CN114438179B - Digital PCR (polymerase chain reaction) kit, primer and probe for detecting chronic lymphocytic leukemia drug resistance related gene mutation - Google Patents

Abstract

The invention relates to the field of biological detection, in particular to a digital PCR kit, primers and probes for detecting drug resistance-related gene mutations in chronic lymphocytic leukemia (CLL). The kit of the present invention contains primers and probes for detecting mutations of BTK C481S site, PLCG2 R655W site, S707Y site, BCL2 G101V site and F104L site. The kit of the invention adopts digital PCR technology to quantitatively detect mutation sites such as human BTK C481S, and provides reference for clinicians to monitor the curative effect of targeted medication and clinical medication for CLL patients.

Description

Digital PCR for detecting drug resistance-related gene mutations in chronic lymphocytic leukemia Kits, primers and probes

Technical Field

The present invention relates to the field of biological detection, and in particular to a digital PCR kit, primers and probes for detecting chronic lymphocytic leukemia drug resistance-related gene mutations.

Background Art

Chronic lymphocytic leukemia (CLL) is a hematological malignancy characterized by the clonal proliferation and accumulation of mature, CD5-positive B cells in peripheral blood, bone marrow, liver, spleen and lymph nodes. It is the most common adult leukemia in Western countries. The median age of CLL patients is 72 years old, and men are more susceptible than women. Although the incidence of CLL in my country is lower than that in Western countries, with the aging of the population, the westernization of lifestyles and the improvement of diagnostic technology, the incidence rate has shown an upward trend. In the past 30 years, the treatment of CLL has gradually moved from immunochemotherapy to targeted therapy, and a variety of new targeted drugs have been put into clinical use, including the Bruton tyrosine kinase (BTK) inhibitor ibrutinib, the PI3K inhibitor idelalisib and the Bcl-2 inhibitor venetoclax.

The continuous activation of the B cell receptor (BCR) signaling pathway plays an important regulatory role in the proliferation and survival of CLL cells. Blocking or inhibiting the BCR pathway is a new strategy for the treatment of CLL. BTK is one of the important components of the BCR signaling pathway and is an important regulatory factor in the B cell maturation, differentiation, proliferation and survival pathways. In CLL, BTK can promote the survival of CLL cells by activating downstream signaling pathways such as NF-κB and MAP kinase. Inhibiting BTK activity can downregulate the BCR downstream signaling pathway, causing CLL cells to undergo apoptosis and produce a significant anti-tumor effect. Therefore, drug treatment strategies targeting BTK have become a research hotspot for hematological malignancies.

Ibrutinib is the first BTK inhibitor approved for the treatment of CLL patients in the world. It irreversibly inhibits the activity of BTK by highly specific covalent binding to cysteine-481 (C481) at the BTK active site, inhibiting the proliferation, chemotaxis and adhesion of B lymphocytes, thereby exerting a good anti-tumor effect. It is a highly efficient and highly selective oral small molecule BTK inhibitor. Ibrutinib was approved by the my country Food and Drug Administration in 2017 for the treatment of relapsed or refractory CLL.

However, during the clinical application of ibrutinib, the phenomenon of ibrutinib resistance in CLL patients was discovered and taken seriously. Many studies have found that the generation of acquired resistance to ibrutinib is related to gene mutations at the covalent binding site of ibrutinib and BTK (such as C481S mutation) and mutations in BTK downstream pathway regulators (such as PLCG2 gene R665W mutation, L846F mutation and S707Y mutation). Among them, BTK C481S mutation plays the most important role in the generation of ibrutinib resistance. Studies have found that the clonal expansion of BTK C481S mutation in the peripheral blood of CLL patients appears 9.3 months earlier than the clinical resistance to ibrutinib. Regular monitoring and timely adjustment of medication can benefit patients' survival. At present, second-generation sequencing technology is often used in clinical practice to perform targeted gene testing on biological samples of CLL patients in order to select the most suitable treatment plan and achieve precision treatment. However, the high economic cost and long detection cycle have become stumbling blocks for the second-generation sequencing technology to serve the clinical medication guidance of CLL patients. Therefore, low-cost, high-precision rapid detection technology is of great value in accurately monitoring and guiding clinical medication for CLL patients treated with ibrutinib.

In view of this, the present invention is proposed.

Summary of the invention

The primary inventive object of the present invention is to provide a digital PCR kit for detecting chronic lymphocytic leukemia drug resistance-related gene mutations.

The second object of the present invention is to provide a method for using the kit.

The third invention objective of the present invention is to provide primers and probes for detecting gene mutations associated with drug resistance in chronic lymphocytic leukemia.

In order to achieve the purpose of the invention, the technical solution adopted is:

The present invention provides a digital PCR kit for detecting chronic lymphocytic leukemia drug resistance-related gene mutations. The kit contains a nucleic acid amplification reagent, and the nucleic acid amplification reagent contains primers and probes for detecting mutations in BCL2 G101V and F104L sites, PLCG2 R655W site, PLCG2 S707Y site, and BTK C481S site.

