Primer blocker composition, kit, method for detecting mutation types of beta thalassemia and application thereof

Abstract

The application belongs to the field of biology and particularly relates to a primer blocker composition, a kit, a method for detecting mutation types of beta thalassemia and application thereof. 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. The specific primer and the blocker are designed based on Gibbs free energy optimization, wild type amplification is selectively inhibited through strict matching of 3' end bases, and high specificity and high sensitivity are shown.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a primer blocker composition, a kit, a method for detecting mutation types of beta thalassemia and application thereof. BACKGROUND

[0002] Beta thalassemia (beta thalassemia for short) is a genetic hemoglobinopathy caused by point mutations or small fragment deletions / insertions of human beta globin gene (HBB) located at 11p15.4 site on chromosome 11, resulting in reduced or complete loss of beta globin chain synthesis. Beta thalassemia is mainly caused by gene point mutations (more than 95%), and a small number of structural variations. The southern region of China is a high incidence area of beta thalassemia, and the carrying rate of the population in Guangxi, Guangdong and other places can reach 2-3%. The severity of beta thalassemia in clinical practice is closely related to the mutation type, mainly manifested as beta + (partial synthesis disorder) and beta 0 (complete synthesis disorder). More than 300 HBB gene mutations have been found, among which CD41-42 (-CTTT), IVS-II-654 (C>T) and CD17 (A>T) are the main high-frequency mutations in East Asian and Southeast Asian populations. The key to the prevention and treatment of thalassemia lies in prenatal diagnosis. Among the current treatment methods, hematopoietic stem cell transplantation can be cured, but the matching is strict and the cost is high, and CRISPR gene therapy is still in the research and development stage. Therefore, under the 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 women is an important means to prevent the birth of severe thalassemia patients and reduce the prevalence of thalassemia. At present, the diagnosis method of thalassemia still has the following defects and deficiencies: 1) The detection methods are all qualitative detection methods, which can only judge the "yes" and "no" of the mutation, and cannot quantitatively analyze the mutation dose; 2) The sensitivity is low; 3) The detection method is complex, the detection time is long or the cost is high. Therefore, it is of great significance to develop quantitative detection technology for rapid typing diagnosis of thalassemia. In addition, the traditional gold standard for prenatal diagnosis (chorionic / amniotic puncture) has a miscarriage risk (0.5-1%) and a late detection window (chorionic puncture 10-15 weeks, amniotic fluid puncture 15-20 weeks), and it is particularly important to promote the development of non-invasive prenatal testing technology (NIPT).

[0003] Thalassemia is a monogenic disease (mainly pathogenic mutations are point mutations / microdeletions), and its non-invasive detection faces three technical bottlenecks: 1) the fetal free DNA in maternal blood accounts for only 5-20%; 2) the cffDNA fragment is short (average 140bp); 3) the difficulty of single nucleotide variation detection caused by the mixture of maternal and fetal DNA. The existing NIPT technology is based on the dosage analysis of chromosomal aneuploidy, which is difficult to detect micro mutations. Emerging technologies such as cSMART, RMD and RHDO have tried to break through, but are still limited by complex bioinformatics analysis and insufficient clinical verification. The core challenge is how to achieve accurate detection of low-frequency fetal pathogenic sites in maternal-fetal mixed DNA, which will become the key technical direction to break through the non-invasive prenatal diagnosis of thalassemia.

[0004] In order to realize the rapid detection and non-invasive prenatal diagnosis of thalassemia, the new molecular diagnostic technology needs to meet the following requirements: 1) it has target enrichment amplification function, and has enough sensitivity and specificity to effectively detect target genes / target mutations (especially point mutations); 2) it needs to be quantitative detection, which can quantitatively analyze the mutation dose, so as to infer the genotype of the patient or fetus; 3) it needs to be a "one-pot" reaction, which can obtain results by one-step detection and data analysis without complicated multi-step detection; 4) on the basis of the above premise, the new technology needs to have good detection efficiency and stability, and at the same time consider the detection cost. SUMMARY

[0005] In view of this, the present application provides a primer blocker composition for detecting beta thalassemia mutation types, a kit, a method and applications thereof.

[0006] In order to realize the above scheme, the technical scheme of the present application is as follows:

[0007] In a first aspect, the present application provides a primer blocker composition, which comprises a primer set and a blocker, the primer set comprising 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 as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the blocker is shown as SEQ ID NO. 3;

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

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

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

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

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

[0014] Optionally, the combination I is used for detecting CD41-42 gene mutation of β-thalassemia, the combination II is used for detecting CD17 gene mutation of β-thalassemia, the combination III is used for detecting IVS-II-654 gene mutation of β-thalassemia, the combination IV is used for detecting CD71-72 gene mutation of β-thalassemia, and the combination V is used for detecting 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 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 types of β-thalassemia mutations, comprising a fluorescent quantitative PCR reagent, water, and the primer blocker composition as described above.

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

[0021] In a fourth aspect, the present application also provides a detection method for detecting the mutation type of beta thalassemia for non-disease diagnosis and / or treatment, comprising the following steps:

[0022] The template DNA of the sample to be detected is subjected to real-time fluorescent quantitative PCR amplification detection by using the primer blocker composition as described above or by using the kit as described above, so as to determine whether the mutation of beta thalasemia exists and the type of the mutation.

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

[0024] As described above, the primer blocker composition, the kit, the method and the application thereof for detecting the mutation type of beta thalasemia of the present application have the following beneficial effects:

[0025] The specific primers and blockers of the present application are designed based on the Gibbs free energy, and the selective inhibition of wild-type amplification is realized by strict matching of the 3' end bases, which shows high specificity and high sensitivity.

[0026] The present application can detect the point mutation of beta thalasemia with high sensitivity and specificity; can perform quantitative detection to realize quantitative analysis of the mutation dose, so as to infer the genotype of the patient or fetus; and the technology is a "one-pot" reaction, which only needs one-step detection and data analysis to obtain the results, without complicated multi-step detection; in addition, the present technology breaks through the time limit of traditional invasive diagnostic technology (chorionic puncture: 10-15 weeks of pregnancy; amniocentesis: 15-20 weeks of pregnancy), and can move the detection window to 8 weeks of pregnancy, only needs 200 μL of maternal peripheral blood sample, can significantly reduce the risk of abortion (0.5-1%) and the physical and mental burden of pregnant women caused by invasive operation, and shows the potential of early diagnosis.

