CN113502332B - Primer, probe and kit for detecting FLT3 gene mutation - Google Patents
Primer, probe and kit for detecting FLT3 gene mutation Download PDFInfo
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Abstract
The invention discloses a primer, a probe and a kit for detecting FLT3 gene mutation, and belongs to the field of gene detection. The primers and probes for detecting FLT3 gene mutation comprise primers and probes for specifically detecting FLT3 gene D835 site mutation and FLT3 gene I836 site mutation. The primer and the kit rely on a digital PCR detection technology, simplify detection steps compared with other existing technologies for detecting FLT3 mutation, and have the advantages of simple and efficient detection process, absolute quantification, high sensitivity and the like. The invention provides powerful technical support for the auxiliary diagnosis and the monitoring of clinical treatment effect of AML patients carrying FLT3 gene D835 or I836del mutation, and has wide application prospect and industrialization prospect.
Description
Technical Field
The invention belongs to the field of gene detection, and particularly relates to a primer, a probe and a kit for detecting FLT3 gene mutation.
Background
The FLT3 gene is located at chromosome 13q12.2, has 24 exons, and the encoded protein belongs to one member of type III receptor tyrosine kinase family, is called FMS-like tyrosine kinase 3 (FMS-like tyrosine kinase 3), and plays an important role in survival, proliferation and differentiation of hematopoietic cells. The FLT3 protein is composed of 5 domains, an immunoglobulin-like extracellular domain located in the extracellular domain, a transmembrane domain, a membrane-proximal domain (JMD), a tyrosine kinase domain disruption (TKD), and a small C-terminal domain. According to the statistics report of Chinese tumors in 2015, the number of onset of leukemia in China is 75300/year, and the number of deaths is 53400/year, wherein the adult Acute Myeloid Leukemia (AML) accounts for about 65% of adult leukemia, and the child AML accounts for about 25% of child leukemia. Clinically, activating mutations in FLT3 gene are the most common gene alterations and poor prognosis factors in Acute Myeloid Leukemia (AML) patients. Mutations in the FLT3 gene occur in about one third of AML patients, with the two most common forms of mutation being internal tandem repeats (ITDs) and point mutations in the Tyrosine Kinase Domain (TKDs), with the most prominent mutation sites being D835 and I836.
In recent years, the therapeutic effect of AML has been greatly improved with the continuous improvement of chemotherapy schemes and the development of molecular targeted drugs, and the targeted therapeutic drug gilteritinib (giritinib) in China is approved by the national drug administration for drug administration (NMPA) and used for treating relapsing or refractory acute myeloid leukemia positive to FLT3 mutation. However, Minimal Residual Disease (MRD) remains a problem affecting the prognosis of AML. The minimal residual disease refers to a state in which a small amount of leukemia cells remain in the body after leukemia-induced chemotherapy is completely alleviated. MRD is a sensitive indicator for evaluating prognosis, and how to accurately detect the amount of leukemia cells mixed in normal hematopoietic cells after the patient is completely relieved by treatment is a key issue. The residual amount of leukemia cells depends on the sensitivity of the detection method. FLT3D 835 and I836 mutations in AML patients, their mutation rates can become one of the important indicators of MRD. Therefore, continuous follow-up detection of FLT3D 835 and I836 mutations during FLT 3-targeted therapy is of great significance for predicting disease progression, guiding drug therapy selection and prognosis in AML patients.
The detection technologies commonly used for FLT3-TKD mutation at present mainly comprise a restriction enzyme + PCR electrophoresis method, a traditional Sanger sequencing method and a second-generation sequencing method. However, these detection methods have some disadvantages. For example, the restriction enzyme method cannot effectively recognize the wild DNA by 100% for cutting, so that false positive detection results are easily caused, the use accuracy of the kit is reduced, and particularly, when the mutation rate in a sample is low, the accuracy and the sensitivity for judging whether the sample has the mutation by using the restriction enzyme cutting are low; the traditional Sanger sequencing has low detection flux and low detection sensitivity: 1% -10%. Although the flux of the detection of the NGS is large, the detection sensitivity is greatly improved compared with that of a capillary electrophoresis method, the cost of the NGS method is high, the data analysis is complex and time-consuming, and the sensitivity is only about 1%. The accuracy of the detection result directly influences the diagnosis of clinical diseases and the judgment of treatment effects, so that a method for detecting FLT3D 835 and I836 mutations more sensitively, simply and accurately is needed in the art. Digital PCR (digital PCR) is a technology that has emerged in recent years, and its principle is to distribute a standard PCR reaction into a large number of tiny reactors, each of which may or may not contain one or more copies of a target molecule (DNA template), to achieve "single-molecule template PCR amplification", and after PCR amplification, to perform fluorescence intensity detection by a biochip analyzer. The intensity of the fluorescence signal of the positive droplets comprising the nucleic acid molecule is increased compared to the negative droplets, and the absolute copy number of the target nucleic acid molecule is calculated by the proportion of positive droplets according to the principle of poisson distribution. The whole process does not depend on a standard curve and a reference sample, and the sample is directly detected to obtain the copy number of the target sequence.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a primer, a probe and a kit for detecting FLT3 gene mutation, wherein the detection kit is simple, efficient, absolute in quantification and high in sensitivity.
