequence of the detection probe P5 is shown in SEQ ID NO.13; and,

[0099] The sequence of the detection probe P6 is shown in SEQ ID NO.14.

[0100] The detection probe of the present application is selected from 1, 2, 3, 4, 5 or 6 of the detection probes P1 to P6.

[0101] In some embodiments of the present application, the detection probe is a self-quenching fluorescent probe, and optionally, the 5' end and 3' end of the self-quenching fluorescent probe are respectively labeled with a fluorescent group and a quenching group. The fluorescent group can be selected from, including but not limited to, FAM, ROX, HEX, and CY5. The quenching group can be selected from, including but not limited to, BHQ1, BHQ2, and BHQ3.

[0102] The second aspect of the present application

[0103] The present application provides a β-thalassemia gene detection primer pair, the amplified fragment of which can be specifically bound by the detection probe described in the first aspect.

[0104] In some embodiments of the present application, the detection primer pair is selected from one or more pairs of the following primer pairs 1 to 4:

[0105] Primer pair 1: forward primer F1 having a sequence as shown in SEQ ID NO.1, and reverse primer R1 having a sequence as shown in SEQ ID NO.2;

[0106] Primer pair 2: forward primer F2 having a sequence as shown in SEQ ID NO.3, and reverse primer R2 having a sequence as shown in SEQ ID NO.4;

[0107] Primer pair 3: forward primer F3 having a sequence as shown in SEQ ID NO.5, and reverse primer R3 having a sequence as shown in SEQ ID NO.6; and,

[0108] Primer pair 4: forward primer F4 having a sequence as shown in SEQ ID NO.7, and reverse primer R4 having a sequence as shown in SEQ ID NO.8.

[0109] The detection primer pair of the present application is selected from 1 pair, 2 pairs, 3 pairs or 4 pairs of primer pairs 1 to 4 below.

[0110] The third aspect of the present application

[0111] The present application provides a β-thalassemia gene detection kit, the kit comprising:

[0112] The detection probe described in the first aspect; or / and,

[0113] The detection primer pair described in the second aspect.

[0114] In some embodiments of the present application, the detection kit further comprises PCR buffer, Mg 2+ , one or more of dNTP and TaqDNA polymerase, or also includes dUTP and UNG enzyme.

[0115] In some embodiments of the present application, the detection kit includes a PCR reaction solution and an enzyme reagent;

[0116] The PCR reaction solution comprises the forward primer F1, the reverse primer R1, the forward primer F2, the reverse primer R2, the forward primer F3, the reverse primer R3, the forward primer F4, the reverse primer R4, the detection probe P1, the detection probe P2, the detection probe P3, the detection probe P4, the detection probe P5, the detection probe P6, and the Mg 2+ , dNTPs, dUTPs and PCR buffer;

[0117] The enzyme reagent comprises Taq DNA polymerase and UNG enzyme.

[0118] In some embodiments of the present application, the PCR reaction solution comprises 0.1 μM to 0.2 μM forward primer F1, 1 μM to 2 μM reverse primer R1, 0.1 μM to 0.2 μM forward primer F2, 1 μM to 2 μM reverse primer R2, 0.1 μM to 0.2 μM forward primer F3, 1 μM to 2 μM reverse primer R3, 0.1 μM to 0.2 μM forward primer F4, 1 μM to 2 μM reverse primer R4, 0.2 μM to 0.3 μM detection probe P1, 0.2 μM to 0.3 μM detection probe P2, 0.2 μM to 0.3 μM detection probe P3, 0.3 μM to 0.4 μM detection probe P4, 0.3 μM to 0.4 μM detection probe P5, 0.2 μM to 0.3 μM detection probe P6, 2 mM to 3 mM Mg 2+ , 0.2mM~0.3mM dNTP, 0.2mM~0.3mM dUTP and PCR buffer, and the fluorescent groups of each detection probe are different; or / and,

[0119] The enzyme reagent includes 3U / μL to 8U / μL Taq DNA polymerase (for example, 3, 3.2, 3.4, 3.6, 3.8, 4.0, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8, 8U / μL) and 1U / μL to 2U / μL (for example, 1, 1.2, 1.4, 1.6, 1.8, 2U / μL) UNG enzyme.