[0027] The BDA (Blocker displacement amplification, BDA, i.e. blocker displacement amplification)-QPCR technology of the present application can complete the targeted analysis (n=52 verification) within 3 h, is suitable for primary medical institutions, upgrades the existing single target detection system to single-pot multiple detection, and can realize the synchronous screening of multiple mutation sites.

[0028] The single detection cost of the application (about 80 yuan) is low, which is reduced by 95% compared with the traditional invasive diagnosis, and can avoid the medical expenses related to complications such as infection.

[0029] The application provides an efficient solution for rapid genotyping diagnosis and non-invasive prenatal diagnosis of single-gene genetic diseases, and shows excellent clinical transformation potential. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The figure is a schematic diagram of the detection of the application. Blocker represents blocker, forward primer represents upstream primer, target represents target, wild type represents wild type, mutant type represents mutant type, control represents control group, and Sanger sequencing represents Sanger sequencing.

[0031] Figure 2 The figure is an agarose gel electrophoresis result diagram. Contrtol represents the control group, and the same below.

[0032] Figure 3 The figure is a BDA-Sanger sequencing feasibility verification result diagram.

[0033] Figure 4 The figure is a BDA-QPCR feasibility verification result diagram. Control represents the control group, +Blocker represents the system adding corresponding blocker, WT represents wild, Cycles represents the number of cycles, and the same below.

[0034] Figure 5 The figure is a result diagram of CD17 blocker end modification comparison. Flurescence represents fluorescence intensity.

[0035] Figure 6 The figure is a result diagram of the blocking effect of BDA technology at different annealing temperatures. Anneal / extend temperature represents annealing temperature.

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

[0037] Figure 8 The figure is a result diagram of BDA-QPCR specificity verification of four genotypes primers and blockers.

[0038] Figure 9 The figure is a BDA-Sanger sequencing peak result diagram of CD41-42 genotype.

[0039] Figure 10 The figure is a BDA-Sanger sequencing peak result diagram of CD17 genotype.

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

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

[0042] Figure 13 BDA-QPCR amplification curve result plot for plasmids with different mutation ratio;

[0043] Figure 14 BDA-QPCR amplification curve result plot for plasmids with different mutation ratio;

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

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

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

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

[0048] Figure 19 BDA technology performance analysis result plot in simulated CD41-42 mutation site non-invasive prenatal diagnosis;

[0049] Figure 20 BDA technology performance analysis result plot in simulated CD17 mutation site non-invasive prenatal diagnosis;

[0050] Figure 21 BDA technology performance analysis result plot in simulated CD71-72 mutation site non-invasive prenatal diagnosis;

[0051] Figure 22 BDA technology performance analysis result plot in simulated IVS-II-654 mutation site non-invasive prenatal diagnosis;

[0052] Figure 23 The figure is the result of the clinical application evaluation of the BDA technology in the paternal mutation non-invasive prenatal diagnosis, PM is paternal mutation, Non-PM is non-paternal mutation, and value is value.

[0053] Figure 24 The figure is the result of the clinical application evaluation of the BDA technology in the CD41-42 paternal mutation non-invasive prenatal diagnosis.

[0054] Figure 25 The figure is the result of the clinical application evaluation of the BDA technology in the CD17 paternal mutation non-invasive prenatal diagnosis.

[0055] Figure 26 The figure is the result of the clinical application evaluation of the BDA technology in the IVS-II-654 paternal mutation non-invasive prenatal diagnosis.

[0056] Figure 27 The figure is the result of the clinical application evaluation of the BDA technology in the CD71-72 paternal mutation non-invasive prenatal diagnosis. DETAILED DESCRIPTION

[0057] The application will be further described below through specific examples, but it should be pointed out that the specific material ratio, process conditions and results described in the embodiments of the application are only used to illustrate the application, and cannot limit the protection scope of the application. Any equivalent changes or modifications made according to the spirit and principle of the application should be covered within the protection scope of the application.

[0058] As shown in Figure 1 The application realizes the accurate detection of paternal mutation of β-thalassemia by constructing and optimizing a non-invasive prenatal diagnosis system based on the blocking displacement amplification (BDA) technology. The system targets the enrichment of low-frequency mutations in maternal peripheral blood fetal cell-free DNA (cffDNA), and specifically:

[0059] Based on Gibbs free energy, the application optimizes the design of specific primers / blockers. Specifically, according to the sequence of β-globin gene, the 3' end base is strictly matched to selectively inhibit wild-type amplification, specific primers are designed at the upstream and downstream of the mutation site, the blocker is completely complementary to the known wild-type sequence and partially overlaps with the specific primer, and the low-frequency mutation in maternal peripheral blood fetal cell-free DNA (cffDNA) is targeted and enriched.

[0060] When the template is a wild-type sequence, the blocking oligo binds to the template more strongly than the primer, and the PCR amplification efficiency is highly inhibited; when the template is a mutant sequence, the primer binds to the template more strongly than the blocking oligo, and the PCR reaction can be effectively carried out; that is, through the dynamic competition of the blocking oligo and the primer, the wild-type amplification in the reaction system is inhibited while the mutant amplification is normally carried out, and the wild-type sequence and the mutant sequence are differentially amplified and amplified; after multiple rounds of PCR reaction, the enrichment degree of the mutant sequence can be up to 1000 times or more;

[0061] That is, the present application uses the technology of sequence selectivity and temperature robust amplification to multiply enrich rare DNA variants, that is, the BDA amplifies and amplifies low-abundance variant DNA, and combines QPCR and Sanger sequencing to detect the non-invasive prenatal diagnosis strategy of paternal mutation of fetal DNA β-thalassemia in pregnant women. Specifically, when the genotype of the maternal mutation is inconsistent with that of the paternal mutation, the paternal mutation in the fetal DNA can be sensitively detected by the BDA combined with the QPCR technology or the Sanger sequencing method, so as to achieve the purpose of exclusion diagnosis. If the paternal mutation is detected, further puncture diagnosis can be performed to verify whether the fetus has severe β-thalassemia; if the paternal mutation is not detected, the possibility of the fetus having severe β-thalassemia can be excluded, and invasive prenatal examination is not needed, which brings convenience to the patient and saves the examination cost; the BDA technology is used to detect the paternal β-thalassemia mutation in the cff DNA, which is simple in operation, low in cost, and easy to read the results. With its wide coverage, high mutation detection sensitivity and low-cost sequencing advantages, the technology shows a wide application prospect.

