CN113817811A - Primer group and kit for detecting FLT3-ITD gene mutation and application of primer group and kit - Google Patents
Primer group and kit for detecting FLT3-ITD gene mutation and application of primer group and kit Download PDFInfo
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Abstract
The invention discloses a primer group and a kit for detecting FLT3-ITD gene mutation and application thereof, belonging to the technical field of genetic engineering. The primer group and the kit provided by the invention are based on a Sanger sequencing technology, comprise an upstream primer and a downstream primer for PCR amplification and a sequencing primer for Sanger sequencing, can be used for detecting FLT3-ITD gene mutation with high accuracy and high sensitivity, are simple to operate and short in detection period, and can meet the requirements of clinical rapid, accurate and high-sensitivity detection.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a primer group and a kit for detecting FLT3-ITD gene mutation and application thereof, in particular to a primer group and a kit for detecting FLT3-ITD gene mutation based on Sanger sequencing and application thereof.
Background
FLT3(Fms-Llike Tyrosine Kinase 3) is a hematopoietic growth factor receptor gene, which is located on the long arm of chromosome 13 (13q12) and is a member of the type III receptor Tyrosine Kinase family. Usually expressed only on early progenitor cells, and has important regulatory effects in promoting the growth and differentiation of early granulocytic and lymphoid hematopoietic cells. Mutation of FLT3 gene can destroy proliferation, differentiation and apoptosis of normal blood cells, even can be combined with other cytokines alone in some cases, provides support for proliferation of leukemia cells, and is an important driving type mutation of bone marrow malignant tumor. The FLT3 gene has internal tandem repeats (ITD) of the sequence of the membrane-proximal region, called FLT3-ITD gene mutation, and the ITD has polymorphism in size and position, and is typically located in 14 th exon, and can also accumulate 14 th intron and 15 th exon.
Acute Myeloid Leukemia (AML) is a cancer characterized by abnormal hematopoietic system, which is caused by rapid infiltration of myeloid cells into bone marrow, blood and other tissues (including spleen, lymph node, liver, etc.) by cloning, proliferation, abnormal differentiation, etc. FLT3-ITD gene mutation occurs in AML of both adults and children, and FLT3-ITD gene mutation is known to have close relation with the occurrence, development and prognosis of AML and is an important gene marker for poor prognosis of AML patients.
In myelodysplastic syndrome (MDS) patients, there is a high likelihood of conversion to AML in FLT3-ITD positive patients, a short time to progression to AML, and a shortened overall survival relative to FLT3-ITD negative patients. After MDS is converted into AML, the positive rate of FLT3-ITD is high, so that FLT3-ITD gene mutation is a high risk factor for converting MDS into AML and is a poor prognosis factor.
Current studies on mutations in the FLT3-ITD gene indicate that FLT3-ITD gene mutations occur most frequently in patients with acute myelogenous leukemia with a mutation incidence of about 15% -30%, followed by myelodysplastic syndrome with a mutation incidence of about 2% -5%. FLT3-ITD gene mutation leads to continuous activation of FLT3 downstream signaling pathway and malignant proliferation of cells, so FLT3 becomes an important target for targeted therapy of AML and MDS. The FLT3-ITD gene mutation shows strong clinical requirements in AML and MDS, and the development of drugs aiming at the FLT3-ITD gene mutation is rapidly developed in recent years. Particularly, in 2016, 19 months, norwalk granted a "breakthrough" low position by the U.S. Food and Drug Administration (FDA) for AML patients who are treated for FLT3-ITD gene mutation against FLT3 inhibitor PKC412, more corroborated the targeted position of FLT3-ITD gene mutation as treatment for AML.
In conclusion, the FLT3-ITD gene mutation can be used as a gene screening before the use of FLT3-ITD targeted drugs, and can also be used as an independent index of poor prognosis of AML and MDS patients. The detection of FLT3-ITD gene mutation can be helpful for in vitro targeted drug screening, classification and targeted drug administration guidance of AML and MDS patients, and can also be helpful for prognosis judgment, treatment guidance, curative effect judgment and disease recurrence judgment, and has very important significance for increasing the long-term survival rate and the survival quality of patients.
