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, a primer and a probe for detecting Chronic Lymphocytic Leukemia (CLL) drug resistance related gene mutation. The kit contains primers and probes for detecting mutation of BTK C481S locus, PLCG2R655W locus, S707Y locus, BCL 2G 101V locus and F104L locus. The kit disclosed by the invention adopts a digital PCR technology to quantitatively detect mutation sites such as human BTK C481S and the like, and provides a reference for clinical doctors to monitor the curative effect of targeted medication of CLL patients and clinical medication.

Description

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

Technical Field

The invention relates to the field of biological detection, in particular to a digital PCR kit, a primer and a probe for detecting chronic lymphocytic leukemia drug resistance related gene mutation.

Background

Chronic Lymphocytic Leukemia (CLL) is a malignant disease of the blood system characterized by the clonal proliferation and accumulation of mature, CD 5-positive B cells in peripheral blood, bone marrow, liver spleen, and lymph nodes, the most common adult leukemia in western countries. The median age of CLL patients was 72 years, with males being more susceptible than females. Although the incidence of CLL in our country is lower than in western countries, the incidence of CLL is on the rise with the aging of population, the westernization of lifestyle and the improvement of diagnostic techniques. Over the last 30 years, CLL therapy has gradually moved from immunotherapy to the age of targeted therapy, and a variety of novel targeted drugs have been put into clinical use, including the Bruton's Tyrosine Kinase (BTK) inhibitor ibuteinib, the PI3K inhibitor idarrarisb (Idelalisib), and the Bcl-2 inhibitor valnecalax (venteclax), among others.

Continuous activation of B Cell Receptor (BCR) signaling pathways plays an important regulatory role in proliferation and survival of CLL cells, and blocking or inhibiting BCR pathways is a new strategy for treating CLL. BTK is one of the important components of BCR signaling pathways, an important regulator of B cell maturation, differentiation, proliferation and survival pathways. In CLL, BTK can promote CLL cell survival by activating downstream signaling pathways such as NF- κ B, MAP kinase. Inhibiting BTK activity can down regulate downstream signal channel of BCR to make CLL cell undergo apoptosis and produce obvious anti-tumor effect. Thus, BTK-targeted drug therapy strategies have become a hotspot in research of hematologic malignancies.

Ibutinib is the first BTK inhibitor in international use for treating CLL patients, which irreversibly inhibits BTK activity by highly specific covalent binding to cysteine-481 (C481) at the BTK active site, inhibits proliferation, chemotaxis and adhesion of B lymphocytes, thereby exerting a good antitumor effect, and is a highly efficient, highly selective, oral small molecule BTK inhibitor. Ibutenib was approved by the drug administration in China for the treatment of recurrent or refractory CLL in 2017.

However, during clinical use of ibutenib, the phenomenon of ibutenib resistance in CLL patients was found and emphasized. Several studies have found that the development of ibutenib acquired resistance is associated with genetic mutations (such as C481S mutation) of the covalent binding site of ibutenib to BTK, BTK downstream pathway regulatory factor mutations (such as PLCG2 gene R665W mutation, L846F mutation and S707Y mutation). Among them, the BTK C481S mutation plays the most important role in the development of ibutinib resistance. According to the research, the cloning and amplification of the BTK C481S mutation in peripheral blood of a CLL patient occur 9.3 months earlier than the clinical drug resistance of ibutinib, and the regular monitoring and timely adjustment of the drug administration can benefit the survival of the patient. At present, a second generation sequencing technology is mostly adopted in clinic to carry out targeted gene detection on biological samples of CLL patients so as to select an optimal treatment scheme and realize accurate treatment. However, the economic cost is high, and the detection period is long, so that the second generation sequencing technology serves as a tripolite for clinical medication guidance of CLL patients. Therefore, the low-cost and high-precision rapid detection technology has very important value for realizing accurate monitoring and guiding clinical medication for CLL patients treated by the ibutenib.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims at providing a digital PCR kit for detecting chronic lymphocytic leukemia drug resistance related gene mutation.

The second invention aims to provide a using method of the kit.

The third object of the present invention is to provide a primer and a probe for detecting a chronic lymphocytic leukemia drug resistance related gene mutation.

In order to accomplish the purpose of the invention, the technical scheme adopted is as follows:

the invention provides a digital PCR kit for detecting chronic lymphocytic leukemia drug resistance related gene mutation, which comprises a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent comprises a primer and a probe for detecting mutation of BCL 2G 101V and F104L sites, PLCG2R655W sites, PLCG2S707Y sites and BTK C481S sites.

