Primer blocker composition, kit and method for detecting beta-thalassemia mutant type and application of primer blocker composition, kit and method

Abstract

The invention belongs to the field of biology, and particularly relates to a primer blocker composition, a kit and a method for detecting a beta-thalassemia mutant type and application of the primer blocker composition, the kit and the method. The primer blocker composition comprises a primer group and a blocker, the primer group comprises an upstream primer and a downstream primer, the upstream primer, the downstream primer and the blocker are selected from at least one of combinations I to V, and the blocker is modified with a primary amino group. According to the specific primer and blocker optimally designed based on Gibbs free energy, wild-type amplification is selectively inhibited through strict matching of a 3'end base, and high specificity and high sensitivity are shown.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a primer blocker composition, a kit, a method and an application for detecting β-thalassemia mutation types. Background Art

[0002] β-thalassemia (hereinafter referred to as β-thal) is an inherited hemoglobinopathy caused by point mutations or small fragment deletions / insertions in the human β-globin gene (HBB) located at locus 11p15.4 on the short arm of chromosome 11, resulting in reduced or complete absence of β-globin chain synthesis. β-thal is mainly characterized by gene point mutations (accounting for more than 95%), and a small number are structural variations. The southern regions of China are high-incidence areas of β-thal, and the carrier rates in populations in Guangxi, Guangdong and other places can reach 2-3%. Clinically, the severity of β-thal is closely related to the mutation type, mainly manifested as β + (partial synthesis disorder) and β 0 (complete synthesis disorder) two types. At present, more than 300 HBB gene mutations have been discovered, and the high-frequency mutations in East Asian and Southeast Asian populations are mainly CD41-42 (-CTTT), IVS-II-654 (C>T) and CD17 (A>T), etc. The key to the prevention and treatment of thalassemia lies in prenatal diagnosis. Among the current treatment methods, although hematopoietic stem cell transplantation can cure the disease, the matching is strict and the cost is high, and CRISPR gene therapy is still in the research and development stage. Therefore, under this current situation, early screening, early diagnosis and early treatment of thalassemia patients are the main means to improve the prognosis of patients, and prenatal diagnosis of pregnant patients is an important means to prevent the birth of severe thalassemia patients and reduce the prevalence of thalassemia. At present, the diagnostic methods for thalassemia still have the following defects and deficiencies: 1) The detection methods are all qualitative detection methods, which can only judge the "presence" and "absence" of mutations, and cannot quantitatively analyze the mutation dose; 2) The sensitivity is relatively low; 3) The detection methods are complex in operation, long in detection time or high in cost. Therefore, the development of quantitative detection technology for rapid typing diagnosis of thalassemia is of great significance. In addition, the traditional gold standard for prenatal diagnosis (chorionic villus / amniocentesis) has a risk of miscarriage (0.5-1%) and a relatively late detection window (chorionic villus sampling at 10-15 weeks, amniocentesis at 15-20 weeks), so it is particularly important to promote the development of non-invasive prenatal testing technology (NIPT).

[0003] As a single-gene disease (the main pathogenic mutation is a point mutation / minor deletion), the non-invasive detection of thalassemia faces three technical bottlenecks: 1) the proportion of fetal free DNA in maternal blood is only 5-20%; 2) cffDNA fragments are short (average 140bp); 3) the mixing of maternal and fetal DNA makes the detection of single nucleotide variants difficult. Existing NIPT technology is based on the dosage analysis of chromosomal aneuploidy, which makes it difficult to detect minor mutations. Although emerging technologies such as cSMART, RMD and RHDO have attempted breakthroughs, they are still limited by complex bioinformatics analysis and insufficient clinical validation. The core challenge lies in how to achieve accurate detection of low-frequency fetal pathogenic sites in maternal-fetal mixed DNA, which will become a key technical breakthrough in the non-invasive prenatal diagnosis of thalassemia.

[0004] To achieve rapid detection and non-invasive prenatal diagnosis of thalassemia, new molecular diagnostic technologies must meet the following requirements: 1) They must have target enrichment and amplification capabilities, with sufficient sensitivity and specificity to effectively detect target genes / target mutations (especially point mutations); 2) They must be quantitative tests that can quantitatively analyze the mutation dose to infer the patient or fetal genotype; 3) They must be "one-tube" reactions that require only one-step testing and data analysis to obtain results, without the need for complicated multiple-step testing; 4) Based on the above basic premises, the new technology must have good detection efficiency and stability, while taking into account the detection cost. Summary of the Invention

[0005] In view of this, the present invention provides a primer blocker composition, a kit, a method and applications thereof for detecting the mutant type of β-thalassemia.

[0006] To achieve the above solution, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present application provides a primer blocker composition, comprising a primer set and a blocker, wherein the primer set comprises an upstream primer and a downstream primer, and the upstream primer, the downstream primer and the blocker are selected from at least one of the following combinations I to V:

[0008] Combination I: the nucleotide sequence of the upstream primer is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.2, and the nucleotide sequence of the blocker is shown in SEQ ID NO.3;

[0009] Combination II: the nucleotide sequence of the upstream primer is shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer is shown in SEQ ID NO.5, and the nucleotide sequence of the blocker is shown in SEQ ID NO.6;

[0010] Combination III: The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.8, and the nucleotide sequence of the blocker is as shown in SEQ ID NO.9;

[0011] Combination IV: The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.10, the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.11, and the nucleotide sequence of the blocker is as shown in SEQ ID NO.12;

[0012] Combination V: The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.13, the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.14, and the nucleotide sequence of the blocker is as shown in SEQ ID NO.15;

[0013] The blocker is modified with a primary amino group.

[0014] Optionally, the combination I is used to detect the CD41-42 gene mutation of β-thalassemia, the combination II is used to detect the CD17 gene mutation of β-thalassemia, the combination III is used to detect the IVS-II-654 gene mutation of β-thalassemia, the combination IV is used to detect the CD71-72 gene mutation of β-thalassemia, and the combination V is used to detect the CD26 gene mutation of β-thalassemia.

[0015] Optionally, the primary amino group is modified at the 3'-end of the blocker.

[0016] Optionally, the molar ratio of the first primer to the second primer is 1-3:1-3.

[0017] Optionally, the molar ratio of the first primer to the blocker is 1-3:5-15.

[0018] In a second aspect, the present application provides the use of the primer-blocker composition as described above in the preparation of a reagent for detecting β-thalassemia.

[0019] In a third aspect, the present application further provides a kit for detecting the mutant type of β-thalassemia, comprising a fluorescence quantitative PCR reagent, water, and the primer-blocker composition as described above.

[0020] Optionally, the mutant types of β-thalassemia include the CD41-42 gene mutation of β-thalassemia, the CD17 gene mutation of β-thalassemia, the IVS-II-654 gene mutation of β-thalassemia, the CD71-72 gene mutation of β-thalassemia, and the CD26 gene mutation of β-thalassemia.

[0021] Fourthly, the present application also provides a method for detecting β-thalassemia mutation types for non-disease diagnosis and / or treatment purposes, including the following steps:

[0022] Using the primer blocker composition as described above, or using the kit as described above to perform real-time fluorescence quantitative PCR amplification detection on the template DNA of the sample to be detected, and determining whether there is a β-thalassemia mutation and the mutation type.

[0023] Optionally, the conditions for the real-time fluorescence quantitative PCR amplification are: pre-denaturation at 92-96°C for 2-4 min, followed by denaturation at 92-96°C for 20 s, with 42-48 denaturation cycles, then annealing and extension at 58-64°C for 40-50 s, with 42-48 annealing and extension cycles, then treatment at 92-96°C for 10-20 s, then treatment at 58-63°C for 58-63 s, and then treatment at 92-96°C for 10-20 s.

[0024] As described above, the primer blocker composition, kit, method and application thereof for detecting β-thalassemia mutation types of the present invention have the following beneficial effects:

[0025] The specific primers and blockers of the present application optimized and designed based on Gibbs free energy selectively inhibit wild-type amplification through strict base matching at the 3' end, showing high specificity and high sensitivity.

[0026] The present application can detect β-thalassemia point mutations with high sensitivity and specificity; it can perform quantitative detection to achieve quantitative analysis of mutation dosage, thereby inferring the genotype of patients or fetuses; and this technology is a "one-tube" reaction, and the result can be obtained only by one-step detection and data analysis without complicated multiple-step detections; in addition, this technology breaks through the time limit of traditional invasive diagnostic techniques (chorionic villus sampling: 10-15 gestational weeks; amniocentesis: 15-20 gestational weeks), and the detection window can be advanced to 8 weeks of pregnancy. Only 200 μL of maternal peripheral blood sample is required, which can significantly reduce the miscarriage risk (0.5-1%) caused by invasive operations and the physical and mental burden of pregnant women, showing the potential for early diagnosis.