In order to achieve the purpose, the invention adopts the technical scheme that: a kit for detecting mutations in FLT3 gene, the kit comprising a primer pair and a specific probe; the sequences of the primer pair are shown as SEQ ID NO. 1 and SEQ ID NO. 2; the sequence of the specific probe is shown as SEQ ID NO. 3.
The kit for detecting FLT3 gene mutation can quickly and efficiently detect whether the FLT3 gene is mutated or not, and the sensitivity and the accuracy of the detection process are higher.
As a preferred embodiment of the kit for detecting a mutation in FLT3 gene according to the present invention, the kit further comprises an internal reference probe; the sequence of the internal reference probe is shown as SEQ ID NO. 4.
As a preferred embodiment of the kit for detecting FLT3 gene mutation, the 5 'end of the specific probe is marked with FAM, and the 3' end of the specific probe is marked with BHQ 1.
As a preferable embodiment of the kit for detecting FLT3 gene mutation, the internal reference probe is labeled with VIC at the 5 'end and BHQ1 at the 3' end.
The difficulty of Drop-off digital PCR is: the digital PCR reaction system can not only completely detect mutant DNA molecules with low mutation ratio in a sample, but also obtain a two-dimensional graph result which is obviously distinguished from a wild type DNA microdroplet fluorescence signal. Aiming at the difficulties, the invention designs different PCR probes aiming at the mutation sites, carries out fluorescence labeling on the PCR probes, carries out the combination test of the two probes with different base lengths, ensures that the mutant DNA template has accurate copy number detection results, and screens out the probe sets.
As a preferred embodiment of the kit for detecting FLT3 gene mutation, the kit comprises a FLT3-TKD detection mixed solution, a reference substance and a PCR reaction premixed solution; the FLT3-TKD detection mixed solution comprises a primer and a probe; the PCR reaction premix comprises buffer, dNTPs, taq enzyme and UDG enzyme.
As a preferable embodiment of the kit for detecting FLT3 gene mutation, the concentration of the primer in the FLT3-TKD detection mixed solution is 900nM, and the concentration of the probe in the FLT3-TKD detection mixed solution is 250 nM.
Through a large number of experimental screens, the primer concentration in the FLT3-TKD detection mixed solution is 900nM, and the probe concentration is 250n, so that a two-dimensional graph with the most obvious result of distinguishing the two mutant types and the wild type of the FLT3 can be obtained.
As a preferred embodiment of the kit for detecting mutations of FLT3 gene according to the present invention, the controls include FLT3D 835 weak positive control, FLT 3I 836del weak positive control and blank control; the FLT3D 835 weak positive control is formed by mixing FLT3D 835 mutant recombinant plasmid and FLT3 wild type plasmid; the FLT 3I 836del weak positive control is formed by mixing FLT 3I 836 mutant recombinant plasmid and FLT3 wild-type plasmid; the blank control is water without ribozyme.
As a preferable embodiment of the kit for detecting FLT3 gene mutation, the nucleotide sequence of FLT3 wild type plasmid is SEQ ID NO. 5; the nucleotide sequence of the FLT3D 835 mutant recombinant plasmid is SEQ ID NO 6; the nucleotide sequence of the FLT 3I 836 mutant recombinant plasmid is SEQ ID NO. 7.
The invention has the beneficial effects that: the primers and the kit rely on a digital PCR detection technology, simplify detection steps compared with other current technologies for detecting FLT3 mutation, and have the advantages of simple and efficient detection process, absolute quantification, high sensitivity and the like. The invention provides powerful technical support for the auxiliary diagnosis and the monitoring of clinical treatment effect of AML patients carrying FLT3 gene D835 or I836del mutation, and has wide application prospect and industrialization prospect.
