Primer probe combination for beta-thalassemia single-tube multi-gene detection and kit thereof

Abstract

The invention discloses a primer probe combination for beta-thalassemia single-tube multi-gene detection and a kit of beta-thalassemia single-tube multi-gene detection. According to the primer probe combination for single-tube multiple nucleic acid detection based on the nucleic acid invasive reaction and the kit thereof, provided by the invention, mutation type, insertion type and deletion type beta-thalassemia can be detected in a single closed tube at the same time through one test, the operation is simple and convenient, the pollution risk is low, and the single base recognition specificity is strong.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to primer and probe combinations and kits for single-tube multiplex gene detection of β-thalassemia. Background Technology

[0002] Thalassemia (also known as Mediterranean anemia, hemoglobinogenesis disorder, or simply thalassemia) is an autosomal recessive hemoglobinopathic disorder caused by a genetic defect in the globin gene (point mutation, deletion, etc.), resulting in a reduction or complete absence of biosynthesis of one or more globin peptide chains. This leads to an inability to maintain the normal balance between globin peptide chains and a decrease in the synthesis of adult-type hemoglobin.

[0003] Thalassemia is currently classified into different types based on the defective globin gene: α-, β-, δ-, and δβ-, with α-thalassemia and β-thalassemia being the most common. The clinical symptoms of this disease vary in severity, but most present as chronic progressive hemolytic anemia. Currently, there is no effective and economical treatment for severe thalassemia; treatment generally relies on regular blood transfusions and iron chelation therapy.

[0004] This disease is prevalent worldwide, but is most common in Mediterranean countries, Central Africa, Asia, and the South Pacific. The World Health Organization estimates that approximately 1.5% of the world's population carries the gene for thalassemia. In my country, it is most prevalent in regions south of the Yangtze River, including Fujian, Guangxi, Guangdong, Hainan, Chongqing, Sichuan, Guizhou, and Yunnan, with the highest incidence rates in Guangdong, Guangxi, and Hainan. It is one of the most common and serious genetic diseases in southern my country, exhibiting significant clinical heterogeneity, ranging from no clinical manifestations to severe anemia and even death (common in severe alpha-thalassemia). Children with beta-thalassemia generally do not show clinical manifestations such as anemia at birth or shortly after.

[0005] Thalassemia involves numerous gene mutation sites, with β-thalassemia primarily caused by these mutations. Globally, over 200 mutation types have been identified, with more than 80 million carriers. Currently, over 40 gene mutations leading to β-thalassemia have been identified in my country. Among them, 15 mutation types—CD41-42(-AAAG), CD17(T>A), IVS-II-654(G>A), -28(T>G), -28(T>C), CD71-72(+C), CD26(C>T), CD26(C>A), IVS-I-1(C>T), IVS-I-1(C>G), IVS-I-1(C>A), CD43(C>A), -29(T>C), CD27-28(+A), and CD14-15(+C)—account for over 99% of all mutations. Therefore, detecting these 15 gene mutations is of great significance for the prevention and treatment of β-thalassemia.

[0006] Different types of thalassemia often represent different treatment methods and varying degrees of risk. Therefore, accurately identifying the mutation type in thalassemia can provide guidance for subsequent medical planning and buy patients valuable early treatment time. Compared with single-mode testing, multiplexing for thalassemia not only greatly reduces the significant human and material costs of single-mode testing but also simplifies experimental procedures, reduces testing time, and provides equally accurate results. It has great practical significance and medical value and deserves further research. Therefore, accurate and effective multiplexing technology is crucial for the diagnosis of thalassemia.

[0007] Current diagnostic methods for β-thalassemia are broadly divided into two categories: hematological screening and genetic testing. Screening tests, including erythrocyte morphology analysis, blood cell analysis, erythrocyte osmotic fragility test, and hemoglobin component analysis, are simple and economical, and are therefore commonly used for large-scale screenings and screening in primary healthcare institutions. Genetic testing, while more complex and expensive, offers a more accurate diagnosis of thalassemia. Currently, the main genetic testing methods for β-thalassemia include: polymerase chain reaction-reverse dot blot (PCR-RDB), real-time fluorescence polymerase chain reaction melting curve analysis (PMCA), gene chip technology, and deoxyribonucleic acid (DNA) sequencing. While all these methods can detect the aforementioned 15 gene mutations, each has its limitations: PCR-RDB, gene chips, and DNA sequencing all require multiple open-tube operations, which are cumbersome and prone to cross-contamination of amplification products; PMCA, although enabling closed-tube detection, mostly requires multiple tubes for separate testing, which remains cumbersome and limits its clinical application. Therefore, developing a multiplex detection method based on single-tube closed-tube polymerase chain reaction (PCR) for β-thalassemia gene detection is more in line with clinical application needs. However, due to the limited number of fluorescence channels in the instrument, conventional PCR methods can only detect 4-6 targets per tube, making simultaneous detection of 15 targets challenging. Although fluorescence coding or melting curve analysis can improve throughput, β-thalassemia-related gene mutations are mostly single-base mutations, and these methods still suffer from insufficient specificity when simultaneously performing multiplex detection of dozens of single-base differential targets; while methods with higher specificity have limited throughput. Therefore, in the field of β-thalassemia gene detection, there is still a lack of a single-tube multiplex high-efficiency detection method that combines high-throughput detection capability with single-base resolution specificity. Summary of the Invention

[0008] Purpose of the invention: The technical problem to be solved by the present invention is to provide a primer-probe combination that can simultaneously detect 15 β-thalassemia gene mutations in a single closed-tube reaction.

[0009] The technical problem that this invention also aims to solve is to provide a detection kit with high specificity and sensitivity for 15 high-frequency β-thalassemia genes.