[0027] The combined detection of the BDA (Blocker displacement amplification, BDA, that is, blocker displacement amplification)-QPCR technology of the present application can complete the targeted analysis within 3 h (verified by n = 52), is applicable to primary medical institutions, upgrades the current single-target detection system to single-tube multiplex detection, and can achieve synchronous screening of multiple mutation sites.

[0028] The single - test cost of this application (about 80 yuan) is low, reducing by 95% compared with traditional invasive diagnosis, and can avoid medical expenditures related to complications such as infection.

[0029] This application provides an efficient solution for the rapid genotyping diagnosis of single - gene genetic diseases and non - invasive prenatal diagnosis, showing excellent clinical transformation potential. Brief Description of the Drawings

[0030] Figure 1 is the detection principle diagram of this application. Blocker represents the blocker, Forward primer represents the upstream primer, target represents the target, wild type represents the wild type, Mutant type represents the mutant type, Control represents the control group, and Sanger sequencing represents Sanger sequencing;

[0031] Figure 2 is the result diagram of agarose gel electrophoresis. Contrtol represents the control group, and the same applies hereinafter;

[0032] Figure 3 is the result diagram for verifying the feasibility of BDA - Sanger sequencing;

[0033] Figure 4 is the result diagram for verifying the feasibility of BDA - QPCR. Control represents the control group, +Blocker represents adding the corresponding blocker to the system, WT represents wild, Cycles represents the number of cycles, and the same applies hereinafter;

[0034] Figure 5 is the result diagram for comparing the terminal modification of the CD17 blocker. Flurescence represents the fluorescence intensity;

[0035] Figure 6 is the result diagram of the blocking effect of the BDA technology at different annealing temperatures. Anneal / extendtemperature represents the annealing temperature;

[0036] Figure 7 is the result diagram of the blocking effect of the BDA technology at different primer / blocker ratios;

[0037] Figure 8 is the result diagram for verifying the specificity of the BDA - QPCR of the primers and blockers for 4 genotypes;

[0038] Figure 9 is the result diagram of the BDA - Sanger sequencing peaks of the CD41 - 42 genotype;

[0039] Figure 10 is the result diagram of the BDA - Sanger sequencing peaks of the CD17 genotype;

[0040] Figure 11 The BDA-Sanger sequencing peak result diagram for the CD71-72 genotype;

[0041] Figure 12 The BDA-Sanger sequencing peak result diagram for the IVS-II-654 genotype;

[0042] Figure 13 The result diagram for the BDA-QPCR amplification curves of plasmids with different mutation ratios;

[0043] Figure 14 The result diagram for the correlation between different mutation ratios and △Ct values, where Variant allele fraction represents the variant allele abundance;

[0044] Figure 15 The result diagram for detecting CD41-42 mutant and wild-type samples by BDA-PCR and Sanger sequencing, where sensitivity represents sensitivity, 1-Specificity represents specificity; Predicted represents predicted;

[0045] Figure 16 The result diagram for detecting CD17 mutant and wild-type samples by BDA-PCR and Sanger sequencing; Predicted represents predicted;

[0046] Figure 17 The result diagram for detecting CD71-72 mutant and wild-type samples by BDA-PCR and Sanger sequencing, Predicted represents predicted;

[0047] Figure 18 The result diagram for detecting IVS-II-654 mutant and wild-type samples by BDA-PCR and Sanger sequencing;

[0048] Figure 19 The result diagram for the performance analysis of the BDA technology in non-invasive prenatal diagnosis of the simulated CD41-42 mutation site;

[0049] Figure 20 The result diagram for the performance analysis of the BDA technology in non-invasive prenatal diagnosis of the simulated CD17 mutation site;

[0050] Figure 21 The result diagram for the performance analysis of the BDA technology in non-invasive prenatal diagnosis of the simulated CD71-72 mutation site;

[0051] Figure 22 The result diagram for the performance analysis of the BDA technology in non-invasive prenatal diagnosis of the simulated IVS-II-654 mutation site;

[0052] Figure 23 This is the result graph for the clinical application evaluation of the BDA technology in non-invasive prenatal diagnosis of paternal mutations. PM stands for Paternal mutation, indicating paternal mutations, Non-PM stands for Non Paternal mutation indicating non-paternal mutations, and value represents the value;

[0053] Figure 24 This is the result graph for the clinical application evaluation of the BDA technology in non-invasive prenatal diagnosis of CD41-42 paternal mutations;

[0054] Figure 25 This is the result graph for the clinical application evaluation of the BDA technology in non-invasive prenatal diagnosis of CD17 paternal mutations;

[0055] Figure 26 This is the result graph for the clinical application evaluation of the BDA technology in non-invasive prenatal diagnosis of IVS-II-654 paternal mutations;

[0056] Figure 27 This is the result graph for the clinical application evaluation of the BDA technology in non-invasive prenatal diagnosis of CD71-72 paternal mutations. Detailed implementation mode

[0057] The present invention will be further described below through specific examples. It should be noted that the specific material ratios, process conditions, results, etc. described in the embodiments of the present invention are only used to illustrate the present invention and cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

[0058] As Figure 1 shown, the present application constructs and optimizes a non-invasive prenatal diagnosis system based on the Blocking Displacement Amplification (BDA) technology to achieve accurate detection of β-thalassemia paternal mutations. This system targets and enriches low-frequency mutations in cell-free fetal DNA (cffDNA) in maternal peripheral blood. Specifically:

[0059] Based on Gibbs free energy, the present application optimally designs specific primers / blockers. Specifically, according to the β-globin gene sequence, wild-type amplification is selectively inhibited by strict base matching at the 3' end. Specific primers are designed upstream and downstream of the mutation site. The blocker is completely complementary to the known wild-type sequence and partially overlaps with the specific primers, targeting and enriching low-frequency mutations in cell-free fetal DNA (cffDNA) in maternal peripheral blood;

[0060] When the template is the wild-type sequence, the binding ability of the blocker to the template is stronger than that of the primer, and the PCR amplification efficiency is highly inhibited; when the template is the mutant sequence, the binding ability of the primer to the template is stronger than that of the blocker, and the PCR reaction can proceed effectively; that is, through the dynamic competition between the blocker and the primer, the wild-type amplification in the reaction system is inhibited while the mutant amplification proceeds normally, and the wild-type sequence and the mutant sequence are differentially amplified and magnified; after multiple rounds of PCR reactions, the enrichment level of the mutant sequence can reach more than 1000 times.

[0061] That is, the present application is a non-invasive prenatal diagnosis strategy for multiplex enrichment of rare DNA variants through sequence selectivity and temperature-robust amplification, that is, blocking displacement amplification to amplify low-abundance variant DNA, and combining QPCR and Sanger sequencing to detect paternal mutations of fetal DNA in pregnant women's plasma. Specifically, when the genotypes of the maternal mutation and the paternal mutation are inconsistent, the paternal mutation in the fetal DNA can be sensitively detected by the method combining BDA with QPCR technology or Sanger sequencing, so as to achieve the purpose of exclusion diagnosis. If a paternal mutation is detected, further puncture diagnosis can be performed to verify whether the fetus has severe β-thalassemia; if no paternal mutation is detected, the possibility of the fetus having severe β-thalassemia can be excluded, and there is no need for invasive prenatal examination, which brings convenience to patients and saves examination costs at the same time; using BDA technology to detect paternal β-thalassemia mutations in cff DNA is simple to operate, low in cost, and easy to interpret the results. With its wide coverage, high mutation detection sensitivity and low-cost sequencing advantages, this technology shows broad application prospects.

[0062] The present invention will be described in detail below through specific examples. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all belong to the protection scope of the present invention. The specific process parameters and the like in the following examples are also only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description in this article, rather than being limited to the specific values in the following examples.

