Primer blocker composition of macroglobulinemia Fahrenheit CXCR4 gene, kit and high-sensitivity detection method of macroglobulinemia Fahrenheit CXCR4 gene

Abstract

The invention discloses a primer blocker composition for detecting a macroglobulinemia Fahrenheit CXCR4 gene, the primer blocker composition comprises a primer group and a blocker, and the primer group comprises a forward primer group and a reverse primer group; the nucleotide sequence of the blocking agent is as shown in SEQ ID NO. 1; the nucleotide sequence of the reverse primer group is as shown in SEQ ID NO. 2; the nucleotide sequence of the forward primer group is as shown in SEQ ID NO. 3. The invention also discloses a kit containing the composition and a detection method. By accurately detecting the CXCR4 gene, the CXCR4 gene can be combined with other markers, and pathological diagnosis and molecular typing of WM are accurate; a plurality of CXCR4 high-frequency mutation sites of WM can be covered by a single reaction, and compared with a digital PCR method only capable of detecting a single site, the cost is reduced by more than 80%, and the method is suitable for being popularized in hospitals.

Description

Technical Field

[0001] This invention belongs to the field of medical testing technology, specifically a primer blocking agent composition, kit, and highly sensitive detection method for the CXCR4 gene in Waldenström macroglobulinemia based on blocking displacement amplification technology. Background Technology

[0002] Waldenström macroglobulinemia (WM) is a rare malignant tumor of the lymphatic system. Precise molecular subtyping and pathological diagnosis can further improve treatment outcomes.

[0003] Molecular subtyping of Waldenström macroglobulinemia depends on the detection results of the CXCR4 and MYD88 genes.

[0004] MYD88 L265P is the most common somatic mutation in Waldenström macroglobulinemia, with a single mutation rate as high as 95%-97%, and can be detected by ARMS-PCR or digital PCR.

[0005] The CXCR4 gene is the second most common somatic mutation gene in Waldenström macroglobulinemia, with an incidence of about 30%-40%. Among them, S338X and R334X are the most common high-frequency mutations, accounting for 70% of all CXCR4 mutations.

[0006] In addition, several other mutation sites exist that can aid in the diagnosis of Waldenström macroglobulinemia. The wide range of mutation sites in the CXCR4 gene increases the difficulty of detection methods.

[0007] While digital PCR can detect low-frequency mutations, it is costly and can only detect one mutation site per reaction. Next-generation sequencing, although capable of detecting mutations in multiple genes simultaneously, is relatively expensive and involves complex data analysis. Traditional Sanger sequencing, a classic gene sequencing technology, offers advantages in mutation site coverage and is relatively inexpensive, but its sensitivity is insufficient, making it unable to effectively detect low-frequency mutations, potentially leading to misdiagnosis or missed diagnosis in clinical applications. Therefore, existing molecular detection technologies all have shortcomings in terms of sensitivity, cost, and mutation site coverage.

[0008] The high cost of digital PCR and next-generation sequencing limits their widespread clinical application, while the low sensitivity of traditional Sanger sequencing makes false negatives common in detecting low-frequency gene variants. These issues not only affect the accurate diagnosis of Waldenström macroglobulinemia but also complicate the selection of subsequent targeted therapies. Therefore, there is an urgent need for a new detection technology that can improve sensitivity while reducing costs and enabling simultaneous detection of multiple sites to meet the clinical needs for molecular subtyping of Waldenström macroglobulinemia.

[0009] Blocker displacement amplification (BDA) is a technique that uses a blocking sequence to tightly bind to a wild-type template sequence, thereby inhibiting PCR amplification of the wild-type template and allowing the mutant sequence to be specifically amplified due to mismatch. BDA Sanger sequencing, combined with traditional Sanger sequencing, not only significantly improves sensitivity but also overcomes the shortcomings of digital PCR (incomplete coverage of gene mutation sites) and next-generation sequencing (NGS) (high cost). Currently, BDA Sanger sequencing has been applied in gene detection for some tumors. Domestic research teams have used BDA Sanger sequencing in gene detection for lung cancer, melanoma, and lymphoma, achieving a sensitivity of 0.2-0.5%, with results highly consistent with digital PCR and NGS. Therefore, BDA technology, by improving the sensitivity of traditional Sanger sequencing, will be a powerful tool for tumor diagnosis. Summary of the Invention

[0010] This invention addresses the technical problems existing in the prior art by providing a technical solution that can improve sensitivity while reducing detection costs and achieve simultaneous detection at multiple sites, thereby meeting the clinical needs for molecular subtyping of Waldenström macroglobulinemia.