Optionally, the nucleotide sequence of the upstream primer for detecting BCL2 G101V and F104L sites is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the probe is shown in SEQ ID NO: 3 to SEQ ID NO: 5; the nucleotide sequence of the upstream primer for detecting the PLCG2 R655W site is shown in SEQ ID NO: 6, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 7, and the nucleotide sequence of the probe is shown in SEQ ID NO: 8 to SEQ ID NO: 9; the nucleotide sequence of the upstream primer for detecting the PLCG2 S707Y site is shown in SEQ ID NO: 10, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 11, and the nucleotide sequence of the probe is shown in SEQ ID NO: 12 to SEQ ID NO: 13; the nucleotide sequence of the upstream primer for detecting the BTK C481S site is shown in SEQ ID NO: 14, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 15, and the nucleotide sequence of the probe is shown in SEQ ID NO: 16 to SEQ ID NO: 17.

Optionally, the 5' end of the probe is connected to a fluorescent group, and the 3' end is connected to a fluorescent quenching group; preferably, the fluorescent group of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:12, and SEQ ID NO:16 is selected from FAM, and the fluorescent quenching group is selected from MGB; the fluorescent group of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:13, and SEQ ID NO:17 is selected from VIC, and the fluorescent quenching group is selected from MGB.

Optionally, the concentration of the upstream primer is 0.17-0.5 μmol/L, preferably 0.35 μmol/L; the concentration of the downstream primer is 0.17-0.5 μmol/L, preferably 0.35 μmol/L; the concentration of the probe is 0.12-0.24 μmol/L, preferably 0.18 μmol/L.

Optionally, the kit further contains a negative control and a positive control, wherein the negative control is process water, and the positive control is a fragmented plasmid standard product containing SEQ ID NO: 18 to SEQ ID NO: 26.

Optionally, the sample applicable to the kit is a plasma specimen or a paraffin specimen.

The present invention also relates to a method for using the digital PCR kit, which at least comprises the following steps:

S1. Sample processing: extract the sample using a commercial extraction kit to obtain a sample processing solution;

S2. preparing a digital PCR reaction mixture: mixing the digital PCR reaction mixture with the sample processing solution to obtain a digital PCR reaction mixture; preferably, the volume ratio of the digital PCR reaction solution to the sample processing solution is 6:14;

S3, adding the digital PCR reaction mixture and droplet generation oil into the droplet generation card, placing it in a droplet generator to generate droplets, sealing the film, and then performing a digital PCR amplification reaction;

S4. Read the fluorescence signal: Place the amplified 96-well plate in a droplet reader and use the software to directly read and analyze the results; automatically calculate the copy number of the mutations at the BTK C481S site, PLCG2R655W site, S707Y site, BCL2 G101V site, and F104L site in the ddPCR reaction system according to the Poisson distribution principle.

Optionally, the conditions of the digital PCR amplification reaction are: first, keep warm at 95°C for 9 to 11 minutes; then keep warm at 94°C for 13 to 16 seconds, and keep warm at 58°C for 58 to 60 seconds, for a total of 39 to 41 cycles; finally, keep warm at 98°C for 10 minutes, and cool to 4°C to stop the reaction; preferably, first, keep warm at 95°C for 10 minutes; then, keep warm at 94°C for 15 seconds, and keep warm at 58°C for 60 seconds, for a total of 40 cycles; finally, keep warm at 98°C for 10 minutes, and cool to 4°C to stop the reaction; more preferably, the heating and cooling rate is ≤2°C/s.

The invention relates to primers and probes for detecting drug resistance-related gene mutations in chronic lymphocytic leukemia by digital PCR. The primers and probes are used for detecting mutations in BCL2 G101V and F104L sites, PLCG2 R655W site, PLCG2 S707Y site, and BTK C481S site.

Optionally, the nucleotide sequence of the upstream primer for detecting BCL2 G101V and F104L sites is shown in SEQ ID NO: 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 2, and the nucleotide sequence of the probe is shown in SEQ ID NO: 3 to SEQ ID NO: 5; the nucleotide sequence of the upstream primer for detecting PLCG2 R655W site is shown in SEQ ID NO: 6, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 7, and the nucleotide sequence of the probe is shown in SEQ ID NO: 8 to SEQ ID NO: 9; the nucleotide sequence of the upstream primer for detecting PLCG2 S707Y site is shown in SEQ ID NO: 10, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 11, and the nucleotide sequence of the probe is shown in SEQ ID NO: 12 to SEQ ID NO: 13; the nucleotide sequence of the upstream primer for detecting BTK C481S site is shown in SEQ ID NO: 14, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 15, and the nucleotide sequence of the probe is shown in SEQ ID NO: 16 to SEQ ID NO: 17; preferably, the nucleotide sequence of SEQ ID NO: The fluorescent group of NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:12, and SEQ ID NO:16 is selected from FAM, and the fluorescence quenching group is selected from MGB; the fluorescent group of SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:13, and SEQ ID NO:17 is selected from VIC, and the fluorescence quenching group is selected from MGB.

The present invention has at least the following beneficial effects:

This kit uses digital PCR technology to quantitatively detect mutations such as human BTK C481S, providing a reference for clinicians to monitor the efficacy of CLL patients and develop individualized treatment plans.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the BCL2 G101V mutation site;

FIG. 2 is a schematic diagram of the interface during the detection process.