[0120] In some embodiments of the present application, the concentration ratio of the forward primer and the reaction primer paired therewith in the PCR reaction solution is 1:(8-12) (for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12).

[0121] In some embodiments of the present application, the detection kit further includes one or more of a β-thalassemia standard control and a β-thalassemia negative control.

[0122] In some embodiments of the present application, the detection kit satisfies one or more of the following conditions:

[0123] (1) The β-thalassemia standard control is a wild-type plasmid containing the β-globin gene or normal human genomic DNA; and,

[0124] (2) The β-thalassemia negative control is 1×TE buffer solution or water.

[0125] The fourth aspect of the present application

[0126] The present application provides a method for detecting a β-globin gene, the detection method comprising the following steps:

[0127] The PCR reaction solution in the kit described in the third aspect and the enzyme reagent are mixed and centrifuged to prepare a PCR reaction premix;

[0128] Putting the DNA of the sample to be tested into the PCR reaction premix and performing PCR amplification;

[0129] Based on the probe melting curve method, the obtained amplified product is detected and compared with the β-thalassemia standard control, and the corresponding Tm values ​​of the test sample and the β-thalassemia standard control are obtained, which are recorded as Tm1 and Tm2, ​​respectively. The mutation status of the β-globin gene in the test sample is determined based on the change of Tm1 relative to Tm2.

[0130] In some embodiments of the present application, judging the mutation of the β-globin gene in the sample to be tested according to the change of Tm1 relative to Tm2 includes:

[0131] The difference between Tm2 and Tm1 is recorded as ΔTm, and judging whether the β-globin gene of the sample to be tested contains a mutation or further determining the genotype of the mutation according to the ΔTm, optionally includes:

[0132] Under the condition of ΔTm=5.03±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β IVS-II-654 ;

[0133] Under the condition of ΔTm=5.51±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-71-72 ;

[0134] Under the condition of ΔTm=11.05±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-41-42 ;

[0135] Under the condition of ΔTm=9.59±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β IVS-I-1 ;

[0136] Under the condition of ΔTm=7.74±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-26 ;

[0137] Under the condition of ΔTm=4.68±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-17 ;

[0138] Under the condition of ΔTm=5.18±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β -28 ;

[0139] Under the condition of ΔTm=3.75±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β -29 .

[0140] See the schematic diagram of the primer pair and probe design in the examples of this application. Fig.19Asymmetric PCR uses a pair of primers in unequal amounts to produce a large amount of single-stranded DNA (SSDNA) after PCR amplification. This pair of primers is called a non-restricting primer and a restricting primer. In the first 10 to 15 cycles of the PCR reaction, the amplified product is mainly double-stranded DNA, but when the restricting primer (low-concentration primer) is consumed, the PCR guided by the non-restricting primer (high-concentration primer) will produce a large amount of single-stranded DNA.

[0141] The present application combines asymmetric PCR and probe melting curve analysis, and then performs melting curve analysis from low temperature to high temperature. In the melting curve analysis stage, each fluorescent probe binds to the corresponding PCR single-stranded product at low temperature. In the process of heating the PCR amplification product, as the temperature rises, the DNA double helix gradually melts. When a certain temperature is reached, a large amount of product melting will occur, and the fluorescence will drop sharply. The temperature at which the total DNA double helix structure degrades by half is called the melting temperature (Tm). DNA of different sequences has different Tm values. Melting curve analysis specifically identifies PCR amplification products by utilizing the differences in the melting performance of different templates and the differences in Tm values.

