CN112410407A - Primer group for simultaneously detecting two or three MPN pathogenic gene mutations and kit containing primer group - Google Patents
Primer group for simultaneously detecting two or three MPN pathogenic gene mutations and kit containing primer group Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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Abstract
The invention belongs to the technical field of gene detection, and particularly relates to a primer group for simultaneously detecting two or three MPN pathogenic gene mutations and a kit containing the primer group. The primer group comprises two pairs of primers, wherein the first pair of primers is JAK2V 617F-F and JAK2V617F-R, the second pair of primers is MPL-F and MPL-R, the sequence of the JAK2V 617F-F is shown in SEQ ID NO.1, and the sequence of the JAK2V617F-R is shown in SEQ ID NO. 2; the sequence of MPL-F is shown in SEQ ID NO.3, and the sequence of MPL-R is shown in SEQ ID NO. 4. The invention has the advantages that the three pairs of specific primers or the kit containing the three pairs of specific primers provided by the invention can quickly and accurately distinguish which mutation of the DNA sample to be detected belongs to JAK2V617F, MPL W515L/K and CALR I/II in one PCR.
Description
Technical Field
The invention belongs to the technical field of gene detection, and particularly relates to a primer group for simultaneously detecting two or three MPN (propylene glycol fatty acid) pathogenic gene mutations and a kit containing the primer group, in particular to a primer group for simultaneously detecting two or three gene mutations in JAK2V617F, MPL W515L/K and CALR I/II and a kit containing the primer group.
Background
Myeloproliferative neoplasms (MPNs) are a class of clonal hematopoietic stem cell diseases characterized primarily by the proliferation of one or more myeloid lineages, including erythroid, granulometric and megakaryoid lineages. Mainly comprises chronic myelogenous leukemia, Polycythemia Vera (PV), Essential Thrombocythemia (ET), and Primary Myelofibrosis (PMF). The excessive proliferation of blood cells of patients can cause hyperviscosity blood, and the blood cells become high-risk factors of various cardiovascular and cerebrovascular diseases. Early risk factors for PV and ET are thromboembolic disorders, mainly myocardial and cerebral infarctions, followed by hemorrhage and intermittent claudication; advanced stages may manifest as bone marrow failure; the acute leukemia, which is the most acute leukemia in 10% -20% of patients, is the myeloid lineage.
MPN-restricted driver genes include JAK2, Calreticulin (CALR) and myeloproliferative leukemia virus (MPL) mutations, which result in abnormal activation of the JAK2/STAT pathway and downstream pathways, causing hyperproliferation of blood cells. The most common mutation, JAK2V617F, activates 3 major myeloid cytokine receptors (erythropoietin receptor, granulocyte colony stimulating factor receptor, and MPL), whereas the CALR or MPL mutations are limited to activation of MPL.
The JAK2V617F mutation is a mutation of the guanine point on exon 14 of the JAK2 gene to thymine (c.1849gnt, GTC → TTC) such that the valine at position 617 of JH2 of JAK2 misencodes phenylalanine. Found in more than 95% PV, 50-60% ET and 55-60% PMF patients (Klampfl T, Gisslinger H, Harutyunyun AS, ET al. organic syndromes of calcium in myeloproliferative neoplasms. N Engl J Med.2013; 369(25):2379-90.)
The somatic mutation of MPL515 was encoded by missense of amino acid 515 in exon 10, the two most common mutations being W515L and W515K, found in 5% ET and 10% PMF (Vannucchi AM, Antonioli E, Guglielmeli P, ET al, Characteristics and clinical certificates of MPL515 WL/K mutation in infectious tissue, which is blood, 2009; 112: 844-7.).
The CALR mutation is seen in 60% to 80% of patients with ET that are mutation-negative for JAK2 and 80% to 88% of patients with PMF that are mutation-negative for JAK2 and MPL, rarely occurs in PV patients, and rarely coexists with the JAK2V617F mutation. The two most common mutations are a deletion of 52 base pairs (p.L367fs 46, type I) and an insertion of 5 base pairs (p.K385fs 47, type II) in exon 9, which form 85% to 90% of all CALR mutations (Nangalia J, Massie CE, Baxter EJ, et al. acidic CALR mutations in myeloablative neural networks with nonmutated JAK2.N Engl. Med. 2013; 369(25): 2391) 405).
