CN107841536A - A kind of Primer composition and its kit and detection method of detection JAK2 V617F gene mutations - Google Patents
A kind of Primer composition and its kit and detection method of detection JAK2 V617F gene mutations Download PDFInfo
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Abstract
The present invention relates to a kind of Primer composition of detection JAK2 V617F gene mutations, it contains SEQ ID NO：2‑SEQ ID NO：The primer and SEQ ID NO of sequence shown in 6：The PNA probe of sequence shown in 7；The invention further relates to a kind of kit and its detection method containing above-mentioned Primer composition.Kit of the present invention and its detection method have good specificity and stability, the sample of gradient mutational load concentration is expanded with the LAMP reaction systems established, it was found that mutational load detection sensitiveness can reach 1%, it is suitable with the real-time PCR of sonde method effect, and it can carry out Visual retrieval to JAK2 V617F gene mutations, compared with traditional detection method, this method is low to equipment and environmental requirement, detection speed is faster, detection sensitivity is higher, contributes to the realization of POCT in future (being detected by bed).
Description
Technical field
The present invention relates to the primer combination of biology field, more particularly to a kind of detection JAK2 V617F gene mutations
Thing and its kit and detection method.
Background technology
Classical bone marrow proliferative tumour (myeloproliferative neoplasm, MPN) is one group with a system or more
It is the Clonal disease that is characterized of mature blood cell amplification, including polycythemia vera (polycythemia vera,
PV), primary thrombocytosis (essential thrombocythemia, ET) and primary myelofibrosis
(primary myelofibrosis, PMF), can cause severe anemia, acute leukemia conversion etc., thrombus be it is most common simultaneously
Send out disease, and the main cause of death of MPN patient.
PV be it is a kind of it is Clonal with erythrocyte abnormality breed based on chronic myeloproliferative disease, patient shows as red
Cell is largely bred, and influences the transport of oxygen, and serum erythropoietin is less than normal limits, and bone marrow biopsy shows hyperplasia
It is active, easily merge bleeding and thrombotic complications and splenomegaly.ET patient show as continuation blood platelet rise (>450×
109/ L), hyperplasia megakaryocytic, splenomegaly, thrombus or bleeding easily occurs.PMF is shown with megacaryocyte in marrow and granulocyte
Hyperplasia accompanied reactive fibre is write, megacaryocyte dysplasia is characterized, and nearly 20% patient finally develops into acute leukemia,
The cause of the death is mostly severe anemia, congestive heart failure, bleeding or repeated infection, acute leukemia conversion etc..This three classes disease it
Between can mutually inversion of phases or merge exist, hypostasis is caused due to patient's haemocyte hyper-proliferative, so as to induce the various hearts
Cranial vascular disease.
PV, PMF and ET routine diagnosis mainly carry out comprehensive according to the analysis of clinical manifestation, laboratory examination and Pathomorphology
Close and judge.There is the increased laboratory examination feature of serum IgG concentation because reactive hyperplasia is same with MPN, in addition MPN
Atypical symptoms, for the antidiastole between different types of MPN and reactive hyperplasia be always perplex it is clinical
Problem.In past nearly half a century, the bone marrow proliferative tumor research slower development about classics, almost one by blood
The corner that educational circles is forgotten, since JAK2 V617F mutation in 2005 are found, particularly in recent years as two generation sequencing technologies exist
Clinical popularization and application, the specific gene mutation pedigrees of MPN are revealed, for examining for classical bone marrow proliferative tumour
Disconnected, prognosis and therapeutic strategy, which are formulated, provides brand-new molecular marker [5].
2005, in the world multiple research groups almost simultaneously be found that exist in the marrow and peripheral blood of MPN patient
JAK2 V617F point mutation, this mutation is to be located at the bit base G of 14 exons 1 of JAK2 genes 849>T point mutation, by by No. 617
The valine of codon, which is replaced into phenylalanine, influences the activity of JH2 domains, and causes JAK2 independently to suppress functional disturbance,
Do not have to make JAK2 remain to the hematopoietic cell proliferation that is activated and promotes under conditions of cell factor stimulation.