[0142] Specifically, the PCR amplification includes the following steps: PCR buffer, amplification primers, probes and enzyme reagents are mixed and centrifuged together with optional additives to prepare a PCR reaction solution. The samples to be tested are placed in the corresponding PCR reaction solutions. Amplification is then performed on an amplification instrument.

[0143] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0144] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0145] Example 1

[0146] According to the β-globin gene sequence, a novel β-thalassemia gene targeting 8 mutation sites (β IVS-II-654 , β CD-71-72 , β CD-41-42 , β IVS-I-1 , β CD-26 , β CD-17 , β -28 , β -29, see Table 1) of the primer pairs and fluorescent probes. After adding all the primer pairs and fluorescent probes designed above into the PCR reaction tube, asymmetric PCR amplification is performed, and a large number of single-stranded PCR products that can complement the corresponding fluorescent probes are generated after PCR amplification.

[0147] The detection range of the β-thalassemia gene mutation of the present invention is shown in the following table.

[0148] Table 1

[0149] serial number HGVS Nomenclature Base changes 1 HBB: c.316-197C>T IVS-II-654(C>T) 2 HBB:c.216_217insA CD71 / 72(+A) 3 HBB:c.126_129delCTTT CD41 / 42(-TTCT) 4 HBB: c.92+1G>T IVS-I-1(G>T) 5 HBB: c.79G>A CD26(G>A) 6 HBB: c.52A>T CD17(A>T) 7 HBB: c.-78A>G -28(A>G) 8 HBB: c.-79A>G -29(A>G)

[0150] 1. Amplification reagents for asymmetric PCR amplification: HBB PCR reaction solution and HBB enzyme mixture. Among them:

[0151] The HBB PCR reaction solution includes: 1×PCR buffer, 2.75mM MgCl2, 0.2mM each of dATP, dCTP, dGTP, dTTP, and dUTP, 0.1μM β-globin gene amplification primer F1 (SEQ ID NO.1), 1μM β-globin gene amplification primer R1 (SEQ ID NO.2), 0.12μM β-globin gene amplification primer F2 (SEQ ID NO.3), 1.2μM β-globin gene amplification primer R2 (SEQ ID NO.4), 0.16μM β-globin gene amplification primer F3 (SEQ ID NO.5), 1.6μM β-globin gene amplification primer R3 (SEQ ID NO.6), 0.12μM β-globin gene amplification primer F4 (SEQ ID NO.7), 1.2μM β-globin gene amplification primer R4 (SEQ ID NO.8), 0.2μM fluorescent probe P1 (SEQ ID NO. NO.9), 0.26μM fluorescent probe P2 (SEQ ID NO.10), 0.2μM fluorescent probe P3 (SEQ ID NO.11), 0.36μM fluorescent probe P4 (SEQ ID NO.12), 0.32μM fluorescent probe P5 (SEQ ID NO.13), and 0.2μM fluorescent probe P6 (SEQ ID NO.14).

[0152] The HBB enzyme mixture includes 5U / μL Taq DNA polymerase and 2U / μL UNG enzyme.

[0153] The length of the amplification primer is an oligonucleotide chain of 16 bp to 35 bp, the Tm is 50° C. to 70° C., and the GC content is 30% to 60%.

[0154] β-globin gene amplification primer F1 is an upstream primer, and its base sequence is shown in SEQ ID NO.1:

[0155] 5'-TGGTAGCTGGATTGTAGCTGCT-3'.

[0156] β-globin gene amplification primer R1 is a downstream primer, and its base sequence is shown in SEQ ID NO.2:

[0157] 5'-AATGTATCATGCCTCTTTGCAC-3'.

[0158] β-globin gene amplification primer F2 is an upstream primer, and its base sequence is shown in SEQ ID NO.3:

[0159] 5'-TGAGGTTGTCCAGGTGAGCCA-3'.