At present, the leukemia gene mutation is widely detected at home and abroad by using the fluorescent quantitative PCR, but the fluorescent quantitative PCR detection technology which can only detect specific sites cannot meet the requirement of clinical work due to the factors of uncertain position of mutation of FLT3-ITD, uncertain size of repeated gene sequence and the like.
Disclosure of Invention
In view of one or more of the problems in the prior art, one aspect of the present invention provides a primer set for detecting FLT3-ITD gene mutation, comprising an upstream primer, a downstream primer and a sequencing primer, wherein the nucleotide sequence of the upstream primer is represented by SEQ ID No.1 in the sequence table, the nucleotide sequence of the downstream primer is represented by SEQ ID No.2 in the sequence table, and the nucleotide sequence of the sequencing primer is represented by SEQ ID No.3 in the sequence table.
In another aspect, the invention provides a kit for detecting FLT3-ITD gene mutation, which comprises the primer set.
When the above kit is used, the final concentration of each of the forward primer and the reverse primer is 0.1 to 1. mu.M.
The kit also comprises a positive control product and a negative control product, wherein the positive control product is DNA of a cell line positive to FLT3-ITD gene mutation, and the negative control product is a system without FLT3-ITD gene mutation.
The application of the primer group or the kit in the detection of FLT3-ITD gene mutation for non-disease diagnosis is also included in the content of the invention.
In still another aspect, the present invention provides a method for detecting FLT3-ITD gene mutation by Sanger sequencing for non-disease diagnostic purposes, which comprises the following steps:
1) carrying out PCR amplification on a sample DNA to be detected by using the upstream primer and the downstream primer according to claim 1 to obtain a PCR product;
2) performing a sequencing reaction by using the sequencing primer of claim 1 and the PCR product obtained in step 1);
3) purifying and denaturing a sequencing reaction product, sequencing, and obtaining a sequencing peak image;
4) analyzing the sequencing peak map, and judging whether FLT3-ITD gene mutation exists in the DNA of the sample to be detected according to the following standards:
(A) if the sequencing peak map has double peaks different from the background signal, determining that FLT3-ITD gene mutation exists in the DNA of the sample to be detected;
(B) if the sequencing peak diagram is a single peak, judging that FLT3-ITD gene mutation exists in the DNA of the sample to be detected when an insertion sequence exists in the sequencing result of the sample to be detected and the wild FLT3 gene sequence in a comparison manner; when the sequencing result of the sample to be detected is consistent with the wild FLT3 gene sequence, FLT3-ITD gene mutation does not exist in the DNA of the sample to be detected.
In the method, after the PCR product is obtained in the step 1), the method further comprises the step of preliminarily judging whether FLT3-ITD gene mutation exists in the DNA of the sample to be detected by performing gel electrophoresis on the PCR product, wherein the judgment standard is as follows: if two strips appear in the gel electrophoresis image, preliminarily judging that FLT3-ITD gene mutation exists in the DNA of the sample to be detected; and if the gel electrophoresis image only has one strip, preliminarily judging that the FLT3-ITD gene mutation does not exist in the DNA of the sample to be detected.
In the method, the concentrations of the upstream primer and the downstream primer in the PCR amplification reaction system in the step 1) are respectively 0.1-1 mu M.
In the above method, the test sample in step 1) includes, but is not limited to, a bone marrow or peripheral blood clinical sample of an acute myeloid leukemia patient (AML) or a myelodysplastic syndrome (MDS) patient, an AML or MDS targeted drug screening model cell sample, or an artificially constructed vector sample containing FLT3-ITD gene mutation.