Optionally, the nucleotide sequence of the upstream primer for detecting BCL 2G 101V and F104L sites is shown in SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 2, the nucleotide sequence of the probe is shown in SEQ ID NO. 3-SEQ ID NO. 5, the nucleotide sequence of the upstream primer for detecting PLCG2R655W site is shown in SEQ ID NO. 6, the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 7, the nucleotide sequence of the probe is shown in SEQ ID NO. 8-SEQ ID NO. 9, the nucleotide sequence of the upstream primer for detecting PLCG2S707Y site is shown in SEQ ID NO. 10, the nucleotide sequence of the downstream primer is shown in SEQ ID NO. 11, the nucleotide sequence of the probe is shown in SEQ ID NO. 12-13, the nucleotide sequence of the upstream primer for detecting BTK C481 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. 16-17.

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

Alternatively, the concentration of the upstream primer is 0.17 to 0.5. Mu. Mol/L, preferably 0.35. Mu. Mol/L; the concentration of the downstream primer is 0.17 to 0.5. Mu. Mol/L, preferably 0.35. Mu. Mol/L; the concentration of the probe is 0.12 to 0.24. Mu. Mol/L, preferably 0.18. Mu. Mol/L.

Optionally, the kit also contains a negative control and a positive control, wherein the negative control is process water, and the positive control is a fragmented plasmid standard substance shown as SEQ ID NO. 18-SEQ ID NO. 26.

Optionally, the sample to which the kit is applied is selected from a plasma sample or a paraffin sample.

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

s1, sample treatment: extracting a sample by using a commercial extraction kit to obtain a sample treatment fluid;

s2, preparing a digital PCR reaction mixed solution: mixing the digital PCR reaction liquid with the sample treatment liquid to obtain a digital PCR reaction mixed liquid; preferably, the volume ratio of the digital PCR reaction liquid to the sample treatment liquid is 6:14;

s3, adding the digital PCR reaction mixed solution and the droplet generation oil into a droplet generation card, placing the droplet generation card into a droplet generator to generate droplets, sealing a film, and then carrying out digital PCR amplification reaction;

s4, reading fluorescent signals: placing the amplified 96-well plate in a droplet reader, and directly reading and analyzing results by using software; and automatically calculating and obtaining the copy numbers of the mutation of the BTK C481S site, the PLCG2R655W site, the S707Y site, the BCL 2G 101V site and the F104L site in the ddPCR reaction system according to the Poisson distribution principle.

Optionally, the conditions of the digital PCR amplification reaction are: firstly, preserving heat for 9-11 min at 95 ℃; then preserving heat at 94 ℃ for 13-16 sec and 58-60 sec for 39-41 cycles; finally, preserving heat for 10min at 98 ℃, cooling to 4 ℃ and stopping the reaction; preferably, the temperature is first kept at 95 ℃ for 10min; then, the temperature is kept at 94 ℃ for 15sec and at 58 ℃ for 60sec, and 40 cycles are carried out in total; finally, preserving heat for 10min at 98 ℃, cooling to 4 ℃ and stopping the reaction; more preferably, the speed of temperature rise and fall is less than or equal to 2 ℃/s.

The invention relates to a primer and a probe for detecting mutation of chronic lymphocytic leukemia drug resistance related genes by adopting digital PCR, which are used for detecting mutation of BCL 2G 101V and F104L locus, PLCG2R655W locus, PLCG2S707Y locus and BTK C481S locus.

Optionally, the nucleotide sequence of the upstream primer for detecting BCL 2G 101V and F104L sites is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, the nucleotide sequence of the probe is shown as SEQ ID NO. 3-SEQ ID NO. 5, the nucleotide sequence of the upstream primer for detecting PLCG2R655W site is shown as SEQ ID NO. 6, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 7, the nucleotide sequence of the probe is shown as SEQ ID NO. 8-SEQ ID NO. 9, the nucleotide sequence of the upstream primer for detecting PLCG2S707Y site is shown as SEQ ID NO. 10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 11, the nucleotide sequence of the probe is shown as SEQ ID NO. 12-13, the nucleotide sequence of the upstream primer for detecting BTK C481 site is shown as SEQ ID NO. 14, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 15, and the nucleotide sequence of the probe is shown as SEQ ID NO. 16-17; preferably, the fluorescent groups of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 8, SEQ ID NO. 12 and SEQ ID NO. 16 are selected from FAM and the fluorescence quenching groups are selected from MGB; the fluorescent groups of SEQ ID NO. 4, SEQ ID NO. 9, SEQ ID NO. 13 and SEQ ID NO. 17 are selected from VIC, and the fluorescence quenching group is selected from MGB.