[0160] β-globin gene amplification primer R2 is a downstream primer, and its base sequence is shown in SEQ ID NO.4:

[0161] 5'-ACCCTTAGGCTGCTGGTGGTC-3'.

[0162] β-globin gene amplification primer F3 is an upstream primer, and its base sequence is shown in SEQ ID NO.5:

[0163] 5'-TGCCCAGTTTTCTATTGGTCTC-3'.

[0164] β-globin gene amplification primer R3 is a downstream primer, and its base sequence is shown in SEQ ID NO.6:

[0165] 5′-ATGGTGCATCTGACTCCTGA-3′.

[0166] β-globin gene amplification primer F4 is an upstream primer, and its base sequence is shown in SEQ ID NO.7:

[0167] 5'-CAGTTGTGTCAGAAGCAAATGTA-3'.

[0168] β-globin gene amplification primer R4 is a downstream primer, and its base sequence is shown in SEQ ID NO.8:

[0169] 5'-CAATCTACTCCCAGGAGCAG-3'

[0170] In the above primer pair: F1 and R1 are for amplifying β IVS-II-654 Forward and reverse primers for gene fragments; F2 and R2 are for amplifying β CD-71-72 , β CD-41-42 Forward and reverse primers for gene fragments; F3 and R3 are for amplifying β IVS-I-1 , βCD-26 , β CD-17 Forward and reverse primers for gene fragments; F4 and R4 are for amplifying β -28 , β -29 Forward and reverse primers for gene fragments.

[0171] The fluorescent probe may be a self-quenching probe with a fluorescent group and a quenching group labeled at the 5' end and the 3' end, respectively.

[0172] The length of the fluorescent probe is an oligonucleotide chain of 16 bp to 35 bp, Tm is 50° C. to 80° C., and the GC content is 40% to 70%.

[0173] The base sequence of the fluorescent probe P1 is shown in SEQ ID NO.9:

[0174] 5'-FAM-ATATTGCTATTGCCTTAACCCAGAA-BHQ1-3'.

[0175] The base sequence of the fluorescent probe P2 is shown in SEQ ID NO.10:

[0176] 5'-ROX-ATCTTACATCACTAAAGGCACCGA-BHQ2-3'.

[0177] The base sequence of the fluorescent probe P3 is shown in SEQ ID NO.11:

[0178] 5'-CY5-TTCCAAAGGACTCAAAGAACCTCTG-BHQ3-3'.

[0179] The base sequence of the fluorescent probe P4 is shown in SEQ ID NO.12:

[0180] 5'-HEX-ATACCAACCTGCCCAGGGCCTCACCAC-BHQ1-3'.

[0181] The base sequence of the fluorescent probe P5 is shown in SEQ ID NO.13:

[0182] 5'-FAM-ACGTTCACCTTGCCCCACAGGGCAGT-BHQ1-3'.

[0183] The base sequence of the fluorescent probe P6 is shown in SEQ ID NO.14:

[0184] 5'-ROX-TGCCCTGACTTTTATGCCCAGCCCTG-BHQ2-3'.

[0185] In the above fluorescent probes: P1 is amplified β IVS-II-654Probe for gene fragment; P2 is amplified β CD-71-72 Probe for gene fragment; P3 is amplified β CD-41-42 Probe for gene fragment; P4 is amplified β IVS-I-1 , β CD-26 Probe for gene fragment; P5 is amplified β CD-17 Probe for gene fragments; P6 is for amplification of β -28 , β -29 Probes for gene fragments.

[0186] Table 2. Correspondence between primer pairs, fluorescent probes and mutation sites

[0187]

[0188] Control reagents: HBB standard control and HBB negative control. Among them:

[0189] HBB standard control: normal human genomic DNA.

[0190] HBB negative control: 1×TE buffer solution without target gene fragment.

[0191] 2. The specific steps are as follows:

[0192] 1. Reagent preparation - liquid preparation area

[0193] First, take all the reagents involved out of the refrigerator and dissolve them at room temperature.

