CN103243153B - JAK2 V617F mutation detection application - Google Patents
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- CN103243153B CN103243153B CN201210030473.7A CN201210030473A CN103243153B CN 103243153 B CN103243153 B CN 103243153B CN 201210030473 A CN201210030473 A CN 201210030473A CN 103243153 B CN103243153 B CN 103243153B
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Abstract
The invention belongs to the field of nucleic acid detection, and particularly relates to the detection of JAK2 (Janus Kinase 2) gene V617F locus DNA mutation (G1849T) and a test kit and a primer pair used for the detection. The present invention discloses a method for detecting V617F mutation in the JAK2 gene, which comprises (1) performing PCR amplification for the samples to be tested, and (2) performing melting analysis by using a melting analyzer. Further, the present invention also discloses the test kit and the primer pair for the detection method.
Description
Technical field
The invention belongs to detection of nucleic acids field, particularly, the present invention relates to the detection of JAK2 gene V617F site thymus nucleic acid sudden change (G1849T) and detection kit and primer equity used.
Background technology
Janus kinase 2 (being called for short JAK2 herein) belongs to Janus kinases family, is tyrosine protein kinase in born of the same parents.JAK2 from N-hold to C-end altogether by JH7-JH1 the homeodomain of totally 7 high conservatives (JH1～JH7) form, by acting on EPO, TPO, IL-3, G-CSG, GM-CSF acceptor, play a part signal conduction in regulating cell, produce cell propagation effect.Yet, the 1849th guanine of JAK2 gene 14 exons replaces with thymus pyrimidine, the 617th phenylalanine that is positioned at the pseudo-kinases of JAK2 albumen region made a variation as α-amino-isovaleric acid (JAK2 V617F sudden change), to cause pseudo-kinases region inactivation, finally cause JAK-STAT, PI3K, the paths such as AKT occur that acellular factor dependency activates, thereby may cause bone marrow proliferative tumour (myeloproliferative neoplasms, MPN), comprise chronic myelocytic leukemia (chronic myeloid leukemia, CML), polycythemia vera (polycythemia vera, PV), primary thrombocytosis (essential thrombocythemia, ET) with PMF (primary myelofibrosis, PMF) etc.From 2008, the World Health Organization was defined as JAK2 V617F sudden change the molecular diagnosis gold standard of MPN in its official classification Case definition.(can be referring to: James C, Ugo V, Le Cou é dic JP, Staerk J, Delhommeau F, et al. (2005) A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera.Nature 434:1144; Swerdlow S, Campo E, Harris NL, Jaffe ES, Pileri SA, et al. (2008) WHO Classifcation of Tumours of Haematopoietic and Lymphoid Tissues.WHO Classification of Tumours.4th ed.Lyon, France:IARC)
The method that detects at present this point mutation is mainly to check order behind polymerase chain reaction (PCR), but the order-checking in this method is consuming time and instrument and the reagent cost of needs are higher, is not suitable for large-scale promotion and uses.The present invention is through long-term and arduous research, and in conjunction with the fortune of some screenings, work out pair of primers and worked out supporting method, can obtain fast by the liquation of pcr amplification product result, wherein without step consuming time, and the maintenance cost of liquation instrument is lower than sequenator.Because the ratio of crowd's mutator gene is very low, the efficiency detecting one by one in practice is very low, therefore often with many people's biased sample, detect, more unexpectedly, the detection method that the inventor finds out can be differentiated the sample that only contains the saltant type of 1% (even lower), and this is to check order to complete, therefore improved detection efficiency.
Summary of the invention
The detection application of the JAK2 V617F sudden change that the technical problem to be solved in the present invention is to provide new, when this application is darker than prior art, cost is lower and/or more efficient.In addition, the present invention also provides test kit and the primer equity for these application.
Particularly, in first aspect, the invention provides the method for the V617F sudden change that detects JAK2 gene, it comprises:
(1) detected sample is carried out to pcr amplification, use therein primer pair sequence is:
5’-TGAAGCAGCAAGTATGATGAG-3’，
5 '-GGGAGTATGTGTCTGTGGAGACTGACACCTAGCTGTGATCCTG-3 '; With
(2) with liquation instrument, carry out liquation.
In this article, detected sample is the vitro samples of the potential people of containing JAK2 gene DNA, comprises blood, body fluid or its purification thing.Because the ratio of crowd's mutator gene is very low, the efficiency detecting one by one in practice is very low, and the inventor finds, the method for first aspect present invention also can be applicable to biased sample.Therefore preferred in the method for first aspect present invention, detected sample is the biased sample from Different Individual.Owing to being biased sample, result can only show in biased sample, whether to contain mutated genes, but cannot accurately judge wherein whether any one individual sample contains mutated genes, therefore can not draw diagnostic result for individuality, and can only draw detected result for biased sample.Preferably the method for first aspect present invention is comprised of above-mentioned (1) and (2), i.e. enclosed method.Like this, more cannot draw for individuality and draw diagnostic result, and can only draw in biased sample, whether to contain this non-diagnostic result of mutated genes.
Because method of the present invention is highly sensitive, can detect gene in biased sample only containing the mutated genes (all the other are wild type gene) of 1% (even lower).In the present invention, content is when referring to the content of gene (comprising wild type gene and mutated genes), based on context, refer to the content of mutated genes in wild type gene and mutated genes total amount, or refer to the content of wild type gene in wild type gene and mutated genes total amount.Therefore more preferably in the method for first aspect present invention, in biased sample, the JAK2 gene content of V617F saltant type is no more than 20%, is preferably no more than 15%, more preferably no more than 10%, more preferably no more than 5%, be more preferably and be no more than 2%, as 1.5 or 1% etc.