[0011] This invention specifically discloses a primer blocking agent composition for detecting the CXCR4 gene in Waldenström macroglobulinemia. The primer blocking agent composition includes a primer set and a blocking agent, wherein the primer set includes a forward primer set and a reverse primer set.

[0012] The nucleotide sequence of the blocking agent is shown in SEQ ID NO.1; the nucleotide sequence of the reverse primer set is shown in SEQ ID NO.2; and the nucleotide sequence of the forward primer set is shown in SEQ ID NO.3.

[0013] Preferably, the primer blocking composition is used to simultaneously detect the R334X and S338X site mutations in the CXCR4 gene of Waldenström macroglobulinemia.

[0014] Preferably, the amount of the blocking agent is 50 nM.

[0015] Preferably, the concentration ratio of the forward primer set, the reverse primer set, and the blocking agent is 1:1:5.

[0016] The present invention also discloses the use of the aforementioned primer blocking composition in the preparation of a kit for detecting Waldenström macroglobulinemia.

[0017] The present invention also discloses a kit for detecting mutant types of Waldenström macroglobulinemia, comprising PCR reagents, enzyme digestion reagents, sequencing reagents and the aforementioned primer blocking agent composition.

[0018] This invention also discloses a method for detecting Waldenström macroglobulinemia mutation types for non-disease diagnosis and / or treatment purposes, comprising the following steps:

[0019] Step 1: Extract DNA from the sample;

[0020] Step 2: Prepare a positive control group and a negative control group;

[0021] Step 3: Simultaneously perform PCR amplification on the DNA extracted in Step 1 and the control group in Step 2 using the kit described in claim 5;

[0022] Step 4: Perform enzymatic digestion on the PCR amplification system from Step 3;

[0023] Step 5: Perform sequencing PCR reaction on the product after enzymatic digestion in step 4;

[0024] Step 6: Purify and sequence the products of the sequencing PCR reaction in Step 5.

[0025] Step 7: Compare the sequenced data with the normal sequence to determine whether mutations have occurred at the R334X and S338X sites.

[0026] Compared with the prior art, the technical effect of the present invention is as follows: The blocking agent independently developed and designed in this application is perfectly matched with the wild-type template of the CXCR4 gene, inhibiting the PCR amplification of the wild-type sequence, so that the mutant template of the CXCR4 gene can be specifically amplified due to mismatch, thereby achieving the purpose of detecting low-frequency hotspot mutations in the CXCR4 gene.

[0027] This invention designs blocking agent sequences covering two high-frequency mutation sites, S338X and R334X, of the CXCR4 gene, enabling the detection of mutation status (missense mutation, deletion mutation, or insertion mutation) of the CXCR4 gene in WM in a single experiment, which is significantly superior to digital PCR, which can only detect a single site.

[0028] This invention enables accurate detection of the CXCR4 gene, which can be combined with other biomarkers for precise pathological diagnosis and molecular subtyping of WM. A single reaction can cover multiple high-frequency CXCR4 mutation sites in WM (including missense mutations, deletion mutations, or insertion mutations). Compared with digital PCR methods that can only detect a single site, this reduces costs by more than 80%, making it suitable for hospital promotion. Attached Figure Description

[0029] Figure 1 The normal base sequence diagram of two hotspot mutant amino acids in the CXCR4 gene is shown using Mutation Surveyor software.

[0030] Dashed box: R334 base sequence CGA, S338 base sequence TCA. Figure 1 The red arrows indicate the positions of the mutated bases when a mutation occurs.

[0031] Figure 2 A schematic diagram of the reverse primers used for BDA Sanger sequencing to determine the CXCR4 gene.

[0032] Figure 3 A schematic diagram of the forward primers used for BDA Sanger sequencing of the CXCR4 gene.

[0033] Figure 4 A schematic diagram for designing CXCR4 gene blockers.

[0034] Figure 5 The image shows the CXCR4 gene base sequence and the wild-type sample base sequence using Mutation Surveyor software. The dashed box in the image represents R334, and the arrow points to the non-specific amplification peak caused by A-rich.

[0035] Figure 6 The image shows the CXCR4 gene base sequence and the wild-type sample base sequence using Mutation Surveyor software. The dashed box indicates S338, and the arrows indicate non-specific amplification peaks.

[0036] Figure 7 and Figure 8 This is a schematic diagram for evaluating the specificity of primer blocking agent compositions.

[0037] Figure 9 This diagram illustrates the optimal ratio of forward primer set (F), reverse primer set (R), and blocker concentration in a PCR amplification system. NGS stands for Next-generation Sequencing, and VAF stands for Variant Allele Frequency.

[0038] Figure 10 This diagram illustrates the optimal amplification annealing temperature for gradient PCR amplification experiments.