Based on the basic principle of Sanger method sequencing, the primer group and the kit for detecting FLT3-ITD gene mutation provided by the technical scheme comprise an upstream primer and a downstream primer which are designed and preferably obtained near the upstream and downstream of the 14 th exon and the 15 th exon of the FLT3-ITD gene, and a sequencing primer which is different from the upstream primer and the downstream primer, wherein the upstream primer and the downstream primer are used for carrying out PCR amplification on sample DNA to be detected, and then the sequencing primer is used for sequencing, so that a target sequence can be identified with high accuracy, and the presented FLIT3-ITD gene mutation detection peak diagram has clear background and high signal value, the analysis difficulty of the mutation can be greatly reduced, the purpose of detecting FLT3-ITD gene mutation with high sensitivity is realized, and the embodiment results show that the primer group and the kit provided by the invention have the lowest detection sensitivity of detecting FLT3-ITD gene mutation and can detect FLT3-ITD gene mutation with 20 percent of the total amount of 5ng DNA. Because the Sanger method sequencing can judge the specific position where the tandem repeat appears and the length of the inserted fragment compared with the traditional fluorescent quantitative PCR method, the primer group and the kit can be used for detecting the FLT3-ITD gene mutation and further analyzing the inserted fragment. The invention can be used for prognosis judgment and relapse of AML, MDS or ALL patients and targeted drug screening, and can play an important role in identification of FLT3-ITD gene mutant cell strains or vectors and other applications, thereby having wide application prospects. Compared with the prior art, the invention also has the following advantages:
1) based on the latest FLT3-ITD gene mutation sequence related to AML and MDS targeted medication and prognosis published by Genbank, the invention designs and preferentially determines a primer capable of comprehensively covering the site, and the primer has strong specificity, wide coverage and more accurate detection result;
2) in the detection method provided by the invention, the PCR preliminary judgment result and the Sanger sequencing result can be mutually verified, so that the detection method provided by the invention has higher accuracy;
3) the invention adopts the inner primer different from the amplification primer as the sequencing primer, can increase the detection accuracy, has lower concentration of the amplification primer, and effectively reduces the detection cost.
Drawings
FIG. 1 is a graph showing the sequencing peaks at the same position of the same sample amplified by primer set 1 and primer set 2 of example 1;
FIG. 2 is the FLT3-ITD PCR electrophoresis gel of leukemia sample in example;
FIG. 3 is a graph showing alignment peaks between sample No.1 and a wild sequence in example;
FIG. 4 is a graph of alignment peaks of sample No.2 in example with wild sequence;
FIG. 5 is a peak diagram of the alignment of sample No.3 with the wild sequence in example;
FIG. 6 is a graph showing alignment peaks of sample No.4 and a wild sequence in example;
FIG. 7 is a peak diagram of alignment of sample No.5 with the wild sequence in example;
FIG. 8 is a graph showing the alignment peaks of the sample containing 20% FLT3-ITD gene mutation at 5ng total DNA in example and the wild type sequence.
Detailed Description
The invention aims to provide a primer group for detecting FLT3-ITD gene mutation, a kit for detecting FLT3-ITD gene mutation based on the primer group, and a method for detecting FLT3-ITD gene mutation based on Sanger method sequencing.
The invention is further illustrated by the following examples. It should be understood that the specific examples are intended to be illustrative of the invention and are not intended to limit the scope of the invention.
The methods used in the following examples are conventional unless otherwise specified, and specific procedures can be found in: a Molecular Cloning Laboratory Manual (Molecular Cloning: A Laboratory Manual, Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).
The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples.
The primers used were synthesized by general Biotechnology Ltd.
Example 1: primer design
This example is intended to identify a Primer set that can detect the mutation of FLT3-ITD gene by Sanger sequencing, and the inventors designed primers by Primer Premier 5.0 software according to the Primer design principle in the vicinity of the upstream and downstream of exons 14 and 15, based on the sequence of mutant genes published by GenBank (GenBank accession No. NG _007066.1) that are relevant to the targeted drug administration and prognosis of AML and MDS.
In this example, the inventors designed a plurality of primer combinations, selected group 1 and group 2 shown in table 1 from among the plurality of combinations, and used the primer combinations with better specificity and amplification efficiency and clear peak pattern background from group 1 and group 2 in the following examples in order to reduce the influence of background signals and non-specific signals of the sequencing peak pattern.