Mutation rates of the JAK2V617F in MPN be respectively：PV 95%, ET 50%~60%, PMF 50%~60%, but
So far it there is no the report for occurring JAK2 V617F mutation in lymphocyte disorder, entity tumor and reactive bone marrow proliferative lesion
Road, therefore now think that diagnosis of the discovery of JAK2 V617F mutation to MPN is revolutionary breakthrough.JAK2 V617F mutation are found
After 1 year, it is found that myeloproliferative leukemia virus oncogene (MPL) is prominent in the negative ET and PMF patients of the mutation
Become, but PV patient finds no this mutation.MPL genes are located at chromosome 1p34, and encoding platelet generates plain acceptor and influenceed huge
The propagation of nucleus and differentiation.MPL mutation be replaced into positioned at the bit codon tryptophan TGG of 10 exon of MPL genes 515 it is bright
Propylhomoserin TTG (515L) or tryptophan TGG replaces with lysine AAG (515K).About 5%~10% ET/PMF patient carries MPL
Mutation, the mutation of MPL 515 have become ET and PMF molecular markers.
At present, JAK2 V617F, MPL W515K/L mutation have been incorporated the Main Diagnosis standard of such disease by WHO, this
The discovery of a little molecular markers not only facilitates MPN diagnosis and differential diagnosis, additionally aids and judges Clinical Outcome and prognosis.Such as
JAK2 V617F mutational loads are higher, and patient age is bigger, and hemoglobin level and white blood cell count(WBC) are then higher, platelet count
Lower, splenomegaly and pruitus are also more obvious.As JAK2 V617F mutational loads increase, PV and ET patients occur thrombus and after
The probability of myelofibrosis after hair ET, PV also significantly increases.In recent years, increasing molecular marker, such as MPL, TET and
CALR mutation etc. are found in succession, and the discovery of these marks is significant for the Molecular pathogenesis for understanding MPN,
Help that the patient of this kind of disease is diagnosed and treated.
In summary, detecting diagnosis, treatment and prognosis of the JAK2 V617F gene mutations to clear and definite MPN has important meaning
Justice.At present, MPN molecular markers object detecting method include quantitative fluorescent PCR (quantitative real-time PCR,
QPCR), restriction fragment length polymorphism (restriction fragment length polymorphis, RFLP) is analyzed,
Sanger is sequenced, high-resolution melting curve analysis (high-resolution melting analysis, HRM) etc..It is above-mentioned
Following limitation be present in method：The principle of normal PCR is all based on, it is necessary to special place, equipment and personnel, testing cost
Height, cycle length, can not carry out in community, the laboratory of basic unit or family.
The content of the invention
The defects of it is an object of the invention to overcome in the prior art, establish one kind and be directed to JAK2 V617F gene mutations
Ring mediated isothermal amplification (Loop-Mediated Isothermal Amplification, the LAMP) body being used for quickly detecting
System.
To achieve the above object, the present invention adopts the following technical scheme that：
First purpose of the present invention is to provide a kind of Primer composition of detection JAK2 V617F gene mutations, and it is wrapped
Include SEQ ID NO：2~SEQ ID NO：The primer of sequence shown in 6.
Further, the Primer composition also includes such as SEQ ID NO：The PNA probe of sequence shown in 7.
Further, the SEQ ID NO：4~SEQ ID NO：At least one 5' ends in the primer of sequence shown in 5
Carry out lock nucleic acid modification；Further, the SEQ ID NO：At least one 5' ends in the primer of sequence shown in 4 are carried out
Lock nucleic acid is modified, and mutational site is arranged at the 5' ends of FIP or BIP primers by the present invention, and carries out lock nucleic acid modification to it, to increase
Its specificity is detected by force.
Second object of the present invention is to provide a kind of detection JAK2 V617F genes containing above-mentioned Primer composition and dashed forward
The kit of change.
In order to further optimize mentioned reagent box, the technical measures that the present invention takes also include：
Further, mentioned reagent box also includes reaction working solution, Bst archaeal dna polymerases, deionized water, DNA profiling.
Further, the composition of the reaction working solution is as follows：1.5 × ThermoPol buffer solutions, 12mM MgSO4,
DNTP 2.1mM/ kinds.
Further, the kit also includes developer.
Further, the developer is selected from hydroxynaphthol blue, calcein, cresol red, phenol red, m-cresol purple, bromine first
One kind in phenol violet, dimethyl diaminophenazine chloride, naphtholphthalein and thymol blue；Further, the developer is dimethyl diaminophenazine chloride.
Further, the kit includes reaction working solution 17 μ L, SEQ ID NO：2~SEQ ID NO：Sequence shown in 6
In each 0.5 μ L, SEQ ID NO of each primer：The μ L of 1 μ L, Bst archaeal dna polymerase of PNA probe 1 of sequence shown in 7, dimethyl diaminophenazine chloride dye
Expect 1 μ L, the μ L of deionized water 0.5, the μ L of DNA profiling 2.
