CN111690749A - Sample processing method and detection kit for detecting MYD88 gene L265P mutation by ddPCR (polymerase chain reaction) - Google Patents
Sample processing method and detection kit for detecting MYD88 gene L265P mutation by ddPCR (polymerase chain reaction) Download PDFInfo
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Abstract
The invention provides a sample processing method for detecting MYD88 gene L265P mutation by ddPCR, which comprises the following steps: s1, extracting sample genome DNA; s2, preparing micro-reaction liquid drops: (1) preparing a micro-reaction solution containing genomic DNA and the following primers and probes: an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2, a MYD88 gene L265P mutant detection probe shown as SEQ ID NO.3, and a MYD88 gene L265P wild type detection probe shown as SEQ ID NO. 4; (2) preparing a micro-reaction liquid drop by using the micro-reaction liquid obtained in the step (1). By using the optimized primers and probes, the sample processing method can effectively reduce non-specific reaction in the detection process, improve the detection sensitivity, specificity and accuracy, has low detection cost, and is favorable for large-scale popularization of MYD88 gene L265P mutation detection.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a sample processing method and a detection kit for detecting MYD88 gene L265P mutation by ddPCR.
Background
In recent years, several studies have demonstrated that the mutation rate at the myeloid differentiation factor 88 (MyD 88) L265P site in lymphoplasmacytic lymphoma/Waldenstrom's Macroglobulinemia, LPL/WM patients is over 90%, while it is very rare in other types of indolent B-cell lymphoma. The patients with MYD88 mutation are more sensitive to BTK inhibitor, and clinical studies show that 80 cases of recurrent or refractory Diffuse Large B Cell Lymphoma (DLBCL) and the BTK inhibitor irrutinib can achieve complete or partial remission of 37% of nonGCB-DLBCL, especially patients with BCR and MYD88 mutation at the same time. In patients with DLBCL of the central nervous system, ibrutinib single drug shows inhibition effect on tumors in 94 percent of cases, and complete remission can be achieved in 86 percent of patients with combined chemotherapy, and at least part of the beneficial patients can be explained by MYD88 mutation. Therefore, the detection of the mutation of the MYD88 gene L265P has important significance for auxiliary diagnosis and concomitant diagnosis in clinical decision.
Currently, researchers mostly use real-time fluorescent quantitative PCR (real-time PCR) or generation sequencing (sanger sequencing) for qualitative detection of MYD88 gene L265P mutation. Real-time fluorescent quantitative PCR (real-time PCR) or first-generation sequencing (Sanger sequencing) has the disadvantages of low sensitivity and/or only qualitative detection, and the like, and cannot meet the requirement of monitoring the curative effect in treatment in clinical practice. The traditional real-time quantitative PCR depends on a reference substance or a standard substance, and the allele frequency of the genetic variation of a detected sample cannot be accurately calculated. The traditional real-time quantitative PCR positive and negative judgment needs to depend on experience, and the background noise and threshold value need to be adjusted according to external data such as standard substances to obtain the Ct value. For example, a cycle threshold (Ct value) <32 of a mutant well response curve is defined as the detection of a mutation; ct of more than or equal to 32 and less than 35 is defined as the recommendation for retest; ct.gtoreq.35 is defined as being lower than the lower detection limit, and is defined as being negative to MyD88 gene L265P mutation. In practice, inhibitors or environmental factors in nucleic acid samples can interfere with the detection greatly, reducing its accuracy. And the NGS sequencing method has higher requirements on instruments and equipment, and cannot be popularized and used in certain areas.