The invention has at least the following beneficial effects:

the kit adopts a digital PCR technology to quantitatively detect the mutation such as human BTK C481S and the like, and provides a reference for clinicians to monitor the curative effect of CLL patients and formulate individual treatment schemes.

Drawings

FIG. 1 is a schematic representation of the mutation site of BCL 2G 101V;

FIG. 2 is a schematic diagram of an interface during detection.

Detailed Description

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms also include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The embodiment of the invention designs the digital PCR detection of five loci of BCL 2G 101V, F104L, PLCG R655W, S707Y and BTK C481S through the comprehensive consideration of the data results.

The BCL2 inhibitor-vitamin Naekla has strong and lasting relieving effect on chronic lymphocytic leukemia patients. However, when BCL 2G 101V is mutated (NM-000633.2: c.302G > T, p. (Gly 101 Val)), resistance to Venezuela is caused in patients with chronic lymphocytic leukemia. Primary cells carrying the G101V mutation in the matrix have significantly increased resistance to valnecalar with higher concentrations than clinically attainable. Whereas in the absence of valnectar, the G101V mutation was as effective as the wild-type BCL2 in protecting the cell line from apoptosis. In the binding experiments, the ability of valicarb to compete with BIM for G101V binding was drastically reduced 180-fold compared to wild-type BCL2, which is likely to be that the larger isopropyl group in VAL occupies the groove for binding valicarb. In cell experiments, valnemulin can easily dissociate BAX and BAK from wild-type BCL2, but fails when these pro-apoptotic molecules bind to G101V. These all suggest that the BCL 2G 101V mutation results in CLL resistance to valnemulin. Therefore, the detection of the mutation condition of the locus can better provide guidance for monitoring the appearance of drug-resistant clones and subsequent clinical medication. A schematic representation of the mutation site of BCL 2G 101V is shown in FIG. 1.

BTK is a member of the Tec family of non-receptor protein tyrosine kinases, is a key kinase in the B cell antigen receptor (BCR) signaling pathway, and can regulate proliferation, differentiation and apoptosis of normal B cells, which fully demonstrates that BTK plays an irreplaceable role in the generation process of B lymphocytes, and is an ideal target for treating blood tumors. The chromosome of the human BTK gene encodes 659 amino acids. The BTK gene includes PH domain, TH domain, SH3 domain, SH2 domain and SH1 domain. Wherein the PH domain consists of about 120 amino acids and comprises the binding sites for the transcription factor BAP-135/TFH-I and the activity downregulating factors PIN1, IBTK, and is also responsible for mediating the actions of BTK and the 2 nd messenger phosphatidylinositol triphosphate (PIP 3). The TH domain consists of about 80 amino acid residues, consisting of two parts, the BTK protein motif (motif) and the proline-rich region. SH3 domains specifically recognize proline-rich fragments in TH domains, inducing intramolecular folding. The SH1 domain comprises an activating loop, an ATP binding site, a catalyst, and an allosteric inhibitory fragment. Activation (phosphorylation) of BTK occurs initially in the activation loop in the SH1 domain, and further activation occurs in SH2 and SH3 domains that contain the primary autophosphorylation sites. The function of the SH2 domain is to specifically recognize the phosphorylation state of tyrosine residues, thereby allowing proteins comprising the SH2 domain to localize to the phosphorylated tyrosine site of other proteins.

Downstream receptors for BTK include growth factors, B cell antigens, chemokines, and non-specific immune receptors, etc., and thus activation energy of BTK triggers such as: cell proliferation, survival, differentiation, angiogenesis, antigen expression, and cytokine synthesis. Whereas BTK activation focuses on migration of BTK to the cell membrane, when some receptors on the cell membrane receive stimulation by the corresponding ligand, the activated receptors recruit and phosphorylate intracellular signal transduction kinase PI3K, which subsequently converts PIP2 on the membrane to the 2 nd messenger PIP3.PIP3 binds to the PH domain of BTK, which is then recruited to the cell membrane, and the Tyr-551 residue is subsequently phosphorylated by Syk and Lyn kinases. BTK is then subjected to autophosphorylation at Tyr-223 residue to thereby exert physiological activity.