[0039] Figure 11 This is a schematic diagram of the repeatability test results.

[0040] Figure 12 A schematic diagram showing the determination of LOD values ​​for CXCR4 R334X and CXCR4 S338X. LOD: Limit of Detection.

[0041] Figure 13 The image shows a comparison of detection patterns for the same sample using traditional Sanger sequencing and BDA Sanger sequencing. Traditional Sanger sequencing can only detect a small portion of the G peaks, as shown in the black peak on the left. In BDA Sanger sequencing, the peak value of the mutation G peak is significantly greater than that of the C peak (blue peak on the right), which can significantly increase the mutation frequency of S338X.

[0042] Figure 14 The image shows a comparison of detection patterns for the same sample using traditional Sanger sequencing and BDA Sanger sequencing. Traditional Sanger sequencing can only detect a small portion of the T peaks, as shown in the red peak on the left. BDA Sanger sequencing significantly increases the peak value of the mutation T peaks (red peak on the right), which can significantly increase the mutation frequency of R334X.

[0043] Figure 15 The image shows a comparison of the detection patterns of the same sample using traditional Sanger sequencing and BDA Sanger sequencing. Traditional Sanger sequencing results in disordered peak patterns due to deletion mutations, requiring careful interpretation (dashed box in the left image). BDA Sanger sequencing, which suppresses wild-type sequences, shows missing peak patterns due to deletion mutations (dashed box in the right image), providing clear and accurate interpretation results. Detailed Implementation

[0044] This invention, through independent innovation, designs an inhibitor sequence covering two high-frequency mutation sites, R334X and S338X, in the CXCR4 gene. Its long enrichment region design significantly improves the efficiency of mutation detection while expanding the coverage of the site. That is, a single experiment can detect both mutation states (missense mutation, deletion mutation, or insertion mutation) of the CXCR4 gene in Waldenström macroglobulinemia, namely R334X and S338X. This is different from the current BDA-Sanger technology, which can only detect single mutation sites of the gene.

[0045] Meanwhile, the applicant further optimized the BDA Sanger sequencing reaction system and amplification conditions, and after performance verification and clinical consistency comparison, it can be used for the auxiliary diagnosis of Waldenström macroglobulinemia.

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments:

[0047] Example 1

[0048] 1. Determine the mutation location of the CXCR4 gene.

[0049] like Figure 1 As shown, the target regions of hotspot mutations in CXCR4 were identified using disease-related gene mutation databases (such as COSMIC and ClinVar). The CXCR4 R334 base sequence is CGA, and the mutation site is c.1000C>T when the amino acid R334 is mutated. The CXCR4 S338 base sequence is TCA, and the mutation site is c.1013C>G / c.1013C>A when the amino acid S338X is mutated.

[0050] 2. Design of blocking molecule compositions

[0051] In this embodiment, the primer blocking composition includes a primer set and a blocking agent, wherein the primer set includes a forward primer set and a reverse primer set.

[0052] Sequence specificity: The blocker involved in this embodiment must be completely complementary to the wild-type template sequence and cover the mutation site region (usually 15-25 nt) to ensure high specificity recognition.

[0053] Meanwhile, the blocking agent and the amplification primers have a certain degree of sequence overlap, so that the amplification primers can replace the blocking agent to achieve amplification in the presence of mutations. To prevent the blocking agent itself from being extended by DNA polymerase, its 3' end needs to be introduced with non-complementary sequences such as AAAA and chemically modified to ensure the high efficiency and specificity of the amplification system.

[0054] 1) Determine the reverse primer approximately 5 bp downstream of the mutation point: melting temperature (Tm) is 58℃;

[0055] See Figure 2 , Figure 2 The red C represents the mutation point TCA of the S338X in the CXCR4 gene; the black box indicates a position 5 bp away from the mutation point.

[0056] 2) Determine the amplification target position (150-200 bp) and determine the forward primer: melting temperature (Tm) is 58℃;

[0057] See Figure 3 , Figure 3 The blue area indicates the forward primer; the pink C indicates the S338X mutation point in the CXCR4 gene; and the green area indicates the reverse primer.

[0058] 3) Design the blocking agent: overlap with the reverse primer by 6-14 bp, melting temperature (Tm) 59 ℃, which is 1 ℃ higher than the upstream and downstream primers; continue to add AAAA at 3' to prevent amplification;

[0059] See Figure 4 The red C represents the S338X mutation point of the CXCR4 gene; the black box represents the overlapping part of the blocking agent and the reverse primer. The first row of sequences is the blocking agent; the second row of sequences is the CXCR4 gene sequence; and the third row of sequences is the reverse primer.