The Droplet digital PCR (ddPCR) is used as a new PCR method, has higher detection sensitivity and specificity, and is convenient to popularize in certain areas which cannot carry out the NGS sequencing method and need to carry out curative effect monitoring due to the characteristic of accurate quantitative detection. ddPCR disperses a large number of diluted nucleic acid solutions into microreactors or microdrops of a chip, with the number of nucleic acid templates per reactor being less than or equal to 1. Thus, after PCR cycling, a reactor with a nucleic acid molecule template will give a fluorescent signal, and a reactor without a template will have no fluorescent signal. Therefore, whether a nucleic acid template exists in each micro-reaction can be determined, uncertainty caused by a Ct value is eliminated, and the detection accuracy is greatly improved. The ddPCR is carried out in a one-tube system for detecting the wild allele and the variant allele, and can calculate respective copy number according to Poisson distribution so as to obtain allele frequency, directly reflect the content of tumor cells in a detected sample and provide the most powerful real-time data for curative effect evaluation.
The specificity and sensitivity of ddPCR are the key points of digital PCR detection, but the chromosome structure, primer design are not good, and the like, so that nonspecific amplification is easily generated, and the nonspecific amplification is often caused by mismatching of primers/probes of variant alleles and wild templates, and the sensitivity and accuracy of detection are reduced.
Disclosure of Invention
Based on the above, the invention aims to provide a sample processing method and a detection kit for detecting MYD88 gene L265P mutation by ddPCR, wherein the sample processing method uses optimally designed primers and probes to prepare micro-reaction liquid drops, so that non-specific amplification can be effectively reduced, false positive results are avoided, and the sensitivity, specificity and accuracy of detection are improved.
The specific technical scheme is as follows:
a sample processing method for detecting MYD88 gene L265P mutation by ddPCR comprises the following steps:
s1, extracting sample genome DNA;
s2, preparing micro-reaction liquid drops: (1) preparing a micro-reaction solution containing genomic DNA and the following primers and probes: an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2, a MYD88 gene L265P mutant detection probe shown as SEQ ID NO.3, and a MYD88 gene L265P wild type detection probe shown as SEQ ID NO. 4; (2) preparing a micro-reaction liquid drop by using the micro-reaction liquid obtained in the step (1).
In some embodiments, step S1 further comprises digesting the extracted genomic DNA with the restriction enzyme HindIII. The restriction enzyme HindIII has a specific cleavage site of 5' -AAGCTT-3', and cleavage occurs between two A's (i.e., 5' -A | AGCTT-3 '). The inventor finds that when the restriction endonuclease HindIII is used for enzyme digestion of the genome DNA, the target DNA fragment can be guaranteed not to be subjected to enzyme digestion, the fragment length of the genome DNA template subjected to enzyme digestion can be in the range capable of effectively improving microdroplet preparation efficiency, and the DNA denaturation efficiency in the subsequent ddPCR process can be improved.
In some embodiments, the concentration of the restriction enzyme HindIII in the enzyme cutting system is 1000-2000 units/ml, and the concentration of the genome DNA is 10-16.5 ng/ul.
In some embodiments, the concentration of the restriction enzyme HindIII in the enzyme cutting system is 1500-2000 units/ml.
In some embodiments, the enzymatic reaction conditions are: incubating at 37 +/-1 ℃ for 5-10 min.
In some embodiments, the molar ratio of the upstream primer, the downstream primer, the mutant detection probe for the MYD88 gene L265P and the wild detection probe for the MYD88 gene L265P in the micro-reaction solution in the step S2 is (1-2): (1-2): (0.5-1): (0.5 to 1).
In some embodiments, the molar ratio of the forward primer to the backward primer in the micro-reaction solution of step S2 is 1: 1; the molar ratio of the mutant detection probe aiming at the MYD88 gene L265P to the wild detection probe aiming at the MYD88 gene L265P is 1: 1.
in some embodiments, the sample of step S1 is peripheral blood or bone marrow.
In some of these embodiments, the sample processing method for ddPCR detection of a mutation at MYD88 gene L265P further comprises the steps of: s3, performing ddPCR amplification to obtain a treated sample, wherein the ddPCR amplification conditions are as follows: 10min at 95 ℃; 35-40 cycles: 30s at 94 ℃ and 60s at 55 ℃; 10min at 98 ℃.
In some of these embodiments, the ddPCR amplification conditions are as follows: 10min at 95 ℃; 40 cycles: 30s at 94 ℃ and 60s at 55 ℃; 10min at 98 ℃.