The ibutinib can be selectively combined with a target protein BTK active site Cys481 to form a covalent bond, so that the self phosphorylation of BTK is inhibited, the effect of inhibiting a BTK signal path is achieved, and the method has the characteristics of high efficiency and irreversibility. Resistance caused by mutation blood RNA analysis of CLL patients (840 mg/d) with resistance to ibrutinib found mutant C481S of BTK, and as can be seen from the docking (docking) result of BTK structure and ibutinib, cysteine at position 481 plays an important role in drug binding, so that mutation from C to S greatly reduces the stability IC50 of the binding. In addition, mutants of the downstream gene PLCG2 of BTK, R665W, L845F and S707Y, were found in CLL patients, which all resulted in ibutinib resistance. But still present life threatening adverse events such as bleeding, infection, bone marrow suppression, nephrotoxicity, secondary tumors, etc. Therefore, the detection of the sites has important clinical medication guidance significance.

The embodiment of the invention relates to a digital PCR kit for detecting chronic lymphocytic leukemia drug resistance related gene mutation, which comprises a nucleic acid amplification reagent, wherein the nucleic acid amplification reagent comprises a primer and a probe for detecting mutation of a BTK C481S locus, a PLCG2R655W locus, a PLCG2S707Y locus, a BCL 2G 101V locus and an F104L locus. Specifically, the nucleotide sequences of the primers and probes are shown in Table 1:

TABLE 1

Primer probe name	Sequence numbering	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

Alternatively, in the nucleic acid amplification reagent, the concentration of the upstream primer is 0.17 to 0.5. Mu. Mol/L, preferably 0.35. Mu. Mol/L; the concentration of the downstream primer is 0.17 to 0.5. Mu. Mol/L, preferably 0.35. Mu. Mol/L; the concentration of the probe is 0.12 to 0.24. Mu. Mol/L, preferably 0.18. Mu. Mol/L.

Optionally, the kit also contains a negative control and a positive control, wherein the negative control is process water. The positive control was a standard containing the fragmented plasmids shown in table 2:

TABLE 2

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Optionally, the sample to which the kit is adapted is selected from a plasma sample or a paraffin sample.

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

s1, sample treatment: extracting a sample by using a commercial extraction kit to obtain a sample treatment fluid;

s2, preparing a digital PCR reaction mixed solution: mixing the digital PCR reaction liquid with the sample treatment liquid to obtain a digital PCR reaction mixed liquid; preferably, the volume ratio of the digital PCR reaction solution to the sample processing solution is 6:14;

s3, adding the digital PCR reaction mixed solution and the droplet generation oil into a droplet generation card, placing the droplet generation card into a droplet generator to generate droplets, sealing a film, and then carrying out digital PCR amplification reaction;

s4, reading fluorescent signals: placing the amplified 96-well plate in a droplet reader, and directly reading and analyzing results by using software; and automatically calculating and obtaining the copy numbers of the mutation of the BTK C481S site, the PLCG2R655W site, the S707Y site, the BCL 2G 101V site and the F104L site in the ddPCR reaction system according to the Poisson distribution principle.

Wherein, the conditions of the digital PCR amplification reaction are as follows: firstly, preserving heat for 9-11 min at 95 ℃; then preserving heat at 94 ℃ for 13-16 sec and 58-60 sec for 39-41 cycles; finally, preserving heat for 10min at 98 ℃, cooling to 4 ℃ and stopping the reaction; preferably, the temperature is first kept at 95 ℃ for 10min; then, the temperature is kept at 94 ℃ for 15sec and at 58 ℃ for 60sec, and 40 cycles are carried out in total; finally, preserving heat for 10min at 98 ℃, cooling to 4 ℃ and stopping the reaction; more preferably, the rate of temperature rise and fall is 2 ℃/sec or less.

The embodiment of the invention also relates to a primer and a probe for detecting mutation of chronic lymphocytic leukemia drug resistance related genes by adopting digital PCR, wherein the primer and the probe are used for detecting mutation of BCL 2G 101V and F104L sites, PLCG2R655W sites, PLCG2S707Y sites and BTK C481S sites, and the nucleotide sequence is specifically shown in the table 1.

Example 1

The composition of the kit is shown in table 3:

TABLE 3 Table 3

The method is applicable to the instrument: QX200 Droplet Digital PCR system (BioRad, usa).

Sample requirements: the method is suitable for detecting human genome free DNA (cfDNA) extracted from plasma samples or human genome DNA extracted from paraffin samples.

Positive judgment value or reference interval: 2% of the BCL2, PLCG2 and BTK gene mutations were detected for 10ng of genomic DNA.

