CN107164474B - Primer composition and kit for detecting CALR gene type 2 mutation - Google Patents
Primer composition and kit for detecting CALR gene type 2 mutation Download PDFInfo
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Abstract
The invention relates to a primer composition for detecting CALR gene type 2 mutation, which comprises a primer containing SEQ ID NO: 2-SEQ ID NO: 7; the invention also relates to a kit containing the primer composition, a detection method thereof, and a CALR gene type 2 mutant target sequence amplified by the primer composition, wherein the target sequence is SEQ ID NO: 1, and (b) is shown in the specification. The kit and the detection method thereof have good specificity and stability, samples with gradient mutation load concentration are amplified by the established LAMP reaction system, the detection sensitivity of the mutation load can reach 3 percent, the detection sensitivity is almost equivalent to the real-time PCR effect of a probe method, and the kit can be used for visually detecting CALR gene type 2 mutation.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a target sequence, an amplification composition and a kit for detecting CALR gene type 2 mutation.
Background
Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem/progenitor cell diseases in which one or more myeloid lineage cells proliferate in the bone marrow and mature and immature cells increase in the peripheral blood. In recent years, a plurality of molecular markers, such as JAK2, MPL, TET mutation and the like, are discovered successively, and the discovery of the markers has important significance for understanding the molecular pathogenesis of MPN and is also helpful for diagnosing and treating patients with the diseases. However, 30-45% of patients with primary thrombocythemia (ET) or Primary Myelofibrosis (PMF) carrying wild-type JAK2/MPL still have diagnosis difficulty, and the newly reported calreticulin gene (CALR) mutation can partially fill the gap and is expected to become a new molecular marker for diagnosing myeloproliferative tumors.
CALR is a multifunctional calcium ion binding and storage protein chaperone located mainly in the endoplasmic reticulum, and participates in the regulation of cell proliferation, apoptosis, adhesion, immunity, etc. by assisting correct protein folding and maintaining cellular calcium ion homeostasis. The CALR gene maps to chromosome 19p13.2, which has 9 exons and 9 protein domains, and mutations in the CALR gene are caused by insertions and deletions of the ninth exon, resulting in a one base pair frame shift, which in turn produces a novel C-carboxy terminal protein with a lack of endoplasmic reticulum retention sequences (KDEL amino acid sequences), and to date, more than 40 different CALR mutants have been detected, the two most common variants being: type 1 variants resulting from deletion of 52 bases (p.l367fs x 46) and type 2 variants resulting from insertion of 5 bases TTGTC (p.k385fs x 47), which account for 53% and 32% of all mutants, respectively, investigators found in vitro experiments that the overexpressed type 1 mutants enhanced interleukin-3 expression, also promoted STAT5 phosphorylation and caused activation of signaling, which could be blocked by JAK inhibitors, suggesting that CALR and JAK2 mutations may have similar mechanisms.
The detection of the CALR gene mutation at present mainly depends on a probe real-time PCR method or a sequencing method and the like, the method is time-consuming and labor-consuming, long in report period, and has defects in sensitivity, required detection equipment is expensive, special equipment and trained technicians are needed during detection, the requirement on a technical platform is high, and the method is not easy to popularize widely.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and establish a Loop-Mediated Isothermal Amplification (LAMP) system for rapidly detecting CALR gene type 2 mutation.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a primer composition for detecting CALR gene 2 type mutation target sequence, which comprises SEQ ID NO: 2-SEQ ID NO: 7.
The invention also provides a kit for detecting CALR gene type 2 mutation, which contains the primer composition.
Preferably, the kit further comprises a reaction buffer solution, Bst DNA polymerase, calcein, deionized water and a DNA template.
Preferably, the kit comprises 25. mu.L of an amplification reaction system comprising 12.5. mu.L of 2 × reaction buffer, SEQ ID NO: 2 to SEQ ID NO: 4, 0.5 μ L of each primer in the sequence shown in SEQ ID NO: 5 to SEQ ID NO: 7, 1. mu.L of each primer, 1. mu.L of Bst DNA polymerase, 1. mu.L of calcein, 4.0. mu.L of deionized water and 2. mu.L of DNA template.