[0062] The application will be described in detail below through specific examples. It should also be understood that the following examples are only used to specifically describe the application, and cannot be understood as limiting the protection scope of the application. Some non-essential improvements and adjustments made by those skilled in the art according to the above content of the application all belong to the protection scope of the application. The specific process parameters and the like in the following examples are only one example in the appropriate range, that is, those skilled in the art can select within the appropriate range through the description herein, and are not limited to the specific values of the examples in the following text.

[0063] (I) Establishment and optimization of BDA-based detection of β-thalassemia mutation method

[0064] 1. Data and experimental methods

[0065] 1.1. Sample source and collection and preservation

[0066] 1.1.1 Sample source

[0067] (1)62 cases of pregnant women who underwent prenatal diagnosis in the First Affiliated Hospital of Guangxi Medical University from January 2023 to December 2024 were selected as the research objects. The inclusion criteria were: ① Both husband and wife were β-thalassemia carriers with different genotypes; ② The pregnant woman had normal genotype, and the husband was a β-thalassemia carrier; ③ The pregnant woman underwent chorionic villus / amniocentesis in our hospital and the fetal genotype was confirmed. The exclusion criteria were: ① The pregnant woman had other blood system diseases or chromosomal abnormalities, malignancies, etc.; ② The husband and wife and the fetus did not carry the genotype of β-thalassemia; ③ The peripheral blood laboratory test was not done in our hospital; ④ Lack of complete case data. 5ml of peripheral blood sample of the research object was collected, and the clinical information such as gender, age, gestational age, genotype of husband and wife and fetus, etc. was recorded. All pregnant women who underwent prenatal diagnosis were performed under the condition of respecting the decision of the pregnant woman and her family, and signed the informed consent form for puncture prenatal diagnosis:

[0068] (2) 52 cases of 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). The inclusion criteria were: ① The genotype of β-thalassemia was clear; ② Peripheral blood was taken for laboratory test in our hospital. The exclusion criteria were: ① Severe thalassemia and received hematopoietic stem cell transplantation; ② Combined with different types of thalassemia or other blood system diseases; ③ Incomplete clinical data. In addition, 52 cases of healthy persons who underwent physical examination in our hospital during the same period were selected as the control group. The results of blood routine examination were all normal, and the blood system diseases, cardiovascular and cerebrovascular diseases, diabetes, hypertension, etc. were excluded. 3mL of peripheral blood sample of the observation group and the control group was collected. There was no statistically significant difference in general information 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: 3mL of peripheral blood was taken from the pregnant woman into an EDTA-K2 anticoagulant tube, the upper plasma was separated, and the plasma free DNA was extracted within 24h. The extracted DNA was placed into a 1.5ml sterile enzyme-free centrifuge tube, and stored at 4℃ for short-term (within one week) and at -20℃ for long-term storage.

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

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

[0073] 1.2.1 Genomic DNA extraction

[0074] (1) Extraction of genomic DNA from whole blood

[0075] The TIANGE blood genomic DNA extraction kit was used to extract peripheral blood gDNA, and the specific extraction process was as follows:

[0076] 1) According to the instructions, prepare the rinse solution PWB with anhydrous ethanol.

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

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

[0079] 4) Discard the supernatant, leave the nuclear pellet, and add 200 μL of buffer GS, mix well by shaking.

[0080] 5) Add 200 μL of buffer GB and 20 μL of proteinase K to the pre-mixed solution obtained in step (4), mix well by inverting.

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

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

[0083] 8) Add 350 μL of buffer BD and mix well by inverting.

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

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

[0086] 11) Add 600 μL of rinse solution PWB, centrifuge at 12000 rpm / min for 30 s, discard the waste in the collection tube, and place the adsorption column back in the collection tube.

[0087] 12) Repeat step (11) of the experiment.

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

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

[0090] 15) Centrifuge at 12000 rpm for 2 min to collect the eluted nucleic acid DNA, and store at -20 °C for standby.

[0091] (2) Extraction of plasma free DNA

[0092] The plasma free DNA was extracted by using the Qiamp DNA Blood Free Kit, and the specific extraction process was as follows:

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

[0094] 2) Preparation of washing solution: add 9 mL of anhydrous ethanol to 21 mL of washing solution A, and mix well; add 21 mL of anhydrous 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, shake well, and treat in a 65 °C water bath for 10 min.

[0096] 4) Add 6 mL of anhydrous ethanol to the centrifuge tube, and mix gently by inverting, if there is a translucent suspension, it does not affect the extraction of DNA 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, and transfer all the remaining solution into the adsorption column, 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, 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, stand for 2 min, centrifuge at 8000 rpm for 2 min, and discard the waste liquid in the collection tube.

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

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

[0103] The gDNA and cfDNA are quantified by ultraviolet spectrophotometer and Qubit 4.0 fluorescence quantitative analyzer respectively. The A260 / A280 of the extracted DNA is between 1.8 and 2.0, which indicates that the purity is good. Record and store at -20°C.

[0104] 1.2.2 Design and synthesis of primers and blockers

[0105] The four most common types of β-thalassemia gene mutations are CD41-42, CD17, CD71-72 and IVS-II-654. The gene sequences of the four types of β-thalassemia gene mutations are searched and extracted through the "Gene" database of NCBI website. According to the BDA design principle, the forward primers, reverse primers and blockers of the four mutation sites are designed and synthesized by a biological company. The blocker sequence is synthesized in the following two forms: four bases (+AAAA) not matching the wild type DNA are connected to the 3' end of the blocker; a chemical modification (+NH2-C6) is connected to the 3' end of the blocker. The primer and blocker sequences are shown in Table 1. The storage concentration of the primers and blockers is prepared as follows: the new primers and blockers are centrifuged at 1500 rpm for 30 s in a small centrifuge. The primers and blockers in dry powder state are collected at the bottom of the tube. According to the nmol number marked on the tube wall, the corresponding microliter number of TE buffer is added to prepare the storage concentration of 100 μM. The primers and blockers are divided into 10 μL / ep tubes and stored in a -20°C refrigerator for standby use. The working concentration is prepared as follows: a clean ep tube is labeled, 5 μL of primer and blocker storage concentration solution is added, and 95 μL of sterile water is added to prepare a working solution of 5 μM for use.