DETAILED DESCRIPTION

It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and/or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and/or combinations thereof.

The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

The embodiment of the present invention has designed digital PCR detection of five sites, namely BCL2 G101V, F104L, PLCG2R655W, S707Y and BTK C481S, by comprehensive consideration of various data results.

The BCL2 inhibitor venetoclax has a potent and durable remission effect on patients with chronic lymphocytic leukemia. However, when BCL2 G101V mutates (NM_000633.2:c.302G>T,p.(Gly101Val)), it can cause resistance to venetoclax in patients with chronic lymphocytic leukemia. Primary cells carrying the G101V mutation in the stroma have significantly increased resistance to venetoclax at concentrations higher than those achievable clinically. In the absence of venetoclax, the G101V mutation is as effective as wild-type BCL2 in protecting cell lines from apoptosis. In binding experiments, venetoclax's ability to compete with BIM for binding to G101V was dramatically reduced by 180-fold compared to wild-type BCL2, which is likely due to the larger isopropyl group in VAL occupying the groove that binds to venetoclax. In cell experiments, venetoclax can easily dissociate BAX and BAK from wild-type BCL2, but these pro-apoptotic molecules become ineffective when bound to G101V. All of these indicate that the BCL2 G101V mutation causes CLL to be resistant to venetoclax. Therefore, detecting the mutation at this site can better provide guidance for monitoring the emergence of drug-resistant clones and subsequent clinical medication. The schematic diagram of the BCL2 G101V mutation site is shown in Figure 1.

BTK is a member of the Tec family of non-receptor protein tyrosine kinases. It is a key kinase in the B cell antigen receptor (BCR) signaling pathway and can regulate the proliferation, differentiation and apoptosis of normal B cells. This fully demonstrates that BTK plays an irreplaceable role in the generation of B lymphocytes and is an ideal target for the treatment of hematological tumors. The chromosome of the human BTK gene encodes 659 amino acids. The BTK gene includes the PH domain, TH domain, SH3 domain, SH2 domain and SH1 domain. The PH domain consists of approximately 120 amino acids and contains the binding sites of the transcription factor BAP-135/TFH-I and the activity down-regulatory factors PIN1 and IBTK. It is also responsible for mediating the interaction between BTK and the second messenger phosphatidylinositol triphosphate (PIP3). The TH domain consists of approximately 80 amino acid residues and consists of two parts: the BTK protein motif and the proline-rich region. The SH3 domain can specifically recognize the proline-rich fragment in the TH domain and induce intramolecular folding. The SH1 domain contains an activation loop, an ATP binding site, a catalytic unit, and an allosteric inhibitory fragment. BTK activation (phosphorylation) initially occurs in the activation loop in the SH1 domain, and further activation occurs in the SH2 and SH3 domains containing the main autophosphorylation sites. The function of the SH2 domain is to specifically recognize the phosphorylation state of tyrosine residues, so that proteins containing the SH2 domain can be localized to the phosphorylated tyrosine sites of other proteins.

BTK's downstream receptors include growth factors, B cell antigens, chemokines, and non-specific immune receptors, so BTK activation can trigger a variety of cellular processes such as cell proliferation, survival, differentiation, angiogenesis, antigen expression, and cytokine synthesis. The focus of BTK activation is that BTK migrates to the cell membrane. When some receptors on the cell membrane receive stimulation from the corresponding ligands, the activated receptors recruit and phosphorylate the intracellular signal transduction kinase PI3K. The phosphorylated PI3K then converts PIP2 on the membrane into the second messenger PIP3. PIP3 binds to the PH domain of BTK, and BTK is then recruited to the cell membrane, and then the Tyr-551 residue is phosphorylated by Syk and Lyn kinases. BTK then undergoes autophosphorylation at the Tyr-223 residue to become physiologically active.

Ibrutinib can selectively bind to the target protein BTK active site Cys481 to form a covalent bond, thereby inhibiting BTK autophosphorylation and achieving the effect of inhibiting the BTK signaling pathway. It has the characteristics of high efficiency and irreversibility. Resistance caused by mutations In the blood RNA analysis of CLL patients (840 mg/d) with resistance to ibrutinib, the BTK mutant C481S was found. From the docking results of BTK structure and ibrutinib, it can be seen that cysteine at position 481 plays an important role in drug binding, so the mutation from C to S greatly reduces the stability of its binding IC50. In addition, mutants of BTK's downstream gene PLCG2, R665W, L845F and S707Y, were found in CLL patients. These mutations can lead to resistance to ibrutinib. However, life-threatening adverse events such as bleeding, infection, bone marrow suppression, nephrotoxicity, and secondary second tumors still occur. Therefore, the detection of these sites has important clinical drug guidance significance.