Preferably, in the kit, SEQ ID NO: 2 to SEQ ID NO: 3, the final concentration of each primer in the sequence shown in the sequence is 0.2 mu mol/L; SEQ ID NO: 4, the final concentration of the primers of the sequence is 0.8 mu mol/L; SEQ ID NO: 5, the final concentration of the primers is 1.6 mu mol/L; SEQ ID NO: 6 to SEQ ID NO: 7, the final concentration of each primer in the sequence shown in 7 is 0.8 mu mol/L; .
The invention also provides a method for detecting CALR gene 2 type mutation for non-diagnosis and non-treatment purposes, which uses the DNA template of the sample to be detected, adopts the primer composition to carry out LAMP reaction, and then carries out visual interpretation to identify whether the sample is positive to CALR gene 2 type mutation.
Preferably, the LAMP reaction conditions are: 60min at 65 ℃.
Preferably, the equipment used for the LAMP reaction is equipment capable of stably providing a constant temperature of 65 ℃, such as a common PCR instrument or a constant temperature metal bath.
Finally, the invention provides a CALR gene type 2 mutant target sequence amplified by the primer composition, which is expressed by SEQ ID NO: 1, and provides a recombinant plasmid containing the CALR gene 2-type mutant target sequence, wherein the recombinant plasmid contains a sequence shown in SEQ ID NO: 1 in sequence shown in figure 1.
The nucleotide sequences of the primers and the target sequence are shown in Table 1 below.
TABLE 1 base sequence table of primers and target sequences for detecting CALR gene type 2 mutation
Compared with the prior art, the invention has the following beneficial effects:
the kit and the loop-mediated isothermal amplification method for rapidly detecting the CALR gene 2 type mutation can be used for visually detecting the CALR gene 2 type mutation, and compared with the traditional detection method, the method has the advantages of low requirements on equipment and environment, higher detection speed and higher detection sensitivity.
Drawings
FIG. 1 is a schematic diagram showing the verification of the specificity of CALR gene type 2 mutant LAMP system;
the reference numbers in the figures are:
b1: clinical CALR-1 mutation positive genomic DNA; b2: clinical CALR-2 mutation positive genomic DNA; b3: clinical CALR wild-type genomic DNA; b4: CALR-1 mutation positive plasmid; b5: CALR-2 mutation positive plasmid; b6: CALR wild-type plasmid; b7: blank control.
Detailed Description
The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.
Example 1
This example shows the target sequence of the CALR gene type 2 mutation used in the present invention, and primers for detecting the CALR gene type 2 mutation.
The target fragment to be detected was selected around the position of CALR gene type 2 mutation, and appropriate specific primers were designed for the target fragment using the online LAMP primer design website PrimeExplorer V5(https:// PrimeExplorer. jp/lampv5/index. html).
(1) The primer sequence is as follows:
F3：TCCTGGTCCTGATGTCGG(SEQ ID NO：2)；
B3：CTCCTCATCCTCCTCATCCT(SEQ ID NO：3)；
FIP：CCTCGTCCTGTTTGTCCTTCATTTGTTTCAAGGCCCTGAGGTGTGT(SEQ ID NO：4)
BIP：GAGGCTTAAGGAGGAGGAAGAAGACACTTTGTCCTCATCATCCTCCGAC(SEQ ID NO：5)；
LF：TGCCTGCAGGCAGAGC(SEQ ID NO：6)；
LB：GAGGAGGCAGAGGACAAT(SEQ ID NO：7)。
(2) the target sequence is specifically:
TCCTGGTCCTGATGTCGGGGGCGGGCAGGGCTGGCAGGGGGCAAGGCCCTGAGGTGTGTGCTCTGCCTGCAGGCAGCAGAGAAACAAATGAAGGACAAACAGGACGAGGAGCAGAGGCTTAAGGAGGAGGAAGAAGACAAGAAACGCAAAGAGGAGGAGGAGGCAGAGGACAATTGTCGGAGGATGATGAGGACAAAGATGAGGATGAGGAGGATGAGGAG(221bp)(SEQ ID NO：1)。
example 2
This example is the kit and method for detecting CALR gene type 2 mutation of the present invention.