Drawings
FIG. 1 shows two TaqMan probes for a pair of primer F/R amplicons identical to the FLT3 mutation region;
FIG. 2 is a two-dimensional graph of digital PCR detection screening of FLT 3I/WT plasmid 10% and reaction premix mix 1;
FIG. 3 is a two-dimensional graph of digital PCR detection screening of FLT 3I/WT plasmid 10% and reaction premix mix 2;
FIG. 4 is a two-dimensional graph of digital PCR assay screening of FLT3D/WT plasmid 10% with reaction premix mix 1;
FIG. 5 is a two-dimensional graph of digital PCR assay screening of FLT3D/WT plasmid 10% with reaction premix mix 2;
FIG. 6 shows the digital PCR detection results of reference samples prepared by mixing FLT3D 835 plasmid with FLT3 wild type plasmid;
FIG. 7 shows the digital PCR detection results of reference samples prepared by mixing FLT 3I 836del plasmid with FLT3 wild type;
FIG. 8 shows the result of digital PCR assay of recombinant plasmid samples containing only wild-type FLT3 gene fragment;
FIG. 9 shows the result of digital PCR detection of DNA samples from peripheral blood of healthy persons;
FIG. 10 shows the result of digital PCR assay of mixed samples of recombinant plasmid containing wild-type FLT3 gene fragment and recombinant plasmid containing FLT3D 835 mutant gene fragment;
FIG. 11 shows the result of digital PCR detection of mixed samples of recombinant plasmid containing wild-type FLT3 gene fragment and recombinant plasmid containing FLT 3I 836del mutant gene fragment.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.
Example 1
The primer, the probe and the kit for detecting FLT3 gene mutation in the embodiment of the invention comprise:
(1) reaction premix for droplet digital PCR: comprises buffer, dNTPs, taq enzyme and UDG enzyme;
(2) FLT3-TKD detection mixture: comprises a pair of primers and two probes for detecting FLT3D 835 and I836 sites; wherein the primers comprise a primer for pertinently detecting the D835 site mutation of the FLT3 gene and a primer for pertinently detecting the I836 site mutation of the FLT3 gene, and the sequences of the primers are shown as SEQ ID NO. 1 and SEQ ID NO. 2; wherein the probes comprise a specific probe and an internal reference probe, and the sequences of the probes are shown as SEQ ID NO. 3 and SEQ ID NO. 4;
(3) FLT3D 835 weak positive control: the recombinant plasmid containing FLT3D 835 mutant gene segment and FLT3 wild type plasmid are diluted by 1 × TE and prepared in proportion; the nucleotide sequence of FLT3 wild type plasmid is shown in sequence table SEQ ID NO. 5; the nucleotide sequence of the FLT3D 835 mutant recombinant plasmid is shown in a sequence table SEQ ID NO. 6;
(4) FLT 3I 836del weak positive control: the recombinant plasmid containing FLT 3I 836 mutant gene segment and FLT3 wild type plasmid are diluted with 1 × TE and prepared in proportion; the nucleotide sequence of the FLT 3I 836 mutant recombinant plasmid is shown in a sequence table SEQ ID NO. 7;
(5) blank control: obtained by commercial ribozyme-free water split charging, and is used for monitoring whether the detection reagent is polluted or not.
In this example, FLT3-TKD assay mixture contains a combination of primers and probes, wherein the reaction concentration of the primers is 900nM and the reaction concentration of the probes is 250 nM. The primer probes of the FLT3-TKD detection mixed solution are prepared into 10 XFLT 3-TKD detection mixed solution according to the using amount of each component. The specific probe designed by the invention is used for detecting D835 and I836del mutations of FLT3 genes. The sequences based on the object to be detected have the following characteristics: the D835 and I836 mutations of FLT3 gene of human genome are located in the gene region "GATATC", so as to design two TaqMan probes for the same pair of primer F/R amplicons of the FLT3 mutation region, as shown in FIG. 1, wherein the length of the primer design is not limited by specific examples, and one of the two probes is a specific probe of FAM fluorescent label, is located in the DNA region of the mutation site, is completely complementary with the wild type sequence and is not completely complementary with the mutation site, and is also called Drop-off probe, and is used for detecting two mutations of FLT 3. Because the sequence of the Drop-off probe and the FLT3D 835 mutant is only 1 base different, while the sequence of the Drop-off probe and the FLT 3I 836del mutant is only 3 base different, when a DNA sample contains mutant DNA, the matching efficiency of the mutant DNA template and the Drop-off probe is reduced due to the base difference, so that the fluorescence value of a droplet containing the mutant DNA template in digital PCR is reduced, and the fluorescence value of the droplet containing the mutant DNA template in the digital PCR is detected differently due to the different mismatched base numbers of the two mutants and the probe in the PCR process, so that the fluorescence value of the droplet containing different mutant DNA templates is detected differently. Wherein the Drop-off probe and wild type match perfectly in droplets containing only wild type DNA, the detected droplets gave the highest fluorescence value of FAM, followed by the lower fluorescence value of droplets containing mutant FLT3D 835 and the lowest fluorescence value of droplets containing mutant FLT 3I 836del, thereby allowing droplet signals to be distinguished between mutant, wild type and two mutants. In addition, another probe designed by the invention is positioned at the upstream of the Drop-off probe, is labeled by VIC fluorescence, is completely complementary to both mutant DNA and wild DNA, is not influenced by mutation points, is called an internal reference probe, and is used for monitoring whether the PCR reaction is normal or not.