Table 1: amplification primer pair and sequencing primer sequence for detecting FLT3-ITD gene mutation
The upstream primer and the downstream primer in the primers of the group 1 and the group 2 listed in the table 1 are respectively used for carrying out PCR amplification on FLT3-ITD negative samples (FLT 3-ITD negative sample DNA confirmed by sequencing detection), after electrophoretic identification, sequencing primers of the group 1 and the group 2 are respectively used for carrying out sequencing reaction, and sequencing reaction products are subjected to purification and denaturation and then sequenced by a sequencer. Sequencing peak profiles were obtained at the same position in the same sample using the primers of set 1 or set 2, respectively (specific steps are described in example 2 below). As shown in FIG. 1, panel A and panel B, wherein panel A is a sequencing peak obtained with the primer set of group 1, and panel B is a sequencing peak obtained with the primer set of group 2. It can be seen that compared with the sequencing peak image of B, the background signal and the nonspecific signal of the sequencing peak image of A are significantly weaker, i.e. the detection peak image has clear background, high signal value and higher detection sensitivity, and can greatly reduce the difficulty of mutation analysis, so that the primer set of group 1 is preferably used as the primer set for detecting FLT3-ITD gene mutation. The primers are positioned in an intron sequence of the FLT3 gene and cover all hot spot regions of FLT3-ITD gene mutation, wherein the sequences shown by SEQ ID NO.1 and SEQ ID NO.2 are used for detecting internal tandem repeat mutation of the FLT3 gene, the sequencing primer shown by SEQ ID NO.3 is used for sequencing reaction, and the sequencing primer is different from a PCR amplification primer, can specifically identify a target sequence, and can enable the detection result to be more accurate.
Example 2: method for detecting FLT3-ITD gene mutation
In this embodiment, the primer set shown in set 1 of example 1 is used to detect FLT3-ITD gene mutation in a sample DNA, which is a DNA in Peripheral Blood Mononuclear Cells (PBMCs) isolated and purified from fresh anticoagulated bone marrow or peripheral blood (total 5 samples, numbered 1-5, respectively) of an Acute Myeloid Leukemia (AML) patient, specifically including the following steps:
1) separating cells: human peripheral blood lymphocyte separation fluid was purchased from solibao and used the principle of cell density gradient centrifugation. Transferring a peripheral blood or bone marrow sample in an anticoagulation tube into a 15ml centrifuge tube, making a name mark of a sample to be detected on the tube wall, and centrifuging at 1500rpm for 5 min; removing the upper plasma layer, adding 2-3 times of PBS into the blood cell sediment, and fully mixing and diluting; taking another 15ml centrifuge tube, adding 6ml human peripheral blood lymphocyte separating medium in advance, adding plasma diluted by PBS obliquely and slowly, obviously layering the plasma and the separating medium, centrifuging for 30min at 400g, raising speed of 2, lowering speed of 0 and 25 ℃, dividing cells in the centrifuge tube into four layers (a PBS layer, an annular milky white lymphocyte layer, a separating liquid layer and a red blood cell layer) from top to bottom after centrifugation, circularly absorbing the annular milky white lymphocyte layer by a pipette into a 15ml centrifuge tube which is added with 5ml PBS in advance, gently mixing uniformly to wash the cells, and centrifuging for 5min at 1500 rpm.
2) And (3) red blood cell lysis: adding 6-10 ml of erythrocyte lysate into the cell sediment, cracking at 4 ℃ for 15-20 min, centrifuging at 1500rpm for 5min, removing supernatant, and adding 1-5 ml of PBS into the cell sediment to resuspend cells to obtain peripheral blood mononuclear cells with high purity.
3) Extracting cell DNA: and (3) performing DNA extraction on the obtained peripheral Blood mononuclear cells with high purity by using a QIAGEN DNeasy Blood and Tissue Kit extraction Kit to obtain a sample DNA to be detected as a template DNA.