[0010] Technical Solution: To solve the above-mentioned technical problems, the present invention provides a primer-probe combination for single-tube multiplex gene detection of β-thalassemia. The primer-probe combination includes: a specific amplification primer pair, an upstream probe, a downstream probe, and a fluorescent reporter probe; the specific amplification primer pair is selected from any one or more of the following combinations: the upstream primer shown in SEQ ID NO:1, the downstream primer shown in SEQ ID NO:2, the upstream primer shown in SEQ ID NO:3, the downstream primer shown in SEQ ID NO:4, the upstream primer shown in SEQ ID NO:5, the downstream primer shown in SEQ ID NO:6, the upstream primer shown in SEQ ID NO:7, the downstream primer shown in SEQ ID NO:8, the upstream primer shown in SEQ ID NO:9, and the downstream primer shown in SEQ ID NO:10; the sequence of the upstream probe is selected from SEQ ID NO:11-19; the sequence of the downstream probe is selected from SEQ ID NO:20-34; and the sequence of the fluorescent reporter probe is selected from SEQ ID NO:35-45.

[0011] The downstream probe is attached to a flap fragment and phosphorylated at the 3' end. The fluorescent reporter probe is labeled with a fluorescent group at the 5' end, a quenching group at the fourth base at the 5' end, and a blocking group at the 3' end.

[0012] The fluorescent reporter probe contains fluorescent groups including various commonly used fluorescent markers, preferably FAM, VIC, ROX, TXR, CY3 or CY5; the quenching group includes various commonly used quenching agents, preferably BHQ-1, BHQ-2, DABCYL or ECLIPE; and the blocking group includes various commonly used blocking groups, preferably C7-NH2 or C6-Spacer.

[0013] The fluorescent labeling method described in this invention can be used for probes with different melting temperatures for n types of targets and m types of fluorescent groups to achieve n×m target detection, where n is 3 to 6 and m is 2 to 6.

[0014] The concentrations of the upstream and downstream primers are both 0.1–1.5 μM, and the concentrations of the upstream probe, downstream probe, and fluorescent reporter probe are all 0.01–1.0 μM.

[0015] The present invention also includes a single-tube multiplex gene detection kit for β-thalassemia, the detection kit comprising the aforementioned primer-probe combination.

[0016] The detection kit also includes PCR Buffer, dNTP mixture, Taq enzyme, and flap endonuclease 1.

[0017] The concentration of the dNTP mixture is 0.1–0.5 mM.

[0018] The concentration of the Taq enzyme is 0.1–5.0 U.

[0019] The concentration of the flap endonuclease 1 is 2000–8000 U.

[0020] The primer and probe combination or kit for single-tube multiplex gene detection of β-thalassemia can be used for single-tube single-target detection, and the single-tube single-reaction system is as follows:

[0021] Components and final concentrations of 1×PCR buffer:

[0022]

[0023] The primer-probe combination or kit for single-tube multiplex gene detection of β-thalassemia can perform single-fluorescent-channel single-tube multiplex target detection. The single-fluorescent-channel single-tube multiplex reaction system is as follows:

[0024] Components and final concentrations of 1×PCR buffer:

[0025]

[0026]

[0027] The primer-probe combination or detection kit for single-tube multiplex gene detection of β-thalassemia can perform multi-fluorescent channel single-tube multiplex target detection. The multi-fluorescent channel single-tube multiplex reaction system is as follows:

[0028] Components and final concentrations of 1×PCR buffer:

[0029]

[0030] The reaction procedure for the single-tube multiplex gene assay for β-thalassemia is as follows:

[0031] a) Pre-denaturation: Pre-denaturate at 90-100℃ for 3-10 minutes;

[0032] b) Amplification cycle: 40-80 cycles, each cycle containing denaturation at 90-95℃ for 5-60s and annealing extension at 55-80℃ for 10-120s;

[0033] c) Melting curve analysis: Subsequently, the temperature was reduced from 60-80℃ to 25-45℃ at a cooling rate of -0.1 to -1℃ / step. Real-time fluorescence data was collected on a fluorescence PCR instrument, and the rate of fluorescence signal change was analyzed to obtain a rate spectrum for target differentiation.

[0034] The β-thalassemia gene mentioned is a common mutant β-thalassemia gene, including the following 15 common mutants: IVS-II-654(G>A), CD41-42(-AAAG), -29(T>C), IVS-I-1(C>T), IVS-I-1(C>G), IVS-I-1(C>A), -28(T>G), -28(T>C), CD17(T>A), CD71-72(+C), CD27-28(+A), CD26(C>T), CD26(C>A), CD14-15(+C), and CD43(C>A).

[0035] In the primer and probe combination or detection kit for single-tube multiplex gene detection of β-thalassemia described in this invention, the multiplex PCR amplification and detection process is completed in a closed single reaction tube.

[0036] Among these methods, the multiplex target detection method based on the temperature at which the maximum enzyme digestion rate is achieved can be coupled with conventional nucleic acid amplification reactions to enable the detection of low-concentration targets. The nucleic acid amplification reaction can be any one of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), sequence-dependent amplification (NASBA), rolling circle amplification (RCA), ligation amplification (LCR), or recombinase polymerase amplification (RPA), with PCR being preferred.

[0037] This invention targets the detection of high-frequency β-thalassemia genes, selects specific gene sequences from relevant gene databases as target fragments, and designs corresponding primer and probe sequences accordingly.