[0060] 4) Using Ape primer design software, primer sequences were determined based on the principle of base complementarity.

[0061] The nucleotide sequence of the blocker, SEQ ID NO.1, is: 5'-TCAGTGGAAACAGATGAATGTCCAC(AAAA)-3';

[0062] The nucleotide sequence of the reverse primer, SEQ ID NO.2, is: 5'-TGAAGACTCAGACTCAGTGGAAAC-3';

[0063] The nucleotide sequence of the forward primer, SEQ ID NO.3, is: 5'-TCTTCCACTGTTGTCTGAACCC-3'.

[0064] 5) Innovative Explanation of Blocker Design

[0065] In other BDA inventions, a single tube can only detect a single gene mutation site. The primer sequence of this invention allows for the detection of two WM hotspot mutations, R334X and S338X, in the CXCR4 gene in a single tube and a single experiment. Furthermore, this primer sequence can detect gene mutations within the R334 and S338 ranges.

[0066] Regarding the base characteristics of the R334 to S338 region of the CXCR4 gene, the inventors discovered, see [link to relevant documentation] Figure 5 In this sequence, the area around R334 is rich in A bases (A-rich), and the area around S338 is rich in T bases. See [link to relevant documentation]. Figure 6 The template strand is rich in A, therefore, non-specific amplification peaks are likely to appear in the experimental results. Figure 6 The middle arrow indicates that the following potential impacts on binding stability may occur during the design of BDA primer sequences.

[0067] Because GC base pairs form three hydrogen bonds, while AT base pairs only form two, their binding stability is much lower than that of GC base pairs with three hydrogen bonds. Therefore, if the Blocker sequence is designed in an A-rich region (corresponding to a T-rich region on the template strand), its overall stability in binding with the wild-type template will decrease, directly leading to a drop in its melting temperature (Tm). This means that at the same PCR annealing temperature, the binding ability of the Blocker to the template is weakened, and the efficiency of its competitive inhibitory primers binding to the wild-type template will decrease, potentially leading to incomplete blocking of wild-type DNA and thus reducing the specificity of enriched mutants.

[0068] Therefore, considering the unique characteristics of the CXCR4 gene sequence, the inventors adopted the following innovative design strategy for the BDA sequence:

[0069] (1) Prioritize lengthening the Blocker

[0070] To compensate for the low stability caused by the A-rich region, the length of the Blocker is increased when designing it. In this application, the Blocker sequence is increased to 25 bases to introduce more base pairs to increase the total binding energy and improve the binding stability.

[0071] (2) Accurate calculation and optimization of thermodynamic parameters

[0072] Using ApE primer design software, the melting temperature (Tm) can still be maintained at 58°C at 25 bases.

[0073] (3) Carefully optimize reaction conditions:

[0074] Since the actual Tm value of Blocker is lower than the software prediction value when A-rich, it is necessary to finely optimize the annealing temperature and the optimal working concentration of Blocker through temperature gradient PCR and Blocker concentration gradient experiments in order to find the strongest wild-type blocking.

[0075] (4) Ensure that the 3' end of the Blocker is strictly modified:

[0076] The 3' end of the blocker must be chemically modified (Blocker sequence + AAAA) to prevent it from being extended by DNA polymerase. This is especially important when binding to the already unstable A-rich region, because any slight extension will directly amplify the wild-type background and completely destroy the experimental specificity.

[0077] In summary, an abundance of A bases around the mutation site challenges the specificity of BDA by reducing binding stability and Tm value. The core strategy is to enhance the Blocker binding force by increasing its length or introducing modifications such as AAAA, and to overcome this challenge through meticulous experimental optimization of annealing temperature and Blocker concentration.

[0078] 3. Balancing primer cross-reactivity

[0079] See Figure 7 and Figure 8 Primer specificity was evaluated using software (Primer-BLAST): [Login] Primer designing tool The specificity of the primer blocking agent composition was evaluated, and the primers designed within the red dashed box were identified as being for the CXCR4 gene.

[0080] 4. Optimization of the reaction system

[0081] Inhibitor concentration gradient test: The optimal blocking concentration of the CXCR4 gene inhibitor was determined through preliminary experiments to balance the wild-type inhibition efficiency and the enrichment of mutation signal. The test showed that in the BDA Sanger assay of the CXCR4 gene, the variant allele frequency (VAF) of 0.2% at the S338X site and 0.4% at the R334X site in CXCR4 could be detected when the inhibitor dosage was 50 nM.