The embodiment of the present invention relates to a digital PCR kit for detecting drug resistance-related gene mutations in chronic lymphocytic leukemia, the kit contains a nucleic acid amplification reagent, and the nucleic acid amplification reagent contains primers and probes for detecting mutations in BTK C481S site, PLCG2 R655W site, PLCG2 S707Y site, BCL2 G101V site and F104L site. Specifically, the nucleotide sequences of the primers and probes are as shown in Table 1:

Table 1

Primer probe name Sequence number Nucleotide sequence Modification BCL2-G101V-F104L-F SEQ ID NO:1 ccacctgtggtccacctga — BCL2-G101V-F104L-R SEQ ID NO:2 caggtgcagctggctgga — BCL2-G101V-P SEQ ID NO:3 caggccgtcgacgact 5'-FAM, 3'-MGB BCL2-G101-F104-WT-P SEQ ID NO:4 cggcgacgacttctcc 5'-VIC, 3'-MGB BCL2-F104L-P SEQ ID NO:5 acgacttgtcccgccg 5'-FAM, 3'-MGB PLCG2-R655W-F SEQ ID NO:6 tactatgacagcctgagcc — PLCG2-R655W-R SEQ ID NO:7 tcgctcccctctcgcttc — PLCG2-R655W-P SEQ ID NO:8 aggattccctgggacg 5'-FAM, 3'-MGB PLCG2-R655-WT-P SEQ ID NO:9 aggattccccgggac 5'-VIC, 3'-MGB PLCG2-S707Y-F SEQ ID NO:10 agcattgtcgcatcaaccgg — PLCG2-S707Y-R SEQ ID NO:11 ctcgtagtaactgacgagct — PLCG2-S707Y-P SEQ ID NO:12 tggggacctacgcctat 5'-FAM, 3'-MGB PLCG2-S707-WT-P SEQ ID NO:13 ctggggacctccgcct 5'-VIC, 3'-MGB BTK-C481S-F SEQ ID NO:14 cgccccatcttcatcatcac — BTK-C481S-R SEQ ID NO:15 agcagctgctgagtctgg — BTK-C481S-P SEQ ID NO:16 aatggctccctcctgaa 5'-FAM, 3'-MGB BTK-C481-WT-P SEQ ID NO:17 aatggctgcctcctgaa 5'-VIC, 3'-MGB

Optionally, in the nucleic acid amplification reagent, the concentration of the upstream primer is 0.17-0.5 μmol/L, preferably 0.35 μmol/L; the concentration of the downstream primer is 0.17-0.5 μmol/L, preferably 0.35 μmol/L; the concentration of the probe is 0.12-0.24 μmol/L, preferably 0.18 μmol/L.

Optionally, the kit also contains negative control and positive control, the negative control is process water. The positive control contains the fragmented plasmid standard shown in Table 2:

Table 2

Optionally, the sample applicable to the kit is a plasma sample or a paraffin sample.

The present invention also relates to a method for using the digital PCR kit, which at least comprises the following steps:

S1. Sample processing: extract the sample using a commercial extraction kit to obtain a sample processing solution;

S2. preparing a digital PCR reaction mixture: mixing the digital PCR reaction mixture with the sample processing solution to obtain a digital PCR reaction mixture; preferably, the volume ratio of the digital PCR reaction solution to the sample processing solution is 6:14;

S3, adding the digital PCR reaction mixture and droplet generation oil into the droplet generation card, placing it in a droplet generator to generate droplets, sealing the film, and then performing a digital PCR amplification reaction;

S4. Read the fluorescence signal: Place the amplified 96-well plate in a droplet reader and use the software to directly read and analyze the results; automatically calculate the copy number of the mutations at the BTK C481S site, PLCG2R655W site, S707Y site, BCL2 G101V site, and F104L site in the ddPCR reaction system according to the Poisson distribution principle.

Among them, the conditions of the digital PCR amplification reaction are: first, keep warm at 95°C for 9 to 11 minutes; then keep warm at 94°C for 13 to 16 seconds, keep warm at 58°C for 58 to 60 seconds, and perform 39 to 41 cycles in total; finally, keep warm at 98°C for 10 minutes, and cool to 4°C to stop the reaction; preferably, first, keep warm at 95°C for 10 minutes; then, keep warm at 94°C for 15 seconds, and keep warm at 58°C for 60 seconds, and perform 40 cycles in total; finally, keep warm at 98°C for 10 minutes, and cool to 4°C to stop the reaction; more preferably, the heating and cooling rate is ≤2°C/sec.

The embodiment of the present invention also relates to primers and probes for detecting chronic lymphocytic leukemia drug resistance-related gene mutations using digital PCR, the primers and probes are used to detect mutations in BCL2 G101V and F104L sites, PLCG2 R655W site, PLCG2S707Y site, and BTK C481S site, and the nucleotide sequences are specifically shown in Table 1.

Example 1

The composition of the kit is shown in Table 3:

Table 3

Applicable instrument: QX200 Droplet Digital PCR System (BioRad, USA).

Sample requirements: Applicable to the detection of human genomic free DNA (cfDNA) extracted from plasma specimens or human genomic DNA extracted from paraffin specimens.

Positive judgment value or reference interval: 2‰ BCL2, PLCG2 and BTK gene mutations can be detected in 10ng genomic DNA.