Example 2
The detection method of the kit for detecting FLT3 gene mutation, disclosed by the embodiment 1, comprises the following steps of:
(1) sample acquisition: suitable sample types are whole blood or bone marrow fluid.
(2) Sample processing and nucleic acid extraction: purchasing a commercialized nucleic acid extraction kit, extracting DNA of a clinical sample according to the instruction of the nucleic acid extraction kit, and measuring the concentration of nucleic acid in an extracted DNA solution by using Nano-Drop;
(3) preparing a reagent:
a. and setting positive quality control and blank control quality control for the detection reaction.
b. Preparation process
1) Thawing all the components to room temperature, fully dissolving all the components, then oscillating and mixing uniformly, and centrifuging for a short time;
2) and determining the reaction number N, wherein N = the number of samples to be detected (N) + the quality control number (3) + 1. The amounts of each reagent added to the reaction mixture were calculated as shown in table 1.
TABLE 1
| Components | Volume (N) |
| Reaction premix for micro-drop digital PCR | 10×Nμl |
| FLT3-TKD detection |
2×Nμl |
| Nuclease- |
1×Nμl |
The reaction premix for the micro-drop digital PCR is a product of Guangdong Yongnuo medical science and technology Limited company, the product number is S02000301, and the micro-drop digital PCR system is matched with a micro drop-100 micro-drop digital PCR system for use.
3) A1.5 ml sterile centrifuge tube is taken to prepare a reaction system, all reagents are added and then shaken and mixed evenly, and the mixture is centrifuged for a plurality of seconds.
4) Then, 13. mu.l/tube of the mixture was dispensed into a 0.2ml PCR reaction tube.
c. Sample application
And (3) respectively adding 7 mul of blank control, positive control and clinical sample DNA into a 0.2ml PCR reaction tube, adding water with the volume less than 7 mul, tightly covering a tube cover, shaking and mixing uniformly, centrifuging for a short time to completely throw liquid on the tube wall to the lower part of the tube (to avoid generating bubbles), and then preparing micro-droplets.
d. Preparation of microdroplets
And taking 20 mu L of the mixed solution in the reaction tube, and generating micro-droplets, wherein the specific operation steps are carried out according to the instruction of a biochip analyzer.
e.PCR amplification
Carrying out PCR amplification on the 96-hole PCR reaction plate with the sealed membrane, wherein the amplification procedure is as follows: reacting at 50 ℃ for 2 min; reacting at 95 ℃ for 10 min; reaction at 95 ℃ for 30sec, at 62 ℃ for 60sec, 45 cycles; reacting at 98 ℃ for 10 min; 16 ℃ was terminated and a ramp rate of 1 ℃/sec was set for each step.
(4) Microdroplet detection and result analysis
a. After PCR amplification, the detection plate is placed in a biochip analyzer, QuantDrop software is started, and the detailed experimental steps are detected according to the instruction of the analyzer.
b. After the detection is complete, click "analyze" to open and analyze the data. Clicking the two-dimensional graph to display the two-dimensional graph, and dividing the wild type microdroplet and the mutant microdroplet groups of the sample according to the fluorescence value of the positive control signal;
c. clicking on "concentration" can indicate the number of copies of DNA added to each well, in copies/ul; mutation level results were obtained by calculating the mutant copy number/(mutant + wild-type copy number) ratio.