In some of these embodiments, the temperature increase and/or decrease rate is set to 2. + -. 0.5 ℃/sec for ddPCR amplification.
The invention also provides a kit for detecting MYD88 gene L265P mutation by ddPCR, which comprises the following primers and probes: an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2, a MYD88 gene L265P mutant detection probe shown as SEQ ID NO.3, and a MYD88 gene L265P wild type detection probe shown as SEQ ID NO. 4.
In some of these embodiments, the kit further comprises a restriction enzyme HindIII.
In some of these embodiments, the kit further comprises a genomic DNA extraction reagent.
Compared with the prior art, the invention has the following beneficial effects:
the primers and the probes used in the preparation of the micro-reaction liquid drop by the sample processing method are obtained by elaborate design of the inventor, and the difference between GC% and delta H (kcal/mol) of the two detection probes is adjusted by controlling thermodynamic parameters of the wild-type detection probe aiming at MYD88 gene L265P and the mutant-type detection probe aiming at MYD88 gene L265P, so that the wild-type detection primer and the probes can be preferentially paired in competition with a wild-type template, and the mutant-type detection primer and the probes are prevented from being mistakenly paired with the wild-type template, thereby effectively reducing non-specific amplification and avoiding false positive results. In addition, the inventor also finds that the genome DNA is firstly subjected to enzyme digestion by using restriction enzyme HindIII, and then the micro-reaction liquid drop is prepared by using the enzyme-digested genome DNA and the primers and the probes, so that the target DNA fragment can be ensured not to be subjected to enzyme digestion, the fragment length of the enzyme-digested genome DNA template can be in a range capable of effectively improving the microdroplet preparation efficiency, the microdroplet generation is prevented from being blocked by the complex structure of the genome chromosome, the DNA denaturation efficiency in the subsequent ddPCR process can be improved, and the fluorescence intensity of detection is improved.
According to the invention, by optimizing the primers and probes used in the sample processing method and performing enzyme digestion treatment on the genome DNA by using the restriction enzyme HindIII, nonspecific reaction in the detection process can be effectively reduced, and the microdroplet preparation efficiency is improved, so that the detection sensitivity, specificity and accuracy are improved, the detection cost is reduced, and the large-scale popularization of MYD88 gene L265P mutation detection is facilitated.
Drawings
FIG. 1 is a microdroplet signal classification diagram of the MYD88 gene L265P mutation assay of the invention described in example 2; wherein, quadrant 1 represents Ch1+Ch2-Type microdroplets containing only mutant templates; quadrant 2 represents Ch1+Ch2+A type microdroplet containing both wild type and mutant templates; quadrant 3 represents Ch1-Ch2-Microdroplets, without any template; quadrant 4 represents Ch1-Ch2+Type microdroplets, containing only wild type template.
FIG. 2 is a graph of a linear regression analysis of the MYD88 gene L265P mutation assay of the invention described in example 2.
FIG. 3 is a graph showing the number of micro-reaction droplets prepared by the sample treatment method described in example 1 and control 1 in example 5.
Detailed Description
The experimental procedures of the present invention, without specifying the specific conditions in the following examples, are generally carried out according to conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.
The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.
Example 1
The embodiment provides a sample processing method for detecting MYD88 gene L265P mutation by ddPCR, which comprises the following steps:
s1, extracting the genome DNA of the peripheral blood or bone marrow sample and carrying out enzyme digestion:
(1) extracting and purifying the genomic DNA of a peripheral Blood or bone marrow sample by using a QIAamp DNA Blood Mini Kit, and strictly performing the operation steps according to the instruction; detecting the DNA concentration by using a Nanodrop system, and diluting the DNA concentration to be within the range of 20-33 ng/ul;
(2) the purified genome DNA was digested with the restriction enzyme HindIII with a specific cleavage site of 5'-A | AGCTT-3': as per NEBuffer 2.1: hind III enzyme: h2The volume ratio of O is 1: 1: 3 preparing a HindIII premix (2x), and taking 2ul HindIAnd (3) uniformly mixing the premixed solution (2x) II and 2ul of diluted DNA solution, wherein the final concentration of HindIII enzyme in the enzyme digestion system is 2000units/ml, and then incubating the enzyme digestion system at 37 ℃ for 5min for enzyme digestion reaction.
