CN107164538A - A kind of the internal reference amplimer composition and its amplification system of detection CALR gene mutations - Google Patents
A kind of the internal reference amplimer composition and its amplification system of detection CALR gene mutations Download PDFInfo
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Abstract
The present invention relates to a kind of internal reference amplimer composition of detection CALR gene mutations, it includes SEQ ID NO：1‑SEQ ID NO：The primer of sequence shown in 6；The invention further relates to a kind of internal reference amplification system containing above-mentioned internal reference amplimer composition and its kit and detection method, and by above-mentioned internal reference amplimer composition expand CALR gene mutations target sequence, the target sequence be SEQ ID NO：Sequence shown in 7.Internal reference amplification system of the present invention and its detection method and the mutation system of CALR genes are supported the use, it can both be used for quickly detecting to the CALR genes of saltant type, the CALR genes of wild type can also be expanded, it is significant in terms of the lifting of quality control as the internal reference system of quality control of CALR mutation detection kits.
Description
Technical field
The present invention relates to biology field, more particularly to a kind of internal reference amplimer group of detection CALR gene mutations
Compound and its amplification system and amplification detection method.
Background technology
Bone marrow proliferative tumour (MPN) be ripe in one or more myeloid cells propagation and peripheral blood in marrow and not into
The ripe increased Clonal hematopoietic stem/progenitor disease of cell.In recent years, multiple molecular markers, such as JAK2, MPL and TET are mutated
Deng being found in succession, the discovery of these marks is significant for the Molecular pathogenesis for understanding MPN, it helps right
The patient of this kind of disease is diagnosed and treated.But, have 30%~45% the primary blood for carrying wild type JAK2/MPL
Platelet increase disease (ET) or primary myelofibrosis (PMF) patient still have difficult diagnosis, the calprotectin reported recently
Gene (CALR) mutation can partly fill up this blank, be expected to as another new molecule mark of diagnosis bone marrow proliferative tumour
Will thing.
CALR is a kind of calcium binding and storage protein molecular companion for having multi-functional for being predominantly located at endoplasmic reticulum, is led to
Assistance protein is crossed correctly to fold and maintain metabolic defect in cellular calcium ion stable state and participate in regulation cell propagation, apoptosis, stick, be immunized and waited
Journey.The CALR assignments of genes gene mapping are in chromosome 19p13.2, and it has 9 extrons and 9 protein structure domains, CALR gene mutations be by
9th extron is inserted and missing causes, and causes the reading frame displacement of a base-pair, and then generation one kind, which has, lacks endoplasm
Net retains the albumen of the new C- carboxyl terminals of sequence (KDEL amino acid sequences).Up to the present, have been detected by and be more than
40 kinds of different CALR mutant, two kinds of variants of most common of which are respectively：1 form variation as caused by 52 base deletions
Body (p.L367fs*46) and the 2 form variation bodies (p.K385fs*47) caused by 5 base TTGTC insertions, they account for institute respectively
Have the 53% and 32% of mutant, researcher is in vitro it was found that, 1 type mutant of overexpression enhances interleukin-3
Expression, also promotes STAT5 phosphorylations and causes the activation of signal transduction, this activation can be by JAK inhibitor
Retardance, this explanation CALR may have similar mechanism with JAK2 mutation.
The detection of CALR gene mutations depends on probe real-time PCR methodology or PCR sequencing PCR etc., above method takes time and effort,
The report cycle is long, and the existing defects in terms of sensitivity, and required detection device is expensive, during detection it is necessary to have special equipment and
Well-trained technical staff, requires high to technology platform, is not easy to be widely popularized.
The content of the invention
It is a kind of for the type of CALR genes 1 and 2 having built up it is an object of the invention to overcome defect of the prior art
Type mutation be used for quickly detecting ring mediated isothermal amplification (Loop-Mediated Isothermal Amplification,
LAMP) on the basis of system, a kind of internal reference amplification system of detection CALR gene mutations, the mutation system with CALR genes are proposed
Support the use, to ensure the accuracy of testing result.
To achieve the above object, the present invention is adopted the following technical scheme that：
First purpose of the present invention is to provide a kind of internal reference amplimer composition of detection CALR gene mutations, and it is wrapped
Include such as SEQ ID NO：F3 primers, such as SEQ ID NO shown in 1：B3 primers, such as SEQ ID NO shown in 2：FIP shown in 3
Primer, such as SEQ ID NO：BIP primers, such as SEQ ID NO shown in 4：LF primers and such as SEQ ID NO shown in 5：6 institutes
The LB primers shown.
Preferably, the CALR gene mutations include the mutation of the type of CALR genes 1 and the mutation of the type of CALR genes 2.