[0082] Example 2

[0083] This embodiment uses the primer blocking agent composition from Example 1 and provides a method for detecting the CXCR4 genotype of Waldenström macroglobulinemia for non-disease diagnosis and / or treatment purposes. The specific steps are as follows:

[0084] 1. Nucleic acid extraction and quality control

[0085] DNA was extracted from the samples using the procedure outlined in the "Blood / Cell / Tissue Genomic DNA Extraction Kit" (catalog number: DP304) from Tiangen Biotech (Beijing) Co., Ltd., and DNA quantification was performed using the Qubit DNA Quantitative Realizer and the Qubit dsDNA HS Assay Kit.

[0086] 2. Polymerase Chain Reaction (PCR)

[0087] 2.1 PCR amplification system:

[0088]

[0089] 2.2 Control group setup

[0090] Positive control group: DNA containing the known CXCR4 gene S338X mutation site was set up as a positive control group for each test;

[0091] Negative control group: DNA from cases known to be CXCR4 wild-type was included as a negative control group for each test;

[0092] 2.3 Amplification Procedure

[0093] Initial denaturation: 95℃ × 10 minutes;

[0094] Cyclic amplification: 42 cycles: 95℃ × 30 seconds → 58℃ × 30 seconds → 72℃ × 45 seconds;

[0095] Final extension: 72℃ × 10 minutes;

[0096] Storage environment: Keep at 4℃ (forever).

[0097] 3. Enzymatic hydrolysis

[0098] 3.1 Reaction system (single tube):

[0099] Add 5 μL of the prepared enzyme digestion reagent (as shown in the table below) to each tube of PCR product.

[0100]

[0101] 3.2 Enzymatic hydrolysis procedure

[0102] Step 1: 37℃ × 30 minutes;

[0103] Step 2: 80℃ × 10 minutes;

[0104] Storage environment: Keep at 4℃ (forever).

[0105] 4. Sequencing reaction

[0106] 4.1 Reaction System (Single Tube):

[0107] Prepare the sequencing reaction solution according to the table below:

[0108]

[0109] 4.2 Sequencing Procedure:

[0110] Step 1: 96℃ × 1 minute;

[0111] Step 2: Repeat 25 times: 95℃ × 10 seconds → 50℃ × 5 seconds → 60℃ × 4 minutes;

[0112] Storage environment: Keep at 4℃ (forever).

[0113] 5. Sequencing product purification

[0114] Add 2 μL of 125 mM EDTA, 2 μL of 3 M NaAc, and 50 μL of 100% anhydrous ethanol to the bottom of each tube, vortex to mix, and let stand at room temperature for 15 minutes. After centrifuging at 4000×g for 30 minutes, immediately invert the 96-well plate and centrifuge to 185×g. The total centrifugation time from start to stop is 1 minute. Then invert the plate back.

[0115] Add 70 μL of 70% alcohol to each tube, shake to mix, centrifuge for 15 minutes, then immediately invert the 96-well plate and centrifuge until it reaches 185×g. The total centrifugation time is 1 minute from start to stop. Then invert the plate back.

[0116] After opening the PCR product, allow it to evaporate the alcohol at room temperature for 1 hour. Add 10 μL of Hi-Di Formamide to dissolve the DNA. After dissolving, denature the sample at 95°C for 4 minutes, then quickly denature at 4°C for 5-8 minutes before proceeding with the subsequent PCR.

[0117] 6. Product sequencing

[0118] Verify that the ABI 3500 instrument and computer are properly connected. Power on the computer. Before entering the username and password, power on the ABI 3500 instrument and perform a system self-test. When you see the message "All 3500 components are connected" displayed in the lower right corner of the computer screen, and the green indicator light in the lower left corner of the ABI 3500 instrument illuminates, open the ABI 3500 software interface. Check the status of all instrument consumables. If the instrument and consumables are functioning normally, begin system warm-up. Create a new 96-well plate, select the Sequence module, edit the layout, check the program and save path, and click the Start button.

[0119] 7. Data Analysis:

[0120] Data analysis was performed using Mutation Surveyor software, and the sequence was compared with CXCR4_NM_003467.2.

[0121] 8. Experimental Results

[0122] See Figure 9The optimal ratio of the forward primer set (F), the reverse primer set (R), and the blocker concentration in the first step PCR amplification system of the CXCR4 gene was determined. The experimental results showed that when F:R:Blocker=1:1:5, the C peak (red arrow) of the sequencing results could achieve the optimal detection frequency.

[0123] 9. Optimization of amplification conditions

[0124] BDA technology maintains stable enrichment efficiency within the range of 56-64℃; therefore, gradient PCR amplification experiments are needed to verify the optimal amplification conditions. Results show that... (See...) Figure 9 and Figure 10 The CXCR4 gene maintains stable performance at 58°C, with an inhibitor concentration of 50 nM and a ratio of 1:1:5 between the forward and reverse primers.