Inspection method:

1. Sample processing:

Extract nucleic acid by yourself (it is recommended to use commercial kits to extract DNA) and use it as a template for PCR reaction. It is recommended to test the extracted nucleic acid immediately, otherwise store it below -20℃.

2. Preparation of amplification reagents and sample addition:

a. Take out the corresponding reaction solution from the kit, melt and mix at room temperature, centrifuge at 2000rpm for 10s, and prepare the PCR premix for each test as follows: 4μL mixed solution ddPCR + 10μL 2×ddPCR MIX3. Dispense the prepared PCR premix into each PCR tube at a volume of 14μL per tube.

b. After the genomic DNA template concentration is determined (qubit determination), dilute it with water to 2 ng/μL, and add 6 μL of the template to the PCR tube containing the above PCR premix.

c. The total volume of each reaction system is 20 μL.

d. Cover the PCR tube tightly, oscillate and mix for more than 20 seconds, and then centrifuge for instantaneous droplet preparation.

3. Preparation of microdroplets:

a. Add 8 20μL reaction systems into the 8 wells in the middle row of the DG8 cartridge.

Note: 1) Samples must be added to the middle row of the DG8 cartridge first (if there are less than 8 samples, add 20μL BX ddPCR Buffer Control to the empty wells; adjust the pipette to 20μL before adding samples, and aspirate all the liquid in the tube when sampling).

2) When adding samples, avoid generating bubbles. If bubbles are visible to the naked eye, use a clean gun tip to puncture the bubbles.

b. Add 70μL of Droplet Generation oil to each of the 8 holes in the bottom row of the DG8 cartridge, cover with a gasket, and gently and steadily place the DG8 cartridge in the droplet generator to start generating droplets. Pay attention to the indicator light on the instrument, which is usually completed within 2 minutes.

c. Droplets are generated in the top row of wells in the cartridge. The generated droplets (approximately 35 to 45 μL) are transferred to a 96-well plate.

5. Film sealing

After the droplets are transferred into the 96-well plate, they are sealed with a preheated PX1 heat sealer. The recommended operating program is: 180°C, 5s, and there is no need to reverse the direction for secondary sealing. After sealing, PCR reaction should be carried out within 30 minutes, or PCR should be carried out within 4 hours in a 4°C refrigerator.

6. PCR amplification:

95℃, 10min; (94℃, 15sec; 58℃, 60sec) 40 cycles; 98℃, 10min; 4℃, 5min, the reaction system is set to 40μL, and the heating and cooling speed is ≤2℃/s.

7. Droplet reading:

a. Turn on your computer first, then turn on the Droplet Reader. Preheat it for at least 30 minutes before use.

b. Place the 96-well plate that has completed PCR into the plate holder and place it steadily into the microdroplet reader.

c. Open QuantaSoft software and set up the sample information in the 96-well plate. Once completed, you can run it.

Note: 1) Select "ddPCR Supermix for probes" in Supermix

2) Select “FAM/VIC” for Dye Set

8. Result analysis:

After the assay is complete, click “2D Amplitude” to view the cluster plot for Channel 1 and Channel 2. This plot allows for manual or automatic adjustment of thresholds to designate positive and negative droplets for each assay channel.

Click "Auto Analyze" to reset the threshold;

Manually specify the threshold:

Use threshold crosshairs to specify classification areas for the entire dot plot (optional only in heatmap mode);

Use the ellipse, rectangle or lasso threshold adjustment tool to classify the dot map area: click the corresponding tool button, then click the area type in "Working cluster selector" and use the tool to select the corresponding area. The interface diagram is shown in Figure 2:

Note: The setting of the fluorescence threshold line refers to the negative control and positive control: In 2D Amplitude, the position of the fluorescence threshold line should make the droplet cluster of the negative control in the "ch1-ch2-" interval, and the four droplet clusters of the positive control are located in four intervals respectively.

Explanation of test results:

1. Validity determination:

Negative control validity determination: the number of points in the "ch1+" area is less than 4 points and the number of points in the "ch2+" area is less than 4 points.

The effectiveness of the positive control was determined as follows: ≥4 points fell in the “ch1+ch2-” region and the mutation ratio was ≥2‰.

Determination of invalid results: The total number of droplets in each reaction tube should be ≥8000. If the total number of droplets is <8000, the droplet generation in the reaction well is not ideal and the droplet generation needs to be repeated.

2. Result determination:

2.1 Qualitative judgment

(1) If the sample has ≥4 points falling in the “ch1+ch2-” region and the mutation ratio is ≥2‰, the BCL2 and BTK loci are determined to be mutated.

(2) If (1) is not met,

If the sample DNA ≥ 50 copies

If the number of points in the sample that fall in the "ch1+ch2-" region is <3 or the mutation ratio is <2‰, it is determined that the BCL2 and BTK sites have no mutation or the mutation is below the minimum detection limit.

If the sample has 3 points falling in the "ch1+ch2-" zone and the mutation ratio is ≥2‰, the BCL2 and BTK site mutations are suspected to be positive and need to be retested. As a result of the retest, if the sample has ≥4 points falling in the "ch1+ch2-" zone and the mutation ratio is ≥2‰, the BCL2 and BTK site mutations are determined. Otherwise, it is determined that there is no mutation in the BCL2 and BTK sites or the mutation is below the minimum detection limit.