[0194] The standard for PCR reaction solution is: take n×19.25μL HBB PCR reaction solution (n is determined by the number of reaction tubes) and n×0.75μL HBB enzyme mixture into a 1.5mL centrifuge tube, shake and mix for a few seconds, and then centrifuge instantly (4000rpm centrifuge for 5 seconds). PCR reaction solution should be used immediately after preparation, and should be stored below -18℃ for overnight use.

[0195] Aliquoting of PCR reaction solution: Dispense 20 μL of HBB reaction solution into PCR thin-walled reaction tubes.

[0196] Transfer the prepared PCR reaction tube to the extraction room and store it in a refrigerator at 2℃~8℃ until the sample is extracted.

[0197] 2. Sample addition - extraction chamber

[0198] Use a micropipette to add 5 μL of the corresponding DNA sample (diluted to 1 ng / μL, a total of 8 DNA samples carrying different mutations) (β-thalassemia gDNA purchased from Jingliang Technology, concentration of 30 ng / μL), HBB standard control, and HBB negative control to each PCR thin-walled reaction tube, and immediately cover the tube tightly.

[0199] Table 3. Reaction system

[0200]

[0201]

[0202] The numbers of reaction tubes corresponding to DNA samples (≥1 ng / μL), HBB standard controls, and HBB negative controls were 8 tubes (200 μL / tube), 1 tube (100 μL / tube), and 1 tube (100 μL / tube), respectively.

[0203] The PCR thin-walled reaction tube is transferred to the PCR amplification area. The amplification instrument used in the present invention is the SLAN-96S fully automatic medical PCR analysis system produced by Shanghai Hongshi.

[0204] 3. PCR amplification and melting curve analysis - amplification region

[0205] (1) PCR reaction procedure and melting curve analysis procedure

[0206] Table 4

[0207]

[0208] (2) After the program is completed, take out the PCR thin-walled reaction tube (closed tube) and place it in a concave-convex bag, seal it tightly, and handle it according to the contamination source.

[0209] 4. Interpretation of results: The specific method is as follows:

[0210] In the results of the fluorescent PCR melting curve analysis: the changes in the Tm values ​​of the melting peaks of the DNA sample and the HBB standard control in each channel can reflect whether the DNA sample contains β-globin gene mutations and the type of gene mutations. It involves reading the Tm values ​​of the sample to be tested and the HBB standard control. The HBB standard control is used as a correction to reduce the Tm error caused by the instrument and operation. Data analysis is carried out in the following steps:

[0211] (1) Read the Tm value of the melting peak of the HBB standard control in each detection channel;

[0212] (2) reading the Tm value of the DNA sample in each detection channel;

[0213] (3) Subtract the Tm value of the DNA sample in each detection channel from the Tm value of the standard control in each detection channel to obtain the ΔTm value of each detection channel. Refer to the table below to determine whether the sample to be tested contains a mutation and the type of mutation.

[0214] Table 5

[0215]

[0216]

[0217] The experimental results are as follows Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 and Fig. 9 In the figure: "1" refers to the melting curve peak of the wild-type sample at the IVS-II-654 site, "1-1" refers to the melting curve peak of the heterozygous sample at the IVS-II-654 site, "2" refers to the melting curve peak of the wild-type sample at the CD71 / 72 site, "2-1" refers to the melting curve peak of the heterozygous sample at the CD71 / 72 site, "3" refers to the melting curve peak of the wild-type sample at the CD41 / 42 site, "3-1" refers to the melting curve peak of the heterozygous sample at the CD41 / 42 site, and "4" refers to the melting curve peak of the heterozygous sample at the IVS-I-1 and CD26 sites. "4-1" refers to the melting curve peak of the wild-type sample at the IVS-I-1 site, "4-2" refers to the melting curve peak of the heterozygous sample at the CD26 site, "5" refers to the melting curve peak of the wild-type sample at the CD17 site, "5-1" refers to the melting curve peak of the heterozygous sample at the CD17 site, "6" refers to the melting curve peaks of the wild-type samples at -28 and -29 sites, "6-1" refers to the melting curve peak of the heterozygous sample at -28 site, and "6-2" refers to the melting curve peak of the heterozygous sample at -29 site.