[0125] Figure 10 In the experiment, gradient PCR amplification was used to verify the optimal amplification and annealing temperature; at 58℃, the G peak (red arrow) in the sequencing results achieved the optimal detection frequency.

[0126] pass Figure 10 It can be seen that the S338X missense mutation in the CXCR4 gene was detected by BDA Sanger sequencing.

[0127] Example 3

[0128] This embodiment evaluates the performance of the BDA Sanger method in the detection of the CXCR4 gene in Waldenström macroglobulinemia.

[0129] 1. Repeatability (Precision):

[0130] See Figure 11 The CXCR4 gene R334X (Next-generation Sequencing, NGS Variant Allele Frequency, VAF=3.6%) weakly positive sample was detected in 10 repeated tests.

[0131] Figure 11 In the study, CXCR4 c.1000C>T (p.R334X) was detected in 10 NGS tests with a VAF level of 3.6%.

[0132] At the point indicated by the red arrow, the C peak (blue) abruptly transforms into the T peak (red).

[0133] 2. Compliance rate:

[0134] Using 29 samples validated by NGS or digital PCR (12 wild-type CXCR4 and 17 mutant CXCR4), the concordance rate was 100%.

[0135] 3. Limit of Detection (LOD):

[0136] Using weakly positive samples of CXCR4 gene R334X (NGS VAF=3.6%) and CXCR4 gene S338X (NGS VAF=10.25%), serial dilutions were performed with wild-type human genomic DNA of CXCR4 to assess the limit of detection. The validation results were: LOD=0.4% for CXCR4 gene R334X and LOD=0.2% for CXCR4 gene S334X. Figure 12 ).

[0137] See Figure 12 LOD: Limit of Detection; VAF: Variant Allele Frequency; The red boxes represent the final determined LOD values ​​for the two loci; The red arrows indicate that the C peak (blue) of CXCR4R334X has mutated into the T peak (red); The C peak (blue) of CXCR4S334X has mutated into the G peak (black).

[0138] 4. Experimental steps:

[0139] 4.1 Nucleic acid extraction and quality control:

[0140] DNA was extracted from the samples using the procedure outlined in the "Blood / Cell / Tissue Genomic DNA Extraction Kit" (catalog number: DP304) from Tiangen Biotech (Beijing) Co., Ltd., and DNA quantification was performed using the Qubit DNA Quantitative Realizer and the Qubit dsDNA HS Assay kit.

[0141] 4.2 Polymerase Chain Reaction (PCR)

[0142] 1) PCR amplification system:

[0143]

[0144] 2) Reference settings:

[0145] Positive control: DNA containing the known CXCR4 S338X mutation site was set up as a positive control for each test;

[0146] Negative control: DNA from a known CXCR4 wild-type case was used as a negative control for each test;

[0147] 3) Amplification procedure:

[0148] Initial denaturation: 95℃ × 10 minutes;

[0149] Cyclic amplification: 42 cycles: 95℃ × 30 sec → 58℃ × 30 sec → 72℃ × 45 sec;

[0150] Final extension: 72℃ × 10 minutes;

[0151] Storage: 4℃ forever.

[0152] 4.3 Enzymatic hydrolysis

[0153] 1) Reaction system (single tube): Add 5 μL of the prepared enzyme digestion reagent (as shown in the table below) to each tube of PCR product.

[0154]

[0155] 2) Enzymatic hydrolysis procedure

[0156] Step 1: 37℃ × 30 minutes;

[0157] Step 2: 80℃ × 10 minutes;

[0158] Storage: 4℃ forever.

[0159] 4.4 Sequencing reaction

[0160] 1) Reaction system (single tube):

[0161]

[0162] 2) Sequencing procedure:

[0163] Step 1: 96℃ × 1 minute;

[0164] Step 2: 25 cycles: 95℃ × 10 seconds → 50℃ × 5 seconds → 60℃ × 4 minutes;

[0165] Storage: 4℃ forever.

[0166] 4.5. Sequencing product purification

[0167] Add 2 μL of 125 mM EDTA, 2 μL of 3 M NaAc, and 50 μL of 100% anhydrous ethanol to the bottom of each tube, vortex to mix, and let stand at room temperature for 15 minutes. After centrifuging at 4000×g for 30 minutes, immediately invert the 96-well plate and centrifuge to 185×g. The total centrifugation time from start to stop is 1 minute. Then invert the plate back.

[0168] Add 70 μL of 70% alcohol to each tube, shake to mix, centrifuge for 15 minutes, then immediately invert the 96-well plate and centrifuge until it reaches 185×g. The total centrifugation time is 1 minute from start to stop. Then invert the plate back.