The technology applied to detecting JAK2, CALR and MPL gene mutation is various, such AS allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), PCR-denaturation high-efficiency liquid chromatography, PCR-HRM, digital PCR, Sanger sequencing, second-generation sequencing and the like. Most of these methods are based on PCR reactions, and only the differences exist in primer design and product analysis means. The second-generation sequencing, digital PCR and chip technology will play an increasingly greater role in molecular diagnosis of MPN, and the PCR-HRM detection sensitivity can be comparable to or even exceed that of some current SNP mutation analysis technologies, is greatly higher than that of sequencing, and is a rapid, closed-tube and low-cost PCR amplification detection technology. Therefore, a product capable of detecting JAK2V617F, MPL W515L/K and CALR mutation rapidly, in a closed tube and at low cost is urgently needed to be developed.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention aims to provide a primer set capable of detecting two or three MPN pathogenic gene mutations rapidly, in a closed tube manner and at a low cost, and a kit comprising the primer set, and in particular, to a primer set capable of detecting two or three gene mutations in JAK2V617F, MPL W515L/K and CALR I/II simultaneously, and a kit comprising the primer set.
The technical scheme adopted by the invention is as follows:
the invention provides a primer group for simultaneously detecting JAK2V617F and MPL W515L/K gene mutation, which comprises two pairs of primers, wherein the first pair of primers is JAK2V 617F-F and JAK2V617F-R, the second pair of primers is MPL-F and MPL-R, the sequence of the JAK2V 617F-F is shown as SEQ ID NO.1, and the sequence of the JAK2V617F-R is shown as SEQ ID NO. 2; the sequence of MPL-F is shown in SEQ ID NO.3, and the sequence of MPL-R is shown in SEQ ID NO. 4.
The invention provides a primer group for simultaneously detecting JAK2V617F and CALR I/II gene mutation, which comprises two pairs of primers, wherein the first pair of primers is JAK2V 617F-F and JAK2V617F-R, the second pair of primers is CALR-F and CALR-R, the sequence of the JAK2V 617F-F is shown as SEQ ID NO.1, the sequence of the JAK2V617F-R is shown as SEQ ID NO.2, the sequence of the CALR-F is shown as SEQ ID NO.5, and the sequence of the CALR-R is shown as SEQ ID NO. 6.
The invention provides a primer group for simultaneously detecting MPL W515L/K and CALR I/II gene mutation, which comprises two pairs of primers, wherein the first pair of primers are MPL-F and MPL-R, the second pair of primers are CALR-F and CALR, the sequence of MPL-F is shown in SEQ ID NO.3, the sequence of MPL-R is shown in SEQ ID NO.4, the sequence of CALR-F is shown in SEQ ID NO.5, and the sequence of CALR-R is shown in SEQ ID NO. 6.
The invention provides a primer group for simultaneously detecting JAK2V617F, MPL W515L/K and CALR I/II gene mutation, which comprises three pairs of primers, wherein the first pair of primers is JAK2V 617F-F and JAK2V617F-R, the second pair of primers is MPL-F and MPL-R, the third pair of primers is CALR-F and CALR-R, the sequence of the JAK2V 617F-F is shown in SEQ ID NO.1, and the sequence of the JAK2V617F-R is shown in SEQ ID NO. 2; the sequence of the MPL-F is shown as SEQ ID NO.3, the sequence of the MPL-R is shown as SEQ ID NO.4, the sequence of the CALR-F is shown as SEQ ID NO.5, and the sequence of the CALR-R is shown as SEQ ID NO. 6.
The invention provides a reagent kit for simultaneously detecting JAK2V617F and MPL W515L/K gene mutation, which comprises the primer group, a PCR amplification reagent, a positive quality control product and a negative quality control product, wherein the positive quality control product is DNA of DL17D583 and MPL W515L/K, and the negative quality control product is DNA of a healthy person.
The invention provides a kit for simultaneously detecting MPL W515L/K and CALR I/II gene mutation, which comprises the primer group, a PCR amplification reagent, a positive quality control product and a negative quality control product, wherein the positive quality control product is DNA of MPL W515L/K and CALR I/II, and the negative quality control product is DNA of a healthy person.