4) PCR amplification of the DNA of the sample to be detected: preparing a PCR amplification system according to the system composition shown in the following table 2, and carrying out PCR amplification reaction according to the PCR amplification program shown in the following table 3 to obtain a PCR amplification product. And then carrying out gel electrophoresis verification on the amplification product of the DNA of the sample to be detected of the No. 1-5 sample, which specifically comprises the following steps: 2% agarose gel is prepared, the gel is run at 80V for about 30-40 minutes, and a DNA electrophoresis strip is observed by a gel imaging system, and the result is shown in figure 2. N is FLT3-ITD negative control, P is FLT3-ITD positive control, and quality control is normal. As preliminary judged from the electropherogram shown in FIG. 2, a band appeared at about 450bp in the FLT3-ITD negative sample (N); if a repetitive tandem structure (ITD) is present inside FLT3, two specific bands are present, i.e., another slightly larger band appears above about 450 bp. Therefore, the electrophoresis bands of the No.1 to No.4 samples are two, so that the samples are positive for FLT3-ITD, namely FLT3-ITD gene mutation exists in DNA of the No.1 to No.4 samples, wherein the second band of the No.2 sample is relatively light and not obvious, and the sample No.2 can be confirmed to be strong positive for FLT3-ITD by combining a sequencing peak diagram (detailed in step 5) of the sample No.2 sample, which indicates that the wild proportion is less.
Table 2: PCR amplification system
Table 3: PCR amplification procedure
In conclusion, the electrophoresis of the PCR product in the step can preliminarily judge the negative and positive of the sample, so that the sample which can obviously distinguish the negative and positive and is only used for the preliminary screening of scientific research can be subjected to no further sequencing by considering time and cost. If the method is used for auxiliary clinical detection or targeted drug screening, the following steps of sequencing are required to ensure the accuracy of the result.
5) Sequencing by the Sanger method: sequencing by the Sanger method can finally confirm whether FLT3-ITD gene mutation exists in sample DNA. And (3) purifying the PCR product (the PCR product of the sample No. 1-5), further Sequencing and purifying, specifically performing Sequencing according to a BigDye Terminator v3.1 Cycle Sequencing Kit Sequencing reagent specification, and performing on-machine Sequencing (3500DX sequencer) by Fertilizer general Biotechnology Co., Ltd to finally obtain a Sequencing peak map of each sample. And (3) comparing the sequencing result of each sample with the wild type sequence by using analysis software Chromas, and analyzing the condition of the gene mutation site to analyze. Wherein the 14 and 15 exon sequences of FLT3 wild type used for alignment are shown in SEQ ID NO: shown at 7.
The results are shown in FIGS. 3-7, which are partial sequencing peak maps corresponding to samples No. 1-5 and FLT3 wild type (i.e. FLT3-ITD negative) sequencing peak maps, wherein A represents the sequencing peak map of the sample, and B represents the normal FLT3 wild type sequence peak map. The positive peak pattern representation forms are generally two: (1) a double peak (generally near 14 exons or 15 exons) which is different from a background signal exists on a sequencing peak image, the FLT3-ITD gene mutation exists in the DNA of a sample, the sample is a heterozygous sample, and the wild type sequence and the mutant type sequence exist at the same time. The method comprises the following specific steps: a. when the sequencing result of the sample is compared with the wild FLT3 gene sequence to form an insertion sequence, the mutant type in the sample has higher comparison, and the position and the size of the insertion fragment can be further analyzed; b. if the sequencing result of the sample is aligned with the wild-type FLT3 gene sequence without an insertion sequence, the mutant ratio in the sample is low, and the FLT3-ITD gene mutation can only be judged to exist in the DNA of the sample. (2) The sequencing peak map is a single peak, and is represented by a single comparison of the whole sequencing peak map, wherein when the sequencing result of the sample is aligned with the wild-type FLT3 gene sequence, the insertion sequence exists, the FLT3-ITD gene mutation exists in the sample DNA, and the proportion of the whole mutant type or the wild type in the sample is very low. The negative peak pattern is expressed as: and (3) judging that the FLT3-ITD gene mutation does not exist in the DNA of the sample if the sequencing peak graph is a single peak and the sequencing result of the sample does not have an insertion sequence when being aligned with the wild FLT3 gene sequence. Therefore, as can be seen from FIGS. 3-7, samples No. 1-4 are FLT3-ITD positive samples (both having two peaks different from the background signal), i.e., the DNA of samples No. 1-4 has FLT3-ITD gene mutation, and sample No.5 is a negative sample, i.e., the DNA of sample No.5 has no FLT3-ITD gene mutation. This is the same as the result of the primary judgment of PCR gel electrophoresis, and further confirms that the sample No.2 which is not obvious in the primary judgment of PCR is indeed the FLT3-ITD strong positive sample.