[0106] 1.2.3 Plasmid synthesis and standard preparation

[0107] (1) Plasmid construction and synthesis

[0108] Using molecular cloning technology, insert the specific gene sequence into the corresponding vector, construct the wild type plasmid and mutant plasmid of CD41-42, CD17, CD71-72, IVS-II-654 four kinds of β-thalassemia gene mutation type, completed by the biological company, the glycerol strain containing the plasmid is stored in the -80℃ ultra-low temperature refrigerator.

[0109] (2) Plasmid DNA extraction

[0110] The glycerol bacteria were taken out from the refrigerator and melted in the clean bench. The glycerol bacteria were evenly coated on the LB plate containing 100 μg / mL ampicillin (i.e. ampicillin) resistance using a inoculation loop. The plate was inverted to avoid condensate water droplets, and cultured in a 37℃ constant temperature incubator overnight. Single colonies were picked and inoculated in 5 mL LB culture solution containing 100 μg / mL ampicillin resistance, and placed in a shaker at 37℃ constant temperature environment at 200 rpm for 16 h.

[0111] 1) Take 5 mL of bacterial culture, centrifuge at 12000 rpm for 1 min, aspirate the supernatant, and add RNase A to solution I before use. Add 250 μL of solution I to the centrifuge tube containing the bacterial precipitate;

[0112] 2) Add 250 μL of solution II to the centrifuge tube, gently invert 8 times to fully lyse the bacteria, and 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 to avoid aspirating the precipitate;

[0114] 4) Take the supernatant of the previous step, add 0.6 times the volume of anhydrous ethanol, and mix well. Add the supernatant / mixture obtained in the previous step to the absorption column (add the absorption column to the collection tube), and place it at room temperature for 2 min. Centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and place the absorption column back into the collection tube;

[0115] 5) Add 750 μL of rinse solution (check if anhydrous ethanol has been added before use) to the absorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the absorption column in the collection tube;

[0116] 6) Add 700 μL of rinse solution to the absorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and place the absorption column in the collection tube. Centrifuge at 12000 rpm for 2 min, and place the absorption column in an open state at room temperature or in a 50℃ incubator for a few minutes;

[0117] 7) Put the adsorption column into a clean centrifuge tube, and add 200 μL 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 then centrifuge at 12000 rpm for 1 min.

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

[0119] Determine the concentration of plasmid DNA by using a microspectrophotometer, calculate the copy number, and dilute it by 10 times with sterile water. 5 Determine the concentration of plasmid DNA by using a microspectrophotometer, calculate the copy number, and dilute it by 10 times with sterile water.

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

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

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

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

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

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

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

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

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

[0129] 1) PCR amplification

[0130] Label 200 μL PCR reaction tubes and place on pre-cooled plate. Prepare reaction system as shown in Table 2, add each component into 1.5 mL centrifuge tube to prepare total tube, mix and centrifuge, then evenly distribute into corresponding PCR reaction tubes, finally add plasmid DNA template with mutation ratio of 100%, 10%, 1%, 0.1%, 0.01%, and 0%. In each batch of detection, add 2 μL sterile water as blank control.

[0131] Table 1 primer and blocker sequences

[0132]

[0133]

[0134] Note: F represents upstream primer, R represents downstream primer, and Blocker represents blocker.

[0135] Table 2 PCR reaction system

[0136] Components BDA volume Control volume TaKaRa Ex Taq (5μ / μL) 0.25μL 0.25μL 10x 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 dd H2O Up to 50μL Up to 50μL

[0137] Note: Buffer represents buffer, Forward Primer represents upstream primer, and Reverse Primer represents downstream primer.

[0138] After sample loading, place on vortex shaker, then centrifuge at 1000 rpm for 1 min. Reaction conditions are shown in Table 3.

[0139] Table 3 PCR reaction conditions

[0140]

[0141]

[0142] 2) Agarose gel electrophoresis

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

[0144] a. Dilute 10x TBE buffer to 0.5x TBE working solution, weigh 1.5 g of agarose powder into a clean conical flask, add 100 mL of 0.5x TBE buffer and mix well, then prepare 1.5% concentration agarose gel;

[0145] b. Put the conical flask into the microwave oven to heat to boiling, shake well, repeat 4 times, each time heating for 30 s, until the agarose powder is completely dissolved, showing clear and transparent;

[0146] c. After cooling the conical flask, add 5 μL of 10000x 4S GelRed nucleic acid dye, mix well, pour into the agarose mold, insert the comb with appropriate number of holes, and place at room temperature for 20 min until the agarose gel is completely cooled;

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

[0148] e. Mix 6x Loading Buffer and PCR product at a ratio of 1:6 by blowing and mixing well, then slowly add to the hole, and add 3 μL of 20 bp DNA Ladder maker to the blank hole as an indicator band;

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

[0150] As can be seen from Figure 2 , the lengths of the target fragments amplified by 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 control group without blocker is single and bright, and the target band of the BDA group with blocker is clear and bright, but due to the limitation of the system, a small amount of non-specific amplification may occur, which has little effect on the detection effect.

[0151] 3) PCR product recovery:

[0152] a. Add 500 μL of equilibration solution BL to the adsorption column CB2 (put the adsorption column into the collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

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

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

[0155] d. Add the solution from the previous step to a spin column CB2 (put the spin column into a collection tube), centrifuge at 12,000 rpm for 1 min, discard the waste in the collection tube, and put the spin column CB2 into the collection tube; add 600 μL of the rinse solution PW (check if anhydrous ethanol has been added before use) to the spin column CB2, centrifuge at 12,000 rpm for 1 min, discard the waste in the collection tube, and put the spin column CB2 into the collection tube, repeat the previous step;

[0156] e. Put the spin column CB2 into the collection tube, centrifuge at 12,000 rpm for 2 min, and try to remove the rinse solution as much as possible. Put the spin column at room temperature for a few minutes and dry it completely;

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

[0158] 3) Sanger sequencing

[0159] The recovered PCR amplification product was outsourced for Sanger sequencing, and the data obtained by sequencing were imported into SnapGene software for analysis to obtain the sequencing results, as shown in Figure 3

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

[0161] (2) Real-time fluorescent quantitative PCR dye method to verify the effectiveness of primers and blockers

[0162] Real-time fluorescent quantitative PCR (QPCR) was used to verify the primers and blockers. Compared with agarose gel electrophoresis, QPCR can more intuitively show whether the primers can effectively amplify through amplification curves and melting curves, and evaluate their amplification efficiency. For mutant templates, their amplification efficiency is theoretically not affected, and the difference (referred to as △Ct value) between the Ct value of the group with added blocker and the Ct value of the group without added blocker 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 proportion of the template is larger, the smaller the △Ct, the more it meets the theoretical trend. Through the melting curve of amplification, it can also be observed whether the designed primers will have primer dimers and other non-specific amplification. If the melting curve presents a single peak, it indicates that the amplification specificity of the primer is good. The specific operation process is as follows:

[0163] ​1) Put QPCR dye method reagent, forward primer, reverse primer, blocker, sterile enzyme-free water and gDNA on ice for standby, prepare QPCR reaction solution, vortex mix for 15s or so, the reaction system is shown in Table 4 below.