[0038] The mechanism of the present invention is as follows Figure 1As shown, in the initial stage of the reaction, the target sequence is amplified by multiplex PCR, and the amplification product increases exponentially with the number of cycles. During this process, the upstream probe and the downstream probe labeled with the Flap sequence form a triple-base overlapping invasion structure under the guidance of the target. The FEN1 enzyme precisely cleaves the downstream probe through structure-specific recognition, generating a Flap fragment with a single base end. After amplification, a cooling stage is entered. The free Flap fragment binds to the FRET fluorescent probe through base complementarity pairing, forming an invasion structure again. The FEN1 enzyme specifically cleaves this structure, separating the fluorescent group from the quenching group, thereby completing the secondary nucleic acid invasion reaction. Since the Flap sequences corresponding to different targets have different Tm values, the temperature at which the second-step nucleic acid invasion reaction reaches its maximum rate also varies. By analyzing the temperature-reaction rate characteristic spectrum, each reaction rate peak is defined as a characteristic peak, and its corresponding temperature is used as the characteristic temperature. Finally, multiplex nucleic acid detection is achieved in a single-fluorescent channel reaction system. This invention includes 15 common mutants and their fluorescence channels and characteristic temperature distributions as shown below. Figure 2 As shown, the FAM channel contains IVS-Ⅱ-654(G>A), CD41-42(-AAAG), -29(T>C), IVS-Ⅰ-1(C>T), IVS-Ⅰ-1(C>G), and IVS-Ⅰ-1(C>A); the VIC channel contains -28(T>G), -28(T>C), CD17(T>A), CD71-72(+C), and CD27-28(+A); and the TXR channel contains CD26(C>T), CD26(C>A), CD14-15(+C), and CD43(C>A).

[0039] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: Based on a reasonable design, this invention has been continuously adjusted through numerous experiments, enabling the entire detection process to be completed within a single real-time fluorescence PCR instrument program. This expands the number of multiplexed detections in a single closed reaction tube, overcoming the bottleneck of common fluorescently labeled probe methods that cannot detect more than six targets in a single tube due to limitations in the fluorescence detection channel. This invention ensures that the designed probe combination exhibits strong single-base recognition specificity for the target gene, low total concentration of specific oligonucleotide probes, and good correlation between probes. It is simple and easy to implement, with low technical requirements and low risk of contamination, allowing for convenient detection of multiple β-thalassemia genes in a single tube according to user requirements. The preferred nucleic acid invasion reaction of this invention recognizes the invasion structure formed by the hybridization of upstream and downstream probes with the target using nuclease recognition, exhibiting no sequence bias and applicability to target detection of any sequence. The method has good versatility and high specificity, achieving single-base difference resolution. The hairpin fluorescently labeled probes are universal, resulting in low detection costs. This invention enables the simultaneous detection of point mutation, insertion, and deletion β-thalassemia in a single, closed tube in a single test. It is simple to operate, has a low risk of contamination, and exhibits high specificity for single-base recognition. Attached Figure Description

[0040] Figure 1 This describes the mechanism of single-tube closed-tube multiplex nucleic acid detection technology.

[0041] Figure 2 The distribution of 15 types of β-thalassemia in different fluorescence channels and characteristic temperatures.

[0042] Figure 3 The real-time fluorescence curves and fluorescence signal rate spectra of the FAM channel single-tube multiplex system for detecting six mutant β-thalassemia are shown in Example 1.

[0043] Figure 4 The real-time fluorescence curves and fluorescence signal rate spectra of the VIC channel single-tube multiplex system for detecting six mutant β-thalassemia are shown in Example 2.

[0044] Figure 5 The real-time fluorescence curves and fluorescence signal rate spectra of the TXR channel single-tube multiplex system for detecting six mutant β-thalassemia are shown in Example 3.

[0045] Figure 6 Real-time fluorescence curves and fluorescence signal rate spectra of a single-tube multiplex system with FAM, VIC, and TXR three-fluorescence channels for the detection of 15 mutant β-thalassemia types are shown in Example 4.

[0046] Figure 7 The real-time fluorescence curves and fluorescence signal rate spectra are for evaluating the sensitivity of a multi-fluorescence channel single-tube multiplex system to 15 mutant β-thalassemia, corresponding to Example 5.

[0047] Figure 8 The real-time fluorescence curves and fluorescence signal rate spectra are for the specificity of a multi-channel single-tube multiplex system against 15 mutant β-thalassemia types, corresponding to Example 6.

[0048] Figure 9 The real-time fluorescence curves and fluorescence signal rate spectra of 15 mutant β-thalassemia complex mutations are shown in Example 7.

[0049] Figure 10 To adjust the fluorescence signal rate spectrum of the characteristic temperature by adjusting the downstream probe concentration, see Example 8. Figure 10 DP17 in the text refers to the downstream probe of CD17, corresponding to SEQ ID NO.28. Detailed Implementation

[0050] The following embodiments, in conjunction with the accompanying drawings, further illustrate the present invention. These embodiments are specific examples and are illustrative only, not representing all possibilities of the invention. The invention is not limited to the materials, reaction conditions, or parameters mentioned in these embodiments. Anyone experienced in the relevant field can implement the β-thalassemia single-tube multiplex gene detection described in this invention using other similar materials or reaction conditions, without departing from the basic concepts described in this invention. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the invention, and should not be construed as limiting the invention.

[0051] This invention uses 15 artificially synthesized nucleic acid fragments with different sequences as detection targets to conduct single-tube multiplex nucleic acid detection to examine the detection capability of reagent combinations for β-thalassemia genes.

[0052] Instrument and reagent instructions:

[0053] The instruments and equipment used in the experiment and their suppliers are as follows: real-time fluorescence PCR system (Q2000B, Langji Scientific Instruments Co., Ltd.), micro spectrophotometer (Nano-100, Hangzhou Aosheng Instrument Co., Ltd.), pH meter (Shanghai Bante Instrument Co., Ltd.). The reagents used in the experiment and their sources are as follows: 10×PCR Buffer (pH 8.5) was prepared in the laboratory, and its components were: 300mM NaCl, 100mM Tris-HCl, 75mM MgCl2 and 0.5% Tween-20; chemical reagents (Tween-20, NaCl, MgCl2·6H2O) were purchased from Sinopharm Chemical Reagent Co., Ltd.; molecular biology reagents included TaqHot Start DNA polymerase (TaKaRa R007A) and dNTP mixture (TaKaRa SD0304) from TaKaRa; flap endonuclease 1 (FEN1) was expressed and purified in the laboratory (Establishment of expression and activity assay method for recombinant flap endonuclease 1, Sheng Nan et al., Journal of Biotechnology); distilled water used in the experiment was Watson's product; the plasmid standard of the present invention was formed by inserting the artificial sequences shown in SEQ ID NO.46 to SEQ ID NO.60 into the EcoRI / HindIII restriction sites of the pUC57 plasmid.Specifically, the plasmid standards are: pUC 57 plasmid containing the synthetic sequence IVS-Ⅱ-654(G>A) SEQ ID NO:46; pUC 57 plasmid containing the synthetic sequence CD41-42(-AAAG) SEQ ID NO:47; pUC 57 plasmid containing the synthetic sequence -29(T>C) SEQ ID NO:48; pUC 57 plasmid containing the synthetic sequence IVS-Ⅰ-1(C>T) SEQ ID NO:49; pUC 57 plasmid containing the synthetic sequence IVS-Ⅰ-1(C>G) SEQ ID NO:50; pUC 57 plasmid containing the synthetic sequence IVS-Ⅰ-1(C>A) SEQ ID NO:51; pUC 57 plasmid containing the synthetic sequence -28(T>G) SEQ ID NO:52; and pUC 57 plasmid containing the synthetic sequence -28(T>C) SEQ ID NO:53. pUC 57 plasmids containing the synthetic CD17(T>A) sequence SEQ ID NO:54; pUC 57 plasmids containing the synthetic CD71-72(+C) sequence SEQ ID NO:55; pUC 57 plasmids containing the synthetic CD27-28(+A) sequence SEQ ID NO:56; pUC 57 plasmids containing the synthetic CD26(C>T) sequence SEQ ID NO:57; pUC 57 plasmids containing the synthetic CD26(C>A) sequence SEQ ID NO:58; pUC 57 plasmids containing the synthetic CD14-15(+C) sequence SEQ ID NO:59; and pUC 57 plasmids containing the synthetic CD43(C>A) sequence SEQ ID NO:60.

[0054] All oligonucleotide probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0055] Specific PCR amplification primers and nucleic acid invasion reaction probes were designed for 15 target nucleic acid molecules and divided into three groups for detection in different fluorescence channels. In each fluorescence channel, the Tm value of the downstream probe of the nucleic acid invasion reaction corresponding to different targets when the flap coding sequence hybridizes with the corresponding fluorescently labeled hairpin probe varies from 4℃ to 6℃. The Tm value of the downstream probe corresponding to each target nucleic acid and the target complementary sequence should be designed with reference to the Tm value when the flap coding sequence hybridizes with the corresponding fluorescently labeled hairpin probe, so that the optimal reaction temperature (i.e., the reaction temperature corresponding to the maximum rate of change of fluorescence signal) of the cascade nucleic acid invasion reaction initiated by different target amplification products varies from 4℃ to 6℃.

[0056] This invention detects common mutant β-thalassemia genes, including the following 15 common mutants: IVS-II-654(G>A), CD41-42(-AAAG), -29(T>C), IVS-I-1(C>T), IVS-I-1(C>G), IVS-I-1(C>A), -28(T>G), -28(T>C), CD17(T>A), CD71-72(+C), CD27-28(+A), CD26(C>T), CD26(C>A), CD14-15(+C), and CD43(C>A).

[0057] The primer and probe sequences corresponding to the 15 targets are as follows (5′-3′):

[0058]

[0059]

[0060]

[0061] The following are 15 artificially synthesized sequences for targets (5′-3′):

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] SEQ ID NO:46 is a synthetically produced IVS-II-654 (G>A) sequence; SEQ ID NO:47 is a synthetically produced CD41-42 (-AAAG) sequence; SEQ ID NO:48 is a synthetically produced -29 (T>C) sequence; SEQ ID NO:49 is a synthetically produced IVS-I-1 (C>T) sequence; SEQ ID NO:50 is a synthetically produced IVS-I-1 (C>G) sequence; SEQ ID NO:51 is a synthetically produced IVS-I-1 (C>A) sequence; SEQ ID NO:52 is a synthetically produced -28 (T>G) sequence; SEQ ID NO:53 is a synthetically produced -28 (T>C) sequence; SEQ ID NO:54 is a synthetically produced CD17 (T>A) sequence; SEQ ID NO:55 is a synthetically produced CD71-72 (+C) sequence; SEQ ID NO:56 is a synthetically produced CD27-28 (+A) sequence; SEQ ID SEQ ID NO:57 is a synthetically produced CD26(C>T) sequence; SEQ ID NO:58 is a synthetically produced CD26(C>A) sequence; SEQ ID NO:59 is a synthetically produced CD14-15(+C) sequence; and SEQ ID NO:60 is a synthetically produced CD43(C>A) sequence. These synthetically produced sequences were inserted into the EcoRI / HindIII restriction sites of the pUC57 plasmid, resulting in 15 different types of plasmid standards.

[0069] SEQ ID NO:1 and SEQ ID NO:2 are upstream and downstream primers for IVS-Ⅱ-654(G>A), SEQ ID NO:3 and SEQ ID NO:4 are upstream and downstream primers for CD41-42(-AAAG) and CD43(C>A), SEQ ID NO:5 and SEQ ID NO:6 are upstream and downstream primers for IVS-Ⅰ-1(C>T / G / A), CD27-28(+A), CD26(C>T / A), CD17(T>A), and CD14-15(+C), SEQ ID NO:7 and SEQ ID NO:8 are upstream and downstream primers for -28(T>G / C) and -29(T>C), and SEQ ID NO:9 and SEQ ID NO:10 are upstream and downstream primers for CD71-72(+C).

[0070] SEQ ID NO:11 is the upstream probe for IVS-Ⅱ-654 (G>A), SEQ ID NO:12 is the upstream probe for CD41-42 (-AAAG) and CD43 (C>A), SEQ ID NO:13 is the upstream probe for -29 (T>C), SEQ ID NO:14 is the upstream probe for IVS-Ⅰ-1 (C>T / G / A), SEQ ID NO:15 is the upstream probe for -28 (T>G / C), SEQ ID NO:16 is the upstream probe for CD17 (T>A), SEQ ID NO:17 is the upstream probe for CD71-72 (+C), SEQ ID NO:18 is the upstream probe for CD27-28 (+A), SEQ ID NO:19 is the upstream probe for CD14-15 (+C), and SEQ ID NO:23-25 ​​is the upstream probe for CD26 (C>T / A).