The inspection method comprises the following steps:

1. sample processing:

nucleic acid extraction (DNA extraction using commercial kits is recommended) is performed on its own as a template for the PCR reaction. The extracted nucleic acid is recommended to be detected immediately, otherwise, stored below-20 ℃.

2. Preparation and sample addition of amplification reagents:

a. the corresponding reaction solution was taken out of the kit, melted and mixed at room temperature, and centrifuged at 2000rpm for 10 seconds, and each of the PCR premix solutions tested was prepared as follows: mu.L of the mixed solution ddPCR+10mu.L of 2 XddPCR MIX3, and the prepared PCR premix was dispensed into each PCR tube in an amount of 14. Mu.L per tube.

b. After the genomic DNA template concentration measurement (qubit measurement) was completed, the sample was diluted to 2 ng/. Mu.L with water, and 6. Mu.L of the template was added to a PCR tube containing the above PCR premix.

c. The total volume of each reaction system was 20. Mu.L.

d. And (3) tightly covering the PCR tube cover, shaking and uniformly mixing for more than 20s, and performing micro-droplet preparation after instantaneous centrifugation.

3. Preparing microdroplets:

a. 8 20. Mu.L of the reaction system was added to the middle row of 8 wells of DG8 cartridge.

Note that: 1) Samples must be added first in row 1 in the middle of DG8 cartridge (if there are less than 8 samples, then 20 μ L BX ddPCR Buffer Control should be added to the wells; the pipette was set to 20 μl before loading and all liquid in the tube was aspirated during sampling).

2) When the sample is added, bubbles are avoided, and if bubbles are visible to naked eyes, the bubbles can be punctured by a clean gun head.

b. 70 mu L Droplet Generation oil is added to each of the 8 holes in the bottom row of DG8 cartridge, a rubber pad (gasset) is covered, the DG8 cartridge is gently and smoothly placed in a droplet generator to start generating droplets, and the state of an indicator lamp on the generator is noted to finish the process within 2 minutes.

c. Droplets are generated in the uppermost row of wells of the cartridge and the generated droplets (approximately 35-45 μl) are transferred into a 96-well plate.

5. Sealing film

After the microdroplet is transferred into a 96-well plate, the microdroplet is sealed by a preheated PX1 heat sealing instrument, and the recommended operation procedure is as follows: 180 ℃ and 5s, and secondary film sealing in the direction of inversion is not needed; after sealing the membrane, the PCR reaction should be performed within 30min or within 4 hours in a refrigerator at 4 ℃.

PCR amplification:

95 ℃ for 10min; (94 ℃,15sec;58 ℃,60 sec) 40 cycles; 98 ℃ for 10min; the temperature is 4 ℃ for 5min, the reaction system is set to 40 mu L, and the temperature rising and falling speed is less than or equal to 2 ℃/s.

7. Droplet reading:

a. firstly, a computer is started, then a Droplet Reader power supply is started, and preheating is needed for at least 30min before use;

b. the 96-well plate with the PCR completed before was placed in a plate holder and smoothly placed in a droplet reader.

c. And opening QuantaSoft software, carrying out Setup on sample information in the 96-well plate, and carrying out Run after completion.

Note that: 1) Supermix selection "ddPCR Supermix for probes"

2) Dye Set selection of "FAM/VIC"

8. Analysis of results:

after the detection is completed, click "2D ampliude" to view channel 1 and channel 2 cluster map. This figure allows manual or automatic adjustment of the threshold for positive and negative droplet assignment for each detection channel.

Clicking on "Auto Analyze" resets the threshold;

manually specifying a threshold:

using a threshold reticle to specify a classification region (selectable only in heat map mode) for the entire dot map;

the dot plot regions are classified using an elliptical, rectangular, or lasso threshold adjustment tool: clicking on the corresponding tool button then clicks on the region type at "Working cluster selector" and the tool is used to select the corresponding region. The interface schematic diagram is shown in fig. 2:

note that: the fluorescent threshold line was set with reference to negative and positive controls: in 2D Amplitude, the fluorescence threshold line should be positioned such that the cluster of droplets of the negative control is within the "ch1-ch2-" interval, and the 4 clusters of droplets of the positive control are within four intervals, respectively.

Interpretation of test results:

1. validity judgment:

negative control validity judgment: the points of the "ch1+" region are < 4 points and the points falling on the "ch2+" region are < 4 points.

Positive control validity determination: the number of points falling in the ch1+ch2- "region is more than or equal to 4, and the mutation ratio is more than or equal to 2 per mill.

Determination of invalid results: the total droplet number of each reaction tube is more than or equal to 8000, and if the total droplet number is less than 8000, the droplet generation of the reaction hole is not ideal, and the droplet generation needs to be carried out again.