S2, preparing micro-reaction liquid drops:
(1) preparing a micro-reaction solution containing the genome DNA after enzyme digestion and the following primers and probes: an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2, a MYD88 gene L265P mutant detection probe (MYD88-L265P-probe) shown as SEQ ID NO.3, and a MYD88 gene L265P wild type detection probe (MYD88-wt-probe) shown as SEQ ID NO. 4. The 5 'end of the MYD88-L265P-probe is modified with a fluorescent group (FAM is preferred in the embodiment), and the 3' end of the MYD88-L265P-probe is modified with a quenching group (HQ 1 is preferred in the embodiment); the 5 'end of the MYD88-wt-probe is modified with a fluorescent group (HEX is preferred in the embodiment) different from the MYD88-L265P-probe, and the 3' end of the MYD88-wt-probe is modified with a quenching group (HQ 1 is preferred in the embodiment). The specific sequence information of the primers and probes are shown in Table 1:
TABLE 1 specific sequence information for primers and probes
The primers and the probe sets are synthesized into nucleic acid sequences by commercial companies, and then the primers and the probes are mixed to make the final concentrations of the primers and the probes in the mixed solution respectively 10pmol/ul and 5pmol/ul, so that MYD88-L265 protection Assay is obtained and used for preparing micro-reaction droplets.
Preparing a micro-reaction solution according to the formula shown in Table 2:
TABLE 2 micro-reaction liquid formulation
| Component name | Dosage of |
| 2x digital PCR supermix for probes(No dUTP) | 10ul |
| MYD88-L265P Mutation Detection Assay | 1ul |
| Digested genomic DNA | 4ul |
| H2O | 5uL |
(2) Preparing a micro-reaction liquid drop by using the micro-reaction liquid obtained in the step (1):
step.1, placing a new DG8 cartridge in the microdroplet generating card device;
step.2, adding 20ul of the micro-reaction solution into the middle row of holes of the DG8 cartridge;
step.3, adding 70ul of microdroplet generating oil into the bottom row of holes of DG8 cartridge, and also not having empty holes;
step.4, covering a rubber mat (gasket) and paying attention to the fact that small holes at two sides are firmly hooked;
step.5, the droplet generation card device is gently and stably placed in a droplet generation instrument, and the generation of droplets is started;
step.6, generating droplets in the uppermost row of pores of the cartridge, carefully and slowly sucking 40ul of droplet reaction liquid by using an 8-channel P-50 row gun of Rainin and a 200ul gun head, and transferring oil droplets into a 96-pore plate;
step.7 the film was sealed using a preheated PX1 heat sealer with the following procedure: 180 ℃ for 10 s.
S3, performing ddPCR amplification to obtain a treated sample:
the amplification conditions were as follows: 10min at 95 ℃; 40 cycles: 30s at 94 ℃ and 60s at 55 ℃; 10min at 98 ℃; the temperature rise and fall rates were set at 2 deg.c/sec.
Example 2
This example provides a method for detecting mutation of L265P in MYD88 gene by ddPCR, which includes the following steps:
s1, extracting the genome DNA of the peripheral blood or bone marrow sample and carrying out enzyme digestion: the same as example 1;
s2, preparing micro-reaction liquid drops: the same as example 1;
s3, performing ddPCR amplification to obtain a treated sample: the same as example 1;
s4, fluorescence detection of the micro-reaction liquid drop: and opening QuantaSoft software, editing sample information including experiment names, experiment types (ABS, CNV and RED), assay, fluorophore information carried by the probes and the like in a Setup menu, running a Run program after the experiment is finished, and detecting FAM and HEX fluorescence signals generated by the target probes.