The invention provides a kit for simultaneously detecting JAK2V617F and CALR I/II gene mutation, which comprises the primer group, a PCR amplification reagent, a positive quality control product and a negative quality control product, wherein the positive quality control product is DNA of DL17D583 and CALR I/II, and the negative quality control product is DNA of a healthy person.
The invention provides a kit for simultaneously detecting JAK2V617F, MPL W515L/K and/or CALR I/II gene mutation, which comprises the primer group, a PCR amplification reagent, a positive quality control product and a negative quality control product, wherein the positive quality control product is DNA of DL17D583, MPL W515L/K and CALR I/II, and the negative quality control product is DNA of a healthy person.
Specifically, the kit for simultaneously detecting two or three gene mutations in JAK2V617F, MPL W515L/K and CALR I/II is described above, and the PCR amplification reagent is 2X HRM PCR Master Mix.
The invention also provides application of the primer group in preparation of a kit for simultaneously detecting two or three gene mutations in JAK2V617F, MPL W515L/K and CALR I/II.
The invention has the beneficial effects that:
by using the three pairs of specific primers provided by the invention, the mutation of a DNA sample to be detected in JAK2V617F, MPL W515L/K and CALR I/II can be quickly and accurately distinguished in one PCR by using a multiple PCR-HRM technology, and the three pairs of specific primers can be applied to the preparation of a kit for simultaneously detecting two or three gene mutations in JAK2V617F, MPL W515L/K and CALR I/II so as to detect the mutation of the JAK2V617F, MPL W515L/K and CALR I/II on various suitable occasions.
Drawings
FIG. 1 is a peak diagram of PCR-HRM in example 4 of the present invention.
FIG. 2 is a peak diagram of PCR-HRM in example 5 of the present invention.
Detailed Description
The invention is further explained below with reference to the drawings and the specific embodiments.
Example 1
The purpose of this example is to provide a primer set for simultaneous detection of two or three of JAK2V617F, MPL W515L/K, and CALR I/II.
According to a human JAK2V617F gene mutation sequence, a human MPL W515L/K gene mutation sequence and a human CALR I/II gene mutation sequence disclosed in NCBI, three pairs of primers containing respective mutation sites are respectively designed according to a primer design principle, wherein the first pair of primers are JAK2V 617F-F and JAK2V617F-R, and the sequences of the primers are respectively shown as SEQ ID NO.1 and SEQ ID NO.2 in Table 1; the second pair of primers is MPL-F and MPL-R, the sequences of which are respectively shown as SEQ ID NO.3 and SEQ ID NO.4 in Table 1, and the third pair of primers is CALR-F and CALR-R, the sequences of which are respectively shown as SEQ ID NO.5 and SEQ ID NO.6 in Table 1. The three pairs of designed specific primers were sent to Invitrogen company for gene synthesis to obtain JAK2V 617F-F, JAK2V617F-R, MPL-F, MPL-R and CALR-F and CALR-R, respectively, and the obtained JAK2V 617F-F, JAK2V617F-R, MPL-F, MPL-R, CALR-F and CALR-R were confirmed by gene sequencing verification.
TABLE 1
Example 2
The purpose of this example is to provide a kit for simultaneous detection of two or three gene mutations in JAK2V617F, MPL W515L/K and CALR I/II.
A kit for simultaneously detecting two or three gene mutations in JAK2V617F, MPL W515L/K and CALR I/II comprises JAK2V 617F-F, JAK2V617F-R, MPL-F, MPL-R, CALR-F, CALR-R, 2X HRM PCR Master Mix, a positive quality control product and a negative quality control product, wherein the positive quality control product is DNA of DL17D583, MPL W515L/K and CALR I/II, and the negative quality control product is DNA of a healthy person.
DL17D583 was collected clinically, MPL W515L/K was synthesized by Invitrogen according to the human MPL W515L/K gene mutation sequence, and CALR I/II DNA was synthesized by Invitrogen according to the human CALR I/II gene mutation sequence.
Two pairs of primers and corresponding positive quality control products are selected randomly to be used as a kit for simultaneously detecting two gene mutations, and three pairs of primers and corresponding positive quality control products are selected to be used as a kit for simultaneously detecting three gene mutations.
Example 3
The purpose of this example is to verify the accuracy of a kit for simultaneously detecting two or three gene mutations in JAK2V617F, MPL W515L/K and CALR I/II, and to separately verify whether each pair of primers can sensitively distinguish the wild type and mutant samples by using a single-tube PCR-HRM technique.