[0063] (1) Establishment and optimization of the method for detecting β-thalassemia mutations based on BDA

[0064] 1 Materials and experimental methods

[0065] 1.1 Sample source, collection and preservation

[0066] 1.1.1 Sample source

[0067] (1) Sixty-two pregnant women who underwent prenatal diagnosis at the First Affiliated Hospital of Guangxi Medical University from January 2023 to December 2024 were selected as the research objects. Inclusion criteria: ① Both husband and wife are β-thalassemia carriers with different genotypes; ② The genotype of the pregnant woman is normal and the husband is a β-thalassemia carrier; ③ The pregnant woman underwent chorionic villus / amniocentesis in this hospital and the fetal genotype was confirmed. Exclusion criteria: ① The pregnant woman has other hematological diseases or chromosomal abnormalities, malignant tumors, etc.; ② The genotypes of both husband and wife and the fetus are not clearly carrying β-thalassemia; ③ Those who have not undergone peripheral blood laboratory tests in this hospital; ④ Those lacking complete case data. 5 ml of peripheral blood samples of the research objects were collected, and their clinical information such as gender, age, gestational week, thalassemia genotypes of the couple and fetus, etc. were recorded. All pregnant women undergoing prenatal diagnosis were carried out under the premise of respecting the decisions of the pregnant woman and her family members, and an informed consent form for prenatal diagnosis by puncture was signed:

[0068] (2) Fifty-two β-thalassemia carriers who visited the First Affiliated Hospital of Guangxi Medical University from January 2023 to December 2024 were selected as the research objects (observation group). Inclusion criteria: ① Those with a clear β-thalassemia genotype; ② Peripheral blood was drawn in this hospital for laboratory tests. Exclusion criteria: ① Those with severe thalassemia who have received hematopoietic stem cell transplantation; ② Those with combined different types of thalassemia or other hematological diseases; ③ Those with incomplete clinical data. In addition, 52 healthy physical examination subjects in this hospital during the same period were selected as the control group. The results of routine blood tests were all normal, and hematological diseases, cardiovascular and cerebrovascular diseases, diabetes, hypertension, etc. were excluded. 3 mL of peripheral blood samples of the observation group and the control group were collected. There was no significant difference in general data such as gender and age between the two groups (P>0.05):

[0069] 1.1.2 Collection and preservation of samples

[0070] (1) Collection of pregnant women's peripheral blood: 3 mL of pregnant women's peripheral blood was drawn into an EDTA-K2 anticoagulant tube, the upper plasma was separated, and plasma-free DNA was extracted within 24 hours. The extracted DNA was loaded into a 1.5 ml sterile and enzyme-free centrifuge tube and stored at 4°C for a short term (within one week) and at -20°C for long-term storage for later use.

[0071] (2) Collection of peripheral blood from β-thalassemia carriers and physical examination population: 3 mL of peripheral blood was drawn into an EDTA-K2 anticoagulant tube, and genomic DNA of whole blood was extracted within 24 hours. If it was not extracted in time, the whole blood was temporarily stored at 4°C for no more than one week. The extracted DNA was loaded into a 1.5 mL sterile and enzyme-free centrifuge tube and stored at 4°C for a short term (within one week) and at -20°C for long-term storage for later use.

[0072] 1.2 Experimental method: Establishment and optimization of the BDA-PCR detection system

[0073] 1.2.1 Genomic DNA Extraction

[0074] (1) Extraction of Genomic DNA from Whole Blood

[0075] The genomic DNA of peripheral blood was extracted using the blood genomic DNA extraction kit from TIANGE. The specific extraction process is as follows:

[0076] 1) Prepare the washing buffer PWB with absolute ethanol according to the instructions.

[0077] 2) Take 200 μL of whole blood and place it in a clean 1.5 mL centrifuge tube. Add 400 μL of lysis buffer CL, invert and mix well, and let it stand at room temperature for 3 min.

[0078] 3) Centrifuge at 10000 rpm for 2 min.

[0079] 4) Discard the supernatant, leave the nuclear precipitate, and add 200 μL of buffer GS. Vortex thoroughly to mix.

[0080] 5) Add 200 μL of buffer GB and 20 μL of proteinase K to the premixed solution obtained in step (4), and invert and mix well.

[0081] 6) Incubate the centrifuge tube in a 56 °C water bath for 15 min, invert and mix every 5 min until the solution becomes clear.

[0082] 7) Centrifuge briefly to remove the liquid droplets on the wall of the centrifuge tube, and let it stand at room temperature for 2 - 5 min.

[0083] 8) Add 350 μL of buffer BD, and invert and mix well.

[0084] 9) Add the liquid obtained in step (8) to the adsorption column CG2, centrifuge at 12000 rpm for 30 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0085] 10) Add 500 μL of buffer GDB to the adsorption column CG2, centrifuge at 12000 rpm for 30 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0086] 11) Add 600 μL of washing buffer PWB, centrifuge at 12000 rpm for 30 s, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0087] 12) Repeat step (11).

[0088] 13) Centrifuge at 12000 rpm for 2 min, discard the waste liquid, let it stand at room temperature for 3 min, and air-dry the adsorption membrane.

[0089] 14) Transfer the adsorption column to a new clean 1.5 mL centrifuge tube, add 35 μL of elution buffer TB to the center of the membrane, and let it stand at room temperature for 3 min.

[0090] 15) Centrifuge at 12,000 rpm for 2 min, collect the eluted nucleic acid DNA, and store it at -20 °C for later use.

[0091] (2) Extraction of plasma-free DNA

[0092] Use the plasma-free DNA extraction kit from BioDee to extract plasma-free DNA. The specific extraction process is as follows:

[0093] 1) Separation of plasma: Collect 2 - 3 mL of EDTA-K2 anticoagulated whole blood samples and separate them within 24 h after sampling; centrifuge in a low-temperature centrifuge at 4 °C and 1600 × g for 10 min, carefully transfer the upper plasma to a new clean 1.5 mL centrifuge tube; centrifuge again in a low-temperature centrifuge at 4 °C and 1600 × g for 10 min, and carefully transfer the upper plasma to a clean 1.5 mL centrifuge tube for later use.

[0094] 2) Preparation of washing solution: Add 9 mL of absolute ethanol to 21 mL of washing solution A and mix well; add 21 mL of absolute ethanol to 9 mL of washing solution B and mix well.

[0095] 3) Add 1.5 mL of sample and 10 μL of DNA Carrier to a 15 mL centrifuge tube, then add 1.5 mL of lysis solution and 150 μL of digestion solution, mix well by oscillation, and incubate in a water bath at 65 °C for 10 min.

[0096] 4) Add 6 mL of absolute ethanol to the centrifuge tube and gently invert to mix well. If there are translucent suspensions, it does not affect the DNA extraction and subsequent experiments.

[0097] 5) Place the adsorption column into the collection tube, transfer 6 mL of the above solution into the adsorption column, centrifuge at 8000 rpm for 2 min, and discard the waste liquid in the collection tube.

[0098] 6) Place the adsorption column back into the collection tube, transfer all the remaining solution into the adsorption column, and repeat step 5).

[0099] 7) Place the adsorption column back into the collection tube, add 2.5 mL of washing solution A to the adsorption column, let it stand for 2 min, centrifuge at 8000 rpm for 2 min, and discard the waste liquid in the collection tube.

[0100] 8) Place the adsorption column back into the collection tube, add 2.5 mL of washing solution B to the adsorption column, let it stand for 2 minutes, centrifuge at 8000 rpm for 2 min, and discard the waste liquid in the collection tube.

[0101] 9) Place the adsorption column back into the manifold and centrifuge at 8000 rpm for 2 min to remove the residual washing solution. During this period, take the eluate at 60 μL per sample (for example, for 10 extracted samples, take 600 μL of eluate), place it in a sterilized 1.5 mL centrifuge tube, and preheat it at 65 °C.

[0102] 10) Take out the adsorption column, put it into a new 15 mL collection tube, add 60 μL of eluate (note that the eluate is evenly added to the middle and around the adsorption membrane), let it stand for 3 minutes, and centrifuge at 8000 rpm for 3 min to collect the DNA solution. The extracted DNA can be used for the next experiment or stored at -20 °C.

[0103] Quantify gDNA and cfDNA using a ultra-micro ultraviolet spectrophotometer and a Qubit 4.0 fluorescence quantitative analyzer respectively. The A260 / A280 of the extracted DNA between 1.8 and 2.0 indicates good purity. After recording, store it at -20 °C.