[0071] SEQ ID NO:20 is the downstream probe for IVS-II-654 (G>A), SEQ ID NO:21 is the downstream probe for CD41-42 (-AAAG), SEQ ID NO:22 is the downstream probe for -29 (T>C), SEQ ID NO:23 is the downstream probe for IVS-I-1 (C>T), SEQ ID NO:24 is the downstream probe for IVS-I-1 (C>G), SEQ ID NO:25 is the downstream probe for IVS-I-1 (C>A), SEQ ID NO:26 is the downstream probe for -28 (T>G), SEQ ID NO:27 is the downstream probe for -28 (T>C), SEQ ID NO:28 is the downstream probe for CD17 (T>A), SEQ ID NO:29 is the downstream probe for CD71-72 (+C), SEQ ID NO:30 is the downstream probe for CD27-28 (+A), SEQ ID NO:31 is the downstream probe for CD26 (C>T), SEQ ID NO:32 is the downstream probe of CD26 (C>A), SEQ ID NO:33 is the downstream probe of CD14-15 (+C), and SEQ ID NO:34 is the downstream probe of CD43 (C>A); the underlined part is the flap sequence.

[0072] SEQ ID NO:35 is the IVS-Ⅱ-654(G>A) fluorescent reporter probe, SEQ ID NO:36 is the CD41-42(-AAAG) fluorescent reporter probe, SEQ ID NO:37 is the -29(T>C) fluorescent reporter probe, SEQ ID NO:38 is the IVS-Ⅰ-1(C>T / G / A) fluorescent reporter probe, SEQ ID NO:39 is the -28(T>G / C) fluorescent reporter probe, SEQ ID NO:40 is the CD17(T>A) fluorescent reporter probe, SEQ ID NO:41 is the CD71-72(+C) fluorescent reporter probe, SEQ ID NO:42 is the CD27-28(+A) fluorescent reporter probe, SEQ ID NO:43 is the CD26(C>T / A) fluorescent reporter probe, SEQ ID NO:44 is the CD14-15(+C) fluorescent reporter probe, SEQ ID NO:45 is the CD43(C>A) fluorescent reporter probe; the underlined part is the sequence corresponding to the flap sequence binding site.

[0073] In the reagent combination for the single-tube multiplex gene detection of β-thalassemia, the PCR reaction procedure is as follows:

[0074] a) Pre-denaturation: Pre-denaturate at 90-100℃ for 3-10 minutes;

[0075] b) Amplification cycle: 40-80 cycles, each cycle containing denaturation at 90-95℃ for 5-60s and annealing extension at 55-80℃ for 10-120s;

[0076] c) Melting curve analysis: Subsequently, the temperature was reduced from 60-80℃ to 25-45℃ at a cooling rate of -0.1 to -1℃ / step. Real-time fluorescence data was collected on a fluorescence PCR instrument, and the rate of fluorescence signal change was analyzed to obtain a rate spectrum for target differentiation.

[0077] Example 1: Detection of FAM channel 6-nucleotide targets using characteristic temperature differences

[0078] FAM channel single-tube 6-fold system preparation: 1×PCR Buffer, 0.25mM dNTP mixture, 0.25U Taq hot-start DNA polymerase, 4000U flap endonuclease 1, 0.25μM IVS-II-654 upstream and downstream primers, 0.25μM CD41-42 upstream and downstream primers, 0.25μM IVS-I-1 upstream and downstream primers, 0.25μM -29 upstream and downstream primers, 0.1μM IVS-II-654 upstream probe, 0.1μM CD41-42 upstream probe, 0.2μM IVS-I-1 upstream probe, 0.05μM -29 upstream probe, 0.1μM IVS-II-654 downstream probe, 0.1μM CD41-42 downstream probe, 0.2μM IVS-I-1 (C>T) downstream probe, 0.2μM The reaction system contains 1 μL of IVS-Ⅰ-1 (C>G) downstream probe, 0.2 μM IVS-Ⅰ-1 (C>A) downstream probe, 0.1 μM IVS-Ⅰ-1 (C>A) downstream probe, 0.1 μM IVS-Ⅱ-654 fluorescent reporter probe, 0.1 μM CD41-42 fluorescent reporter probe, 0.1 μM IVS-Ⅰ-1 fluorescent reporter probe, and 0.05 μM IVS-Ⅱ-29 fluorescent reporter probe. 3 Plasmid standards in copies / μL, final volume 20μL.

[0079] The plasmid standards in this embodiment are pUC 57 plasmids containing the artificially synthesized sequence IVS-Ⅱ-654(G>A) SEQ ID NO:46; pUC 57 plasmids containing the artificially synthesized sequence CD41-42(-AAAG) SEQ ID NO:47; pUC 57 plasmids containing the artificially synthesized sequence -29(T>C) SEQ ID NO:48; pUC 57 plasmids containing the artificially synthesized sequence IVS-Ⅰ-1(C>T) SEQ ID NO:49; pUC 57 plasmids containing the artificially synthesized sequence IVS-Ⅰ-1(C>G) SEQ ID NO:50; and pUC 57 plasmids containing the artificially synthesized sequence IVS-Ⅰ-1(C>A) SEQ ID NO:51.

[0080] The following temperature program was used: ① Pre-denaturation: 94℃ × 2 min; ② Cycling parameters: 60 cycles (94℃ 15s → 68℃ 40s, fluorescence acquisition); ③ Gradient analysis: 68℃ → 35℃ (Δ1℃ / 20s), with real-time monitoring of fluorescence signals at each temperature point. Experimental results are as follows: Figure 3 , Figure 3It can be seen that IVS-Ⅱ-654(G>A) peaks during the amplification stage and the signal reaches a plateau. CD41-42(-AAAG), -29(T>C), and IVS-Ⅰ-1(C>T / G / A) peak during the cooling stage and the characteristic temperatures decrease sequentially. The characteristic temperatures are 60℃, 54~52℃, and 45℃, respectively, and the characteristic temperatures of each target are well distinguished.