If the sample DNA is less than 50 copies, it indicates that the added DNA is of poor quality or contains PCR inhibitors, and it is necessary to re-extract the DNA or re-sample. After re-testing, if the sample DNA is less than 50 copies and does not meet (1), it is determined that the DNA quality does not meet the requirements. Otherwise, make the corresponding judgment according to the above conditions.

2.2 Quantitative determination

If the sample BTK site is mutated, the mutation percentage can be calculated according to the formula Ch1/(Ch1+Ch2).

Experimental Example 1 Primer screening test

Prepare the primer-probe reaction solution according to general principles, and prepare 20 small samples for each site that needs to be tested for preliminary experiments.

1. Prepare the components in Table 4 according to the BCL2 G101V-F104L locus. Then replace the primers F1/R2, F2/R1, and F2/R2 with F1/R1 in Table 3 for experiments. Use standards with known mutation frequencies obtained by other detection methods such as NGS sequencing to perform droplet reaction experiments to compare the quality of each primer combination and select the optimal combination.

Table 4: BCL2 reaction solution

name concentration Serves 1 Serves 20 BCL2-F1 50μmol/L 0.36μL; 7.2μL BCL2-R1 50μmol/L 0.36μL 7.2μL Probe G101V 100μmol/L 0.03μL 0.6μL Probe F104L 100μmol/L 0.03μL 0.6μL Probe BCL2-WT 100μmol/L 0.05μL 1μL TE 3.17μL 63.4μL TOTAL 4μL 80μL

The primer sequences are shown in Table 5:

Table 5

Primer name Sequence number Nucleotide sequence BCL2-F1 SEQ ID NO:27 ccggtgccacctgtggtc BCL2-R1 SEQ ID NO:28 gcggtgaagggcgtcagg BCL2-F2 SEQ ID NO:1 ccacctgtggtccacctga BCL2-R2 SEQ ID NO:2 caggtgcagctggctgga PLCG2-R655W-F1 SEQ ID NO:6 tactatgacagcctgagcc PLCG2-R655W-R1 SEQ ID NO:29 cgctcccctctcgcttcc PLCG2-R655W-F2 SEQ ID NO:30 caagccgtggtactatgacagc PLCG2-R655W-R2 SEQ ID NO:7 tcgctcccctctcgcttc PLCG2-S707Y-F1 SEQ ID NO:31 tcagggctaggggcaagg PLCG2-S707Y-R1 SEQ ID NO:32 aatgcttctcgtagtaactgacg PLCG2-S707Y-F2 SEQ ID NO:10 agcattgtcgcatcaaccgg PLCG2-S707Y-R2 SEQ ID NO:11 ctcgtagtaactgacgagct BTK-C481S-F1 SEQ ID NO:14 cgccccatcttcatcatcac BTK-C481S-R1 SEQ ID NO:15 agcagctgctgagtctgg BTK-C481S-F2 SEQ ID NO:33 cccatcttcatcatcactgagtac BTK-C481S-R2 SEQ ID NO:34 catccttgcacatctctagcag

The results of the BCL2 primer screening test are shown in Tables 6 to 9:

Table 6: BCL2 F2/R2 primer screening test results

Table 7: BCL2 F1/R1 primer screening test results

Table 8: BCL2 F2/R1 primer screening test results

Table 9: BCL2 F1/R2 primer screening test results

It can be seen from the droplet reading results shown in Tables 6 to 9 that the reaction solution mixed with BCL2F2/R2 primers has the highest accuracy in detecting the mutation rate of positive standard samples, and has good detection specificity in negative samples. At the same time, no non-specific mutation points (ch1+ch2-points) were found.

According to the same method, 20 small sample reagents were prepared for the detection sites PLCG2R655W, PLG2 S707Y and BTK C481S according to the conventional reaction solution ratios shown in Tables 10, 11, and 12, and then the primers F1/R2, F2/R1, and F2/R2 were replaced with F1/R1 in the table to conduct experiments, compare the quality of each primer, and select the best combination according to the same standard.

Table 10: PLCG2 R655W reaction solution

name concentration Serves 1 Serves 20 PLCG2-R655F1 50μmol/L 0.36 7.2 PLCG2-R655R1 50μmol/L 0.36 7.2 Probe PLCG2 R655W 100μmol/L 0.03 0.6 Probe PLCG2 R655-WT 100μmol/L 0.05 1 TE 3.2 64 TOTAL 4 80

Table 11: PLCG2 S707Y reaction solution

name concentration Serves 1 Serves 20 PLCG2-S707F1 50μmol/L 0.36 7.2 PLCG2-S707R1 50μmol/L 0.36 7.2 Probe PLCG2 S707Y 100μmol/L 0.03 0.6 Probe PLCG2 S707-WT 100μmol/L 0.05 1 TE 3.2 64 TOTAL 4 80

Table 12: BTK C481S reaction solution

name concentration Serves 1 Serves 20 BTK C481F1 50μmol/L 0.36 7.2 BTK C481F1 50μmol/L 0.36 7.2 Probe BTK C481S 100μmol/L 0.03 0.6 Probe BTK C481-WT 100μmol/L 0.05 1 TE 3.2 64 TOTAL 4 80