1. Preparation of DNA sample: JAK2V617F mutation specimen is clinical collection (DL17D583), a Lab-Aid 820 nucleic acid extractor is adopted, a Lab-Aid nucleic acid (DNA) separation kit (Xiamen high Biotechnology, Inc.) is matched for whole blood DNA extraction, the concentration of the obtained DNA nucleic acid is controlled to be 20-40 ng/mu l, OD260/280 is between 1.8-2.0, and the DNA is frozen and stored in a low-temperature freezing refrigerator at-80 ℃; MPL W515L/K, CALR I type mutation (c1092-1143del), II type mutation (c1154-1155ins) were synthesized by Invitrogen according to the mutation sequence of human MPL W515L/K gene and the mutation sequence of human CALR I/II gene disclosed in NCBI, and were frozen in plasmids and glycerobacteria containing the corresponding plasmids, plasmid DNA was extracted according to the procedure of plasmid extraction kit, the concentration of plasmid DNA nucleic acid was controlled at 20-40 ng/. mu.l, OD260/280 was between 1.8-2.0, and the cells were frozen at-80 ℃ in a low temperature freezer.
2. Preparing a specific primer:
primers JAK2V 617F-F and JAK2V617F-R, MPL-F and MPL-R and CALR-F and CALR-R, designed and verified as in example 1, were diluted to 0.3. mu.M/L with double distilled water.
3. Preparing a PCR reaction solution:
8 PCR reaction tubes are taken, numbered 1-8 PCR reaction tubes are sequentially numbered, the reagents of each component are respectively added into each PCR reaction tube according to the sample adding amount shown in the following table 2, wherein DNA samples to be detected in the 1-8 PCR reaction tubes are shown in the table 3, and JAK2V617F wild-type DNA, JAK2V617F mutant-type DNA, CALR I mutant-type DNA, CALR II mutant-type DNA, CALR wild-type DNA, MPL W515K mutant-type DNA, MPL W515L mutant-type DNA and MPL wild-type DNA are sequentially added into the 1-8 PCR reaction tubes. The final volume of each PCR reaction was set to 25. mu.L. In this example JAK2V617F, MPL and CALR represent JAK2V 617F-F and JAK2V617F-R, MPL-F and MPL-R and CALR-F and CALR-R, respectively.
TABLE 2
TABLE 3
4. PCR reaction
And (3) centrifuging and mixing the prepared PCR reaction solution uniformly, and carrying out PCR reaction according to the following conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing at a certain temperature for 30s, and extension at 72 ℃ for 20s for 45 cycles; extension at 72 ℃ for 5 min. The annealing temperatures for the individual PCR primers are shown in Table 1.
After completion of PCR amplification, HRM was performed using HR-1 high detection rate melting curve analysis system (Idaho Technology, USA) in combination with PCR capillary (Daizhiki corporation). HRM system: mu.l of the amplification product, blocked with 10. mu.l of paraffin oil (Sigma, USA), and the melting curve fluorescence signal collection temperature was set at 65 ℃ to 98 ℃ at 0.5 ℃/s. The resulting melting curves were analyzed using the matched software HR-1 analysis tool.
5. Detection of
The results of PCR for each gene alone are shown in FIG. 1, where the curves labeled 1 to 8 show the melting peak of JAK2V617F primer for detecting the melting peak of JAK2V617F wild-type DNA, JAK2V617F primer for detecting the melting peak of JAK2V617F mutant DNA, CALR primer for detecting the melting peak of CALR I mutant DNA, CALR primer for detecting the melting peak of CALR II mutant DNA, CALR primer for detecting the melting peak of CALR wild-type DNA, MPL primer for detecting the melting peak of MPL W515K mutant DNA, MPL primer for detecting the melting peak of MPL W515L mutant DNA, and MPL primer for detecting the melting peak of MPL wild-type DNA, respectively.
As shown in FIG. 1, the difference between the melting temperatures of the JAK2V617F wild type and mutant type is about 1.5 ℃, the difference between the melting temperatures of CALR wild type and I type and II type mutation peaks is about 2 ℃ and 0.2 ℃, the difference between the melting temperatures of MPL wild type and mutant type is about 1 ℃, and the difference between the melting temperatures of two mutant types is about 0.1 ℃, which indicates that the wild type and mutant type of each gene can be separated, i.e., the wild type and mutant type of three genes can be identified by the three pairs of primers.