2. And (3) result judgment:

2.1 qualitative determination

(1) If the number of points of the sample falling on the 'ch1+ch2-' region is more than or equal to 4 and the mutation proportion is more than or equal to 2 per mill, the mutation of the BCL2 and the BTK sites is judged.

(2) If not (1),

if the sample DNA is more than or equal to 50 copies

If the point of the sample falling on the 'ch1+ch2-' region is <3 or the mutation proportion is < 2%o, judging that the BCL2 and BTK sites have no mutation or the mutation is lower than the lowest detection limit value.

If the number of points of the sample falling on the 'ch1+ch2-' region is 3 and the mutation ratio is more than or equal to 2 per mill, judging that the mutation of the BCL2 and BTK sites is suspected to be positive, and detecting again. And (3) according to the re-detection result, if the points of the sample falling on the 'ch1+ch2-' region are more than or equal to 4 and the mutation proportion is more than or equal to 2 per mill, judging the mutation of the BCL2 and the BTK sites. In contrast, it was determined that BCL2 and BTK sites were free of mutation or mutation below the minimum detection limit.

If the sample DNA is less than 50 copies, it is suggested that the added DNA is of poor quality or contains PCR inhibitors, and it is necessary to re-extract the DNA or re-sample it before doing so. After re-detection, the sample DNA is <50 copies and does not meet (1), and the DNA quality is judged to be unsatisfactory. Otherwise, the corresponding judgment is carried out according to the conditions.

2.2 quantitative determination

If the sample BTK site is mutated, the mutation percentage can be calculated according to the formula Ch 1/(Ch1+Ch2).

Experimental example 1 primer screening test

Preparing primer probe reaction liquid according to a general principle, and preparing 20 human hand samples on each site to be detected for pre-experiment.

1. The components in Table 4 were formulated according to the BCL 2G 101V-F104L sites. Then, the primers F1/R2, F2/R1 and F2/R2 are subjected to experiments to replace F1/R1 in Table 3, and droplet reaction experiments are performed by using standards with known mutation frequencies obtained through other detection methods such as NGS sequencing to compare the quality of each primer matching, so that an optimal matching scheme is selected.

Table 4: BCL2 reaction solution

Name of the name	Concentration of	1 person	20 human parts
				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 numbering	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 assay are shown in tables 6-9:

table 6: results of the BCL2F2/R2 primer screening test

Table 7: results of the BCL2F 1/R1 primer screening test

Table 8: results of the BCL2F 2/R1 primer screening test

Table 9: results of the BCL2F 1/R2 primer screening test

As can be seen from the results of the droplet readings shown in tables 6 to 9, the reaction solution mixed with the BCL2F2/R2 primer has the highest accuracy in detecting mutation rate of the positive standard, has better detection specificity in the negative sample, and does not find nonspecific mutation points (ch1+ch2-points).

According to the same method, 20 human sample reagents are prepared according to the conventional reaction liquid ratios shown in tables 10, 11 and 12 for the detection sites PLCG2R655W, PLG S707Y and BTK C481S respectively, then F1/R2, F2/R1 and F2/R2 are subjected to experiments for replacing F1/R1 in the tables, the quality of the primers is compared, and the optimal matching is selected according to the same standard.

Table 10: PLCG2R655W reaction liquid

Name of the name	Concentration of	1 person	20 human parts
				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: PLCG2S707Y reaction liquid

Name of the name	Concentration of	1 person	20 human parts
				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 of the name	Concentration of	1 person	20 human parts
				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 PLCG2R655W primer screening test are shown in tables 13 to 16:

table 13: PLCG2R 655W-F1/PLCG 2R 655W-R2 primer screening test results

Table 14: PLCG2R 655W-F2/PLCG 2R 655W-R2 primer screening test results

Table 15: PLCG2R 655W-F2/PLCG 2R 655W-R1 primer screening test results

Table 16: PLCG2R 655W-F1/PLCG 2R 655W-R1 primer screening test results

The results of PLCG2S707Y primer screening test are shown in tables 17 to 20:

table 17: PLCG2S 707Y-F2PLCG 2S 707Y-R2 primer screening test results

Table 18: PLCG2S 707Y-F1/PLCG 2S 707Y-R1 primer screening test results

Table 19: PLCG2S 707Y-F2/PLCG 2S 707Y-R1 primer screening test results

TABLE 20 PLCG2S 707Y-F1/PLCG 2S 707Y-R2 primer screening test results

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

table 21: results of the BTK C481S-F1/BTK C481S-R1 primer screening test

Table 22: results of the BTK C481S-F2/BTK C481S-R2 primer screening assay

Table 23: results of the BTK C481S-F2/BTK C481S-R1 primer screening test

Table 24: results of the BTK C481S-F1/BTK C481S-R2 primer screening assay

As can be seen from the observation of the results of the droplet readings (tables 13 to 24), the reaction mixtures of the PLCG2R655W F1/R2, PLCG2S707Y F/R2, BTK C481S-F1/R1 primers gave the highest accuracy in the detection of the mutation rate of the positive standard, and had better detection specificity in the negative samples, while no non-specific mutation point (ch1+ch2-point) was found, and as a formulation for the selection of the pre-experiments.

The primer concentration and the probe concentration are interrelated in the whole PCR reaction, so that the two factors are selected to carry out cross experiments on plasmid references diluted to 10% to optimize the PCR reaction system.

Table 25: results of BTK C481S primer concentration and probe concentration experiments

The experimental results show that the combination with the primer concentration of 0.35 mu M and the probe concentration of 0.18 mu M is the most effective detection collocation on the premise of saving raw materials and ensuring the detection accuracy. Indicating that the mutation rate detected without affecting the copy number is consistent with the reference. Corresponding experiments were also performed on the other four sites in the same manner, and the results were consistent. Tests of sensitivity, accuracy, stability, etc. were performed according to this formulation.

Experimental example 2 limit of detection experiment

And determining the detection sensitivity, and carrying out gradient dilution after selecting a plasmid reference containing mutation to be mixed with a wild plasmid according to a certain proportion so as 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: test results of BTK C481S detection limit setting

As can be seen from the observation of the droplet reading results, the BTK C481S reaction solution can still be detected for the sample with the mutation rate of 0.1% or more. However, in the range of 0.1% -0.2%, a large deviation between the quantitative result and the expected mutation rate occurs; when the mutation rate is reduced to less than 0.1%, detection results are easy to miss detection (such as negative detection). Therefore, the qualitative detection range is more than or equal to 0.2 percent. The same detection sensitivity determination test is carried out on other PLCG2R655W, S707Y, BCL G101V and G104L loci by the same method, and the mutation detection range of all loci is determined to be more than or equal to 0.2%.

Detection results of detection limit: according to the experimental results, the detection is repeated 20 times by adopting the BTK C481S mutation proportion as a determined detection limit, and the detection results are as follows:

table 27: BTK C481S detection limit results

From the above results, it can be seen that the experiment was performed using the BTK C481S detection limit reference (mutation rate=0.2%) with a 95% confidence interval, and the positive coincidence rate of the experimental results repeated 20 times was 100%, which showed effective detection sensitivity. The same determination experiment was performed on other PLCG2R655W, S707Y, BCL G101V and G104L sites by the same method, and the positive coincidence rate of all sites is 100%.

Experimental example 3 accuracy experiment and results

3 cases of clinical DNA samples of chronic lymphocyte leukemia patients with positive BTK C481S are selected for detection experiments and compared with mutation rates detected as positive by a high-throughput sequencing method.

Table 28: results of BTK C481S clinical sample accuracy experiments

From the results, samples 9047, 4915, and 4672 showed BTK C481S positivity with mutation rates of 24.1%, 44.7%, and 43.3%, respectively, consistent with the frequencies calculated by NGS detection. In order to further confirm the correctness of the mutation ratio, two identical repeated experiments are carried out, and the result is consistent with the first result, so that the possibility that the result is inaccurate due to operation errors is eliminated.

Similarly, a total of 5 samples positive to the clinical tests PLCG2R655W, S707Y, BCL G101V and G104L were selected for testing (see table 29), and the results were consistent with the frequencies calculated by NGS testing, and each set of experiments was repeated three times to ensure the effect of experimental errors on the results.

Table 29: clinical sample accuracy test results

Experimental example 4 specificity experiments and results

Two examples of clinical samples of which the BTK C481S, BCL G101V, BCL G104L, PLCG2R655W and PLCG2S707Y are negative through high-throughput sequencing detection and DNA samples of the diffuse tissue lymphoma cell line SU-DHL2 are selected to be detected by using the kit of the 5 sites respectively. The results of the BTK C481S kit experiments are shown in table 30.

Table 30: BTK C481S kit specificity results

The above results show that: the detection result of the BTK C481S kit is negative and the result of the cell line sample is negative, which indicates that the kit has good specificity. The same determination experiment was performed on other PLCG2R655W, S707Y, BCL G101V and G104L sites by the same method, and the detection results of all the sites are negative.