[0081] Example 2: Detection of VIC channel 5-fold nucleic acid target using characteristic temperature differences

[0082] VIC channel single-tube 5-fold system preparation: 1×PCR Buffer, 0.25mM dNTP mixture, 0.25U Taq hot-start DNA polymerase, 4000U flap endonuclease 1, 0.5μM -28 upstream and downstream primers, 0.75μM MCD17 / CD27-28 upstream and downstream primers, 0.125μM CD71-72 upstream and downstream primers, 0.25μM -28 upstream probe, CD17 upstream probe, CD27-28 upstream probe, 0.5μM -28 (T>G) downstream probe, 0.5μM -28 (T>C) downstream probe, 0.3μM CD17 downstream probe, 0.1μM CD71-72 downstream probe, 0.5μM The downstream probes CD27-28, 0.25 μM CD27-28 fluorescent reporter probe, CD17 fluorescent reporter probe, CD71-72 fluorescent reporter probe, and CD27-28 fluorescent reporter probe were added to the reaction system at a concentration of 1×10⁻⁶. 3 Plasmid standards in copies / μL, final volume 20μL.

[0083] The plasmid standards in this embodiment are pUC57 plasmid containing the synthetically produced -28(T>G) sequence SEQ ID NO:52; pUC57 plasmid containing the synthetically produced -28(T>C) sequence SEQ ID NO:53; pUC57 plasmid containing the synthetically produced CD17(T>A) sequence SEQ ID NO:54; pUC57 plasmid containing the synthetically produced CD71-72(+C) sequence SEQ ID NO:55; and pUC57 plasmid containing the synthetically produced CD27-28(+A) sequence SEQ ID NO:56.

[0084] The following temperature program was used: ① Pre-denaturation: 94℃ × 2 min; ② Cycling parameters: 60 cycles (94℃ 15s → 68℃ 40s, fluorescence acquisition); ③ Gradient analysis: 68℃ → 35℃ (Δ1℃ / 20s), with real-time monitoring of fluorescence signals at each temperature point. Experimental results are as follows: Figure 4 ,from Figure 4It can be seen that -28 (T>G / C) peaks during the amplification stage and the signal reaches a plateau. CD17 (T>A), CD71-72 (+C), and CD27-28 (+A) peak during the cooling stage and their characteristic temperatures decrease sequentially, with characteristic temperatures of 59℃, 55℃, and 50℃, respectively. The characteristic temperatures of each target are well distinguished.

[0085] Example 3: Detection of TXR channel quadruple nucleic acid targets using characteristic temperature differences

[0086] TXR channel single-tube 4-fold system preparation: 1×PCR Buffer, 0.25mM dNTP mixture, 0.25U Taq hot-start DNA polymerase, 4000U flap endonuclease 1, 0.25μM CD26 / CD14-15 upstream and downstream primers, CD43 upstream and downstream primers, 0.1μM IVS-I-1 (C>T) downstream probe, DP IVS-I-1 (C>G) downstream probe, DPIVS-I-1 (C>A) downstream probe (all serving as CD26 upstream probes), 0.25μM CD14-15 upstream probe, CD43 upstream probe, 0.25μM CD26 (C>T) downstream probe, CD26 (C>A) downstream probe, CD14-15 downstream probe, CD43 downstream probe, 0.25μM CD26 fluorescent reporter probe, CD14-15 fluorescent reporter probe, and CD43 fluorescent reporter probe were added to the reaction system at a concentration of 1×10⁻⁶ μL. 3 Plasmid standards in copies / μL, final volume 20μL.

[0087] The plasmid standards in this embodiment contain pUC57 plasmids with the artificially synthesized CD26(C>T) sequence SEQ ID NO:57; pUC57 plasmids with the artificially synthesized CD26(C>A) sequence SEQ ID NO:58; pUC57 plasmids with the artificially synthesized CD14-15(+C) sequence SEQ ID NO:59; and pUC57 plasmids with the artificially synthesized CD43(C>A) sequence SEQ ID NO:60.

[0088] The following temperature program was used: ① Pre-denaturation: 94℃ × 2 min; ② Cycling parameters: 60 cycles (94℃ 15s → 68℃ 40s, fluorescence acquisition); ③ Gradient analysis: 68℃ → 35℃ (Δ1℃ / 20s), with real-time monitoring of fluorescence signals at each temperature point. Experimental results are shown below. Figure 5 ,from Figure 5This indicates that CD26 (C>T / A) peaks during the amplification stage and the signal reaches a plateau, while CD14-15 (+C) and CD43 (C>A) peak during the cooling stage and their characteristic temperatures decrease sequentially, with characteristic temperatures of 60℃ and 54℃, respectively. The characteristic temperatures of each target are well distinguished.

[0089] Example 4: Detection of 15-fold nucleic acid targets with multi-fluorescent channels using characteristic temperature differences

[0090] Preparation of a 15-fold multi-fluorescence channel system: 1×PCR Buffer, 0.25mM dNTP mixture, 1.5U Taq hot-start DNA polymerase, 4000U flap endonuclease 1, 0.5μM all upstream and downstream primers (SEQ ID NO.1~SEQ ID NO.10), 0.2μM all upstream probes (SEQ ID NO:11~19), 0.3μM IVS-II-654 downstream probe, 0.08μM MCD41-42 downstream probe, 0.08μM IVS-I-1 (C>T) downstream probe, 0.08μM IVS-I-1 (C>G) downstream probe, 0.12μM The following downstream probes were added to the reaction system: IVS-Ⅰ-1 (C>A), 0.1 μM CD29, 0.05 μM CD28 (T>G), 0.05 μM CD28 (T>C), 0.2 μM MCD17, 0.1 μM CD71-72, 0.05 μM CD27-28, 0.3 μM MCD26 (C>T), 0.3 μM CD26 (C>A), 0.3 μM CD14-15, 0.1 μM CD43, 0.125 μM each of four fluorescent reporter probes from the FAM channel, 0.1 μM each of four fluorescent reporter probes from the VIC channel, and 0.125 μM each of three fluorescent reporter probes from the TXR channel. 1 μL of a 1×10⁻⁶ concentration was added to the reaction system. 3 Plasmid standards in copies / μL.