Example 4
The purpose of this example is to verify the accuracy of a kit for simultaneous detection of two or three mutations in JAK2V617F, MPL W515L/K and CALR I/II. Namely, the method verifies whether the three pairs of primers can sensitively distinguish the mutation of three different genes and the wild type and mutant type samples of the same gene when the final concentration of the primer reaction is 0.3 mu M/L and the annealing temperature is 55 ℃.
1. Preparation of DNA sample: JAK2V617F mutation specimen is clinical collection (DL17D583), a Lab-Aid 820 nucleic acid extractor is adopted, a Lab-Aid nucleic acid (DNA) separation kit (Xiamen high Biotechnology, Inc.) is matched for whole blood DNA extraction, the concentration of the obtained DNA nucleic acid is controlled to be 20-40 ng/mu l, OD260/280 is between 1.8-2.0, and the DNA is frozen and stored in a low-temperature freezing refrigerator at-80 ℃; MPL W515L/K, CALRI type mutation (c1092-1143del), II type mutation (c1154-1155ins) are synthesized by Invitrogen company according to the mutation sequence of human MPL W515L/K gene and the mutation sequence of human CALR I/II gene disclosed on NCBI, and are frozen and stored in plasmid and glycerol bacteria containing corresponding plasmid, plasmid DNA is extracted according to the operation steps of a plasmid extraction kit, the nucleic acid concentration of the plasmid DNA is controlled to be 20-40 ng/mu l, OD260/280 is between 1.8-2.0, and the plasmid DNA is frozen and stored in a low-temperature freezing refrigerator at-80 ℃;
2. preparing a specific primer:
primers JAK2V 617F-F and JAK2V617F-R, MPL-F and MPL-R and CALR-F and CALR-R, designed and verified as in example 1, were diluted to 0.3. mu.M/L with double distilled water.
3. Preparing a PCR reaction solution:
three pairs of primers and other reagents were added to 1 PCR reaction tube in the amounts shown in Table 4. Wherein the DNA sample comprises JAK2V617F wild type DNA, JAK2V617F mutant DNA, CALR I type mutant DNA, CALR II type mutant DNA, CALR wild type DNA, MPL W515K type mutant DNA, MPL W515L type mutant DNA and MPL wild type DNA. The sample adding amount of the 2X HRM PCR Master Mix and the primers is constant, and the sample adding amount of the double distilled water is adjusted to control the total volume of the PCR reaction solution to be 25 mu L. In this example JAK2V617F, MPL and CALR represent JAK2V 617F-F and JAK2V617F-R, MPL-F and MPL-R and CALR-F and CALR-R, respectively.
TABLE 4
4. PCR reaction
And (3) centrifuging and mixing the prepared PCR reaction solution uniformly, and carrying out PCR reaction according to the following conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 20s for 45 cycles; extension at 72 ℃ for 5 min.
After completion of PCR amplification, HRM was performed using HR-1 high detection rate melting curve analysis system (Idaho Technology, USA) in combination with PCR capillary (Daizhiki corporation). HRM system: mu.l of the amplification product, blocked with 10. mu.l of paraffin oil (Sigma, USA), and the melting curve fluorescence signal collection temperature was set at 65 ℃ to 98 ℃ at 0.5 ℃/s. The resulting melting curves were analyzed using the matched software HR-1 analysis tool.
5. Detection of
The multiplex PCR-HRM result is shown in FIG. 1, wherein a yellow curve in the graph shows that three different peak values exist, the mark 1 is a JAK2V617 peak, the mark indicates that the dissolution temperature of JAK2V617 is near 78 ℃, the mark 2 is a CALR peak, the mark indicates that the dissolution temperature of CALR is near 82 ℃, the mark 3 is an MPL peak, the mark indicates that the dissolution temperature of MPL is near 90 ℃, and the dissolution temperature difference of primers of three genes is above 4 ℃, which indicates that three pairs of primers can be completely separated in a one-tube multiple PCR-HRM system, thereby indicating that the detection result of the kit is accurate and reliable.