Signal type of divided droplet: the Ch1 channel detects FAM fluorophores (i.e., fluorophores for MYD88-L265P-probe), and the Ch2 channel detects HEX fluorophores (i.e., fluorophores for MYD 88-wt-probe).
Droplet signal classification: as shown in FIG. 1, quadrant 1 represents Ch1+Ch2-Type microdroplets containing only mutant templates; quadrant 2 represents Ch1+Ch2+A type microdroplet containing both wild type and mutant templates; quadrant 3 represents Ch1-Ch2-Microdroplets, without any template; quadrant 4 represents Ch1-Ch2+Type microdroplets, containing only wild type template.
And (4) judging a result standard: ch1+Ch2-If the number of droplets is more than 3, the mutant type is judged to be positive. Then, the copy numbers of the wild-type template and the mutant template are calculated according to the Poisson distribution, and the allele frequency is calculated.
Example 3
In this example, 61 peripheral blood samples (including peripheral blood samples of 25 healthy persons and peripheral blood samples of 36 lymphoma patients) with known MYD88 gene L265P mutation were tested by the ddPCR method for testing MYD88 gene L265P mutation as described in example 2, and the specific test results are shown in table 3:
TABLE 3 test results
The results show that when the method is used for detecting 61 exceptional peripheral blood samples, 25 healthy human samples have no mutation, and the display specificity is 100%; positive mutations were detected in 13 lymphoma patient samples identified as carrying the mutation in MYD88 gene L265P, showing a sensitivity of 100%. The detection method has good sensitivity and specificity, the detection result is completely consistent with the known mutation condition, and the accuracy is up to 100%.
Example 4
This example tests the detection sensitivity of the detection method described in example 2. Genomic DNAs of a No. 55 sample (mutation ratio is 32.28%) with positive mutation of MYD88 gene L265P and a No. 56 sample (mutation ratio is 0) with negative mutation of MYD88 gene L265P in example 3 are selected and mixed according to a certain ratio to obtain 4 gradient standards: the mutation ratios were 5%, 1% and 0.5% respectively, and then 4-step markers of MYD88 gene L265P were tested using the test method described in example 2, each standard was tested in duplicate for 4 replicates, and the results were analyzed.
The specific test results for the 4 gradient standards are shown in table 4:
TABLE 4 test results
The results show that the lower limit of detection of the MYD88 gene L265P mutation detection method is 1 per mill, which indicates that the detection method has higher sensitivity. To the pre-estimationPerforming linear regression analysis on the mutation ratio (i.e. the mutation ratio of the gradient standard) and the actual mutation ratio data, and displaying the regression coefficient R2>0.99 (fig. 2), which indicates that the detection method of the present invention has high data fitting degree, which indicates that the detection method of the present invention has high accuracy.
Example 5
This example investigates the effect of digestion of genomic DNA with the restriction enzyme HindIII on droplet preparation efficiency.