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

[0165] Components BDA volume Control 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 ddH2O Up to 25μL Up to 25μL DNA 2μL 2μL Total volume 25μL 25μL

[0166] Note: Up to means add.

[0167] Put clean eight-tube on pre-cooling plate, add reaction solution and cover eight-tube cover, put eight-tube on small centrifuge at 1000rpm for 1min, spin dry the liquid on tube wall and prevent bubble generation, put eight-tube on QPCR instrument, reaction conditions are shown in Table 5, and amplification results are analyzed by Bio-Rad CFX Manager3.0 software, and results are shown in Figure 4

[0168] Table 5 Reaction conditions of real-time fluorescent quantitative PCR

[0169] Step Temperature (°C) Time Cycle number Pre-denaturation 95 3min 1 Denaturation 95 20s 45 Annealing and extension 62 40s 45 Melting curve analysis 95 15s 1 Melting curve analysis 60 60s 1 Melting curve analysis 95 15s 1

[0170] As shown in Figure 4 , using CD41-42 system to amplify MT △Ct value is 0.315±0.210, and WT △Ct value is 8.385±0.067; using CD17 system to amplify MT △Ct value is 3.300±0.417, and WT △Ct value is 7.805±0.400; using CD71-72 system to amplify MT △Ct value is 0.982±0.027, and WT △Ct value is 10.110±0.088; using IVS-II-654 system to amplify MT △Ct value is 1.013±0.027, and WT △Ct value 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 wild type.

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

[0172] Taking two most common β-thalassemia genotypes CD41-42 and CD17 as examples, the forward and reverse primers of corresponding target genes, blocker (+AAAA / +NH2-C6), TB Green Premix Ex Taq II, enzyme-free water, and 10 5 ​The wild type / mutant plasmid DNA of copy number was oscillated, centrifuged at 1000 r / min for a short time, and placed on ice for standby. The total pipe was configured according to the system of Table 6 in the clean bench, the eight-pipe was labeled and placed on the pre-cooling plate, and the solution in the total pipe was evenly added to the corresponding eight-pipe hole. Finally, the plasmid DNA template was added. In each batch of detection, 2 μL of sterile water without enzyme was added to one hole as a blank control.

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

[0174] Components BDA volume (+AAAA) [CAT] BDA group (+NH2-C6) volume Control 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 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, it was placed on a vortex shaker and shaken at 1000 rpm for 1 min. The reaction was carried out according to the reaction conditions of Table 5, and the results were analyzed by Bio-Rad CFX Manager 3.0 software, as shown in Figure 5 .

[0177] As shown in Figure 5 , compared with the Control group without adding the blocker, after adding the blocker with 4 unmodified oligonucleotides connected at the 3' end (+AAAA), the amplification was obviously inhibited, and the cycle number difference was 4.89. With the blocker with chemical modification connected at the 3' end (+NH2-C6), the PCR amplification was more significantly inhibited, and the cycle number difference with the Control group increased to 11.13. The results show that the blocker with chemical modification connected at the 3' end has better blocking effect and does not affect the stability of the system.

[0178] (4) Optimization of reaction conditions

[0179] 1) Optimal annealing temperature exploration:

[0180] Taking CD41-42 genotype as an example, the robustness of BDA reaction system in the temperature range of 52-66℃ was explored, and the optimal reaction temperature was explored to achieve the best effect. The reaction system was added according to Table 4, and 100%, 10%, 1%, 0% plasmid was used as template for amplification. The amplification conditions are shown in Table 5. The annealing temperature was set to 52℃, 54℃, 56℃, 58℃, 60℃, 62℃, 64℃, 66℃, respectively, and the rest of the reaction conditions were unchanged. The results are shown in Figure 6 .

[0181] As shown in Figure 6 , when the annealing and extension temperature was 52-66℃, the Ct values of different mutant proportion plasmid DNA were differentFigure 6 A), wherein the ACt value at 62°C is the largest, the blocking effect is the most significant, and the agarose gel electrophoresis result shows that the bands are bright and single at 52-66°C, and no primer dimer is generated Figure 6 B).

[0182] 2) Optimal primer / blocker ratio

[0183] The primer / blocker concentration ratio has a certain influence on the BDA system. In order to explore the optimal primer / blocker ratio, the wild type plasmid was used as the template, the primer / blocker ratio was designed as 1:1, 1:2, 1:5, 1:8, 1:10, and the rest of the conditions were unchanged, the reaction system was as shown in Table 4, and the results were as shown in Figure 7

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

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

[0186] 1. Materials and experimental methods

[0187] 2.1. Samples were as above.

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

[0189] 2.2.1 Specificity verification of BDA detection system

[0190] According to the construction and optimization of the BDA reaction system in the early stage, the optimal reaction condition was selected, and the detection system of CD41-42, CD17, CD71-72 and IVS-II-654 primer blockers was used for BDA-QPCR detection of the four genotypes of mutant samples. The reaction system and QPCR reaction condition of Table 4 were added and run, each sample was repeated three times, the detection results were analyzed by Bio-Rad CFX Manager3.0 software, and the difference (i.e. ACt value) between the Ct value without blocker and the Ct value with blocker was compared. In theory, the CD41-42 detection system can only successfully detect the CD41-42 mutant sample, i.e. the ACt value tends to 0, while the other genotypes of mutant samples are inhibited, and the ACt value increases significantly, indicating that the specificity of the detection system is good, and the other three detection systems are the same. The detection results are as shown in Figure 8

[0191] As shown in Figure 8 ​​As shown in

[0192] 2.2.2 BDA-Sanger sequencing minimum detection limit

[0193] According to the optimal reaction conditions, the plasmid DNA of 4 kinds of gene mutations was prepared into different mutation proportions (100%, 50%, 10%, 1%, 0.1%, 0.01%, 0%) for BDA-PCR, which was divided into control group without blocker and experimental group with blocker. The recovered PCR amplification products were sent to the company for Sanger sequencing. The sequencing results were analyzed by SnapGene software, and the differences between the two groups were compared to explore the minimum detection limit of BDA detection of mutations. The results are shown in Figure 9-12 .