Further, the SEQ ID NO：2~SEQ ID NO：The final concentration of each primer in sequence shown in 3 is
0.2μmol/L；SEQ ID NO：4~SEQ ID NO：The final concentration of each primer in sequence shown in 5 is 1.6 μm of ol/L；
SEQ ID NO：Final concentration of 0.8 μm of ol/L of the primer of sequence shown in 6；SEQ ID NO：The end of the PNA probe of sequence shown in 7
Concentration is 0.8 μm of ol/L.
Third object of the present invention is to provide a kind of for the non-diagnostic and detection JAK2 V617F bases of non-treatment purpose
Because of the method for mutation, it carries out LAMP reactions, so with the DNA profiling of tested sample using above-mentioned Primer composition or kit
Nephelometry or visualization interpretation are used afterwards to identify whether sample is mutated the positive for JAK2 V617F.
Further, the condition of the LAMP reactions is：55 DEG C -70 DEG C, 30-60min；Further, the LAMP
The condition of reaction is：58 DEG C -68 DEG C, 30-60min
Further, LAMP reactions equipment used is that can stablize the equipment for providing 55 DEG C of -70 DEG C of constant temperature, such as
Regular-PCR instrument or constant-temperature metal bath etc..
Fourth object of the present invention is to provide a kind of JAK2 V617F gene mutations of above-mentioned Primer composition amplification
Target sequence, it is by SEQ ID NO：Sequence shown in 1 forms.
The base sequence of above-mentioned primer, probe and target sequence is as shown in table 1 below.
Table 1 detects the base sequence table of the target sequences of JAK2 V617F gene mutations, primer and probe
Wherein, at least one 5' in upper table in amplimer FIP and amplimer BIP carries out lock nucleic acid modification.
Compared with prior art, the invention has the advantages that：
Kit of the present invention and detection method are lower without special place, equipment and personnel, testing cost
Honest and clean, detection sensitivity is higher, and can complete quick detection within 30-40 minutes, contributes to POCT's in future (being detected by bed)
Realize.
Brief description of the drawings
Fig. 1 is the specific checking schematic diagram of JAK2 V617F gene mutation LAMP systems in one embodiment of the invention；
Fig. 2 is the LAMP systems of the JAK2 V617F gene mutation samples of various concentrations gradient in one embodiment of the invention
Testing result schematic diagram；
Reference is in figure：
B1-B4 is the testing result that clinical JAK2 V617F are mutated positive sample, and B5 is that clinical CALR-1 is mutated positive sample
This testing result, B6 are the testing result that clinical CALR-2 is mutated positive sample, and B7 is the detection knot that MPL is mutated positive sample
Fruit, B8 are the testing result of negative control (the wild gene group DNA of normal person is template).
1~7 is respectively that concentration is 108copies/ml、107copies/m、106copies/ml、105Copies/ml is mould
Plate, 104copies/ml、103copies/ml、102The testing result figure of copies/ml JAK2 V617F mutation positive samples,
8 be negative control (the wild gene group DNA of normal person is template).
Embodiment
, should the invention provides the Primer composition and its kit and detection method of a kind of detection JAK2 V617F mutation
Primer composition includes SEQ ID NO：2~SEQ ID NO：The primer of sequence shown in 6, it may also include such as SEQ ID NO：7
The PNA probe of shown sequence.
With reference to the accompanying drawings and examples, the embodiment of the present invention is further described.Following examples are only
For clearly illustrating technical scheme, and can not be limited the scope of the invention with this.
Embodiment 1
The present embodiment is the target sequence for the JAK2 V617F mutation chosen in a better embodiment of the invention, and is used for
Detect the primer and probe sequence of JAK2 V617F mutation.
Website PrimerExplorer V5 (https are designed using online LAMP primer://
Primerexplorer.jp/lampv5/index.html target fragment) is directed to, designs appropriate specific primer：
(1) primer sequence is：
F3：GTCAAACAACAATTCTTTGTACT(SEQ ID NO：2)；
B3：CAGTTTCAAAAATACTTAACTCCT(SEQ ID NO：3)；
FIP：AACATACTCCATAATTTAAAACCAAGTCTTTCTTTGAAGCAGC(SEQ ID NO：4), its 5' A
Base has carried out lock nucleic acid modification；
BIP：AACTACAGGCTTTCTAATGCCTTTCTCATTACACTGACACCTAGCT(SEQ ID NO：5)；
LF：AATGCTTGTGAGAAAGCTTGCTC(SEQ ID NO：6).