[0218] Similarly, the above method can also be used to detect DNA in oral swab samples, and the test results are accurate.

[0219] The sequences of primer probe screening during the experiment are as follows:

[0220] Referring to the reaction system in Table 3 and the reaction procedure in Table 4, detection was performed using the control primer pair and control probe shown in Table 6.

[0221] Table 6

[0222]

[0223]

[0224] Results Fig.10 , Fig.11 , Fig.12 , Fig.13 , Fig.14 , Fig.15 , Fig.16 , Fig.17 as well as Fig.18 .

[0225] Figures 10 to 18 The experimental result graph shown is a partial result graph of the primer probe screening during the experiment. Fig.10 , Fig.11 , Fig.12 , Fig.13 , Fig.14 It can be seen that: Table 6 is a part of the primers and probes designed and screened in the primer and probe research of the present application. Combined with the results shown in the figure, it can be seen that the listed primers and probes cannot completely distinguish the wild type, homozygous mutant type, and heterozygous type.

[0226] Fig.15 , Fig.16 , Fig.17 as well as Fig.18 This is a graph showing the experimental results of optimizing the ratio and concentration of primers and probes during the experiment after the primer and probe sequences are confirmed.

[0227] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0228] The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solutions of the present invention in detail, but cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. A β-thalassemia gene detection probe, characterized in that: The detection probe is selected from one or more of the following detection probes P1 to P6: The sequence of the detection probe P1 is shown in SEQ ID NO.9; The sequence of the detection probe P2 is shown in SEQ ID NO.10; The sequence of the detection probe P3 is shown in SEQ ID NO.11; The sequence of the detection probe P4 is shown in SEQ ID NO.12; The sequence of the detection probe P5 is shown in SEQ ID NO.13; and, The sequence of the detection probe P6 is shown in SEQ ID NO.

14.

2. The β-thalassemia gene detection probe according to claim 1, characterized in that: The detection probe is a self-quenching fluorescent probe, and the 5' end and 3' end of the self-quenching fluorescent probe are respectively labeled with a fluorescent group and a quenching group.

3. A primer pair for detecting β-thalassemia gene, characterized in that: The amplified fragment of the detection primer pair can be specifically bound by the detection probe described in claim 1 or 2.

4. The β-thalassemia gene detection primer pair according to claim 3, characterized in that: The detection primer pair is selected from one or more pairs of the following primer pairs 1 to 4: Primer pair 1, comprising: a forward primer F1 having a sequence as shown in SEQ ID NO.1, and a reverse primer R1 having a sequence as shown in SEQ ID NO.2; Primer pair 2, comprising: a forward primer F2 having a sequence as shown in SEQ ID NO.3, and a reverse primer R2 having a sequence as shown in SEQ ID NO.4; Primer pair 3, comprising: a forward primer F3 having a sequence as shown in SEQ ID NO.5, a reverse primer R3 having a sequence as shown in SEQ ID NO.6; and, Primer pair 4 includes: a forward primer F4 whose sequence is shown as SEQ ID NO.7, and a reverse primer R4 whose sequence is shown as SEQ ID NO.

8.

5. A β-thalassemia gene detection kit, characterized in that: The kit comprises: The detection probe according to claim 1 or 2; or / and, The detection primer pair according to claim 3 or 4.

6. The detection kit according to claim 5, characterized in that The detection kit also includes PCR buffer, Mg 2+ , one or more of dNTP and Taq DNA polymerase, and also includes dUTP and UNG enzyme.