As shown in FIGS. 3-7, through comparison with the wild-type FLT3 gene sequence, the positions of mutations of four FLT3-ITD positive samples are all on 14 exons, an insertion sequence exists, the starting position of the insertion sequence is at the beginning or middle position of the 14 exon sequence, the size of the insertion fragment is 57-93 bp, and the specific steps are as follows: the sequencing peak pattern of sample No.1 is a single peak and is consistent with the alignment of the normal wild-type FLT3 gene sequence, and the result is shown in FIG. 7, which indicates that sample No.5 is negative for FLT3-ITD gene mutation.
In conclusion, the method for detecting FLT3-ITD gene mutation based on Sanger method sequencing provided by the invention can not only detect whether FLT3-ITD gene mutation exists in sample DNA, but also can determine the specific position of FLT3-ITD gene mutation and inserted base fragment, is convenient to detect, can provide more information for clinical work, and can be used for prognosis judgment and relapse of AML, MDS or Acute Lymphoblastic Leukemia (ALL) patients, targeted drug screening and identification of FLT3-ITD mutant cell strains or vectors.
Example 3 sensitivity of the method for detecting FLT3-ITD Gene mutation
In this embodiment, on the basis of the method for detecting FLT3-ITD gene mutation provided in example 2, the detection sensitivity of the method is evaluated by using DNA of a FLT3-ITD positive cell line Molm-13 (purchased from Bai Biotech, Inc., Nanjing) and DNA of a FLT3-ITD negative sample confirmed by sequencing detection, and the method specifically comprises the following steps:
1) the FLT3-ITD positive cell line Molm-13 and the DNA of the FLT3-ITD negative sample confirmed by sequencing detection are mixed according to a certain proportion to obtain a series of mixed samples with different total DNA amounts and different mutation proportions, and then PCR amplification and Sanger sequencing are sequentially carried out according to the operations of steps 4) and 5) in the above example 2, so as to analyze the total DNA amount and the detection limit of distinguishable mutation proportions according to the representation of a sequencing peak diagram.
As shown in FIG. 8, it is a sequencing peak diagram when the total amount of DNA in a sample is 5ng and the mutation ratio of FLT3-ITD gene is 20%, and it is obvious that there is a section of double peaks (around the exon 14 of FLT3 gene) on the sequencing peak diagram, which is different from the background signal, so that the sample is judged to be positive, i.e. FLT3-ITD gene mutation exists in the sample DNA, therefore, the method for detecting FLT3-ITD gene mutation provided by the present invention can be determined to have high sensitivity, and FLT3-ITD gene mutation of 20% in the minimum 5ng total amount of DNA can be detected.
Example 4 kit for detecting FLT3-ITD Gene mutation
The kit for detecting FLT3-ITD gene mutation provided by the invention comprises:
the primer group for detecting FLT3-ITD gene mutation shown in group 1 in example 1 comprises an upstream primer, a downstream primer and a sequencing primer, wherein the upstream primer and the downstream primer are used for PCR amplification reaction, and the sequencing primer is used for sequencing by Sanger method; the method specifically comprises the following steps: the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1 in the sequence table, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.2 in the sequence table, and the nucleotide sequence of the sequencing primer is shown as SEQ ID NO.3 in the sequence table.
In use, a 20 μ L reaction system for a PCR amplification reaction comprises: template 2. mu. L, Prime
10. mu.L of HS (premix), 0.5. mu.L of forward primer (final concentration of 0.1-1.0. mu.M, preferably 0.25. mu.M), 0.5. mu.L of reverse primer (final concentration of 0.1-1.0. mu.M, preferably 0.25. mu.M), RNase Free ddH2O7. mu.L. The PCR amplification procedure was: 5sec at 98 ℃ for 1 cycle; 10sec at 98 ℃, 5sec at 66 ℃, 35 cycles; 1min at 72 ℃, 10min at 12 ℃ and 1 cycle.