[0104] 1.2.2 Design and synthesis of primers and blockers

[0105] The 4 types of β-thalassemia gene mutations, CD41-42, CD17, CD71-72, and IVS-II-654, are the most common. Through the "Gene" database on the NCBI website, search for and extract the gene sequences of these 4 types of β-thalassemia gene mutations. According to the BDA design principle, design the forward primers, reverse primers, and blockers for these 4 mutation sites respectively, and synthesize them by a biological company. The blocker sequences are synthesized in the following two forms: 4 bases mismatched with the wild-type DNA are connected to the 3' end of the blocker (+AAAA); a chemical modification (+NH2-C6) is connected to the 3' end of the blocker. The primer and blocker sequences are shown in Table 1. Prepare the storage concentration of primers and blockers: Place the new primers and blockers in a small centrifuge and centrifuge at 1500 rpm for 30 s to concentrate the dry powder state of primers and blockers at the bottom of the tube. Add the corresponding microliters of TE buffer according to the amount of nmol marked on the tube wall multiplied by 10 to prepare a storage concentration of 100 μM. Aliquot it into 10 μL / ep tubes and store it in a -20 °C refrigerator for standby. Prepare the working concentration: Take a clean ep tube, label it, add 5 μL of the primer and blocker storage concentration solution, and then add 95 μL of sterile and enzyme-free water to prepare a 5 μM working solution for use.

[0106] 1.2.3 Plasmid synthesis and standard product configuration

[0107] (1) Plasmid construction and synthesis

[0108] Using molecular cloning technology, specific gene sequences were inserted into the corresponding vectors to construct wild-type plasmids and mutant plasmids of four β-thalassemia gene mutation types: CD41-42, CD17, CD71-72, and IVS-II-654. This was completed by a biological company, and the glycerol strains containing the plasmids were stored in an ultra-low temperature freezer at -80°C.

[0109] (2) Plasmid DNA extraction

[0110] Using a commercially available kit, the glycerol bacteria were taken out of the refrigerator. After it melted, a inoculation loop was used to dip the glycerol bacteria in the ultra-clean workbench and evenly spread them on an LB plate containing 100 μg / mL Ampicillin (i.e., ampicillin) resistance. The plate was inverted to avoid condensation water droplets from falling, and it was cultured overnight in a 37°C constant temperature incubator; single colonies were picked and inoculated into 5 mL of LB culture medium containing 100 μg / ml Ampicillin resistance, and placed on a shaker at 200 rpm in a 37°C constant temperature environment for 16 h.

[0111] 1) Take 5 mL of the bacterial culture, centrifuge at 12000 rpm for 1 min, aspirate the supernatant, add RNase A to Solution I before use, and add 250 μL of Solution I to the centrifuge tube with the bacterial cell pellet remaining.

[0112] 2) Add 250 μL of Solution II to the centrifuge tube, gently invert 8 times to fully lyse the bacterial cells, add 350 μL of Solution III to the centrifuge tube, immediately gently invert 8 times, and mix well. At this time, a white flocculent precipitate will appear.

[0113] 3) Centrifuge at 12000 rpm for 10 min, carefully transfer the supernatant to another clean centrifuge tube, avoiding sucking out the precipitate.

[0114] 4) Take the supernatant from the previous step, add 0.6 times the volume of absolute ethanol, and mix well; add the supernatant / mixed solution obtained in the previous step to the adsorption column (the adsorption column is placed in the collection tube), let it stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0115] 5) Add 750 μL of the washing solution to the adsorption column (please check whether absolute ethanol has been added before use), centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column into the collection tube.

[0116] 6) Add 700 μL of the washing solution to the adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and put the adsorption column into the collection tube. Centrifuge at 12000 rpm for 2 min, and place the adsorption column with the opening facing up at room temperature or in a 50°C incubator for several minutes.

[0117] (7) Place the adsorption column into a clean centrifuge tube, suspend and add 200 μL of eluent preheated in a 65 °C water bath to the center of the adsorption membrane, let it stand at room temperature for 2 min, and centrifuge at 12,000 rpm for 1 min.

[0118] (3) Preparation of samples with different mutation ratios

[0119] Use a micro-spectrophotometer to detect the concentration of plasmid DNA, calculate the copy number, and serially dilute it with sterile and enzyme-free water to 10 5 copy number (to simulate the plasma sample concentration). Mix wild-type DNA and mutant DNA to prepare plasmid samples with mutation ratios of 100%, 50%, 10%, 1%, 0.1%, 0.01%, and 0%. The specific operations are as follows:

[0120] 1) Take 50 μL of mutant DNA (plasmid sample with 100% mutation ratio) and add it to 50 μL of wild-type DNA (plasmid sample with 0% mutation ratio), and mix well by shaking to obtain a plasmid sample with a mutation ratio of 50%.

[0121] 2) Take 10 μL of mutant DNA (plasmid sample with 100% mutation ratio) and add it to 90 μL of wild-type DNA (plasmid sample with 0% mutation ratio), and mix well by shaking to obtain a plasmid sample with a mutation ratio of 10% (sample A).

[0122] 3) Take 10 μL from sample A and add it to 90 μL of wild-type DNA, and mix well by shaking to obtain a plasmid sample with a mutation ratio of 1% (sample B).

[0123] 4) Take 10 μL from sample B and add it to 90 μL of wild-type DNA, and mix well by shaking to obtain a plasmid sample with a mutation ratio of 0.1% (sample C).

[0124] 5) Take 10 μL from sample C and add it to 90 μL of wild-type DNA, and mix well by shaking to obtain a plasmid sample with a mutation ratio of 0.01%.

[0125] 6) Store the prepared plasmid samples in a -20 °C refrigerator.

[0126] 1.2.4 Optimization of the BDA-PCR reaction system conditions

[0127] (1) Verification of the effectiveness of primers and blockers by agarose gel electrophoresis

[0128] Perform single-tube amplification of the designed forward and reverse primers and blockers of each genotype (the nucleotide sequences are shown in Table 1). Select plasmids with different mutation ratios as templates. After amplification, perform agarose gel electrophoresis on the PCR products and analyze the bands using a UV imager. The specific operation procedure is as follows:

[0129] 1) PCR amplification

[0130] Label 200 μL PCR reaction tubes and place them on a pre-cooled plate for later use. Configure the reaction system as shown in Table 2. Add each component to a 1.5 mL centrifuge tube in sequence to prepare the master mix, mix well and centrifuge. Then, evenly distribute it into the corresponding PCR reaction tubes. Finally, add plasmid DNA templates with mutation ratios of 100%, 10%, 1%, 0.1%, 0.01%, and 0%. In each batch of tests, add 2 μL of sterile and enzyme-free water to one well as a blank control.

[0131] Table 1 Primer and blocker sequences

[0132]

[0133]

[0134] Note: F represents the forward primer, R represents the reverse primer, and Blocker represents the blocker.

[0135] Table 2 PCR reaction system

[0136] Component BDA Volume Control Group Volume TaKaRa Ex Eaq(5μ / μL) 0.25μL 0.25μL 10×Ex Taq Buffer(20mM) 5μL 5μL dNTP Mixture(2.5mM each) 4μL 4μL Forward Primer(5μM) 1μL 1μL Reverse Primer(5μM) 1μL 1μL Blocker(5μM) 5μL / DNA 2μL 2μL <![CDATA[dd H2O]]> Up to 50μL Up to 50μL

[0137] Note: Buffer represents buffer solution, Forward Primer represents the forward primer, and Reverse Primer represents the reverse primer.

[0138] After loading the samples, place them on a vortex oscillator and shake, then centrifuge horizontally at 1000 rp for 1 min. The reaction conditions are shown in Table 3.

[0139] Table 3 PCR reaction conditions

[0140]

[0141]

[0142] 2) Agarose gel electrophoresis

[0143] Take 5 μL of the PCR amplification product for agarose gel electrophoresis. If the target fragment shows a bright single band in the electrophoresis gel imaging, it indicates successful amplification. The specific operation is as follows:

[0144] a. Dilute the 10×TBE buffer to prepare a 0.5×TBE working solution. Weigh 1.5 g of agarose powder and pour it into a clean conical flask. Add 100 mL of 0.5×TBE buffer and gently shake to mix well, then a 1.5% agarose gel can be prepared.

[0145] b. Place the conical flask in a microwave oven and heat it to boiling. Shake well and repeat 4 times, heating for 30 s each time until the agarose powder is completely dissolved and appears clear and transparent.

[0146] c. After cooling the conical flask, add 5 μL of 10,000× 4S GelRed nucleic acid dye, mix well, pour it into the agarose mold, insert a comb with an appropriate number of wells, and let it stand at room temperature for 20 min until the agarose gel is completely cooled.

[0147] d. After cooling, pull out the comb, place the gel block in the electrophoresis tank, and add 0.5× TBE buffer until the buffer covers the gel surface.