7. The detection kit according to claim 6, characterized in that The detection kit includes PCR reaction solution and enzyme reagent; The PCR reaction solution comprises the forward primer F1, the reverse primer R1, the forward primer F2, the reverse primer R2, the forward primer F3, the reverse primer R3, the forward primer F4, the reverse primer R4, the detection probe P1, the detection probe P2, the detection probe P3, the detection probe P4, the detection probe P5, the detection probe P6, and the Mg 2+ , dNTPs, dUTPs and PCR buffer; The enzyme reagent comprises Taq DNA polymerase and UNG enzyme.

8. The detection kit according to claim 7, characterized in that The PCR reaction solution comprises 0.1 μM to 0.2 μM forward primer F1, 1 μM to 2 μM reverse primer R1, 0.1 μM to 0.2 μM forward primer F2, 1 μM to 2 μM reverse primer R2, 0.1 μM to 0.2 μM forward primer F3, 1 μM to 2 μM reverse primer R3, 0.1 μM to 0.2 μM forward primer F4, 1 μM to 2 μM reverse primer R4, 0.2 μM to 0.3 μM detection probe P1, 0.2 μM to 0.3 μM detection probe P2, 0.2 μM to 0.3 μM detection probe P3, 0.3 μM to 0.4 μM detection probe P4, 0.3 μM to 0.4 μM detection probe P5, 0.2 μM to 0.3 μM detection probe P6, and 2 mM to 3 mM Mg 2+ , 0.2mM to 0.3mM dNTP, 0.2mM to 0.3mM dUTP and PCR buffer, and the fluorescent groups of the detection probes are different; or / and, The enzyme reagent comprises 3U / μL to 8U / μL Taq DNA polymerase and 1U / μL to 2U / μL UNG enzyme.

9. The detection kit according to claim 8, characterized in that The concentration ratio of the forward primer and the reverse primer paired therewith in the PCR reaction solution is 1:(8-12).

10. The detection kit according to any one of claims 5 to 9, characterized in that The detection kit also includes one or more of a β-thalassemia standard control and a β-thalassemia negative control; The β-thalassemia standard control is a wild-type plasmid containing the β-globin gene or normal human genomic DNA; The β-thalassemia negative control is 1×TE buffer solution or water.

11. A method for detecting β-globin gene, characterized in that: The detection method comprises the following steps: Providing a kit according to any one of claims 7 to 10; The PCR reaction solution and the enzyme reagent are mixed and centrifuged to prepare a PCR reaction premix; placing the DNA of the sample to be tested into the PCR reaction premix, and performing PCR amplification to obtain an amplified product of the sample to be tested; and, Based on the probe melting curve method, the amplified product of the test sample is detected and compared with the β-thalassemia standard control, and the corresponding Tm values ​​of the test sample and the β-thalassemia standard control are obtained, which are recorded as Tm1 and Tm2, ​​respectively. The mutation status of the β-globin gene in the test sample is determined based on the change of Tm1 relative to Tm2.

12. The method for detecting the β-globin gene according to claim 11, characterized in that: Determining the mutation of the β-globin gene in the sample to be tested according to the change of Tm1 relative to Tm2 includes: The difference between Tm2 and Tm1 is recorded as ΔTm, and judging whether the β-globin gene of the sample to be tested contains a mutation according to the ΔTm, and / or determining the genotype of the mutation, optionally includes: Under the condition of ΔTm=5.03±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β IVS-II-654 ; Under the condition of ΔTm=5.51±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-71-72 ; Under the condition of ΔTm=11.05±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-41-42 ; Under the condition of ΔTm=9.59±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β IVS-I-1 ; Under the condition of ΔTm=7.74±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-26 ; Under the condition of ΔTm=4.68±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β CD-17 ; Under the condition of ΔTm=5.18±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β -28 ; Under the condition of ΔTm=3.75±1.0, it is determined that there is a mutation in the β-globin gene in the sample to be tested, and the mutation site is β -29 .

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