[0091] The plasmid standards in this embodiment are as follows: pUC 57 plasmid containing the artificially synthesized sequence IVS-II-654(G>A) SEQ ID NO:46; pUC 57 plasmid containing the artificially synthesized sequence CD41-42(-AAAG) SEQ ID NO:47; pUC 57 plasmid containing the artificially synthesized sequence -29(T>C) SEQ ID NO:48; pUC 57 plasmid containing the artificially synthesized sequence IVS-I-1(C>T) SEQ ID NO:49; pUC 57 plasmid containing the artificially synthesized sequence IVS-I-1(C>G) SEQ ID NO:50; pUC 57 plasmid containing the artificially synthesized sequence IVS-I-1(C>A) SEQ ID NO:51; pUC 57 plasmid containing the artificially synthesized sequence -28(T>G) SEQ ID NO:52; and pUC 57 plasmid containing the artificially synthesized sequence -28(T>C) SEQ ID NO:53. pUC 57 plasmids containing the synthetic CD17(T>A) sequence SEQ ID NO:54; pUC 57 plasmids containing the synthetic CD71-72(+C) sequence SEQ ID NO:55; pUC 57 plasmids containing the synthetic CD27-28(+A) sequence SEQ ID NO:56; pUC 57 plasmids containing the synthetic CD26(C>T) sequence SEQ ID NO:57; pUC 57 plasmids containing the synthetic CD26(C>A) sequence SEQ ID NO:58; pUC 57 plasmids containing the synthetic CD14-15(+C) sequence SEQ ID NO:59; and pUC 57 plasmids containing the synthetic CD43(C>A) sequence SEQ ID NO:60.

[0092] The following temperature program was used: ① Pre-denaturation: 94℃ × 2 min; ② Cycling parameters: 60 cycles (94℃ 15s → 68℃ 40s, fluorescence acquisition); ③ Gradient analysis: 68℃ → 35℃ (Δ1℃ / 20s), with real-time monitoring of fluorescence signals at each temperature point. Experimental results for the six targets in the FAM channel of the 15-fold detection system are shown below. Figure 6In the 15-target detection system, IVS-Ⅱ-654 (G>A) showed a peak and plateaued during the amplification phase. CD41-42 (-AAAG), -29 (T>C), and IVS-Ⅰ-1 (C>T / G / A) exhibited characteristic peaks during the cooling phase, with characteristic temperatures of 61℃, 54℃, and 49–47℃, respectively, demonstrating good differentiation. Among the five targets in the VIC channel of the 15-target detection system, -28 (T>G / C) showed a peak and plateaued during the amplification phase, while CD17 (T>A), CD71-72 (+C), and CD27-28 (+A) showed similar peaks. During the cooling phase, characteristic peaks were observed at temperatures of 62℃, 56℃, and 50℃, demonstrating good differentiation. Among the four targets in the TXR channel of the 15-fold detection system, CD26 (C>T / A) showed a peak and reached a plateau during the amplification phase, while CD14-15 (+C) and CD43 (C>A) showed characteristic peaks during the cooling phase at temperatures of 56℃ and 48℃, respectively, demonstrating good differentiation. Thus, a single-tube multi-fluorescent channel multiplex nucleic acid detection system has been established, enabling the detection of 15 β-thalassemia mutation genes using three fluorescent labels within one tube.

[0093] Example 5

[0094] The sensitivity of multi-fluorescent channel 15-fold nucleic acid target detection was investigated using characteristic temperature differences. The preparation and reaction procedure of the multi-fluorescent channel 15-fold system were the same as in Example 4.

[0095] like Figure 7 In the 15-fold detection system, the detection sensitivity of six mutant genotypes, namely IVS-Ⅱ-654(G>A), CD41-42(-AAAG), -29(T>C), IVS-Ⅰ-1(C>A), -28(T>G), and CD71-72(+C), reached 10^1 copies / reaction; the detection sensitivity of nine mutant genotypes, namely IVS-Ⅰ-1(C>T), IVS-Ⅰ-1(C>G), -28(T>C), CD17(T>A), CD27-28(+A), CD26(C>T), CD26(C>A), CD14-15(+C), and CD43(C>A), was 10^2 copies / reaction.

[0096] Example 6

[0097] The specificity of multi-fluorescent channel 15-fold nucleic acid target detection was investigated using characteristic temperature differences. The preparation and reaction procedure of the multi-fluorescent channel 15-fold system were the same as in Example 4.

[0098] No obvious positive signal was observed in any fluorescence channel for genomic DNA. Figure 8 .

[0099] In my country, high-frequency mutations account for over 90% of β-thalassemia cases, but compound mutations (mutations at two sites simultaneously) also occur. An ideal detection protocol needs to cover both high-frequency and compound mutations. Therefore, it is necessary to examine the ability of a 15-fold detection system to simultaneously detect both types of mutations.

[0100] Example 7

[0101] Complex mutations of 15-fold multi-fluorescent-channel nucleic acid targets were performed using characteristic temperature differences. The preparation and reaction procedures for the 15-fold multi-fluorescent-channel system were the same as in Example 4.

[0102] Based on the genotype distribution of β-thalassemia in 7223 children (analysis of genotypes of thalassemia in 30417 children, Li Dongming et al., Chinese Journal of Contemporary Pediatrics), cases with a double heterozygote frequency greater than 0.05% were detected, with 12 different mutation site combinations and 20 different genotype combinations all detectable. The results are shown below. Figure 9 .