While the preferred embodiment has been described, it is not intended to limit the scope of the claims, and any person skilled in the art can make several possible variations and modifications without departing from the spirit of the invention, so the scope of the invention shall be defined by the claims.

Sequence listing

<110> Jiangsu province people hospital

SUZHOU YUNTAI BIOLOGICAL PHARMACEUTICAL Co.,Ltd.

<120> digital PCR kit, primer and probe for detecting chronic lymphocytic leukemia drug resistance related gene mutation

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Claims (6)

1. A digital PCR kit for detecting chronic lymphocytic leukemia drug resistance related gene mutation is characterized in that the kit contains a nucleic acid amplification reagent, and the nucleic acid amplification reagent contains a primer and a probe for detecting mutation of BCL 2G 101V and F104L sites, PLCG2R655W sites, PLCG2S707Y sites and BTK C481S sites;

the nucleotide sequence of the upstream primer for detecting the BCL 2G 101V and F104L sites is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, the nucleotide sequence of the probe is shown as SEQ ID NO. 3-SEQ ID NO. 5,

the nucleotide sequence of the upstream primer for detecting the PLCG2R655W locus is shown as SEQ ID NO. 6, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 7, the nucleotide sequence of the probe is shown as SEQ ID NO. 8-SEQ ID NO. 9,

the nucleotide sequence of the upstream primer for detecting the PLCG2S707Y locus is shown as SEQ ID NO. 10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 11, the nucleotide sequence of the probe is shown as SEQ ID NO. 12-SEQ ID NO. 13,

the nucleotide sequence of the upstream primer for detecting the BTK C481S locus is shown as SEQ ID NO. 14, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 15, and the nucleotide sequence of the probe is shown as SEQ ID NO. 16-SEQ ID NO. 17.

2. The digital PCR kit of claim 1, wherein the probe has a fluorescent group attached to the 5 'end and a fluorescence quenching group attached to the 3' end;

the fluorescent groups of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 8, SEQ ID NO. 12 and SEQ ID NO. 16 are FAM, and the fluorescence quenching group is MGB; the fluorescent groups of SEQ ID NO. 4, SEQ ID NO. 9, SEQ ID NO. 13 and SEQ ID NO. 17 are VIC and the fluorescence quenching group is MGB.

3. The digital PCR kit according to claim 1, wherein the concentration of the upstream primer is 0.35 μmol/L; the concentration of the downstream primer is 0.35 mu mol/L; the concentration of the probe was 0.18. Mu. Mol/L.

4. The digital PCR kit according to claim 1, further comprising a negative control and a positive control, wherein the negative control is process water and the positive control is a fragmented plasmid standard containing SEQ ID NO. 18-SEQ ID NO. 26.

5. The digital PCR kit according to claim 1, wherein the sample to which the kit is adapted is selected from a plasma sample or a paraffin sample.

6. The primer and the probe for detecting the mutation of the chronic lymphocytic leukemia drug resistance related gene by adopting digital PCR are characterized by being used for detecting the mutation of BCL 2G 101V and F104L sites, PLCG2R655W sites, PLCG2S707Y sites and BTK C481S sites;

the nucleotide sequence of the upstream primer for detecting the BCL 2G 101V and F104L sites is shown as SEQ ID NO. 1, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 2, the nucleotide sequence of the probe is shown as SEQ ID NO. 3-SEQ ID NO. 5,

the nucleotide sequence of the upstream primer for detecting the PLCG2R655W locus is shown as SEQ ID NO. 6, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 7, the nucleotide sequence of the probe is shown as SEQ ID NO. 8-SEQ ID NO. 9,

the nucleotide sequence of the upstream primer for detecting the PLCG2S707Y locus is shown as SEQ ID NO. 10, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 11, the nucleotide sequence of the probe is shown as SEQ ID NO. 12-SEQ ID NO. 13,

the nucleotide sequence of the upstream primer for detecting the BTK C481S locus is shown as SEQ ID NO. 14, the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 15, and the nucleotide sequence of the probe is shown as SEQ ID NO. 16-SEQ ID NO. 17;

the fluorescent groups of SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 8, SEQ ID NO. 12 and SEQ ID NO. 16 are FAM, and the fluorescence quenching group is MGB; the fluorescent groups of SEQ ID NO. 4, SEQ ID NO. 9, SEQ ID NO. 13 and SEQ ID NO. 17 are VIC and the fluorescence quenching group is MGB.

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