When any two gene mutations of JAK2V617F, MPL W515L/K and CALR I/II need to be detected, adding two corresponding pairs of primers when preparing PCR reaction liquid, namely, simultaneously detecting the two gene mutations; when three gene mutations of JAK2V617F, MPL W515L/K and CALR I/II need to be detected, three pairs of primers are added to prepare PCR reaction liquid, so that the three gene mutations can be simultaneously detected, the sample adding amounts of a DNA sample and a 2X HRM PCR Master Mix are fixed, the total volume of the PCR reaction liquid is controlled to be 25 mu L, when the sample adding amount of the primers is reduced, the sample adding amount of double distilled water is increased, and when the sample adding amount of the primers is increased, the sample adding amount of the double distilled water is reduced.
Example 5
The purpose of this example is to verify the accuracy of a kit for simultaneous detection of two or three mutations in JAK2V617F, MPL W515L/K and CALR I/II. Namely, the method verifies whether the three pairs of primers can sensitively distinguish the mutation of three different genes and the wild type and mutant type samples of the same gene when the final concentration of the primer reaction is 0.4 mu M/L and the annealing temperature is 55 ℃.
1. Preparation of DNA sample: JAK2V617F mutation specimen is clinical collection (DL17D583), a Lab-Aid 820 nucleic acid extractor is adopted, a Lab-Aid nucleic acid (DNA) separation kit (Xiamen high Biotechnology, Inc.) is matched for whole blood DNA extraction, the concentration of the obtained DNA nucleic acid is controlled to be 20-40 ng/mu l, OD260/280 is between 1.8-2.0, and the DNA is frozen and stored in a low-temperature freezing refrigerator at-80 ℃; MPL W515L/K, CALRI type mutation (c1092-1143del), II type mutation (c1154-1155ins) are synthesized by Invitrogen company according to the mutation sequence of human MPL W515L/K gene and the mutation sequence of human CALR I/II gene disclosed on NCBI, and are frozen and stored in plasmid and glycerol bacteria containing corresponding plasmid, plasmid DNA is extracted according to the operation steps of a plasmid extraction kit, the nucleic acid concentration of the plasmid DNA is controlled to be 20-40 ng/mu l, OD260/280 is between 1.8-2.0, and the plasmid DNA is frozen and stored in a low-temperature freezing refrigerator at-80 ℃;
2. preparing a specific primer:
primers JAK2V 617F-F and JAK2V617F-R, MPL-F and MPL-R and CALR-F and CALR-R, designed and verified as in example 1, were diluted to 0.3. mu.M/L with double distilled water.
3. Preparing a PCR reaction solution:
1 PCR reaction tube was taken, and each component reagent was added to each PCR reaction tube in the amount indicated in Table 5. Wherein the DNA sample comprises JAK2V617F wild type DNA, JAK2V617F mutant DNA, CALR I type mutant DNA, CALR II type mutant DNA, CALR wild type DNA, MPL W515K type mutant DNA, MPL W515L type mutant DNA and MPL wild type DNA. The sample adding amount of the 2X HRM PCR Master Mix and the primers is constant, and the sample adding amount of the double distilled water is adjusted to control the total volume of the PCR reaction solution to be 25 mu L. In this example JAK2V617F, MPL and CALR represent JAK2V 617F-F and JAK2V617F-R, MPL-F and MPL-R and CALR-F and CALR-R, respectively.
TABLE 5
4. PCR reaction
And (3) centrifuging and mixing the prepared PCR reaction solution uniformly, and carrying out PCR reaction according to the following conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 20s for 45 cycles; extension at 72 ℃ for 5 min.
After completion of PCR amplification, HRM was performed using HR-1 high detection rate melting curve analysis system (Idaho Technology, USA) in combination with PCR capillary (Daizhiki corporation). HRM system: mu.l of the amplification product, blocked with 10. mu.l of paraffin oil (Sigma, USA), and the melting curve fluorescence signal collection temperature was set at 65 ℃ to 98 ℃ at 0.5 ℃/s. The resulting melting curves were analyzed using the matched software HR-1 analysis tool.
5. Detection of
The multiplex PCR-HRM result is shown in FIG. 2, the purple red curve in the figure shows that three different peak values exist, the label is 1, the JAK2V617 peak indicates that the dissolving temperature of JAK2V617 is near 78 ℃, the label is 2, the CALR peak indicates that the dissolving temperature of CALR is near 82 ℃, the label is 3, the MPL peak indicates that the dissolving temperature of MPL is near 90 ℃, and the difference of the dissolving temperatures of the primers of three genes is above 4 ℃, which indicates that the three pairs of primers can be completely separated in a one-tube multiple PCR-HRM system, thereby indicating that the detection result of the kit is accurate and reliable.