[0148] e. Pipette and mix the 6× Loading Buffer and the PCR product evenly at a ratio of 1:6, then slowly add it into the wells. Add 3 μL of 20 bp DNA Ladder maker into the blank well as an indicator band.

[0149] f. Perform electrophoresis at a constant voltage of 180 V for 25 min. After electrophoresis, place the gel block on a BioRad gel imager for imaging, and observe and photograph the electrophoresis results. The results are as Figure 2 shown.

[0150] As Figure 2 can be seen, the lengths of the amplified target fragments of CD41-42, CD17, CD71-72, and IVS-II-654 are 153 bp, 165 bp, 163 bp, and 223 bp respectively. The electrophoresis band of the non-blocking subgroup (Control group) is single and bright, and the target band of the blocking subgroup (BDA group) is clear and bright. However, due to the inherent limitations of the system, there will be a small amount of non-specific amplification, which has little impact on the detection effect.

[0151] 3) Recovery of PCR products:

[0152] a. Add 500 μL of equilibration buffer BL to the adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and place the adsorption column back into the collection tube.

[0153] b. Cut out the single target DNA band from the agarose gel (try to cut off the excess part) and put it into a clean centrifuge tube, and weigh it.

[0154] c. Add an equal volume of solution PC to the gel block (if the gel weighs 0.1 g, its volume can be regarded as 100 μL, then add 100 μL of PC solution), place it in a 50 °C water bath for about 10 min, and gently invert the centrifuge tube up and down continuously during this period to ensure that the gel block is completely dissolved.

[0155] d. Add the solution obtained in the previous step to an adsorption column CB2 (the adsorption column is placed in a collection tube), centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube; add 600 μL of wash buffer PW to the adsorption column CB2 (please check whether absolute ethanol has been added before use), centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube, and repeat the previous step;

[0156] e. Place the adsorption column CB2 in the collection tube, centrifuge at 12,000 rpm for 2 min to remove as much wash buffer as possible. Place the adsorption column at room temperature for several minutes to dry completely;

[0157] f. Place the adsorption column CB2 in a clean centrifuge tube, add (an appropriate amount of) elution buffer EB dropwise to the middle position of the adsorption membrane, and let it stand at room temperature for 2 min. Centrifuge at 12,000 rpm for 2 min to collect the DNA solution.

[0158] 3) Sanger sequencing

[0159] The recovered PCR amplification products are entrusted to a biological company for Sanger sequencing. The sequencing data obtained are imported into the SnapGene software for analysis to obtain the sequencing results, and the results are as Figure 3 shown.

[0160] As Figure 3 shown, mutations were successfully detected in all genotype MT samples, and the mutation detection effect of the BDA group after adding blockers was better. The BDA technology played a role in expanding mutations and made it easier to detect mutation sites by Sanger sequencing.

[0161] (2) Verification of the effectiveness of primers and blockers by real-time fluorescence quantitative PCR dye method

[0162] Verify the primers and blockers using real-time fluorescence quantitative PCR (QPCR). Compared with agarose gel electrophoresis, QPCR can more intuitively show whether the primers can be effectively amplified through amplification curves and melting curves, and evaluate their amplification efficiency. For mutant templates, their amplification efficiency is theoretically not affected, and the difference in Ct values (abbreviated as △Ct value) between the group with blockers and the group without blockers is close to 0; for wild-type templates, the larger the △Ct value, the better the blocking effect and the higher the feasibility; when the mutation ratio of the template is larger, the smaller the △Ct, which conforms to the theoretical trend of blocking. Whether there is non-specific amplification such as primer dimers in the designed primers can also be observed through the melting curves of the amplification. If the melting curve shows a single peak, it indicates that the amplification specificity of the primer is good. The specific operation process is as follows:

[0163] 1) Place the QPCR dye method reagent, forward primer, reverse primer, blocker, sterile and enzyme-free water, and gDNA on ice for later use. Prepare the QPCR reaction solution and vortex for about 15 s. The reaction system is shown in Table 4 below.

[0164] Table 4 Real-time fluorescence quantitative PCR system

[0165] Component BDA Group Volume Control Group Volume Forward Primer(5uM) 0.5μL 0.5μL Reverse Primer(5uM) 0.5μL 0.5μL Blocker(5uM) 2.5μL / TB Green Premix Ex Taq II 12.5μL 12.5μL <![CDATA[ddH2O]]> Up to 25μL Up to 25μL DNA 2μL 2μL Total volume 25μL 25μL

[0166] Note: Up to means add up to.

[0167] Place the clean eight-well strip tube on a pre-cooled plate. After adding the reaction solution, cover the eight-well strip tube lid. Place the eight-well strip tube on a small centrifuge and centrifuge at 1000 rpm for 1 min to spin dry the liquid on the tube wall and prevent the generation of bubbles. Place the eight-well strip tube on the QPCR instrument. The reaction conditions are shown in Table 5. The amplification results are analyzed using the Bio-Rad CFX Manager 3.0 software, and the results are as Figure 4 shown.

[0168] Table 5 Reaction conditions for real-time fluorescence quantitative PCR

[0169] Step Temperature(℃) Time Number of Cycles Pre-denaturation 95 3min 1 Denaturation 95 20s 45 Annealing Extension 62 40s 45 Melting Curve Analysis 95 15s 1 Melting Curve Analysis 60 60s 1 Melting Curve Analysis 95 15s 1

[0170] As Figure 4 shown, when using the CD41-42 system to amplify, the MT △Ct value is 0.315 ± 0.210, and the △Ct value of WT is 8.385 ± 0.067; when using the CD17 system to amplify, the MT △Ct value is 3.300 ± 0.417, and the △Ct value of WT is 7.805 ± 0.400; when using the CD71-72 system to amplify, the MT △Ct value is 0.982 ± 0.027, and the △Ct value of WT is 10.110 ± 0.088; when using the IVS-II-654 system to amplify, the MT △Ct value is 1.013 ± 0.027, and the △Ct value of WT is 8.996 ± 0.049. The results show that the actual sample detection effect is consistent with the theoretical trend, and the blocker has a good inhibitory effect on the wild type.

[0171] (3) Selection of 3'-end modification of the blocker

[0172] Taking the two most common β-thalassemia genotypes, CD41-42 and CD17, as examples, the forward and reverse primers of the corresponding target gene, blocker (+AAAA / +NH2-C6), TB Green Premix Ex Taq II, enzyme-free water, and 10 5Vortex and mix the wild-type / mutant plasmid DNA copies, briefly centrifuge at 1000 rpm, and place on ice until ready for use. In a clean hood, configure a master reaction tube according to the system in Table 6. Label the eight-tube strips and place them on a pre-cooled plate. Add the solution from the master tube evenly to the corresponding wells of the eight-tube strips, and finally add the plasmid DNA template. For each batch of testing, add 2 μL of sterile, enzyme-free water to one well as a blank control.

[0173] Table 6 Real-time fluorescence quantitative PCR system-2

[0174] Component BDA Group(+AAAA) Volume <![CDATA[Volume of BDA group (+NH2-C6)]]> Control Group Volume Forward Primer(5uM) 0.5μL 0.5μL 0.5μL Reverse Primer(5uM) 0.5μL 0.5μL 0.5μL Blocker(5uM) 2.5μL 2.5μL / TB Green Premix Ex Taq II 12.5μL 12.5μL 12.5μL <![CDATA[ddH2O]]> Up to 25μL Up to 25μL Up to 25μL Wild-type / Mutant Plasmid DNA 2μL 2μL 2μL Total volume 25μL 25μL 25μL

[0175] Note: Total volume indicates the total amount.

[0176] After the sample was added, the mixture was placed on a vortex shaker and centrifuged horizontally at 1000 rpm for 1 minute. The reaction was carried out according to the reaction conditions in Table 5, and the results were analyzed using Bio-Rad CFX Manager 3.0 software. Figure 5 shown.

[0177] like Figure 5 As shown, compared to the control group without a blocker, the addition of a blocker with four unmodified oligonucleotides attached to the 3' end (+AAAA) significantly inhibited amplification, with a cycle difference of 4.89. The inhibition of PCR amplification by a chemically modified blocker attached to the 3' end (+NH2-C6) was even more pronounced, with the cycle difference compared to the control group increasing to 11.13. This result demonstrates that chemically modified blockers attached to the 3' end of the blocker have a more effective blocking effect without compromising the stability of the system.