The results of the PLCG2 R655W primer screening test are shown in Tables 13 to 16:

Table 13: PLCG2 R655W-F1/PLCG2 R655W-R2 primer screening test results

Table 14: PLCG2 R655W-F2/PLCG2 R655W-R2 primer screening test results

Table 15: PLCG2 R655W-F2/PLCG2 R655W-R1 primer screening test results

Table 16: PLCG2 R655W-F1/PLCG2 R655W-R1 primer screening test results

The results of the PLCG2 S707Y primer screening test are shown in Tables 17 to 20:

Table 17: PLCG2 S707Y-F2PLCG2 S707Y-R2 Primer Screening Test Results

Table 18: PLCG2 S707Y-F1/PLCG2 S707Y-R1 primer screening test results

Table 19: PLCG2 S707Y-F2/PLCG2 S707Y-R1 primer screening test results

Table 20: PLCG2 S707Y-F1/PLCG2 S707Y-R2 primer screening test results

The results of the BTK C481S primer screening test are shown in Tables 21 to 24:

Table 21: BTK C481S-F1/BTK C481S-R1 primer screening test results

Table 22: BTK C481S-F2/BTK C481S-R2 primer screening test results

Table 23: BTK C481S-F2/BTK C481S-R1 Primer Screening Test Results

Table 24: BTK C481S-F1/BTK C481S-R2 primer screening test results

By observing the droplet reading results, it can be seen (Tables 13 to 24) that the reaction solution mixed with PLCG2 R655W F1/R2, PLCG2S707YF2/R2, and BTK C481S-F1/R1 primers has the highest accuracy in detecting the mutation rate of the positive standard, and has good detection specificity in negative samples. At the same time, no non-specific mutation points (ch1+ch2-points) were found, and it was selected as the preparation scheme for preliminary experiments.

Primer concentration and probe concentration are interrelated in the entire PCR reaction, so these two factors were selected to conduct cross-experiments on plasmid reference products diluted to 10% to optimize the PCR reaction system.

Table 25: BTK C481S primer concentration and probe concentration test results

The above experimental results show that the combination of primer concentration of 0.35μM and probe concentration of 0.18μM is the most effective detection combination under the premise of saving raw materials and ensuring detection accuracy. It shows that the mutation rate detected is consistent with the reference product under the premise that the copy number is not affected. Similarly, corresponding experiments were conducted on the other four sites, and the results were consistent. According to this preparation scheme, sensitivity, accuracy, stability and other tests were carried out.

Experimental Example 2 Detection Limit Experiment

Determination and basis of detection sensitivity Select a plasmid reference containing mutations and mix it with wild plasmids in a certain proportion and then perform gradient dilution to determine the reference value of the mutation rate of the reaction solution. Taking the BTK C481S site as an example, the detection results are as follows:

Table 26: BTK C481S detection limit setting test results

By observing the droplet reading results, it can be seen that the BTK C481S reaction solution can still detect samples with a mutation rate greater than or equal to 0.1%. However, in the range of 0.1% to 0.2%, the quantitative results deviate greatly from the expected mutation rate; when the mutation rate drops below 0.1%, the test results are prone to missed detection (such as negative detection). Therefore, the qualitative detection range is set to ≥0.2%. The same method was used to determine the same detection sensitivity for other PLCG2 R655W, S707Y, BCL2 G101V and G104L sites, and the mutation detection range of all sites was set to ≥0.2%.

Detection limit test results: Based on the above experimental results, the BTK C481S mutation ratio was used as the detection limit, repeated 20 times, and the test results are as follows:

Table 27: BTK C481S detection limit results

From the above results, it can be seen that under the condition of 95% confidence interval, the positive coincidence rate of the experimental results repeated 20 times was 100%, using the BTK C481S detection limit reference (mutation rate = 0.2%), showing effective detection sensitivity. The same method was used to perform the same determination experiment on other PLCG2 R655W, S707Y, BCL2 G101V and G104L sites, and the positive coincidence rate of all sites was 100%.

Experimental Example 3 Accuracy Experiment and Results

DNA samples from three patients with chronic lymphocytic leukemia who were clinically positive for BTK C481S were selected for detection experiments and compared with the mutation rate detected positive by high-throughput sequencing method.

Table 28: BTK C481S clinical sample accuracy test results

According to the results, samples 9047, 4915, and 4672 were BTK C481S positive, with mutation rates of 24.1%, 44.7%, and 43.3%, respectively, which were consistent with the frequencies calculated by NGS testing. In order to further confirm the correctness of the mutation rate, the same experiment was repeated twice, and the results were consistent with the first result, eliminating the possibility that the operation error would cause inaccuracy in the results.

Similarly, a total of 5 samples that were clinically positive for PLCG2 R655W, S707Y, BCL2 G101V, and G104L were selected for testing (see Table 29). The results were consistent with the frequencies calculated by NGS testing. Each group of experiments was repeated three times to ensure the impact of experimental errors on the results.