[0194] As shown in Figure 9-12 , the minimum detection limit of CD41-42, CD71-72, IVS-II-654 sites in the Control group was 10% VAF, and the CD17 site was 1% VAF Figure 9-12 . The detection sensitivity of all sites in the BDA group was significantly improved to 0.1% VAF, and the mutation signal peak diagram was clear and distinguishable Figure 9-12 .

[0195] 2.2.3 BDA-QPCR amplification curve and standard curve drawing

[0196] According to the optimal reaction conditions, the plasmid DNA of 4 kinds of gene mutations was prepared into different mutation proportions (100%, 50%, 10%, 1%, 0.1%, 0.01%, 0%) for BDA-PCR, which was divided into control group without blocker and experimental group with blocker. The recovered PCR amplification products were sent to the company for Sanger sequencing. The sequencing results were analyzed by SnapGene software, and the differences between the two groups were compared to explore the minimum detection limit of BDA detection of mutations. The results are shown in Figure 13 .

[0197] As shown inFigure 13 As shown, the ΔCt value is negatively correlated with the mutation ratio (decreasing mutation ratio → increasing 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, consistent with the Sanger sequencing results.

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

[0199] Based on the previous construction and optimization of the BDA reaction system, the optimal reaction conditions were selected to perform BDA-PCR detection on 52 clinical samples from four common β-thalassemia genotypes: 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 participants underwent physical examination during the same period; 20 carriers of the CD17 genotype and 20 participants underwent physical examination during the same period; 6 carriers of the CD71-72 genotype and 6 participants underwent physical examination during the same period; and 6 carriers of the IVS-II-654 genotype and 6 participants underwent physical examination during the same period. Using extracted genomic DNA as a template, forward and reverse primers and blocking agents designed for the corresponding genotypes were used for the reaction. The detection results were analyzed using Bio-Rad CFX. Manager 3.0 software was used for analysis to compare the differences in ΔCt values ​​between the experimental group and the control group, thereby verifying the effectiveness of the BDA system in the clinical application of β-thalassemia. Simultaneously, the PCR amplification products were sent to a biotechnology company for Sanger sequencing, and the detection results were analyzed. The results are as follows... Figure 14 to 18 As shown.

[0200] like Figure 14 As shown, the lowest effective detection limit is 0.1%. Standard curves were plotted for 100%, 10%, 1%, and 0.1% versus ΔCt values. The correlations between the mutation rates of CD41-42, CD17, CD71-72, and IVS-II-654 and ΔCt values ​​were 0.9964, 0.9550, 0.9805, and 0.9773, respectively.

[0201] like Figure 15 As shown, in the CD41-42 cohort (n=20 patients + 20 controls), BDA-QPCR results showed that the mutation group had a ΔCt of 2.312±0.602 vs. the control group had a ΔCt of 7.951±0.194 (P<0.001), and the sensitivity / specificity was 1.0 at a cutoff value of ΔCt=5.357 (AUC=1.0). Sanger sequencing results showed that both the 20 mutation cases and the 20 normal sequences were accurately detected.

[0202] like Figure 16As shown, in the CD17 cohort (n=20 patients + 20 controls), BDA-QPCR results showed that the mutation group had a ΔCt of 2.613±0.415 vs. the control group had a ΔCt of 6.219±0.600 (P<0.001), and the sensitivity / specificity was 1.0 at a cutoff value of ΔCt=4.815 (AUC=1.0). Sanger sequencing results showed that both the 20 mutation cases and the 20 normal sequences were accurately detected.

[0203] like Figure 17 As shown, in the CD71-72 cohort (n=6 patients + 6 controls), BDA-QPCR results showed that the mutation group had a ΔCt of 1.332±0.209 vs. the control group had a ΔCt of 8.631±0.284 (P<0.001), and the sensitivity / specificity was 1.0 at a cutoff value of ΔCt=4.9265 (AUC=1.0). Sanger sequencing results showed that all 6 mutations and 6 normal sequences were accurately detected.

[0204] 2.5.4 BDA-qPCR and Sanger sequencing results of the IVS-II-654 cohort

[0205] like Figure 18 As shown, for the IVS-II-654 cohort (n=6 patients + 6 controls), BDA-QPCR results showed that the mutation group had a ΔCt of 1.643±1.094 vs. the control group had a ΔCt of 8.249±4.399 (P=0.005), and the sensitivity / specificity was 1.0 at a cutoff value of ΔCt=3.9225 (AUC=1.0). Sanger sequencing results showed that all 6 mutations and 6 normal sequences were accurately detected.

[0206] (III) Clinical efficacy validation of the BDA non-invasive prenatal testing system

[0207] 3. Materials and Experimental Methods

[0208] 3.1. Same as above.

[0209] 3.2. Experimental Methods: Clinical efficacy verification of the BDA non-invasive prenatal testing system

[0210] 3.2.1. Detection of cf-DNA in plasma from artificially simulated pregnant women

[0211] (1) Fragmented DNA

[0212] The gDNA with a volume of 100 μL and a concentration of 50 ng / μL is fragmented using a non-contact ultrasonic disruptor, and the instrument is operated at a fixed frequency for 15 min; the gDNA is fragmented into small fragments with a length of less than 300 bp, thereby simulating small fragments of plasma cfDNA; the distribution of the DNA fragments after fragmentation is analyzed by agarose gel electrophoresis, the gel block with a DNA fragment length of less than 300 bp is cut in an ultraviolet illuminator, and the gel block is recovered and purified using an agarose gel DNA recovery kit, and the detailed operation process is as follows:

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

[0214] 2) The DNA fragment band of interest is cut from the agarose gel and placed in a clean centrifuge tube, and the weight is measured;