Second object of the present invention is to provide a kind of detection CALR genes containing above-mentioned internal reference amplimer composition
The internal reference amplification system of mutation.
Preferably, the internal reference amplification system also includes reaction buffer, Bst archaeal dna polymerases, calcein, deionization
Water, DNA profiling.
Preferably, the cumulative volume of the internal reference amplification system is 25 μ L, and it includes the μ L of 2 × reaction buffer (RM) 12.5,
Primers F 3：1 μ L, primer B3：1 μ L, primers F IP：0.5 μ L, primer BIP：1 μ L, primer LF：1 μ L, primer LB：1 μ L, Bst DNA
The μ L of polymerase 1, the μ L of calcein (FD) 1, the μ L of deionized water 3.0, the μ L of DNA profiling 2.
Preferably, in the internal reference amplification system, the final concentration of the F3 primers and B3 primers is 0.2 μm of ol/L；
The final concentration of the FIP primers and BIP primers is 1.6 μm of ol/L；The final concentration of the LF primers and LB primers is 0.8 μ
mol/L。
It will be appreciated that under conditions of isothermal amplification reactions are met, the concentration of each reagent in above-mentioned reaction mixture
And consumption can carry out appropriate adjustment, but using the reagent of above-mentioned volume and concentration, its expanding effect is optimal, and testing result is more
To be accurate.
Third object of the present invention is the kit of the detection CALR gene mutations containing above-mentioned internal reference amplification system.
Fourth object of the present invention be to provide it is a kind of using above-mentioned internal reference amplimer composition be used for non-diagnostic and
The method of the detection CALR gene mutations of therapeutic purposes, this method is with the DNA profiling of tested sample, using SEQ ID NO：1~
SEQ ID NO：Primer described in 6 carries out LAMP reactions, then carries out visualization interpretation to identify whether sample is CALR genes
Mutation is positive.
Preferably, the condition of the LAMP reactions is：55~70 DEG C, 30~60min；It is highly preferred that the LAMP reactions
Condition be：63~68 DEG C, 60min；Most preferably, the condition of the LAMP reactions is：65 DEG C, 60min.
Preferably, LAMP reactions equipment used is the equipment that can stably provide 65 DEG C of constant temperature, such as regular-PCR
Instrument or constant-temperature metal bath etc..
The 5th purpose of the present invention is to provide a kind of target of the CALR gene mutations of above-mentioned internal control primer composition amplification
Sequence, it is by SEQ ID NO：Sequence shown in 7 is constituted, and provides a kind of weight of the target sequence containing above-mentioned CALR gene mutations
Group plasmid, the recombinant plasmid is the NO of ID containing SEQ：The TA cloned plasmids of sequence shown in 7.
The base sequence of above-mentioned primer and target sequence is as shown in table 1 below.
The internal control primer of the detection CALR gene mutations of table 1 and the nucleotides sequence list of target sequence
Compared with prior art, the invention has the advantages that：
The internal reference amplification system and its amplification method of detection CALR gene mutations of the present invention, it is prominent with CALR genes
Change system is supported the use, and the internal reference amplification system can be both used for quickly detecting to the CALR genes of saltant type, can also be to open country
The CALR genes of raw type are expanded, as the internal reference system of quality control of CALR mutation detection kits, in the lifting of quality control
Aspect is significant.
Brief description of the drawings
Fig. 1 is the specific checking schematic diagram of CALR gene mutation LAMP systems；
Reference is in figure：
A1：Clinical wildtype genomic DNA is template；A2：Clinical CALR-1 types mutator group DNA is template；A3：Face
Bed CALR-2 type mutator groups DNA is template；A4：108Copies/ml plasmid sample；A5：105Copies/ml plasmid
Sample；A6：104Copies/ml plasmid sample；A7：103Copies/ml plasmid sample；A8：Negative control sample.
Embodiment
With reference to the accompanying drawings and examples, the embodiment to the present invention is further described.Following examples are only
For clearly illustrating technical scheme, and it can not be limited the scope of the invention with this.
Embodiment 1
The present embodiment is the target sequence of CALR gene mutations of the present invention, and for detecting CALR gene mutations
Internal reference amplimer.
In the relative conserved sequence region of CALR genes, target fragment design primer is chosen, and is carried out after optimal screening, is designed
Appropriate specific primer.