Table 29: Clinical sample accuracy test results

Experimental Example 4: Specificity Experiment and Results

Two clinical samples that were negative for BTK C481S, BCL2 G101V, BCL2 G104L, PLCG2 R655W, and PLCG2 S707Y by high-throughput sequencing and a DNA sample of diffuse tissue lymphoma cell line SU-DHL2 were selected for detection using the above five loci kits. The experimental results of the BTK C481S kit are shown in Table 30.

Table 30: BTK C481S kit specific results

The above results show that the BTK C481S kit tested negative and the cell line samples were negative, indicating that the kit has good specificity. The same method was used to perform the same determination experiment on other PLCG2 R655W, S707Y, BCL2 G101V and G104L sites, and the test results of all sites were negative.

Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the claims. Any technical personnel in this field may make several possible changes and modifications without departing from the concept of the present application. Therefore, the scope of protection of the present application shall be based on the scope defined by the claims of the present application.

Sequence Listing

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Suzhou Yuntai Biopharmaceutical Technology Co., Ltd.

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Claims (6)

1. A digital PCR kit for detecting drug resistance-related gene mutations in chronic lymphocytic leukemia, characterized in that, the nucleic acid amplification reagent is contained in the kit, and the nucleic acid amplification reagent contains a reagent for detecting BCL2 G101V and F104L site, PLCG2 R655W site, PLCG2 S707Y site, BTK C481S site mutation primers and probes; The nucleotide sequence of the upstream primer used to detect BCL2 G101V and F104L sites is shown in SEQ ID NO:1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2, and the nucleotide sequence of the probe is shown in Shown in SEQ ID NO:3~SEQ ID NO:5, The nucleotide sequence of the upstream primer used to detect the PLCG2 R655W site is shown in SEQ ID NO: 6, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 7, and the nucleotide sequence of the probe is shown in SEQ ID Shown in NO:8~SEQ ID NO:9, The nucleotide sequence of the upstream primer used to detect the PLCG2 S707Y site is shown in SEQ ID NO:10, the nucleotide sequence of the downstream primer is shown in SEQ ID NO:11, and the nucleotide sequence of the probe is shown in SEQ ID Shown in NO:12~SEQ ID NO:13, The nucleotide sequence of the upstream primer used to detect the BTK C481S site is shown in SEQ ID NO: 14, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 15, and the nucleotide sequence of the probe is shown in SEQ ID Shown in NO:16~SEQ ID NO:17. 2. digital PCR kit according to claim 1, is characterized in that, the 5 ' end of described probe is connected with fluorescent group, and 3 ' end is connected with fluorescence quenching group; The fluorescent group of said SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16 is FAM, and the fluorescent quenching group is MGB; said SEQ ID The fluorescent group of NO:4, SEQ ID NO:9, SEQ ID NO:13, and SEQ ID NO:17 is VIC, and the fluorescent quenching group is MGB. 3. digital PCR kit according to claim 1, is characterized in that, the concentration of described upstream primer is 0.35 μ mol/L; The concentration of described downstream primer is 0.35 μ mol/L; The concentration of described probe is 0.18 μmol/L. 4. digital PCR kit according to claim 1, is characterized in that, also contains negative control and positive control in the kit, and described negative control is process water, and described positive control is to contain SEQ ID NO:18 ~ Fragmented plasmid standard shown in SEQ ID NO:26. 5. The digital PCR kit according to claim 1, characterized in that, the sample to which the kit is applied is selected from plasma samples or paraffin samples. 6. A primer and probe for detecting chronic lymphocytic leukemia drug resistance-related gene mutations by digital PCR, characterized in that, the primers and probes are used to detect BCL2 G101V and F104L sites, PLCG2 R655W sites, PLCG2S707Y site, BTK C481S site mutation primers and probes; The nucleotide sequence of the upstream primer used to detect BCL2 G101V and F104L sites is shown in SEQ ID NO:1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO:2, and the nucleotide sequence of the probe is shown in Shown in SEQ ID NO:3~SEQ ID NO:5, The nucleotide sequence of the upstream primer used to detect the PLCG2 R655W site is shown in SEQ ID NO: 6, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 7, and the nucleotide sequence of the probe is shown in SEQ ID Shown in NO:8~SEQ ID NO:9, The nucleotide sequence of the upstream primer used to detect the PLCG2 S707Y site is shown in SEQ ID NO:10, the nucleotide sequence of the downstream primer is shown in SEQ ID NO:11, and the nucleotide sequence of the probe is shown in SEQ ID Shown in NO:12~SEQ ID NO:13, The nucleotide sequence of the upstream primer used to detect the BTK C481S site is shown in SEQ ID NO: 14, the nucleotide sequence of the downstream primer is shown in SEQ ID NO: 15, and the nucleotide sequence of the probe is shown in SEQ ID Shown in NO:16~SEQ ID NO:17; The fluorescent group of said SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:16 is FAM, and the fluorescent quenching group is MGB; said SEQ ID The fluorescent group of NO: 4, SEQ ID NO: 9, SEQ ID NO: 13, and SEQ ID NO: 17 is VIC, and the fluorescent quenching group is MGB.

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