The sample was treated by the sample treatment method described in example 1, and a control 1 was provided, and the sample treatment method of control 1 was the same as the sample treatment method described in example 1 except that the purified genomic DNA was not digested in step S1 with the restriction enzyme HindIII having a specific cleavage site of 5'-A | AGCTT-3'. The sample treatment methods described in example 1 and control 1 were used to treat 30 identical peripheral blood samples to prepare microreaction droplets, and the number of microreaction droplets prepared was then measured, with the specific results shown in table 5 and fig. 3:
TABLE 5 detection results of the number of micro-reaction droplets
As can be seen from Table 5 and FIG. 3, the number of the micro-reaction droplets prepared by the sample treatment method described in example 1 is significantly higher than that of the sample treatment method described in control 1, which indicates that the efficiency of droplet preparation can be effectively improved by performing the enzyme digestion treatment on the genomic DNA using the restriction enzyme HindIII. In the research process, the inventor finds that the space structure of the chromosome can influence the preparation of the droplets due to the small droplets, usually in the nl class, and even can sometimes cause the pipeline to be blocked, so that the preparation of the droplets fails. The restriction enzyme HindIII is used for carrying out enzyme digestion treatment on the genome DNA, so that the adverse effect of a chromosome space structure on droplet preparation can be improved, the efficiency and the success rate of droplet preparation are improved, the successful realization of subsequent detection is ensured, the fluorescence intensity of detection is improved, and the sensitivity of detection is improved.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Guangzhou gold-area medical inspection center, Inc
<120> sample processing method and detection kit for detecting MYD88 gene L265P mutation by ddPCR
<130>2020-07-07
<160>4
<170>PatentIn version 3.3
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Claims (12)
1. A sample processing method for detecting MYD88 gene L265P mutation by ddPCR is characterized by comprising the following steps:
s1, extracting sample genome DNA;
s2, preparing micro-reaction liquid drops: (1) preparing a micro-reaction solution containing genomic DNA and the following primers and probes: an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2, a MYD88 gene L265P mutant detection probe shown as SEQ ID NO.3, and a MYD88 gene L265P wild-type detection probe shown as SEQ ID NO. 4; (2) preparing a micro-reaction liquid drop by using the micro-reaction liquid obtained in the step (1).
2. The sample processing method for ddPCR detection of MYD88 gene L265P mutation as claimed in claim 1, wherein step S1 further comprises enzyme digestion of the extracted genomic DNA with restriction enzyme HindIII.
3. The sample processing method for ddPCR detection of MYD88 gene L265P mutation according to claim 2, wherein the concentration of restriction enzyme HindIII in the enzyme digestion system is 1000-2000 units/ml, and the concentration of genomic DNA is 10-16.5 ng/ul.
4. The sample processing method for ddPCR detection of MYD88 gene L265P mutation according to claim 3, wherein the enzyme digestion reaction conditions are as follows: incubating at 37 +/-1 ℃ for 5-10 min.
5. The sample processing method for ddPCR detection of MYD88 gene L265P mutation according to claim 1, wherein the molar ratio of an upstream primer, a downstream primer, a detection probe for MYD88 gene L265P mutation and a detection probe for MYD88 gene L265P wild type in the micro-reaction solution in step S2 is (1-2): (1-2): (0.5-1): (0.5 to 1).
6. The sample processing method for ddPCR detection of MYD88 gene L265P mutation according to claim 5, wherein the molar ratio of the upstream primer to the downstream primer in the micro-reaction solution in the step S2 is 1: 1; the molar ratio of the mutant detection probe for MYD88 gene L265P to the wild-type detection probe for MYD88 gene L265P is 1: 1.
7. The sample processing method for ddPCR detection of a mutation in MYD88 gene L265P of claim 1, wherein the sample in step S1 is peripheral blood or bone marrow.
8. The sample processing method for ddPCR detection of the L265P mutation of the MYD88 gene according to any one of claims 1-7, further comprising the following steps: s3, performing ddPCR amplification to obtain a treated sample, wherein the ddPCR amplification conditions are as follows: 10min at 95 ℃; 35-40 cycles: 30s at 94 ℃ and 60s at 55 ℃; 10min at 98 ℃.
9. The sample processing method for ddPCR detection of a mutation at MYD88 gene L265P of claim 8, wherein the temperature rise and/or decrease rate is set to 2 ± 0.5 ℃/sec during ddPCR amplification.
10. A kit for detecting MYD88 gene L265P mutation by ddPCR, which is characterized by comprising the following primers and probes: an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2, a mutation type detection probe for MYD88 gene L265P shown as SEQ ID NO.3, and a wild type detection probe for MYD88 gene L265P shown as SEQ ID NO. 4.
11. The kit for ddPCR detection of a mutation at MYD88 gene L265P as described in claim 10, wherein the kit further comprises restriction enzyme HindIII.
12. The kit for detecting the L265P mutation of the MYD88 gene by ddPCR as claimed in any one of claims 10-11, wherein the kit further comprises a genomic DNA extraction reagent.
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