(5) Determination of detection result
a. Judging droplet generation effectiveness: the total number of droplets in each reaction tube is more than or equal to 50000, and if the total number of droplets is less than 50000, the droplets in the reaction holes are not ideal to be generated, and the droplets are required to be generated again;
b. and (3) blank comparison validity judgment: (ii) fewer positive droplets falling in the "channel 2 +" region than 3, and fewer positive droplets falling in the "channel 1+, channel 2 +" region than 3;
c. and (3) judging the effectiveness of the positive control: the detection result is in accordance with the mutation rate at about 10% mutation level;
d. the sample detection hole falls between the channel 1+ and 2 +' areas to form a droplet group, the number of droplets is more than or equal to 3, otherwise, the detection is invalid, and the detection needs to be carried out again.
e. If the sample falls below the wild-type microdroplet group and the positive microdroplet with the fluorescence value of 'channel 1 +' being below 25000 is more than or equal to 1 and less than 3, the FLT3-TKD mutation is judged to be positive, the sample amount is required to be detected again, the positive microdroplet with the fluorescence value of 'channel 1 +' being 25000-15000 is judged to be more than or equal to 3, the FLT3D 835 mutation positive microdroplet is judged, the positive microdroplet with the fluorescence value of 'channel 1 +' being below 15000 is judged to be more than or equal to 3, the FLT 3I 836del mutation positive microdroplet is judged to be lower than the lowest detection limit, and the other point is judged to be lower than the lowest detection limit.
In the PCR reaction process, both a Drop-off probe and an internal reference probe are hybridized based on the combination of products amplified by a pair of primers, 5-10 ten thousand microdroplets can be generated by using the Yongnuo medical Microdrop-100B digital PCR platform, and sample DNA molecules are randomly and singly distributed in each generated independent microdroplet. In a sample detection system with only wild-type DNA molecules, droplets generated by the digital PCR platform contain only wild-type DNA or no DNA, the fluorescence signal values of the droplets detected by the digital PCR are concentrated in a stable central range, and the negative droplets without DNA are also stable in a lower fluorescence value range. In the sample detection system containing mutant DNA molecules, droplets generated by the digital PCR platform exist as droplets containing only mutant DNA molecules and droplets containing only wild type DNA molecules and negative droplets, as described in the above primer probe design, the detected fluorescence values of the droplets containing different mutant types are different, finally, corresponding copy numbers are obtained through the droplets with different fluorescence values, and the mutation ratio is obtained by calculating the ratio of the copy number of the mutant type droplets/(the copy number of the mutant type + the copy number of the wild type droplets).
Example 3 optimization of reaction premix composition
The components of the reaction premix for droplet-type digital PCR of the kit for detecting FLT3 gene mutation of example 1 of the present invention were screened by assay. Two different reaction premix mix1 and mix2 for digital PCR were prepared for testing, and the specific steps were as follows:
(1) samples of different mutants were prepared with plasmids:
a. taking out the diluted FLT3D 835 plasmid, FLT 3I 836del plasmid and FLT3 wild plasmid solution from a refrigerator at the temperature of-20 ℃, dissolving, and then shaking and mixing uniformly;
b. the mutant FLT3D 835 plasmid solution is diluted 10 times with FLT3 wild type plasmid solution to prepare: 30ul of mutant FLT3D 835 plasmid is added into 270ul of wild type plasmid and mixed evenly, and the plasmid is named as FLT3D/WT plasmid 10 percent;
c. mutant FLT 3I 836del plasmid solution was diluted 10-fold with FLT3 wild type plasmid solution to make up: 30ul of mutant FLT 3I 836del plasmid is added into 270ul of wild type plasmid and mixed evenly, and the plasmid is named as FLT 3I/WT plasmid 10 percent;
(2) using two different reaction premixed solutions mix1 and mix2 for droplet-type digital PCR as digital PCR reaction buffer systems, respectively, using the 5 plasmid solutions as templates and two mix for combined testing, and performing digital PCR detection according to the steps of the example 2;
(3) the results of the digital PCR assay are shown in FIGS. 2-5.
From the results of the two-dimensional graphs detected, it was found that the signal interference between the mutant droplet and the wild-type droplet obtained by using mix2 as the reaction premix for the digital PCR was the least, and the distinction between the mutant droplet and the wild-type droplet was the most concentrated.