Target fragment length is 214bp, and particular sequence is：
TTGCAGACAAGCCAGGATGCACGCTTTTATGCTCTGTCGGCCAGTTTCGAGCCTTTCAGCAACAAAGGCCAGACGCT
GGTGGTGCAGTTCACGGTGAAACATGAGCAGAACATCGACTGTGGGGGCGGCTATGTGAAGCTGTTTCCTAATAGTT
TGGACCAGACAGACATGCACGGAGACTCAGAATACAACATCATGTTTGGTCCCGACATCT(SEQ ID NO：7)；
Primer sequence is：
F3：5'-TTGCAGACAAGCCAGGATG-3'(SEQ ID NO：1)；
B3：5'-AGATGTCGGGACCAAACATG-3'(SEQ ID NO：2)；
FIP：5'-TGAACTGCACCACCAGCGTCCTCTGTCGGCCAGTTTCG-3'(SEQ ID NO：3)
BIP：5'-GCAGAACATCGACTGTGGGGGTCTGAGTCTCCGTGCATGT-3'(SEQ ID NO：4)；
LF：5'-GCCTTTGTTGCTGAAAGGCT-3'(SEQ ID NO：5)；
LB：5'-CGGCTATGTGAAGCTGTTTCC-3'(SEQ ID NO：6).
Embodiment 2
The present embodiment detects internal reference amplification system, kit and the amplification detection method of CALR gene mutations for the present invention.
The kit of detection CALR gene mutations of the present invention includes internal reference amplification system, the internal reference amplification system
Cumulative volume is 25 μ L, and it includes the μ L of 2 × reaction buffer (RM) 12.5, primers F 3：1 μ L (0.2 μm of ol/L of final concentration), primer
B3：1 μ L (0.2 μm of ol/L of final concentration), primers F IP：0.5 μ L (1.6 μm of ol/L of final concentration), primer BIP：The 1 μ L (μ of final concentration 1.6
Mol/L), primer LF：1 μ L (0.8 μm of ol/L of final concentration), primer LB：1 μ L (0.8 μm of ol/L of final concentration), Bst archaeal dna polymerases 1
μ L (8U), the μ L of calcein (FD) 1, the μ L of deionized water 3.0, the μ L of DNA profiling 2.
LAMP reactions are carried out using mentioned reagent box, LAMP reaction conditions are：65 DEG C, 60min；Equipment used is common
PCR instrument or constant-temperature metal bath etc. can stablize the equipment for providing 65 DEG C of constant temperature；Then carry out visualization interpretation is to identify sample
No is CALR positive gene mutations, and specially result judges：After reaction terminates, the color of reaction solution faint yellow is changed into from original
Green, that is, be determined as reacting positive.
The TA cloned plasmids containing target sequence are built, the double sample of gradient concentration is prepared, specific concentration is 103copies/
Ml, 104Copies/ml, 105Copies/ml, 106Copies/ml, 107Copies/ml, 108Copies/ml, with what is set up
The general LAMP reaction systems of CALR genes (internal reference amplification system) are detected, can be detected；It is logical with the CALR genes set up
With LAMP reaction systems (internal reference amplification system) to clinical wildtype and the sample of saltant type, it can efficiently stablize detection.Inspection
Survey functional, after as a result showing as shown in figure 1, the system of A1-A7 samples is expanded through 60min, equal greening becomes cloudy, be presented positive
As a result, A8 is negative control, after being expanded through 60min, is not any change, still faint yellow clarification, and negative findings is presented.
From above-described embodiment, the internal reference amplification system of detection CALR gene mutations of the present invention and its amplification side
Method, is supported the use with the mutation system of CALR genes, and the internal reference amplification system can both carry out fast to the CALR genes of saltant type
Speed detection, can also be expanded to the CALR genes of wild type, as the internal reference system of quality control of CALR mutation detection kits,
It is significant in terms of the lifting of quality control.
The specific embodiment of the present invention is described in detail above, but it is only used as example, and the present invention is not intended to limit
In particular embodiments described above.To those skilled in the art, it is any to the practicality carry out equivalent modifications and replace
In generation, is also all among scope of the invention.Therefore, the equalization made without departing from the spirit and scope of the invention is converted and repaiied
Change, all should be contained within the scope of the invention.