[0215] 3) The corresponding volume of the solution PC is added to the gel block (if the gel weighs 0.1 g, 100 μL of the PC solution is added, and the gel block is placed in a 50°C water bath for 10 min, and the centrifuge tube is constantly and gently turned upside down during the process to ensure that the gel block is fully dissolved;

[0216] 4) The liquid obtained in step 3) is added to the adsorption column CB2, centrifuged at a speed of 12,000 rpm for 1 min, the waste liquid in the collection tube is discarded, and the adsorption column CB2 is placed in the collection tube;

[0217] 5) 600 μL of the rinse solution PW (anhydrous ethanol is added before use) is added to the adsorption column CB2, centrifuged at a speed of 12,000 rpm for 1 min, the waste liquid in the collection tube is discarded, and the adsorption column CB2 is placed in the collection tube;

[0218] 6) Repeat the previous step;

[0219] 7) The adsorption column CB2 is placed in the collection tube, centrifuged at a speed of 12,000 rpm for 2 min, and the adsorption column is placed at room temperature for several minutes to completely dry;

[0220] 8) The adsorption column CB2 is placed in a clean centrifuge tube, and 30 μL of the elution buffer EB is added to the middle of the adsorption membrane, and the DNA solution is collected after centrifugation at a speed of 12,000 rpm for 2 min.

[0221] (2) Construction of a paternal mutation pregnant woman plasma DNA model

[0222] The application constructs a maternal plasma free DNA model containing paternal origin mutations, uses normal human (βN / βN) plasma cfDNA to simulate maternal plasma cfDNA, and uses β-thalassemia carrier fragmented DNA to simulate free fetal DNA; the fragmented DNA (50 ng / μL) of the β-thalassemia carrier is added to the plasma cfDNA of the normal sample in an amount of 8 μL, 6 μL, 4 μL, and 2 μL in turn to simulate the plasma samples of pregnant women with fetal mutation proportions of 20%, 15%, 10%, and 5%, respectively, and each concentration is repeated three times; similarly, the fragmented DNA of normal humans (βN / βN) is added to the normal sample plasma cfDNA at the same proportion to simulate the plasma samples of pregnant women without paternal origin. Then the free DNA of the pregnant woman's plasma is extracted to construct the plasma DNA model of the pregnant woman.

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

[0224] The plasma models of the pregnant women of the four common genotypes CD41-42, CD17, CD71-72, and IVS-II-654 are constructed, the extracted plasma free DNA is subjected to the experimental procedures of BDA-QPCR, and statistical analysis is performed to compare the differences in △Ct between the paternal mutation group and the non-paternal origin group, △Ct = Ct (with blocker group) - Ct (without blocker group); the ROC curve is drawn to determine the diagnostic efficiency of the BDA detection method in the artificial model, i.e. the accuracy, sensitivity, and specificity of the detection; BDA-PCR combined with Sanger sequencing is performed to verify whether the model is successfully detected, and the results are shown in Figure 19-22 , wherein Figure 19 is the BDA-QPCR and BDA-Sanger detection result graph of 10 CD41-42 mutant samples and 10 wild type simulation samples, respectively; Figure 20 is the BDA-QPCR and BDA-Sanger detection result graph of 10 CD17 mutant samples and 10 wild type simulation samples, respectively; Figure 21 is the BDA-QPCR and BDA-Sanger detection result graph of 5 CD71-72 mutant samples and 5 wild type simulation samples, respectively; Figure 22 is the BDA-QPCR and BDA-Sanger detection result graph of 5 IVS-II-654 mutant samples and 5 wild type simulation samples, respectively.

[0225] As shown in Figure 19 , the △Ct value of BDA in the CD41-42 mutant thalassemia simulation plasma sample 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), and with ΔCt=2.345 as the cutoff value, all CD41-42 mutant samples could be distinguished from wild-type samples. Figure 19 C), the ROC curve shows that the detection sensitivity is 1.0, the specificity is 1.0, and the AUC value is 1.0 (C). Figure 19 D). BDA-Sanger can detect the corresponding mutant sequence in mutant simulated samples, while it detects the normal sequence in wild-type samples. Figure 19 E).

[0226] like Figure 20 As shown, the ΔCt value of BDA in CD17 mutant thalassemia simulated plasma samples was 3.084±0.428, while the ΔCt 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), and with ΔCt=3.996 as the cutoff value, all CD17 mutant samples could be distinguished from wild-type samples. Figure 20 C), the ROC curve shows that the detection sensitivity is 1.0, the specificity is 1.0, and the AUC value is 1.0 (C). Figure 20 D). BDA-Sanger can detect the corresponding mutant sequence in mutant simulated samples, while it detects the normal sequence in wild-type samples. Figure 20 E).

[0227] like Figure 21 As shown, the ΔCt value of BDA in simulated plasma samples of CD71-72 mutant thalassemia was 1.479±0.217, while the ΔCt value in the wild-type control group was 7.976±0.563. Figure 21 The differences between the two groups (A and 21B) were statistically significant (P < 0.001). Using ΔCt = 4.413 as the cutoff value, all CD71-72 mutant samples could be distinguished from wild-type samples. Figure 21 C), the ROC curve shows that the detection sensitivity is 1.0, the specificity is 1.0, and the AUC value is 1.0 (C). Figure 21 D). BDA-Sanger can detect the corresponding mutant sequence in mutant simulated samples, while it detects the normal sequence in wild-type samples. Figure 21 E).

[0228] like Figure 22 As shown, the ΔCt value of BDA in the IVS-II-654 mutant thalassemia simulated plasma sample was 1.165±0.365, while the ΔCt 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), with a cut-off value of ΔCt = 4.257, all IVS-II-654 mutant samples could be distinguished from wild-type samples Figure 22 C), 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 sequence in mutant samples, and normal sequence in wild-type samples Figure 22 E).