The kit adopted by the invention comprises an amplification reaction system, the total volume of the amplification reaction system is 25 mu L, and the amplification reaction system comprises 2 multiplied by 12.5 mu L of reaction buffer solution (RM), a primer F3: 0.5. mu.L (final concentration 0.2. mu. mol/L), primer B3: 0.5. mu.L (final concentration 0.2. mu. mol/L), primer FIP: 0.5. mu.L (final concentration 0.8. mu. mol/L), primer BIP: 1 μ L (final concentration 1.6 μmol/L), primer LF: 1 μ L (final concentration 0.8 μmol/L), primer LB: mu.L (final concentration 0.8. mu. mol/L), Bst DNA polymerase 1. mu.L (8U), calcein (FD) 1. mu.L, deionized water 4.0. mu.L, DNA template 2. mu.L.
The kit is adopted to carry out LAMP reaction, and the LAMP reaction conditions are as follows: 60min at 65 ℃; the used equipment is equipment which can stably provide constant temperature of 65 ℃ such as a common PCR instrument or a constant temperature metal bath; and then carrying out visual interpretation to identify whether the sample is positive to CALR gene type 2 mutation, specifically judging the result: after the reaction, the reaction solution was judged to be positive when the color of the reaction solution changed from pale yellow to green.
Constructing TA cloning plasmid containing target sequence, preparing gradient concentration double samples with specific concentration of 101copies/ml，102copies/ml，103copies/ml，104copies/ml，105copies/ml，106copies/ml，107copies/ml，108copies/ml, can be detected by using the established CALR gene type 2 mutation LAMP reaction system for detection; the specificity detection of the genomic DNA of the whole blood sample of the clinical CALR gene type 2 mutant patient and the constructed recombinant plasmid can be realized, and the non-specificity amplification of the genomic DNA of the whole blood sample of the clinical CALR gene type 1 mutant patient, the constructed recombinant plasmid or the wild type genomic DNA of a normal person and the constructed recombinant plasmid is avoided, so that the detection effect is good, and detailed picture 1 is shown.
293T cells into which CALR-2 type mutant plasmids were transferred and 293T cells into which wild type plasmids were transferred were 106Extracting DNA by using a genome DNA extraction kit, diluting the DNA in proportion to prepare a sample with gradient mutation load concentration, and amplifying the sample by using the established LAMP reaction system, wherein the detection sensitivity of the mutation load can reach 3 percent and is slightly lower than the real-time PCR effect of a probe method.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications or alterations to this practice will occur to those skilled in the art and are intended to be within the scope of this invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> Huashan Hospital affiliated with the university of Compound Dan; university of Compound Dan
(ii) a Shanghai Rapid diagnosis products Co Ltd
<120> primer composition for detecting CALR gene type 2 mutation and kit thereof
<160>7
<170>PatentIn version 3.5
<210>1
<211>221
<212>DNA
<213>Artificial Sequence
<220>
<223> target sequence
<400>1
tcctggtcct gatgtcgggg gcgggcaggg ctggcagggg gcaaggccct gaggtgtgtg 60
ctctgcctgc aggcagcaga gaaacaaatg aaggacaaac aggacgagga gcagaggctt 120
aaggaggagg aagaagacaa gaaacgcaaa gaggaggagg aggcagagga caattgtcgg 180
aggatgatga ggacaaagat gaggatgagg aggatgagga g 221
<210>2
<211>18
<212>DNA
<213>Artificial Sequence
<220>
<223> amplification primer F3
<400>2
tcctggtcct gatgtcgg 18
<210>3
<211>20
<212>DNA
<213>Artificial Sequence
<220>
<223> amplification primer B3
<400>3
ctcctcatcc tcctcatcct 20
<210>4
<211>46
<212>DNA
<213>Artificial Sequence
<220>
<223> amplification primer FIP
<400>4
cctcgtcctg tttgtccttc atttgtttca aggccctgag gtgtgt 46
<210>5
<211>49
<212>DNA
<213>Artificial Sequence
<220>
<223> amplification primer BIP
<400>5
gaggcttaag gaggaggaag aagacacttt gtcctcatca tcctccgac 49
<210>6
<211>16
<212>DNA
<213>Artificial Sequence
<220>
<223> amplification primer LF
<400>6
tgcctgcagg cagagc 16
<210>7
<211>18
<212>DNA
<213>Artificial Sequence
<220>
<223> amplification primer LB
<400>7
gaggaggcag aggacaat 18
Claims (6)
1. A kit for detecting CALR gene type 2 mutation is characterized by comprising SEQ ID NO: 2-SEQ ID NO: 7.
2. The kit for detecting CALR genotype 2 mutations as recited in claim 1, further comprising reaction buffer, BstDNA polymerase, calcein, and deionized water.