For the convenience of detection, the kit can also comprise a positive control substance and a negative control substance, wherein the positive control substance is a cell line Molm-13 positive to FLT3-ITD, and the negative control substance is DNA negative to FLT3-ITD, and the PCR process and/or the sequencing process are monitored to determine whether the process is normal or not.
For convenient detection, the kit may further comprise the detection method description of example 2 and the judgment standard of the sequencing peak map, wherein the judgment standard is specifically: and if the sequencing results of the negative control substance and the positive control substance are normal, the detection result is reliable. On the basis of the above, if a doublet (generally near 14 exons or 15 exons) which is different from a background signal exists on a sequencing peak image, the FLT3-ITD gene mutation exists in the DNA of the sample, and the sample is a heterozygous sample, and wild-type and mutant sequences exist at the same time. The method comprises the following specific steps: a. when the sequencing result of the sample is aligned with the wild-type FLT3 gene sequence (the 14 and 15 exon sequences of FLT3 gene) and an insertion sequence exists, the mutant sequence in the sample is higher, and the position and the size of the insertion fragment can be further analyzed; b. if the sequencing result of the sample is aligned with the wild-type FLT3 gene sequence without an insertion sequence, the mutant ratio in the sample is low, and the FLT3-ITD gene mutation can only be judged to exist in the DNA of the sample. (2) If the sequencing peak image is a single peak, the sequencing peak image is characterized in that the whole sequencing peak image is single, and the sequencing peak image is divided into: when the sequencing result of the sample is compared with the wild FLT3 gene sequence, if the insertion sequence exists, the FLT3-ITD gene mutation exists in the sample DNA, and the ratio of all the mutant types or the wild types in the sample is very low; and when the sequencing result of the sample is aligned with the wild FLT3 gene sequence, judging that FLT3-ITD gene mutation does not exist in the DNA of the sample.
Example 5 clinical sample testing
This example used the kit provided in example 4 to detect DNA extracted from bone marrow samples (respectively numbered No.1, No.2, No.3, No.4, No.5) of 5 AML patients collected from the hematology of the first subsidiary hospital of the university of medical science at anhui, DNA of the AML 3-ITD gene mutation positive cell line Molm-13 was used as a positive control, DNA of AML patient FLT3-ITD negative cell detected by sequencing was used as a negative control, PCR amplification and Sanger sequencing were sequentially performed on the above DNA samples according to the detection method provided in the above kit, and whether there was a FLT3-ITD gene mutation in the clinical sample was judged from the comparison result of the sequencing peak map and the wild-type FLT3 gene sequence, that is, the FLT3-ITD gene mutation negative-positive was judged.
The detection results are shown in the following table 4, and it can be seen that FLT3-ITD gene mutation exists in AML clinical bone marrow sample DNA with the numbers of No.1, No.2, No.3 and No.4, and the AML clinical bone marrow sample DNA is a FLT3-ITD gene mutation positive sample; the DNA of AML clinical bone marrow sample No.5 has no FLT3-ITD gene mutation and is negative to FLT3-ITD mutation. The PCR preliminary judgment results of the samples numbered No.1, No.2, No.3 and No.5 are consistent with the Sanger sequencing result, and the PCR preliminary judgment result of the sample numbered No.4 is not obvious, but is confirmed to be a positive sample of FLT3-ITD gene mutation after Sanger sequencing.
Therefore, the kit provided by the invention can be used for carrying out FLT3-ITD gene mutation detection on clinical samples of AML or MDS bone marrow or peripheral blood, and mutual mapping between a PCR primary judgment result and a Sanger sequencing result can be formed in the detection method, so that the detection method provided by the invention has higher accuracy, and can play an important role in AML or MDS prognosis judgment and relapse, AML or MDS targeted drug screening, identification of FLT3-ITD mutant cell strains or vectors and other processes.