[0103] Example 8

[0104] The optimization process utilizes adjustments to the downstream probe concentration to optimize the characteristic temperature, and the results are as follows: Figure 10 Preparation of the 15-fold multi-channel fluorescence system: 1×PCR Buffer, 0.25mM dNTP mixture, 0.25U Taq hot-start DNA polymerase, 4000U flap endonuclease 1, 0.3μM all upstream and downstream primers (SEQ ID NO: 1~10), 0.2μM all upstream probes (SEQ ID NO: 11~19), 0.3μM Downstream probes for IVS-II-654, 0.1 μM CCD41-42, 0.2 μM IVS-I-1 (C>T), 0.3 μM IVS-I-1 (C>G), 0.3 μM IVS-I-1 (C>A), and different concentrations of -29 downstream probes (0.5 μM, 0.75 μM, 1 μM, 1.25 μM); 0.3 μM -28 (T>G), 0.3 μM -28 (T>C), 0.1 μM CD17, 0.1 μM CD71-72, 0.1 μM CD27-28, 0.3 μM CD26 (C>T), 0.3 μM CD26 (C>A), 0.3 μM CD14-15, and 0.3 μM... The downstream probe of CD43, 0.125 μM each of four fluorescent reporter probes in the FAM channel, 0.1 μM each of four fluorescent reporter probes in the VIC channel, and 0.125 μM each of three fluorescent reporter probes in the TXR channel were added to the reaction system in 1 μL of a 1×10⁻⁶ concentration. 3Plasmid standards were prepared in copies / μL. The following temperature program was used: ① Pre-denaturation: 94℃ × 2 min; ② Cycling parameters: 60 cycles (94℃ 15s → 68℃ 40s, fluorescence acquisition); ③ Gradient analysis: 68℃ → 35℃ (Δ1℃ / 20s), with real-time monitoring of fluorescence signal at each temperature point. The results are as follows: Figure 10 As the downstream probe concentration decreased in gradient (0.7, 0.5, 0.3, 0.1 μM), the peak temperatures of CD17 were 62℃, 60℃, 59℃ and 57℃, respectively. Therefore, the target characteristic temperature can be adjusted by changing the downstream probe concentration.

Claims

1. A primer-probe combination for single-tube multiplex gene detection of β-thalassemia, characterized in that, The primer-probe combination comprises: a specific amplification primer pair, an upstream probe, a downstream probe, and a fluorescent reporter probe; the specific amplification primer pair is selected from any one or more of the following combinations: the upstream primer shown in SEQ ID NO:1, the downstream primer shown in SEQ ID NO:2, the upstream primer shown in SEQ ID NO:3, the downstream primer shown in SEQ ID NO:4, the upstream primer shown in SEQ ID NO:5, the downstream primer shown in SEQ ID NO:6, the upstream primer shown in SEQ ID NO:7, the downstream primer shown in SEQ ID NO:8, the upstream primer shown in SEQ ID NO:9, and the downstream primer shown in SEQ ID NO:10; the sequence of the upstream probe is selected from SEQ ID NO:11-19; the sequence of the downstream probe is selected from SEQ ID NO:20-34; and the sequence of the fluorescent reporter probe is selected from SEQ ID NO:35-45.

2. The primer-probe combination for single-tube multiplex gene detection of β-thalassemia according to claim 1, characterized in that, The downstream probe is attached to a flap fragment and phosphorylated at the 3' end. The fluorescent reporter probe is labeled with a fluorescent group at the 5' end, a quenching group at the fourth base at the 5' end, and a blocking group at the 3' end.

3. The primer-probe combination for single-tube multiplex gene detection of β-thalassemia according to claim 1, characterized in that, The fluorescent reporter probe contains fluorescent groups including various commonly used fluorescent markers. Preferably, the fluorescent groups include FAM, VIC, ROX, TXR, CY3, or CY5. The quenching groups include various commonly used quenchers. Preferably, the quenching groups include BHQ-1, BHQ-2, DABCYL, or ECLIPE. The blocking groups include various commonly used blocking groups. Preferably, the blocking groups include C7-NH2 or C6-Spacer.

4. The primer-probe combination for single-tube multiplex gene detection of β-thalassemia according to claim 1, characterized in that, The concentrations of the upstream and downstream primers are both 0.1–1.5 μM, and the concentrations of the upstream probe, downstream probe, and fluorescent reporter probe are all 0.01–1.0 μM.

5. A single-tube multiplex gene detection kit for β-thalassemia, characterized in that, The detection kit includes the primer-probe combination as described in any one of claims 1 to 3.

6. The β-thalassemia single-tube multiplex gene detection kit according to claim 5, characterized in that, The test kit also includes PCR Buffer, dNTP mixture, Taq enzyme, and flap endonuclease 1.

7. The β-thalassemia single-tube multiplex gene detection kit according to claim 6, characterized in that, The concentration of the dNTP mixture is 0.1~0.5 mM.

8. The β-thalassemia single-tube multiplex gene detection kit according to claim 6, characterized in that, The concentration of the Taq enzyme is 0.1~5.0 U.

9. The β-thalassemia single-tube multiplex gene detection kit according to claim 6, characterized in that, The concentration of the flap endonuclease 1 is 2000~8000 U.

10. The β-thalassemia single-tube multiplex gene detection kit according to claim 6, characterized in that, The reaction procedure for the single-tube multiplex gene assay for β-thalassemia is as follows: a) Pre-denaturation: Pre-denaturate at 90~100℃ for 3-10 min; b) Amplification cycle: 40-80 cycles, each cycle containing denaturation at 90-95℃ for 5-60 s and annealing extension at 55-80℃ for 10-120 s; c) Melting curve analysis: Subsequently, the temperature was reduced from 60~80℃ to 25~45℃ at a cooling rate of -0.1~-1℃ / step. Real-time fluorescence data was collected on a fluorescence PCR instrument, and the rate of fluorescence signal change was analyzed to obtain a rate spectrum for target differentiation.

Images