[0178] (4) Optimization of reaction conditions

[0179] 1) Optimal annealing temperature exploration:

[0180] Taking the CD41-42 genotype as an example, the robustness of the BDA reaction system in the temperature range of 52-66°C was explored, and the optimal reaction temperature was explored to achieve the best effect. According to the reaction system shown in Table 4, TB Green Premix Ex TaqII, forward and reverse primers, 3'-end modified blocker and enzyme-free water were added in sequence, and 100%, 10%, 1% and 0% plasmid were used as templates for amplification. The amplification conditions are shown in Table 5. The annealing temperatures were set at 52°C, 54°C, 56°C, 58°C, 60°C, 62°C, 64°C and 66°C respectively. The other reaction conditions remained unchanged. The results are shown in Table 5. Figure 6 shown.

[0181] like Figure 6 As shown, when the annealing extension temperature is 52-66℃, the Ct values of plasmid DNA with different mutation ratios are different (Figure 6 A), where the ΔCt value is the largest at 62 °C, and the blocking effect is the most significant. Moreover, the results of agarose gel electrophoresis show that the bands are bright and single at 52 - 66 °C, and no primer dimers are generated. Figure 6 B).

[0182] 2) Optimal primer / blocker ratio

[0183] The primer / blocker concentration ratio will have a certain impact on the BDA system. To explore the optimal primer / blocker ratio, using the wild-type plasmid as the template, five groups with primer / blocker ratios of 1:1, 1:2, 1:5, 1:8, and 1:10 were designed in the reaction system, and the other conditions remained unchanged. The reaction system is shown in Table 4, and the results are as Figure 7 shown.

[0184] As Figure 7 shown, with the increase of the primer / blocker ratio, the Ct values of the 4 common mutant genotypes also show a gradually increasing trend (7A). No non-specific amplification was observed in BDA-QPCR, and the electrophoresis bands were bright and single, and no primer dimers appeared (7B).

[0185] (II) Performance verification of the BDA-PCR detection system

[0186] 1 Materials and experimental methods

[0187] 2.1. Samples are the same as above.

[0188] 2.2. Experimental method: Performance verification of the BDA-PCR detection system

[0189] 2.2.1 Specificity verification of the BDA detection system

[0190] According to the construction and optimization of the previous BDA reaction system, the optimal reaction conditions were selected. The BDA-QPCR detection of these 4 genotype mutant samples was carried out using the detection systems of CD41 - 42, CD17, CD71 - 72, and IVS-II-654 primer blockers. Sampling and running were carried out according to the reaction system in Table 4 and the QPCR reaction conditions in Table 5. Each sample was repeated three times, and the detection results were analyzed by Bio-Rad CFX Manager 3.0 software to compare the differences in Ct values between the group without blocker and the group with blocker (i.e., ΔCt value). Theoretically, the CD41 - 42 detection system can only successfully detect CD41 - 42 mutant samples, that is, the ΔCt value tends to 0, while it has an inhibitory effect on other genotype mutant samples, and the ΔCt value increases significantly, indicating that the specificity of this detection system is good. The same applies to the other 3 detection systems, and the detection results are as Figure 8 shown.

[0191] As Figure 8As shown, the average ΔCt value of the CD41-42 system for detecting samples with this genotype mutation is 2.153. For samples with other genotype mutations, the detected ΔCt values are all > 8, showing an obvious blocking effect. Similar to the CD41-42 genotype, the average ΔCt values of the CD17, CD71-72, and IVS-II-654 systems for detecting corresponding mutant samples are 3.233, 1.400, and 0.960 respectively, but they have an obvious inhibitory effect on the other three mutant genotypes, and the difference is statistically significant. This result indicates that the four common genotype detection systems designed and optimized in this application have good specificity and can be used in subsequent experiments.

[0192] 2.2.2 Minimum Detection Limit of BDA-Sanger Sequencing

[0193] According to the optimal reaction conditions, plasmid DNA of four gene mutation types was prepared into different mutation ratios (100%, 50%, 10%, 1%, 0.1%, 0.01%, 0%) for BDA-PCR. It was divided into a control group without blockers and an experimental group with blockers. The recovered PCR amplification products were sent to the company for Sanger sequencing. The sequencing results were analyzed by SnapGene software to compare the differences between the two groups and explore the minimum detection limit of BDA for detecting mutations. The results are as Figures 9 - 12 shown.

[0194] As Figures 9 - 12 shown, in the Control group, the minimum detection limits of the CD41-42, CD71-72, and IVS-II-654 loci are 10% VAF, and the CD17 locus is 1% VAF ( Figures 9 - 12 ), while in the BDA group, the detection sensitivity of all loci is significantly improved to 0.1% VAF, and the mutant signal peak maps are clearly distinguishable ( Figures 9 - 12 ).

[0195] 2.2.3 Plotting of BDA-QPCR Amplification Curve and Standard Curve

[0196] According to the optimal reaction conditions, plasmid DNA of four gene mutation types was proportionally prepared into different mutation concentrations (100%, 10%, 1%, 0.1%, 0.01%, 0%) for fluorescence quantitative PCR detection (BDA-QPCR). It was divided into a control group without blockers and an experimental group with blockers, and three repeated experiments were carried out. The amplification efficiency differences of different mutation ratios were analyzed, and a standard curve was established through the cycle number difference (ΔCt) of plasmid DNA at each mutation ratio reaching the threshold. The calculation formula for the cycle number difference: ΔCt of plasmid samples at each mutation ratio = Ct (experimental group) - Ct (control group of 100% mutant plasmid DNA). The results are as Figure 13 shown.

[0197] AsFigure 13 As shown, the ΔCt value is negatively correlated with the mutation ratio (decrease in mutation ratio → increase in ΔCt), verifying the dose-dependent inhibitory effect of BDA on wild-type amplification; the sensitivity results show that the BDA-QPCR system can stably identify 0.1% VAF mutations, which is consistent with the Sanger sequencing results.

[0198] 2.2.4 Detection of β-thalassemia in whole blood genomic DNA

[0199] According to the construction and optimization of the previous BDA reaction system, the best reaction conditions were selected to perform BDA-PCR detection on 52 clinical samples of four common β-thalassemia genotypes, namely CD41-42, CD17, CD71-72, and IVS-II-654. The reaction system is shown in Table 4, and the amplification conditions are shown in Table 5. Twenty carriers of the CD41-42 genotype and 20 healthy controls during the same period, 20 carriers of the CD17 genotype and 20 healthy controls during the same period, 6 carriers of the CD71-72 genotype and 6 healthy controls during the same period, and 6 carriers of the IVS-II-654 genotype and 6 healthy controls during the same period were selected. Using the extracted genomic DNA as a template, forward and reverse primers and blockers designed for the corresponding genotypes were used for the reaction respectively. The detection results were analyzed by Bio-Rad CFX Manager 3.0 software to compare the differences in ΔCt values between the experimental group and the control group, and to verify the effectiveness of the BDA system in the application of β-thalassemia clinical samples. At the same time, the PCR amplification products were sent to a biological company for Sanger sequencing to analyze the detection results, and the results are as Figures 14 to 18 shown.

[0200] As Figure 14 shown, the lowest effective detection limit is 0.1%. A standard curve of 100%, 10%, 1%, 0.1% and ΔCt value was drawn. The correlations between the mutation ratios of CD41-42, CD17, CD71-72, and IVS-II-654 and ΔCt value are 0.9964, 0.9550, 0.9805, and 0.9773 respectively.

[0201] As Figure 15 shown, for the CD41-42 cohort (n = 20 patients + 20 controls), the BDA-QPCR detection results show that the ΔCt of the mutant group = 2.312 ± 0.602 vs the ΔCt of the control group = 7.951 ± 0.194 (P < 0.001). When the cut-off value ΔCt = 5.357, the sensitivity / specificity is both 1.0 (AUC = 1.0). The Sanger sequencing results show that 20 mutant and 20 normal sequences were accurately detected.

[0202] As Figure 16As shown, for the CD17 cohort (n = 20 patients + 20 controls), the BDA-QPCR test results showed that in the mutant group, ΔCt = 2.613 ± 0.415 vs. the control group with ΔCt = 6.219 ± 0.600 (P < 0.001). When the cut-off value of ΔCt was 4.815, both the sensitivity and specificity were 1.0 (AUC = 1.0). The Sanger sequencing results showed that all 20 mutant and 20 normal sequences were accurately detected.

[0203] As Figure 17 shown, for the CD71-72 cohort (n = 6 patients + 6 controls), the BDA-QPCR test results showed that in the mutant group, ΔCt = 1.332 ± 0.209 vs. the control group with ΔCt = 8.631 ± 0.284 (P < 0.001). When the cut-off value of ΔCt was 4.9265, both the sensitivity and specificity were 1.0 (AUC = 1.0). The Sanger sequencing results showed that all 6 mutant and 6 normal sequences were accurately detected.