[0169] After opening the PCR product, allow it to evaporate the alcohol at room temperature for 1 hour. Add 10 μL of Hi-Di Formamide to dissolve the DNA. After dissolving, denature the sample at 95°C for 4 minutes, then quickly denature at 4°C for 5-8 minutes before proceeding with the subsequent PCR.

[0170] 4.6. Product sequencing

[0171] Verify that the ABI 3500 instrument and computer are properly connected. Power on the computer. Before entering the username and password, power on the ABI 3500 instrument and perform a system self-test. When you see the message "All 3500 components are connected" displayed in the lower right corner of the computer screen, and the green indicator light in the lower left corner of the ABI 3500 instrument illuminates, open the ABI 3500 software interface. Check the status of all instrument consumables. If the instrument and consumables are functioning normally, begin system warm-up. Create a new 96-well plate, select the Sequence module, edit the layout, check the program and save path, and click the Start button.

[0172] 4.7 Data Analysis: Data analysis was performed in Mutation Surveyor software, and the sequence was compared with CXCR4_NM_003467.2.

[0173] Example 4

[0174] This embodiment verifies the multi-detection capability of the present invention.

[0175] See Figure 13 The mutation G peak of BDA Sanger was significantly higher than that of C peak (blue peak in the right figure); this significantly increased the mutation frequency of S338X.

[0176] See Figure 14 The mutation T peak of BDA Sanger was significantly increased (red peak in the right figure); the mutation frequency of R334X was significantly increased.

[0177] See Figure 15BDA Sanger technology suppresses wild-type sequences, allowing for clearer and more accurate interpretation of frameshift mutations (dashed box in the right figure).

[0178] The specific experimental steps in this embodiment are as follows:

[0179] 1. Nucleic acid extraction and quality control:

[0180] DNA was extracted from the samples using the procedure outlined in the "Blood / Cell / Tissue Genomic DNA Extraction Kit" (catalog number: DP304) from Tiangen Biotech (Beijing) Co., Ltd., and DNA quantification was performed using the Qubit DNA Quantitative Realizer and the Qubit dsDNA HS Assay kit.

[0181] 2. Polymerase Chain Reaction (PCR)

[0182] 1) PCR amplification system:

[0183]

[0184] 2) Reference settings:

[0185] Positive control: DNA containing the known CXCR4 S338X mutation site was set up as a positive control for each test;

[0186] Negative control: DNA from a known CXCR4 wild-type case was used as a negative control for each test;

[0187] 3) Amplification procedure:

[0188] Initial denaturation: 95℃ × 10 minutes;

[0189] Cyclic amplification: 42 cycles: 95℃ × 30 sec → 58℃ × 30 sec → 72℃ × 45 sec;

[0190] Final extension: 72℃ × 10 minutes;

[0191] Storage: 4℃ forever.

[0192] 3. Enzymatic hydrolysis

[0193] 1) Reaction system (single tube): Add 5 μL of the prepared enzyme digestion reagent (as shown in the table below) to each tube of PCR product.

[0194]

[0195] 2) Enzymatic hydrolysis procedure

[0196] Step 1: 37℃ × 30 minutes;

[0197] Step 2: 80℃ × 10 minutes;

[0198] Storage: 4℃ forever.

[0199] 4. Sequencing reaction

[0200] 1) Reaction system (single tube):

[0201]

[0202] 2) Sequencing procedure:

[0203] Step 1: 96℃ × 1 minute;

[0204] Step 2: 25 cycles: 95℃ × 10 seconds → 50℃ × 5 seconds → 60℃ × 4 minutes;

[0205] Storage: 4℃ forever.

[0206] 5. Sequencing product purification

[0207] Add 2 μL of 125 mM EDTA, 2 μL of 3 M NaAc, and 50 μL of 100% anhydrous ethanol to the bottom of each tube, vortex to mix, and let stand at room temperature for 15 minutes. After centrifuging at 4000×g for 30 minutes, immediately invert the 96-well plate and centrifuge to 185×g. The total centrifugation time from start to stop is 1 minute. Then invert the plate back.

[0208] Add 70 μL of 70% alcohol to each tube, shake to mix, centrifuge for 15 minutes, then immediately invert the 96-well plate and centrifuge until it reaches 185×g. The total centrifugation time is 1 minute from start to stop. Then invert the plate back.

[0209] After opening the PCR product, allow it to evaporate the alcohol at room temperature for 1 hour. Add 10 μL of Hi-Di Formamide to dissolve the DNA. After dissolving, denature the sample at 95°C for 4 minutes, then quickly denature at 4°C for 5-8 minutes before proceeding with the subsequent PCR.