(2) PNA probe sequence is：
TGGAGTATGTGTCTGT(SEQ ID NO：7).
(3) target sequence, it is specially：
gtcaaacaacaattctttgtacttttttttttccttagTCTTTCTTTGAAGCAGCAAGTATGATGAGCAAGCTTTCT
CACAAGCATTTGGTTTTAAATTATGGAGTATGTTTCTGTGGAGACGAGAgtaagtaaaactacaggctttctaatgc
ctttctcagagcatctgtttttgtttatatagaaaattcagtttcaggatcacagctaggtgtcagtgtaaactata
atttaacaggagttaagtatttttgaaactg(262BP)(SEQ ID NO：1).
In above-mentioned primer sequence, the design section of LAMP primer is：
tctcactttgatctccatattccaggcttacacaggggtttcctcagaacgttgatggcagttgcaggtccatataa
agggaccaaagcacattgtatcctcatctatagtcatgctgaaagtaggagaaagtgcatctTtattatggcagaga
gaattttctgaactatttatggacaacagtcaaacaacaattctttgtacttttttttttccttagTCTTTCTTTGA
AGCAGCAAGTATGATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAATTATGGAGTATGTTTCTGTGGAGACGAGA
gtaagtaaaactacaggctttctaatgcctttctcagagcatctgtttttgtttatatagaaaattcagtttcagga
tcacagctaggtgtcagtgtaaactataatttaacaggagttaagtatttttgaaactgaaaacactgtaggactat
tcagttatatcttgtgaaaaaggaaagcaatgaagttaaaagtagaaggttacaatgcccaaacaatagagtattat
agtaaacaaatgtctataaaacattttgtgttcatgata；
PNA closing probe design section be：
aattctttgtacttttttttttccttagTCTTTCTTTGAAGCAGCAAGTATGATGAGCAAGCTTTCTCACAAGCATT
TGGTTTTAAATTATGGAGTATGTTTCTGTGGAGACGAGAgtaagtaaaactacaggctttctaatgcctttctcaga
gcatctgtttttgtttatatagaaaattcagtttcaggatcacagctaggtgtca。
Embodiment 2
The present embodiment is the present invention detection JAK2 V617F kits being mutated and its method.
Kit of the present invention includes amplification reaction system, and the cumulative volume of the amplification reaction system is 25 μ L, its
It includes reacting the μ L of working solution 17, primers F 3：0.5 μ L (0.2 μm of ol/L of final concentration), primer B3：The 0.5 μ L (μ of final concentration 0.2
Mol/L), primers F IP：0.5 μ L (1.6 μm of ol/L of final concentration), primer BIP：0.5 μ L (1.6 μm of ol/L of final concentration), primer LF：
0.5 μ L (0.8 μm of ol/L of final concentration), PNA probe：1 μ L (0.8 μm of ol/L of final concentration), the μ L (8U) of Bst archaeal dna polymerases 1, it is neutral
The μ L of red 1, the μ L of deionized water 0.5, the μ L of DNA profiling 2；The composition for wherein reacting working solution is as follows：1.5 × ThermoPol delays
Fliud flushing, 12mM MgSO4, dNTP 2.1mM/ kinds.
LAMP reactions are carried out using mentioned reagent box, LAMP reaction conditions are：58 DEG C -68 DEG C, 60min；Equipment used
It can stablize the equipment that 58 DEG C of -68 DEG C of constant temperature are provided for regular-PCR instrument or constant-temperature metal bath etc.；Then using nephelometry or visual
Change interpretation to identify whether sample is JAK2 V617F positive gene mutations, specially result judges：After reaction terminates, reaction solution
Color from it is original it is faint yellow be changed into red, that is, be determined as reacting positive.