[0204] 2.5.4 BDA-QPCR and Sanger Sequencing Results of the IVS-II-654 Cohort

[0205] As Figure 18 shown, for the IVS-II-654 cohort (n = 6 patients + 6 controls), the BDA-QPCR test results showed that in the mutant group, ΔCt = 1.643 ± 1.094 vs. the control group with ΔCt = 8.249 ± 4.399 (P = 0.005). When the cut-off value of ΔCt was 3.9225, both the sensitivity and specificity were 1.0 (AUC = 1.0). The Sanger sequencing results showed that all 6 mutant and 6 normal sequences were accurately detected.

[0206] (3) Clinical Efficacy Verification of the BDA Non-invasive Prenatal Detection System

[0207] 3 Materials and Experimental Methods

[0208] 3.1. The samples are the same as above.

[0209] 3.2. Experimental Method: Clinical Efficacy Verification of the BDA Non-invasive Prenatal Detection System

[0210] 3.2.1. Detection of Artificially Simulated cf-DNA in Pregnant Women's Plasma

[0211] (1) Fragmented DNA

[0212] Fragment 100 μL of gDNA with a concentration of 50 ng / μL using a non-contact ultrasonic disruptor, and run the instrument at a fixed frequency for 15 min; fragment the gDNA into small fragments with a length less than 300 bp to simulate small-fragment plasma cfDNA; analyze the distribution of the fragmented DNA fragments by agarose gel electrophoresis, cut out the gel block with a DNA fragment length less than 300 bp in a UV developer, and recover and purify the gel block using an agarose gel DNA recovery kit. The detailed operation process is as follows:

[0213] Add 500 μL of equilibration buffer BL to the adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0214] 2) Cut out the target DNA fragment band from the agarose gel and put it into a clean centrifuge tube, and weigh it;

[0215] 3) Add the corresponding volume of solution PC to the gel block (if the gel weighs 0.1 g, add 100 μL of PC solution, place it in a 50 °C water bath for 10 min, and gently invert the centrifuge tube up and down continuously during this period to ensure that the gel block is fully dissolved;

[0216] 4) Add the liquid obtained in step 3) to the adsorption column CB2, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube;

[0217] 5) Add 600 μL of wash buffer PW (add absolute ethanol before use) to the adsorption column CB2, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column CB2 into the collection tube;

[0218] 6) Repeat the previous step;

[0219] 7) Put the adsorption column CB2 into the collection tube, centrifuge at 12,000 rpm for 2 min, place the adsorption column at room temperature for several minutes, and dry it thoroughly;

[0220] 8) Put the adsorption column CB2 into a clean centrifuge tube, suspend and add 30 μL of elution buffer EB to the middle position of the adsorption membrane, place it at room temperature for 2 min, centrifuge at 12,000 rpm for 2 min, and collect the DNA solution.

[0221] (2) Construct a paternal mutation pregnant women plasma DNA model

[0222] This application constructs a maternal plasma-free DNA model containing paternal mutations. The plasma cfDNA of normal individuals (βN / βN) is used to simulate maternal plasma cfDNA, and fragmented DNA from β-thalassemia carriers is used to simulate free fetal DNA. The fragmented DNA (50 ng / μL) of β-thalassemia carriers is added to the plasma cfDNA of normal samples in amounts of 8 μL, 6 μL, 4 μL, and 2 μL in sequence to simulate maternal plasma samples with fetal mutation ratios of 20%, 15%, 10%, and 5%, and each concentration is repeated three times. Similarly, fragmented DNA of normal individuals (βN / βN) is incorporated into the plasma cfDNA of normal samples in the same proportion to simulate non-paternal maternal plasma samples. Then, the free DNA in maternal plasma is extracted to construct a maternal plasma DNA model.

[0223] (3) Detection of simulated maternal plasma-free DNA samples

[0224] Maternal plasma models of 4 common genotypes, namely CD41-42, CD17, CD71-72, and IVS-II-654, are constructed respectively. The extracted plasma-free DNA is experimented according to the operation process of BDA-QPCR, and statistical analysis is carried out to compare the differences in △Ct between the paternal mutation group and the non-paternal group. △Ct = Ct (group with blocker) - Ct (group without blocker); an ROC curve is made to judge the diagnostic efficacy of the BDA detection method in this artificial model, that is, the accuracy, sensitivity, and specificity of the detection; at the same time, BDA-PCR combined with Sanger sequencing is carried out to verify whether the incorporation model is successfully detected. The results are as Figures 19 - 22 shown, among which, Figure 19 is the result diagram of BDA-QPCR and BDA-Sanger detection of 10 CD41-42 mutant and 10 wild-type simulated samples respectively; Figure 20 is the result diagram of BDA-QPCR and BDA-Sanger detection of 10 CD17 mutant and 10 wild-type simulated samples respectively; Figure 21 is the result diagram of BDA-QPCR and BDA-Sanger detection of 5 CD71-72 mutant and 5 wild-type simulated samples respectively; Figure 22 is the result diagram of BDA-QPCR and BDA-Sanger detection of 5 IVS-II-654 mutant and 5 wild-type simulated samples respectively.

[0225] As Figure 19 shown, the △Ct value of BDA in the simulated plasma samples of CD41-42 mutant thalassemia is 1.845 ± 0.452, and the △Ct value in the wild-type control group is 4.082 ± 1.367 ([[]] Figure 19 A and Figure 19B), the difference between the two groups was statistically significant (P < 0.001). Taking △Ct = 2.345 as the cut-off value, all CD41-42 mutant samples could be distinguished from wild-type samples ( Figure 19 C), the ROC curve showed that the sensitivity of the detection was 1.0, the specificity was 1.0, and the AUC value was 1.0( Figure 19 D). BDA-Sanger could detect the corresponding mutant sequence in the mutant mimic samples, while it was a normal sequence in the wild-type samples( Figure 19 E).

[0226] As Figure 20 shown, the △Ct value of BDA in CD17 mutant thalassemia mimic plasma samples was 3.084 ± 0.428, and the △Ct value in the wild-type control group was 5.825 ± 1.004( Figure 20 A and Figure 20 B), the difference between the two groups was statistically significant (P < 0.001). Taking △Ct = 3.996 as the cut-off value, all CD17 mutant samples could be distinguished from wild-type samples( Figure 20 C), the ROC curve showed that the sensitivity of the detection was 1.0, the specificity was 1.0, and the AUC value was 1.0( Figure 20 D). BDA-Sanger could detect the corresponding mutant sequence in the mutant mimic samples, while it was a normal sequence in the wild-type samples( Figure 20 E).

[0227] As Figure 21 shown, the △Ct value of BDA in CD71-72 mutant thalassemia mimic plasma samples was 1.479 ± 0.217, and the △Ct value in the wild-type control group was 7.976 ± 0.563( Figure 21 A and 21B), the difference between the two groups was statistically significant (P < 0.001). Taking △Ct = 4.413 as the cut-off value, all CD71-72 mutant samples could be distinguished from wild-type samples( Figure 21 C), the ROC curve showed that the sensitivity of the detection was 1.0, the specificity was 1.0, and the AUC value was 1.0( Figure 21 D). BDA-Sanger could detect the corresponding mutant sequence in the mutant mimic samples, while it was a normal sequence in the wild-type samples( Figure 21 E).

[0228] As Figure 22 shown, the △Ct value of BDA in IVS-II-654 mutant thalassemia mimic plasma samples was 1.165 ± 0.365, and the △Ct value in the wild-type control group was 7.610 ± 0.566( Figure 22A and 22B), the difference between the two groups was statistically significant (P < 0.001). Using △Ct = 4.257 as the cut-off value, all IVS-II-654 mutant samples could be distinguished from wild-type samples ( Figure 22 C), the ROC curve showed that the sensitivity of the detection was 1.0, the specificity was 1.0, and the AUC value was 1.0 ( Figure 22 D). BDA-Sanger could detect the corresponding mutant sequences in mutant mimic samples, while the sequences were normal in wild-type samples ( Figure 22 E).