[0210] 6. Product sequencing

[0211] Verify that the ABI 3500 instrument and computer are properly connected. Power on the computer. Before entering the username and password, power on the ABI 3500 instrument and perform a system self-test. When you see the message "All 3500 components are connected" displayed in the lower right corner of the computer screen, and the green indicator light in the lower left corner of the ABI 3500 instrument illuminates, open the ABI 3500 software interface. Check the status of all instrument consumables. If the instrument and consumables are functioning normally, begin system warm-up. Create a new 96-well plate, select the Sequence module, edit the layout, check the program and save path, and click the Start button.

[0212] 7. Data Analysis: Data analysis was performed using MutationSurveyor software, and the sequence was compared with CXCR4_NM_003467.2.

[0213] The validation experiments in Examples 3 and 4 showed that the BDA Sanger detection method has a detection sensitivity of 0.2% for the high-frequency WM mutation site CXCR4 S338X, which is superior to the digital PCR method (1%) (Table 1), but the cost is only 1 / 5 to 1 / 10 of the latter. Furthermore, it has the advantage of equipment compatibility, requiring only a conventional qPCR instrument and capillary electrophoresis system, without the need for dedicated digital PCR instruments or NGS equipment.

[0214]

[0215]

[0216] The treatment of Waldenström macroglobulinemia (WM) has shifted from traditional chemotherapy to targeted therapy. This invention combines BDA technology with traditional Sanger sequencing, overcoming the technical bottlenecks of traditional Sanger sequencing. It has successfully developed an innovative BDA Sanger sequencing method suitable for detecting the CXCR4 gene. Through clinical consistency and performance validation, the sensitivity of BDA Sanger sequencing has been improved from 10% to 0.2%. Its long enriched region design significantly improves the efficiency and coverage of CXCR4 gene mutation detection. BDA-Sanger technology truly and effectively realizes the policy of using molecular markers to assist in pathological diagnosis, and is expected to become a new standard for WM molecular diagnosis, enabling personalized and precise medication in clinical treatment and benefiting more patients. The widespread application of this sequencing technology will provide sensitive, accurate, and economical detection methods and theoretical support for the auxiliary diagnosis of WM.

[0217] Any aspects of this invention not described in detail are well-known to those skilled in the art.

[0218] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A primer blocking agent composition for detecting the CXCR4 gene in Waldenström macroglobulinemia, characterized in that: The primer blocking agent composition includes a primer set and a blocking agent, wherein the primer set includes a forward primer set and a reverse primer set; The nucleotide sequence of the blocking agent is shown in SEQ ID NO.1; The nucleotide sequence of the reverse primer set is shown in SEQ ID NO.2; The nucleotide sequence of the forward primer set is shown in SEQ ID NO.

3.

2. The primer blocking agent composition for detecting the CXCR4 gene in Waldenström macroglobulinemia as described in claim 1, characterized in that: The primer-blocker composition is used to simultaneously detect the R334X and S338X site mutations in the CXCR4 gene of Waldenström macroglobulinemia.

3. The primer blocking agent composition for detecting the CXCR4 gene in Waldenström macroglobulinemia as described in claim 1, characterized in that: The amount of the blocking agent used is 50 nM.

4. The primer blocking agent composition for detecting the CXCR4 gene in Waldenström macroglobulinemia as described in claim 1, characterized in that: The concentration ratio of the forward primer set, the reverse primer set, and the blocking agent is 1:1:

5.

5. The use of the primer blocking composition according to any one of claims 1 to 4 in the preparation of a kit for detecting Waldenström macroglobulinemia.

6. A kit for detecting mutant types of Waldenström macroglobulinemia, characterized in that: Includes PCR reagents, enzyme digestion reagents, sequencing reagents, and primer blocking agent compositions as described in any one of claims 1 to 4.

7. A method for detecting Waldenström macroglobulinemia mutation types for purposes other than disease diagnosis and / or treatment: characterized by: Includes the following steps: Step 1: Extract DNA from the sample; Step 2: Prepare a positive control group and a negative control group; Step 3: Simultaneously perform PCR amplification on the DNA extracted in Step 1 and the control group in Step 2 using the kit described in claim 5; Step 4: Perform enzymatic digestion on the PCR amplification system from Step 3; Step 5: Perform sequencing PCR reaction on the product after enzymatic digestion in step 4; Step 6: Purify and sequence the products of the sequencing PCR reaction in Step 5. Step 7: Compare the sequenced data with the normal sequence to determine whether mutations have occurred at the R334X and S338X sites.

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