1) clinical JAK2 V617F mutation positive sample is gathered, clinical CALR-1 is mutated positive sample, and clinical CALR-2 dashes forward
Become positive sample, MPL mutation positive samples and negative control, carry out pcr amplification reaction respectively using the kit and to the examination
The specificity of agent box is verified.Testing result is as shown in figure 1, its result can be adopted using nephelometry interpretation or visualization interpretation
During with nephelometry interpretation, the developer of any visual method detection is not added in reaction system, wherein clinical JAK2 V617F are mutated
Positive sample testing result is muddy-positive, and it is clear-feminine gender with pattern detection；During using visualization interpretation,
The dyestuff of visual method detection is added in reaction system：Dimethyl diaminophenazine chloride, clinical JAK2 V617F mutation positive sample testing result are equal
To be red-positive, clinical CALR-1 mutation positive sample, clinical CALR-2 mutation positive sample, MPL mutation positive samples and the moon
Property control test result be yellow-feminine gender, as from the foregoing the present embodiment use kit JAK2 V617F mutation LAMP bodies
System's specificity is high.
2) the TA cloned plasmids (i.e. JAK2 V617F types mutant plasmid) containing target sequence are built, have been transferred to JAK2 V617F types
The 293T cells of mutant plasmid, are 106Individual cell, DNA is extracted with genome DNA extraction kit, is then diluted in proportion,
The sample of gradient mutational load concentration is made, specific concentration is 101Copies/ml, 102Copies/ml, 103Copies/ml,
104Copies/ml, 105Copies/ml, 106Copies/ml, 107Copies/ml, 108Copies/ml, with the JAK2 established
V617F mutation LAMP reaction systems are detected, and can be detected；Expanded, found prominent with the LAMP reaction systems established
Varying duty detection sensitiveness can reach 1%, suitable with the real-time PCR of sonde method effect, and its testing result uses visualization interpretation,
Refer to Fig. 2.
The structure of the collection and processing of DNA sample in the present embodiment, TA cloned plasmids and 293T cells uses
Method commonly used in the art, the genomic DNA in blood is extracted for example with Tiangeng genome DNA extraction kit, using T-
Vector pMD19 carry out structure of TA cloned plasmids etc..
From above-described embodiment, the kit and detection method of JAK2 V617F gene mutations of the present invention are relative
Improved in the detection method detection sensitivity of prior art, and detection method is simple, cost is cheap, and the report cycle is short, is applied to
General examination.
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 impartial conversion made without departing from the spirit and scope of the invention and repair
Change, all should be contained within the scope of the invention.
Sequence table
<110>Huashan Hospital Affiliated To Fudan Univ
<120>A kind of Primer composition and its kit and detection method of detection JAK2 V617F gene mutations
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gtcaaacaac aattctttgt actttttttt ttccttagtc tttctttgaa gcagcaagta 60
tgatgagcaa gctttctcac aagcatttgg ttttaaatta tggagtatgt ttctgtggag 120
acgagagtaa gtaaaactac aggctttcta atgcctttct cagagcatct gtttttgttt 180
atatagaaaa ttcagtttca ggatcacagc taggtgtcag tgtaaactat aatttaacag 240
gagttaagta tttttgaaac tg 262
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cagtttcaaa aatacttaac tcct 24
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aacatactcc ataatttaaa accaagtctt tctttgaagc agc 43
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aactacaggc tttctaatgc ctttctcatt acactgacac ctagct 46
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aatgcttgtg agaaagcttg ctc 23
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Claims (10)
1. a kind of Primer composition of detection JAK2V617F gene mutations, it is characterised in that including SEQ ID NO：2~SEQ
ID NO：The primer of sequence shown in 6.
A kind of 2. Primer composition of detection JAK2V617F gene mutations according to claim 1, it is characterised in that
Also include such as SEQ ID NO：The PNA probe of sequence shown in 7.
A kind of 3. Primer composition of detection JAK2V617F gene mutations according to claim 1, it is characterised in that
The SEQ ID NO：4~SEQ ID NO：At least one 5' ends in the primer of sequence shown in 5 carry out lock nucleic acid modification.
A kind of 4. examination of the detection JAK2V617F gene mutations containing Primer composition according to any one of claims 1 to 3
Agent box.
5. the kit of detection JAK2V617F gene mutations according to claim 4, it is characterised in that also include reaction
Working solution, Bst archaeal dna polymerases, deionized water, DNA profiling.
6. the kit of detection JAK2V617F gene mutations according to claim 5, it is characterised in that also include colour developing
Agent.