Table 4: examination of bone marrow samples from 5 AML patients
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (9)
1. The primer group for detecting FLT3-ITD gene mutation is characterized by comprising an upstream primer, a downstream primer and a sequencing primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.1 in a sequence table, the nucleotide sequence of the downstream primer is shown as SEQ ID NO.2 in the sequence table, and the nucleotide sequence of the sequencing primer is shown as SEQ ID NO.3 in the sequence table.
2. A kit for detecting a mutation in FLT3-ITD gene, comprising the primer set according to claim 1.
3. The kit according to claim 2, wherein the final concentration of each of the forward primer and the reverse primer is 0.1 to 1. mu.M when the kit is used.
4. The kit according to claim 2 or 3, further comprising a positive control and a negative control, wherein the positive control is DNA of a cell line positive for mutation of FLT3-ITD gene, and the negative control is a system not containing mutation of FLT3-ITD gene.
5. Use of the primer set of claim 1 or the kit of any one of claims 2 to 4 for the detection of FLT3-ITD gene mutations for non-disease diagnostic purposes.
6. A method for detecting non-disease diagnostic purposes of FLT3-ITD gene mutation by Sanger sequencing, comprising the steps of:
1) carrying out PCR amplification on a sample DNA to be detected by using the upstream primer and the downstream primer according to claim 1 to obtain a PCR product;
2) performing a sequencing reaction by using the sequencing primer of claim 1 and the PCR product obtained in step 1);
3) purifying and denaturing a sequencing reaction product, sequencing, and obtaining a sequencing peak image;
4) analyzing the sequencing peak map, and judging whether FLT3-ITD gene mutation exists in the DNA of the sample to be detected according to the following standards:
(A) if the sequencing peak map has double peaks different from the background signal, determining that FLT3-ITD gene mutation exists in the DNA of the sample to be detected;
(B) if the sequencing peak diagram is a single peak, judging that FLT3-ITD gene mutation exists in the DNA of the sample to be detected when an insertion sequence exists in the sequencing result of the sample to be detected and the wild FLT3 gene sequence in a comparison manner; when the sequencing result of the sample to be detected is consistent with the wild FLT3 gene sequence, FLT3-ITD gene mutation does not exist in the DNA of the sample to be detected.
7. The method according to claim 6, wherein after obtaining the PCR product in step 1), the method further comprises a step of performing gel electrophoresis on the PCR product to preliminarily determine whether FLT3-ITD gene mutation exists in the sample DNA to be tested, and the determination criteria are as follows: if two strips appear in the gel electrophoresis image, preliminarily judging that FLT3-ITD gene mutation exists in the DNA of the sample to be detected; and if the gel electrophoresis image only has one strip, preliminarily judging that the FLT3-ITD gene mutation does not exist in the DNA of the sample to be detected.
8. The method according to claim 6, wherein the concentrations of the forward primer and the backward primer in the reaction system of the PCR amplification in step 1) are 0.1-1 μ M, respectively.
9. The method according to any one of claims 6 to 8, wherein the test sample in step 1) includes, but is not limited to, a bone marrow or peripheral blood clinical sample from an acute myeloid leukemia patient (AML) or myelodysplastic syndrome (MDS) patient, an AML or MDS targeted drug screening model cell sample, or an artificially constructed vector sample containing a mutation in FLT3-ITD gene.
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| CN106566875A (en) * | 2016-09-20 | 2017-04-19 | 上海荻硕贝肯医学检验所有限公司 | Primers, kit and method for detecting myelodysplastic syndromes (MDS) gene mutation |
| CN108374044A (en) * | 2018-01-04 | 2018-08-07 | 广州金域医学检验集团股份有限公司 | Primer group, kit and method for detecting c-kit gene mutation |
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| CN106566875A (en) * | 2016-09-20 | 2017-04-19 | 上海荻硕贝肯医学检验所有限公司 | Primers, kit and method for detecting myelodysplastic syndromes (MDS) gene mutation |
| CN108374044A (en) * | 2018-01-04 | 2018-08-07 | 广州金域医学检验集团股份有限公司 | Primer group, kit and method for detecting c-kit gene mutation |
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