[0229] 3.2.2 Detection of clinical maternal plasma samples

[0230] Plasma samples of pregnant women who underwent prenatal diagnosis in our hospital (the fetal genotype was determined by amniocentesis) were collected, and were divided into CD41-42, CD17, CD71-72, IVS-II-654, etc. four groups according to the genotype of the husband of the pregnant woman. Clinical information was collected, and the maternal plasma free DNA was extracted for BDA-QPCR experiment, and the results were statistically analyzed. At the same time, Sanger sequencing was used to detect the paternal mutation in the maternal plasma to evaluate the accuracy, sensitivity and specificity of the BDA non-invasive prenatal diagnosis strategy in detecting fetal mutations in maternal plasma, and the results are shown in Figure 23-27 , wherein, Figure 23 is the detection result graph of 62 β-thalassemia clinical maternal plasma samples; Figure 24 is the detection result graph of 26 high-risk families whose husbands are CD41-42 genotype, and whose pregnant women are other genotypes or normal, and whose fetal genotype is known after amniocentesis. Among them, 11 cases of amniocentesis results are paternal mutations (41-42M), and 15 cases of amniocentesis results are non-paternal mutations (other genotypes or normal); Figure 25 is the detection result graph of 26 high-risk families whose husbands are CD17 genotype, and whose pregnant women are other genotypes or normal, and whose fetal genotype is known after amniocentesis. Among them, 17 cases of amniocentesis results are paternal mutations (17M), and 9 cases of amniocentesis results are non-paternal mutations (other genotypes or normal);

[0231] Figure 26 is the detection result graph of 8 high-risk families whose husbands are IVS-II-654 genotype, and whose pregnant women are other genotypes or normal, and whose fetal genotype is known after amniocentesis. Among them, 3 cases of amniocentesis results are paternal mutations (654M), and 5 cases of amniocentesis results are non-paternal mutations (other genotypes); Figure 27This image shows the test results for two high-risk families where the husbands had the CD71-72 genotype, the pregnant women had other genotypes or were normal, and both underwent amniocentesis with known fetal genotypes. One amniocentesis result showed a paternal mutation (CD71-72), and the other result showed a non-paternal mutation (normal).

[0232] like Figure 23 As shown, BDA-qPCR results indicated that the ΔCt value for the paternal mutant group was 2.219±1.448, while the ΔCt value for the non-paternal mutant group was 4.920±1.630, showing a significant difference between the two groups (P<0.001). Sanger sequencing results were consistent with these findings. Figure 23 A and Figure 23 B). The ROC curve showed that, compared with the results of puncture diagnosis, the BDA-PCR paternal non-invasive prenatal diagnosis strategy exhibited 85.5% accuracy, 78.1% sensitivity, and 93.3% specificity. Figure 23 C).

[0233] like Figure 24 As shown, the BDA-qPCR results indicated that the ΔCt value in the paternal mutation group was 2.260±1.650, while the ΔCt value in the non-paternal mutation group was 4.138±1.183, showing 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 results of puncture diagnosis, the BDA-QPCR paternal non-invasive prenatal diagnosis strategy exhibited 80.8% accuracy, 72.7% sensitivity, and 86.7% specificity. Figure 24 D). Using ΔCt = 3.166 as the optimal cutoff 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] like Figure 25 As shown, the BDA-QPCR results indicated that the ΔCt value in the paternal mutation group was 2.270±1.415, while the ΔCt value in the non-paternal mutation group was 5.935±1.933 (25A and 25B), showing a significant difference between the two groups (P<0.001). The ROC curves showed that, compared with the results of amniocentesis, the BDA-QPCR non-invasive prenatal diagnostic strategy for paternal mutations exhibited 84.6% accuracy, 76.5% sensitivity, and 100% specificity. Figure 25 D). Using ΔCt = 2.999 as the optimal cutoff value, 13 positive cases (carrying 17M) and 9 negative cases (without 17M) were successfully detected. Figure 25 C). Sanger sequencing results were consistent with qPCR results.Figure 25 E).

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

[0236] like Figure 27 As shown, the BDA-qPCR results indicated that each sample was tested in triplicate. The ΔCt value for paternal mutations was 1.603±1.163, and the ΔCt value for non-paternal mutations was 3.790±1.216, consistent with the ideal trend. Sanger sequencing successfully detected one paternal mutation sequence and one normal sequence, consistent with the puncture results. Figure 27 B).

[0237] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in 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 comprises 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 IV: Combination I: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the blocker is shown as SEQ ID NO. 3; Combination II: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 5, and the nucleotide sequence of the blocker is shown as SEQ ID NO. 6; Combination III: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 8, and the nucleotide sequence of the blocker is shown as SEQ ID NO. 9; Combination IV: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 11, and the nucleotide sequence of the blocker is shown as SEQ ID NO. 12; The blocker is modified with a primary amino group.

2. The primer blocker composition of claim 1, wherein, The combination I is used for detecting CD41-42 gene mutation of beta thalassemia, the combination II is used for detecting CD17 gene mutation of beta thalassemia, the combination III is used for detecting IVS-II-654 gene mutation of beta thalassemia, and the combination IV is used for detecting CD71-72 gene mutation of beta thalassemia.

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

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

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

6. Use of a primer blocker composition according to any one of claims 1 to 5 for the preparation of a reagent for the detection of a mutation type of beta-thalassemia, characterized in that, The beta thalassemia mutation types include CD41-42 gene mutation of beta thalassemia, CD17 gene mutation of beta thalassemia, IVS-II-654 gene mutation of beta thalassemia, and CD71-72 gene mutation of beta thalassemia.

7. A kit for detecting a mutation type of β-thalassemia, characterized by, The primer blocker composition comprises a primer set and a blocker, the primer set comprises 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 IV:

8. The kit of claim 7, wherein Combination I: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, and the nucleotide sequence of the blocker is shown as SEQ ID NO. 3; Combination II: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 4, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 5, and the nucleotide sequence of the blocker is shown as SEQ ID NO. 6; Combination III: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 7, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 8, and the nucleotide sequence of the blocker is shown as SEQ ID NO. 9; Combination IV: the nucleotide sequence of the upstream primer is shown as SEQ ID NO. 10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 11, and the nucleotide sequence of the blocker is shown as SEQ ID NO. 12; The blocker is modified with a primary amino group. The combination I is used for detecting CD41-42 gene mutation of beta thalassemia, the combination II is used for detecting CD17 gene mutation of beta thalassemia, the combination III is used for detecting IVS-II-654 gene mutation of beta thalassemia, and the combination IV is used for detecting CD71-72 gene mutation of beta thalassemia. The primary amino group is modified at the 3' end of the blocker. The molar ratio of the first primer to the second primer is 1-3: 1-3. The molar ratio of the first primer to the blocker is 1-3: 5-15. The beta thalassemia mutation types include CD41-42 gene mutation of beta thalassemia, CD17 gene mutation of beta thalassemia, IVS-II-654 gene mutation of beta thalassemia, and CD71-72 gene mutation of beta thalassemia.

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