Example 4 detection of recombinant plasmid formulated Linear reference
In this example, the kit for detecting FLT3 gene mutation in example 1 and the detection method in example 2 are used to detect a linear reference sample prepared from recombinant plasmid, and the specific detection method is as follows:
(1) a reference sample with mutation ratio is prepared by mixing FLT3D 835 plasmid and FLT 3I 836del plasmid with FLT3 wild type respectively, and FLT3D 835/FLT 3%: 0.01%, 0.1%, 1%, 10%, FLT 3I 836del/FLT 3%: 0.01%, 0.1%, 1%, 10%, respectively;
(2) preparing a reagent:
a. setting blank control quality control for the detection reaction;
b. the preparation process comprises the following steps:
1) thawing all the components to room temperature, fully dissolving all the components, then oscillating and mixing uniformly, and centrifuging for a short time;
2) and determining the reaction number N, wherein N = the number of samples to be detected (N) + the quality control number (1) + 1. Calculating the amount of each reagent added to the reaction mixture as shown in table 2;
3) preparing a reaction system by taking a 1.5ml sterile centrifuge tube, adding all reagents, shaking and uniformly mixing, and centrifuging for several seconds;
4) then, 18. mu.l/tube of the mixture was dispensed into 0.2ml PCR reaction tubes.
c. Sample adding: respectively adding 2 mul of blank control and linear reference sample DNA into a 0.2ml PCR reaction tube, covering a tube cover tightly, shaking and mixing uniformly, centrifuging for a short time to completely throw liquid on the tube wall to a lower part (to avoid generating bubbles), and then preparing micro-droplets.
TABLE 2
| Components | Volume (N) |
| Reaction premix for micro-drop digital PCR | 10×Nμl |
| FLT3-TKD detection |
2×Nμl |
| Nuclease-free water | 6×Nμl |
(3) Preparation of microdroplets (sample preparation area)
a. Taking 20 mu L of mixed liquor in the reaction tube, and generating micro-droplets, wherein the specific operation steps are carried out according to the instruction of a biochip analyzer;
PCR amplification: taking a prepared detection plate containing micro-droplets of a sample to be detected, and putting the detection plate into a PCR amplification instrument for amplification, wherein the amplification procedure is as follows: reacting at 50 ℃ for 2 min; reacting at 95 ℃ for 10 min; reaction at 95 ℃ for 30sec, at 62 ℃ for 60sec, 45 cycles; reacting at 98 ℃ for 10 min; 16 ℃ was terminated and a ramp rate of 1 ℃/sec was set for each step.
(4) Microdroplet detection and result analysis
after PCR amplification, placing a 96-hole PCR reaction plate in a MicroDrop-100B droplet detector for detection;
b. after the detection is complete, click "analyze" to open and analyze the data. Clicking the two-dimensional graph to display the two-dimensional graph, dividing the wild type microdroplet and the mutant microdroplet groups of the sample according to the fluorescence value of the positive control signal, and analyzing the result;
(5) the results of the digital PCR assays are shown in tables 3-4, FIGS. 6-7.
TABLE 3
| Linear range reference FLT3D 835/2503G>T/FLT3 | Total number of droplets | Mutant copy number | Wild type copy number | Mutant/wild type copy number% | Logarithmic copy number of mutant/wild type |
| 0.01% | 71312 | 3.9 | 14008.0 | 0.02% | -3.7 |
| 0.1% | 69460 | 10.0 | 11947.8 | 0.10% | -3.0 |
| 1% | 68772 | 137.7 | 13650.8 | 1.00% | -2.0 |
| 10% | 70363 | 1609.5 | 15355.1 | 10.50% | -1.0 |
TABLE 4
| Linear range reference FLT 3I 836del/FLT3 | Total number of droplets | Mutant copy number | Wild type copy number | Mutant/wild type copy number% | Logarithmic copy number of mutant/wild type |
| 0.01% | 65799 | 0.7 | 11462.7 | 0.01% | -4.2 |
| 0.1% | 68394 | 4.1 | 11471.2 | 0.04% | -3.5 |
| 1% | 58333 | 26.2 | 11380.4 | 0.23% | -2.6 |
| 10% | 61069 | 436 | 11591.3 | 3.76% | -1.4 |
The kit and the detection method are adopted to carry out linear reference on two mutant types with 0.01% -10% mutation ratio, the result is linear within the range of the mutation ratio, and the correlation coefficient R2 is more than 0.98. The method and the kit have the advantages of relatively simple detection process, absolute quantification, high sensitivity and the like, and lay a foundation for the auxiliary diagnosis and treatment, related research and a finished kit suitable for industrialization of FLT3D 835 and I836del mutation patients in leukemia by using a digital PCR technology at present.