7. the kit of detection JAK2V617F gene mutations according to claim 6, it is characterised in that the kit bag
Include reaction working solution 17 μ L, SEQ ID NO：2~SEQ ID NO：Each 0.5 μ L, SEQ ID of each primer in sequence shown in 6
NO：1 μ L, Bst archaeal dna polymerase of PNA probe 1 the μ L, the neutral μ L of red 1, the μ L of deionized water 0.5 of sequence shown in 7, DNA profiling
2μL。
8. the kit of detection JAK2V617F gene mutations according to claim 7, it is characterised in that the SEQ ID
NO：2~SEQ ID NO：The final concentration of each primer in sequence shown in 3 is 0.2 μm of ol/L；
SEQ ID NO：4~SEQ ID NO：The final concentration of each primer in sequence shown in 5 is 1.6 μm of ol/L；
SEQ ID NO：Final concentration of 0.8 μm of ol/L of the primer of sequence shown in 6；
SEQ ID NO：Final concentration of 0.8 μm of ol/L of the PNA probe of sequence shown in 7.
9. a kind of be used for the non-diagnostic and method of the detection JAK2V617F gene mutations of non-treatment purpose, it is characterised in that with quilt
The DNA profiling of test sample sheet, LAMP reactions are carried out using Primer composition according to any one of claims 1 to 3, then used
Nephelometry visualizes interpretation to identify whether sample is that JAK2V617F mutation are positive.
10. the method for detection JAK2V617F gene mutations according to claim 9, it is characterised in that the LAMP reactions
Condition be：55 DEG C -70 DEG C, 30-60min.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022101259A1 (en) * | 2020-11-10 | 2022-05-19 | MultiplexDX, s.r.o. | Loop mediated isothermal nucleic acid amplification (lamp) using lna-modified primers and a metal-based colorimetric method for detecting an amplification product |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102234690A (en) * | 2010-04-30 | 2011-11-09 | 爱科来株式会社 | Method for detecting mutation in exon 12 of JAK2 gene, and nucleic acid probe and kit therefor |
| US20120171676A1 (en) * | 2009-07-10 | 2012-07-05 | Daniel Adlerstein | HIGHLY SENSITIVE RAPID ISOTHERMAL METHOD FOR THE DETECTION OF POINT MUTATIONS AND SNPs, A SET OF PRIMERS AND A KIT THEREFOR |
| CN103243153A (en) * | 2012-02-10 | 2013-08-14 | 复旦大学附属华山医院 | JAK2 V617F mutation detection application |
| CN106119386A (en) * | 2016-08-18 | 2016-11-16 | 上海睿昂生物技术有限公司 | The PCR kit of detection people's JAK2 gene mutation |
| CN106520517A (en) * | 2016-10-09 | 2017-03-22 | 上海交通大学医学院附属仁济医院 | Portable micro-fluidic chip LAMP (loop-mediated isothermal amplification) visible detector and detection method thereof |
-
2017
- 2017-10-24 CN CN201711002034.4A patent/CN107841536A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120171676A1 (en) * | 2009-07-10 | 2012-07-05 | Daniel Adlerstein | HIGHLY SENSITIVE RAPID ISOTHERMAL METHOD FOR THE DETECTION OF POINT MUTATIONS AND SNPs, A SET OF PRIMERS AND A KIT THEREFOR |
| CN102234690A (en) * | 2010-04-30 | 2011-11-09 | 爱科来株式会社 | Method for detecting mutation in exon 12 of JAK2 gene, and nucleic acid probe and kit therefor |
| CN103243153A (en) * | 2012-02-10 | 2013-08-14 | 复旦大学附属华山医院 | JAK2 V617F mutation detection application |
| CN106119386A (en) * | 2016-08-18 | 2016-11-16 | 上海睿昂生物技术有限公司 | The PCR kit of detection people's JAK2 gene mutation |
| CN106520517A (en) * | 2016-10-09 | 2017-03-22 | 上海交通大学医学院附属仁济医院 | Portable micro-fluidic chip LAMP (loop-mediated isothermal amplification) visible detector and detection method thereof |
Non-Patent Citations (4)
| Title |
|---|
| HUA WANG等: "Toward point-of-care testing for JAK2 V617F mutation on a microchip", 《JOURNAL OF CHROMATOGRAPHY A》 * |
| MASAHIRO ITONAGA等: "Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification", 《PLOS ONE》 * |
| 张阳等: "环介导等温基因扩增技术检测 JAK2 V617 F基因突变", 《中华检验医学杂志》 * |
| 雷光焰等: "《现代肿瘤理论与临床治疗决策》", 30 November 2014, 中国科学技术出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022101259A1 (en) * | 2020-11-10 | 2022-05-19 | MultiplexDX, s.r.o. | Loop mediated isothermal nucleic acid amplification (lamp) using lna-modified primers and a metal-based colorimetric method for detecting an amplification product |
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