3. A method for detecting CALR gene type 2 mutation for non-diagnosis and non-treatment purposes, which is characterized in that a DNA template of a sample to be detected is adopted, a reaction system is prepared by adopting the kit of claim 1, LAMP reaction is carried out, and then visual interpretation is carried out to identify whether the sample is positive to CALR gene type 2 mutation.
4. The method for detecting CALR genotype 2 mutations according to claim 3, characterized in that the LAMP reaction conditions are: 60min at 65 ℃.
5. The method for detecting CALR genotype 2 mutations as recited in claim 3, wherein the reaction system comprises 12.5 μ L of 2 x reaction buffer, SEQ ID NO: 2 to SEQ ID NO: 4, 0.5 μ L of each primer in the sequence shown in seq id NO: 5 to SEQ ID NO: 7, 1. mu.L of each primer, 1. mu.L of Bst DNA polymerase, 1. mu.L of calcein, 4.0. mu.L of deionized water and 2. mu.L of DNA template.
6. The method for detecting CALR genotype 2 mutations as recited in claim 5, wherein SEQ ID NO: 2 to SEQ ID NO: 3, the final concentration of each primer in the sequence shown in the sequence is 0.2 mu mol/L;
SEQ ID NO: 4, the final concentration of the primers of the sequence is 0.8 mu mol/L;
SEQ ID NO: 5, the final concentration of the primers is 1.6 mu mol/L;
SEQ ID NO: 6 to SEQ ID NO: 7 was added to the mixture at a final concentration of 0.8. mu. mol/L.
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| CN109161582A (en) * | 2018-08-14 | 2019-01-08 | 关明 | It is a kind of for the reagent and its kit of ring mediated isothermal amplification and application |
| CN111471768B (en) * | 2020-04-15 | 2023-12-26 | 内蒙古医科大学附属医院(内蒙古自治区心血管研究所) | PCR primer set and kit for detecting JAK2V617F and CALR ninth exon gene mutation |
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| CN104561287A (en) * | 2014-12-26 | 2015-04-29 | 南京艾迪康医学检验所有限公司 | Reagent and method for detecting ninth exon mutation of CALR gene |
| CN105441562A (en) * | 2015-12-31 | 2016-03-30 | 德赛诊断系统(上海)有限公司 | Detection method for CALR (Calreticulin) gene deletion and insertion mutation and kit |
| WO2016077409A1 (en) * | 2014-11-12 | 2016-05-19 | Neogenomics Laboratories, Inc. | Determining tumor load and biallelic mutation in patients with calr mutation using peripheral blood plasma |
| CN105916876A (en) * | 2013-09-16 | 2016-08-31 | 分子医学研究中心责任有限公司 | Mutant calreticulin for the diagnosis of myeloid malignancies |
| WO2016172505A2 (en) * | 2015-04-23 | 2016-10-27 | Quest Diagnostics Investments Incorporated | Methods and compositions for the detection of calr mutations in myeloproliferative diseases |
| KR20160137785A (en) * | 2015-05-21 | 2016-12-01 | (의료)길의료재단 | Composition for diagnosing myeloproliferative disorder |
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| CN105916876A (en) * | 2013-09-16 | 2016-08-31 | 分子医学研究中心责任有限公司 | Mutant calreticulin for the diagnosis of myeloid malignancies |
| WO2016077409A1 (en) * | 2014-11-12 | 2016-05-19 | Neogenomics Laboratories, Inc. | Determining tumor load and biallelic mutation in patients with calr mutation using peripheral blood plasma |
| CN104561287A (en) * | 2014-12-26 | 2015-04-29 | 南京艾迪康医学检验所有限公司 | Reagent and method for detecting ninth exon mutation of CALR gene |
| WO2016172505A2 (en) * | 2015-04-23 | 2016-10-27 | Quest Diagnostics Investments Incorporated | Methods and compositions for the detection of calr mutations in myeloproliferative diseases |
| KR20160137785A (en) * | 2015-05-21 | 2016-12-01 | (의료)길의료재단 | Composition for diagnosing myeloproliferative disorder |
| CN105441562A (en) * | 2015-12-31 | 2016-03-30 | 德赛诊断系统(上海)有限公司 | Detection method for CALR (Calreticulin) gene deletion and insertion mutation and kit |
Non-Patent Citations (1)
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