Example 5
In this example, the kit for detecting FLT3 gene mutation of example 1 and the detection method of example 2 were used to detect a recombinant plasmid sample containing only the wild-type FLT3 gene fragment, a mixed plasmid sample containing the wild-type FLT3 gene fragment and a recombinant plasmid containing FLT3D 835 mutant gene fragment, a mixed plasmid sample containing the wild-type FLT3 gene fragment and a recombinant plasmid containing FLT 3I 836del mutant gene fragment, and a DNA sample of peripheral blood of a healthy person, respectively, and the detection results are shown in fig. 8 to 11.
As can be seen from FIG. 8, in the recombinant plasmid sample containing only the wild-type FLT3 gene fragment, the fluorescence values of the droplet population were concentrated in a stable central range, the fluorescence value of the channel 1 of the Drop-off probe, the fluorescence value of the channel 2 of the reference probe, and the fluorescence value of the negative droplet were clearly distinguished; as can be seen from FIG. 9, the DNA sample of peripheral blood of healthy people does not contain the mutant microdroplet signal group, and the fluorescence value distribution of the two-dimensional graph detected by the plasmid sample in FIG. 8 is more consistent; as can be seen from FIG. 10, in the samples containing the wild-type recombinant plasmid containing FLT3 gene fragment and the recombinant plasmid containing FLT3D 835 mutant gene fragment, the fluorescence of the channel 1 of the droplet Drop-off probe containing FLT3D 835 droplet signal group is reduced: as can be seen from FIG. 11, the fluorescence values of the channel 1 of the droplet Drop-off probes of the droplet population containing FLT 3I 836del were reduced in the samples containing the recombinant plasmid of the wild-type FLT3 gene fragment and the recombinant plasmid of the FLT 3I 836del mutant gene fragment, and were lower than the fluorescence value of the channel 1 of the droplet FLT3D 835 shown in FIG. 10.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Guangdong Yongno medical science and technology Co., Ltd, Guangzhou Yongno Biotechnology Co., Ltd
<120> primer, probe and kit for detecting FLT3 gene mutation
<130> 2021.08.17
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> FLT3 TKD-F1
<400> 1
tcacagagac ctggccgc 18
<210> 2
<211> 18
<212> DNA
<213> FLT3 TKD-R2
<400> 2
cagcctcaca ttgcccct 18
<210> 3
<211> 29
<212> DNA
<213> specific probe
<400> 3
tggctcgaga tatcatgagt gattccaac 29
<210> 4
<211> 27
<212> DNA
<213> internal reference Probe
<400> 4
tcacccacgg gaaagtggtg aagatat 27
<210> 5
<211> 400
<212> DNA
<213> FLT3 wild type plasmid
<400> 5
ttccatcacc ggtacctcct actgaagttg agtctagaag aaagattgca ctccaggata 60
atacacatca cagtaaataa cactctggtg tcattcttga cagtgtgttc acagagacct 120
ggccgccagg aacgtgcttg tcacccacgg gaaagtggtg aagatatgtg actttggatt 180
ggctcgagat atcatgagtg attccaacta tgttgtcagg ggcaatgtga ggctgctatt 240
tcctacttat ttttatacgg ctattttgtg ttgtgtcgtt atcatggtaa acaactgcac 300
tcactgtggt gcatttttga tttatggtaa catcaaaaaa ccctcacagc agtctgctta 360
cttatgctta aaaggttttt ctgcagcttc agggaatctt 400
<210> 6
<211> 400
<212> DNA
<213> FLT3D 835 mutant recombinant plasmid
<400> 6
ttccatcacc ggtacctcct actgaagttg agtctagaag aaagattgca ctccaggata 60
atacacatca cagtaaataa cactctggtg tcattcttga cagtgtgttc acagagacct 120
ggccgccagg aacgtgcttg tcacccacgg gaaagtggtg aagatatgtg actttggatt 180
ggctcgatat atcatgagtg attccaacta tgttgtcagg ggcaatgtga ggctgctatt 240
tcctacttat ttttatacgg ctattttgtg ttgtgtcgtt atcatggtaa acaactgcac 300
tcactgtggt gcatttttga tttatggtaa catcaaaaaa ccctcacagc agtctgctta 360
cttatgctta aaaggttttt ctgcagcttc agggaatctt 400
<210> 7
<211> 397
<212> DNA
<213> FLT 3I 836 mutant recombinant plasmid
<400> 7
ttccatcacc ggtacctcct actgaagttg agtctagaag aaagattgca ctccaggata 60
atacacatca cagtaaataa cactctggtg tcattcttga cagtgtgttc acagagacct 120
ggccgccagg aacgtgcttg tcacccacgg gaaagtggtg aagatatgtg actttggatt 180
ggctcgagat atgagtgatt ccaactatgt tgtcaggggc aatgtgaggc tgctatttcc 240
tacttatttt tatacggcta ttttgtgttg tgtcgttatc atggtaaaca actgcactca 300
ctgtggtgca tttttgattt atggtaacat caaaaaaccc tcacagcagt ctgcttactt 360
atgcttaaaa ggtttttctg cagcttcagg gaatctt 397
Claims (5)
1. A Drop-off digital PCR detection kit for FLT3 gene mutation is characterized by comprising a primer pair, a specific probe and an internal reference probe; the sequences of the primer pair are shown as SEQ ID NO. 1 and SEQ ID NO. 2; the sequence of the specific probe is shown as SEQ ID NO. 3; the sequence of the internal reference probe is shown as SEQ ID NO. 4; the 5 'end of the specific probe is marked with FAM, and the 3' end of the specific probe is marked with BHQ 1; the internal reference probe is marked with VIC at the 5 'end and is marked with BHQ1 at the 3' end.