SEQUENCE LISTING
<110>Huashan Hospital Affiliated To Fudan Univ；Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences
<120>A kind of the internal reference amplimer composition and its amplification system of detection CALR gene mutations
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223>F3 primers, 5'-3'
<400> 1
ttgcagacaa gccaggatg 19
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>B3 primers, 5'-3'
<400> 2
agatgtcggg accaaacatg 20
<210> 3
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223>FIP primers, 5'-3'
<400> 3
tgaactgcac caccagcgtc ctctgtcggc cagtttcg 38
<210> 4
<211> 40
<212> DNA
<213> Artificial Sequence
<220>
<223>BIP primers, 5'-3'
<400> 4
gcagaacatc gactgtgggg gtctgagtct ccgtgcatgt 40
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223>LF primers, 5'-3'
<400> 5
gcctttgttg ctgaaaggct 20
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223>LB primers, 5'-3'
<400> 6
cggctatgtg aagctgtttc c 21
<210> 7
<211> 214
<212> DNA
<213> Artificial Sequence
<220>
<223>Target sequence
<400> 7
ttgcagacaa gccaggatgc acgcttttat gctctgtcgg ccagtttcga gcctttcagc 60
aacaaaggcc agacgctggt ggtgcagttc acggtgaaac atgagcagaa catcgactgt 120
gggggcggct atgtgaagct gtttcctaat agtttggacc agacagacat gcacggagac 180
tcagaataca acatcatgtt tggtcccgac atct 214
Claims (10)
1. a kind of internal reference amplimer composition of detection CALR gene mutations, it is characterised in that including such as SEQ ID NO：1 institute
F3 primers, such as SEQ ID NO shown：B3 primers, such as SEQ ID NO shown in 2：FIP primers, such as SEQ ID NO shown in 3：4
Shown BIP primers, such as SEQ ID NO：LF primers and such as SEQ ID NO shown in 5：LB primers shown in 6.
2. a kind of internal reference amplification system of detection CALR gene mutations, it is characterised in that contain internal reference as claimed in claim 1
Amplimer composition.
3. the internal reference amplification system of a kind of detection CALR gene mutations according to claim 2, it is characterised in that also include
Reaction buffer, Bst archaeal dna polymerases, calcein, deionized water, DNA profiling.
4. the internal reference amplification system of a kind of detection CALR gene mutations according to claim 3, it is characterised in that described
In internal reference amplification system, the final concentration of the F3 primers and B3 primers is 0.2 μm of ol/L；The FIP primers and BIP primers
Final concentration is 1.6 μm of ol/L；The final concentration of the LF primers and LB primers is 0.8 μm of ol/L.
5. a kind of kit of detection CALR gene mutations, its feature exists, containing as any one of claim 2~4
The kit of internal reference amplification system.
6. a kind of method of detection CALR gene mutations for non-diagnostic and therapeutic purposes, it is characterised in that with tested sample
DNA profiling, using internal reference amplimer composition as claimed in claim 1 carry out LAMP reactions, then visualized
Interpretation identifies whether sample is CALR positive gene mutations.
7. the method for a kind of detection CALR gene mutations according to claim 6, it is characterised in that the LAMP reactions
Condition is：55~70 DEG C, 30~60min.
8. the method for a kind of detection CALR gene mutations according to claim 7, it is characterised in that the LAMP reactions
Condition is：63~68 DEG C, 60min.
9. a kind of target sequence for the CALR gene mutations that internal reference amplimer composition as described in claim 1 is expanded, it is special
Levy and be, by SEQ ID NO：Sequence shown in 7 is constituted.
10. a kind of recombinant plasmid of the target sequence of the CALR gene mutations containing described in claim 9.
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| CN107988358A (en) * | 2017-12-28 | 2018-05-04 | 湖北工业大学 | A kind of the internal reference amplimer composition and its amplification system of detection NPPA gene mutations c.T413C |
| CN112011633A (en) * | 2020-10-14 | 2020-12-01 | 河南智泰生物科技有限公司 | Primer group, kit and method for LAMP (loop-mediated isothermal amplification) simultaneous detection of mycobacterium tuberculosis complex group and rpoB gene mutation |
| CN112195275A (en) * | 2020-10-14 | 2021-01-08 | 河南智泰生物科技有限公司 | Primer group, kit and method for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107699564A (en) * | 2017-10-23 | 2018-02-16 | 中国科学院苏州生物医学工程技术研究所 | Long-chain non-coding RNA sequence and its application are used in human prostata cancer early diagnosis |
| CN107699564B (en) * | 2017-10-23 | 2019-09-20 | 中国科学院苏州生物医学工程技术研究所 | Human prostata cancer early diagnosis long-chain non-coding RNA sequence and its application |
| CN107988358A (en) * | 2017-12-28 | 2018-05-04 | 湖北工业大学 | A kind of the internal reference amplimer composition and its amplification system of detection NPPA gene mutations c.T413C |
| CN112011633A (en) * | 2020-10-14 | 2020-12-01 | 河南智泰生物科技有限公司 | Primer group, kit and method for LAMP (loop-mediated isothermal amplification) simultaneous detection of mycobacterium tuberculosis complex group and rpoB gene mutation |
| CN112195275A (en) * | 2020-10-14 | 2021-01-08 | 河南智泰生物科技有限公司 | Primer group, kit and method for LAMP combined detection of influenza A virus, influenza B virus and novel coronavirus |
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