When any two gene mutations of JAK2V617F, MPL W515L/K and CALR I/II need to be detected, adding two corresponding pairs of primers when preparing PCR reaction liquid, namely, simultaneously detecting the two gene mutations; when three gene mutations of JAK2V617F, MPL W515L/K and CALR I/II need to be detected, three pairs of primers are added to prepare PCR reaction liquid, so that the three gene mutations can be simultaneously detected, the sample adding amounts of a DNA sample and a 2X HRM PCR Master Mix are fixed, the total volume of the PCR reaction liquid is controlled to be 25 mu L, when the sample adding amount of the primers is reduced, the sample adding amount of double distilled water is increased, and when the sample adding amount of the primers is increased, the sample adding amount of the double distilled water is reduced.
Effect example 1
The purpose of this effect example is to provide the detection results of a kit for simultaneously detecting two or three gene mutations in JAK2V617F, MPL W515L/K and CALR I/II.
1. Preparing DNA to be detected: collecting EDTA anticoagulation blood of MPN patients to be diagnosed, adopting a Lab-Aid 820 nucleic acid extractor, matching with a Lab-Aid nucleic acid (DNA) separation kit (Xiamen-derived good biotechnology, Inc.) to extract whole blood DNA, controlling the concentration of the obtained DNA nucleic acid to be 20-40 ng/mu l, controlling the OD260/280 to be between 1.8-2.0, and freezing and storing in a low-temperature freezing refrigerator at-80 ℃.
2. Preparing a PCR reaction solution:
taking 2 PCR reaction tubes, wherein the numbers are 1 and 2, respectively adding each component reagent into each PCR reaction tube according to the sample adding amount shown in table 4 or table 5, and adding three pairs of primers into each PCR reaction tube. Wherein the DNA sample added in the PCR with the number of 1 comprises JAK2V617F wild-type DNA, JAK2V617F mutant-type DNA, CALR I type mutant DNA, CALR II type mutant DNA, CALR wild-type DNA, MPL W515K type mutant DNA, MPL W515L type mutant DNA and MPL wild-type DNA, and the DNA sample added in the PCR with the number of 2 is DNA to be detected. The sample adding amount of the 2X HRM PCR Master Mix and the primers is constant, and the sample adding amount of double distilled water is adjusted to control the total volume of the PCR reaction solution to be 25 mu L.
4. PCR reaction
And (3) centrifuging and mixing the prepared PCR reaction solution uniformly, and carrying out PCR reaction according to the following conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 10s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 20s for 45 cycles; extension at 72 ℃ for 5 min.
After completion of PCR amplification, HRM was performed using HR-1 high detection rate melting curve analysis system (Idaho Technology, USA) in combination with PCR capillary (Daizhiki corporation). HRM system: mu.l of the amplification product, blocked with 10. mu.l of paraffin oil (Sigma, USA), and the melting curve fluorescence signal collection temperature was set at 65 ℃ to 98 ℃ at 0.5 ℃/s. The resulting melting curves were analyzed using the matched software HR-1 analysis tool.
5. Detection of
The present invention is not limited to the above-described alternative embodiments, and various other forms of products can be obtained by anyone in light of the present invention. The above detailed description should not be taken as limiting the scope of the invention, which is defined in the following claims, and which description is intended to be interpreted accordingly.