[0229] 3.2.2 Detection of clinical pregnant women's plasma samples

[0230] Collect plasma samples of pregnant women undergoing prenatal diagnostic examinations in our hospital (the fetal genotypes have been determined through puncture diagnosis). Divide them into 4 cohorts according to the β-thalassemia genotypes of the pregnant women's husbands, namely CD41-42, CD17, CD71-72, and IVS-II-654. Collect clinical information, extract cell-free DNA from the plasma of pregnant women for BDA-QPCR experiments, and perform statistical analysis on the results. At the same time, combine Sanger sequencing to detect the fetal paternal mutations in the plasma of pregnant women to evaluate the accuracy, sensitivity, and specificity of the BDA non-invasive prenatal diagnosis strategy in detecting fetal mutations in maternal plasma. The results are as Figures 23 - 27 shown, among which, Figure 23 is the detection result graph of 62 plasma samples of clinical pregnant women with β-thalassemia; Figure 24 is the detection result graph of 26 high-risk families where the husbands have the CD41-42 genotype, the pregnant women have other genotypes or are normal, and have undergone puncture diagnosis and known fetal genotypes. Among them, 11 puncture results are paternal mutations (41-42M), and 15 puncture results are non-paternal (other genotypes or normal) samples; Figure 25 is the detection result graph of 26 high-risk families where the husbands have the CD17 genotype, the pregnant women have other genotypes or are normal, and have undergone puncture diagnosis and known fetal genotypes. Among them, 17 puncture results are paternal mutations (17M), and 9 puncture results are non-paternal mutations (other genotypes or normal) samples;

[0231] Figure 26 is the detection result graph of 8 high-risk families where the husbands have the IVS-II-654 genotype, the pregnant women have other genotypes or are normal, and have undergone puncture diagnosis and known fetal genotypes. Among them, 3 puncture results are paternal mutations (654M), and 5 puncture results are non-paternal mutations (other genotypes) samples; Figure 27Detection result graphs of two high-risk families where the husband has the CD71-72 genotype, the pregnant woman has other genotypes or is normal, and prenatal diagnosis by chorionic villus sampling has been performed and the fetal genotype is known. Among them, the chorionic villus sampling result of one family showed a paternal mutation (CD71-72), and the result of the other family showed a non-paternal mutation (normal).

[0232] As Figure 23 shown, the BDA-QPCR detection results showed that the ΔCt value of the paternal mutation group was 2.219±1.448, and the ΔCt value of the non-paternal group was 4.920±1.630. There was a significant difference between the two groups (P<0.001). The Sanger sequencing results were consistent with it ( Figure 23 A and Figure 23 B). The ROC curve showed that compared with the chorionic villus sampling diagnosis results, the BDA-PCR non-invasive prenatal diagnosis strategy for paternal mutations showed 85.5% accuracy, 78.1% sensitivity, and 93.3% specificity ( Figure 23 C).

[0233] As Figure 24 shown, the BDA-QPCR detection results showed that the ΔCt value of the paternal mutation group was 2.260±1.650, and the ΔCt value of the non-paternal group was 4.138±1.183. There was a significant difference between the two groups (P = 0.002)( Figure 24 A and Figure 24 B). The ROC curve showed that compared with the chorionic villus sampling diagnosis results, the BDA-QPCR non-invasive prenatal diagnosis strategy for paternal mutations showed 80.8% accuracy, 72.7% sensitivity, and 86.7% specificity ( Figure 24 D). Using the ΔCt value = 3.166 as the optimal cut-off value, 8 positive cases (carrying 41-42M) and 13 negative cases (without 41-42M) were successfully detected ( Figure 24 C). The Sanger sequencing results were consistent with the QPCR results ( Figure 24 E).

[0234] As Figure 25 shown, the BDA-QPCR detection results showed that the ΔCt value of the paternal mutation group was 2.270±1.415, and the ΔCt value of the non-paternal group was 5.935±1.933 (25A and 25B). There was a significant difference between the two groups (P<0.001). The ROC curve showed that compared with the chorionic villus sampling diagnosis results, the BDA-QPCR non-invasive prenatal diagnosis strategy for paternal mutations showed 84.6% accuracy, 76.5% sensitivity, and 100% specificity ( Figure 25 D). Using the ΔCt value = 2.999 as the optimal cut-off value, 13 positive cases (carrying 17M) and 9 negative cases (without 17M) were successfully detected ( Figure 25 C). The Sanger sequencing results were consistent with the QPCR resultsFigure 25 E).

[0235] As Figure 26 shown, the BDA-QPCR test results showed that the ΔCt value of the paternal mutation group was 1.984 ± 1.592, and the ΔCt value of the non-paternal group was 5.668 ± 1.127 (26A and 26B), with a significant difference between the two groups (P = 0.008). The ROC curve showed that compared with the puncture diagnosis results, the BDA-QPCR non-invasive prenatal diagnosis strategy for paternal origin showed 100% accuracy, 100% sensitivity and 100% specificity ( Figure 26 D). Taking the ΔCt value = 4.039 as the optimal cut-off value, 3 positive cases (carrying 654M) and 5 negative cases (without 654M) were successfully detected ( Figure 26 C). The Sanger sequencing results were consistent with the QPCR results ( Figure 26 E)

[0236] As Figure 27 shown, the BDA-QPCR test results showed that three replicates were performed for each sample. The ΔCt value of the paternal mutation was 1.603 ± 1.163, and the ΔCt value of the non-paternal origin was 3.790 ± 1.216, conforming to the ideal trend. The Sanger sequencing results successfully detected 1 paternal mutation sequence and 1 normal sequence, which were consistent with the puncture results ( Figure 27 B).

[0237] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A primer blocker composition, characterized in that, The primer blocker composition comprises a primer set and a blocker. The primer set includes an upstream primer and a downstream primer. The upstream primer, the downstream primer, and the blocker are selected from at least one of the following combinations I to V: Combination I: The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2, and the nucleotide sequence of the blocker is as shown in SEQ ID NO.3; Combination II: The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.5, and the nucleotide sequence of the blocker is as shown in SEQ ID NO.6; Combination III: The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.7, the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.8, and the nucleotide sequence of the blocker is as shown in SEQ ID NO.9; Combination IV: The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.10, the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.11, and the nucleotide sequence of the blocker is as shown in SEQ ID NO.12; Combination V: The nucleotide sequence of the upstream primer is as shown in SEQ ID NO.13, the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.14, and the nucleotide sequence of the blocker is as shown in SEQ ID NO.15; The blocker is modified with a primary amino group.

2. The primer blocker composition according to claim 1, wherein Combination I is used for detecting β-thalassemia CD41-42 gene mutation, Combination II is used for detecting β-thalassemia CD17 gene mutation, Combination III is used for detecting β-thalassemia IVS-II-654 gene mutation, Combination IV is used for detecting β-thalassemia CD71-72 gene mutation, and Combination V is used for detecting β-thalassemia CD26 gene mutation.

3. The primer blocker composition according to claim 1, wherein The primary amino group is modified at the 3'-end of the blocker.

4. The primer blocker composition according to claim 1, characterized in that, The molar ratio of the first primer to the second primer is 1-3:1-3.

5. The primer blocker composition according to claim 1, wherein The molar ratio of the first primer to the blocker is 1-3:5-15.

6. Use of the primer blocker composition according to any one of claims 1-5 in the preparation of a reagent for detecting β-thalassemia.

7. A kit for detecting β-thalassemia mutation types, characterized in that, Comprising a fluorescence quantitative PCR reagent, water, and the primer blocker composition according to any one of claims 1-5.

8. The kit according to claim 7, wherein The β-thalassemia mutant types include β-thalassemia CD41-42 gene mutation, β-thalassemia CD17 gene mutation, β-thalassemia IVS-II-654 gene mutation, β-thalassemia CD71-72 gene mutation, and β-thalassemia CD26 gene mutation.

9. A method for detecting β-thalassemia mutation types for non-disease diagnosis and / or treatment purposes, characterized in that, Comprising the following steps: Use the primer blocker composition described in any one of claims 1-5, or use the kit described in claim 7 or 8 to perform real-time fluorescence quantitative PCR amplification detection on the template DNA of the sample to be detected, and determine whether there is a β-thalassemia mutation and the type of the mutation.

10. The detection method according to claim 9, wherein The conditions for the real-time fluorescence quantitative PCR amplification are: pre-denaturation at 92-96 °C for 2-4 min, then denaturation at 92-96 °C for 20 s, with 42-48 denaturation cycles, followed by annealing and extension at 58-64 °C for 40-50 s, with 42-48 annealing and extension cycles, then treatment at 92-96 °C for 10-20 s, then treatment at 58-63 °C for 58-63 s, and then treatment at 92-96 °C for 10-20 s.

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