2. The Drop-off digital PCR detection kit for FLT3 gene mutation according to claim 1, comprising FLT3-TKD detection mixture, a control and PCR reaction premix; the FLT3-TKD assay mixture comprises the primers and probes of claim 1; the PCR reaction premix solution comprises buffer solution, dNTPs, taq enzyme and UDG enzyme.
3. The Drop-off digital PCR detection kit for FLT3 gene mutation according to claim 2, wherein the concentration of the primer in the FLT3-TKD detection mixture is 900nM, and the concentration of the probe in the FLT3-TKD detection mixture is 250 nM.
4. The Drop-off digital PCR detection kit for mutation of FLT3 gene according to claim 2, wherein the controls comprise FLT3D 835 weak positive control, FLT 3I 836del weak positive control and blank control; the FLT3D 835 weak positive control is formed by mixing FLT3D 835 mutant recombinant plasmid and FLT3 wild type plasmid; the FLT 3I 836del weak positive control is formed by mixing FLT 3I 836 mutant recombinant plasmid and FLT3 wild-type plasmid; the blank reference substance is non-ribozyme water; the FLT3D 835 weak positive control is prepared by diluting mutant FLT3D 835 plasmid solution by 10 times with FLT3 wild type plasmid solution; the FLT 3I 836del weak positive control is prepared by diluting a mutant FLT 3I 836del plasmid solution by a FLT3 wild-type plasmid solution by 10 times.
5. The Drop-off digital PCR detection kit for FLT3 gene mutation according to claim 4, wherein the nucleotide sequence of FLT3 wild-type plasmid is SEQ ID NO: 5; the nucleotide sequence of the FLT3D 835 mutant recombinant plasmid is SEQ ID NO 6; the nucleotide sequence of the FLT 3I 836 mutant recombinant plasmid is SEQ ID NO. 7.
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| CN108410981B (en) * | 2018-01-23 | 2021-05-25 | 北京旌准医疗科技有限公司 | Kit for detecting FLT3-D835 gene and application |
| CN108676876A (en) * | 2018-05-23 | 2018-10-19 | 苏州锐讯生物科技有限公司 | A kind of primer sets and its application based on digital pcr detection saltant type FLT3-ITD |
| EP3860655A4 (en) * | 2018-10-01 | 2022-08-03 | CTI Biopharma Corp. | Methods of treating flt3-mutated hematologic cancers |
| CN110592198B (en) * | 2019-09-10 | 2023-09-29 | 中山大学附属第七医院(深圳) | Method, system and kit for detecting FLT3/D835Y gene mutation with high sensitivity |
| CN110982884B (en) * | 2019-12-05 | 2022-02-18 | 阅尔基因技术（苏州）有限公司 | Primer group, kit and method for detecting AML related gene mutation |
| CN111154881A (en) * | 2020-03-09 | 2020-05-15 | 南京实践医学检验有限公司 | Detection kit for gene mutation in acute myeloid leukemia and application |
| CN111560438B (en) * | 2020-06-11 | 2024-01-19 | 迈杰转化医学研究(苏州)有限公司 | Primer composition and kit for detecting AML prognosis related gene mutation and application thereof |
| CN113249475B (en) * | 2021-04-30 | 2022-02-11 | 镇江市第一人民医院 | Drop-off ddPCR method and kit for quantitatively detecting NPM1 gene mutation |
| CN113186283B (en) * | 2021-04-30 | 2022-02-22 | 镇江市第一人民医院 | Drop-off ddPCR method and kit for quantitatively detecting U2AF1 gene mutation |
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