Sequence listing
<110> lie prostrate
<120> primer set for simultaneously detecting two or three MPN pathogenic gene mutations and kit comprising the same
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> homo-JAK 2V 617F-F (homo sapiens)
<400> 1
<210> 4
<211> 23
<212> DNA
<213> homo-JAK 2V617F-R (homo sapiens)
<400> 4
agaaaggcat tagaaagcct gta 23
<210> 4
<211> 21
<212> DNA
<213> homo-MPL-F (homo sapiens)
<400> 4
tctcctagcc tggatctcct t 21
<210> 4
<211> 19
<212> DNA
<213> homo-MPL-R (homo sapiens)
<400> 4
cagagcgaac caagaatgc 19
<210> 5
<211> 20
<212> DNA
<213> homo-CALR-F (homo sapiens)
<400> 5
<210> 6
<211> 20
<212> DNA
<213> homo-CALR-R (homo sapiens)
<400> 6
Claims (10)
1. The primer group for simultaneously detecting JAK2V617F and MPL W515L/K gene mutation is characterized in that: the primer group comprises two pairs of primers, wherein the first pair of primers is JAK2V 617F-F and JAK2V617F-R, the second pair of primers is MPL-F and MPL-R, the sequence of the JAK2V 617F-F is shown in SEQ ID NO.1, and the sequence of the JAK2V617F-R is shown in SEQ ID NO. 2; the sequence of MPL-F is shown in SEQ ID NO.3, and the sequence of MPL-R is shown in SEQ ID NO. 4.
2. The primer group for simultaneously detecting JAK2V617F and CALR I/II gene mutation is characterized in that: the primer group comprises two pairs of primers, wherein the first pair of primers is JAK2V 617F-F and JAK2V617F-R, the second pair of primers is CALR-F and CALR-R, the sequence of the JAK2V 617F-F is shown in SEQ ID NO.1, the sequence of the JAK2V617F-R is shown in SEQ ID NO.2, the sequence of the CALR-F is shown in SEQ ID NO.5, and the sequence of the CALR-R is shown in SEQ ID NO. 6.
3. The primer group for simultaneously detecting MPL W515L/K and CALR I/II gene mutation is characterized in that: the primer group comprises two pairs of primers, wherein the first pair of primers are MPL-F and MPL-R, the second pair of primers are CALR-F and CALR-R, the sequence of MPL-F is shown as SEQ ID NO.3, the sequence of MPL-R is shown as SEQ ID NO.4, the sequence of CALR-F is shown as SEQ ID NO.5, and the sequence of CALR-R is shown as SEQ ID NO. 6.
4. The primer group for simultaneously detecting JAK2V617F, MPL W515L/K and CALR I/II gene mutation is characterized in that: the primer group comprises three pairs of primers, wherein the first pair of primers is JAK2V 617F-F and JAK2V617F-R, the second pair of primers is MPL-F and MPL-R, the third pair of primers is CALR-F and CALR-R, the sequence of the JAK2V 617F-F is shown in SEQ ID NO.1, and the sequence of the JAK2V617F-R is shown in SEQ ID NO. 2; the sequence of the MPL-F is shown as SEQ ID NO.3, the sequence of the MPL-R is shown as SEQ ID NO.4, the sequence of the CALR-F is shown as SEQ ID NO.5, and the sequence of the CALR-R is shown as SEQ ID NO. 6.
5. A kit for simultaneously detecting JAK2V617F and MPL W515L/K gene mutation is characterized in that: the kit comprises the primer group, a PCR amplification reagent, a positive quality control product and a negative quality control product, wherein the positive quality control product is DNA of DL17D583 and MPL W515L/K, and the negative quality control product is DNA of a healthy person.
6. A kit for simultaneously detecting MPL W515L/K and CALR I/II gene mutation is characterized in that: the kit comprises the primer group, a PCR amplification reagent, a positive quality control product and a negative quality control product, wherein the positive quality control product is DNA of MPL W515L/K and CALR I/II, and the negative quality control product is DNA of a healthy person.
7. A kit for simultaneously detecting JAK2V617F and CALR I/II gene mutation is characterized in that: the kit comprises the primer group, a PCR amplification reagent, a positive quality control product and a negative quality control product, wherein the positive quality control product is DNA of DL17D583 and CALR I/II, and the negative quality control product is DNA of a healthy person.
8. A kit for simultaneously detecting JAK2V617F, MPL W515L/K and CALR I/II gene mutation is characterized in that: the kit comprises the primer group, a PCR amplification reagent, a positive quality control product and a negative quality control product, wherein the positive quality control product is DNA of DL17D583, MPL W515L/K and CALR I/II, and the negative quality control product is DNA of a healthy person.
9. The kit according to any one of claims 5 to 8, characterized in that: the PCR amplification reagent is 2X HRM PCR Master Mix.
10. Use of the primer set of any one of claims 1 to 4 in the preparation of a kit for simultaneously detecting mutations in two or three genes selected from JAK2V617F, MPL W515L/K and CALR I/II.
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