In the method for first aspect present invention, the system of pcr amplification can also comprise Taq enzyme, damping fluid and dNTP etc.Preferably, in the method for first aspect present invention, the system of pcr amplification also comprises SYTO-9 fluorescence dye.SYTO-9 fluorescence dye can mix in double-stranded DNA and send fluorescence, thereby can indicate the degree of melting.These compositions can be prepared voluntarily, also can buy by commercial sources, preferably by commercial sources, buy.
In the method for first aspect present invention, pcr amplification is incomplete pcr amplification, is convenient to like this carry out liquation with liquation instrument.Incomplete pcr amplification refers to sex change (as with 95 ℃ of sex change), the annealing (as with 55 ℃ of annealing) in each circulation in pcr amplification and the time of (as with 72 ℃ of extensions) of extending is no more than respectively 8 seconds, preferably be no more than 5 seconds, more preferably no more than 3 seconds, be more preferably and be no more than 2 seconds, as 1.5 seconds or 1 second.According to the product characteristics of amplification, the inventor has optimized the step of pcr amplification.Preferably, in the method for first aspect present invention, the step of pcr amplification is: 95 ℃ of 2min of denaturation, 70 thermal cyclings, wherein each circulation be 95 ℃ 1 second, 55 ℃ 1 second, 72 ℃ 1 second.
In the method for first aspect present invention, liquation is to generate fluorescence-melting temperature (Tm) derivative curve.This can and map by reading voluntarily, also can automatically generate by commercially available liquation instrument, for example, in the specific embodiment of the present invention, by the generation automatically of Qiagen Rotor-Gene Q high resolving power liquation instrument.
In second aspect, the invention provides primer pair, its nucleotides sequence is classified as:
5’-TGAAGCAGCAAGTATGATGAG-3’，
5’-GGGAGTATGTGTCTGTGGAGACTGACACCTAGCTGTGATCCTG-3’。
Primer pair generally passes through synthetic.This primer pair can the method for first aspect present invention in.
In the third aspect, the invention provides the test kit for detection of the V617F sudden change of JAK2 gene, it comprises the primer pair of second aspect present invention.In test kit, each composition is all separated placement.
The test kit of third aspect present invention can also comprise the compositions such as Taq enzyme, damping fluid and dNTP.Preferably the test kit of third aspect present invention also comprises SYTO-9 fluorescence dye.
In yet another aspect, the invention provides the primer pair of second aspect present invention and/or the application of the test kit of third aspect present invention in the reagent of the method for the preparation of first aspect present invention.For example, the application of the primer pair that the invention provides second aspect present invention in the test article of the method for the preparation of first aspect present invention, preferred wherein test article comprises the test kit of third aspect present invention, can be also the test kit of third aspect present invention.And for example, the application of the test kit that the invention provides third aspect present invention in the reagent of the method for the preparation of first aspect present invention, preferably wherein test article comprises the test kit of third aspect present invention and has recorded the specification sheets of the method for first aspect present invention.
The beneficial effect that the present invention obtains is, detect save time, cost is low and/or can detect biased sample, highly sensitive, efficiency is high.
For the ease of understanding, below the drawings and specific embodiments by concrete are described in detail the present invention.It needs to be noted, these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification sheets, many variations of the present invention, change have been all obviously concerning one of ordinary skill in the art.The open source literature that the present invention quotes is in addition in order more clearly to describe the present invention, and their full text content is all included in and carried out reference herein, just looks like that repeated description is excessively the same in this article for their full text.
Accompanying drawing explanation:
Fig. 1: fluorescence-melting temperature (Tm) derivative curve of different testing samples, wherein the curve in legend (explanation) from top to bottom sample be successively mixture (saltant type accounts for 10%) and the saltant type of mixture (saltant type accounts for 1%), wild-type and saltant type of mixture (saltant type accounts for 0.1%), wild-type and the saltant type of wild-type, wild-type and saltant type.
Embodiment
The present invention will carry out exemplary explanation by specific embodiment, wherein, if any not using up part, can be referring to the laboratory manuals such as < < molecular cloning experiment guide (third edition) > > (Science Press, Beijing) and commercially available reagent and the operation instruction of instrument.
The inventor entrusts Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to synthesize the JAK2 gene (referred to as wild-type) of wild-type and the JAK2 V617F mutator gene (referred to as saltant type) suddenling change with G 1849T according to the sequence shown in Genbank accession number NM_004972.3, and by mutated genes by a certain percentage (0.1%, 1%, 10%) be mixed into wild type gene, form different detected samples.
The inventor entrusts Shanghai Sangon Biological Engineering Technology And Service Co., Ltd to synthesize following primer simultaneously:
Primer 1:5 '-TGAAGCAGCAAGTATGATGAG-3 ';
Primer 2: 5 '-GGGAGTATGTGTCTGTGGAGACTGACACCTAGCTGTGATCCTG-3 '.
Use TaKaRa PCR test kit (purchased from TaKaRa company) to carry out pcr amplification in Qiagen Rotor-Gene Q high resolving power liquation instrument (purchased from Qiagen company), wherein pcr amplification system is:
Detected sample solution 1 μ l;
RTaq enzyme (5U/ μ l) 0.1 μ l;
10 * PCR damping fluid (Mg
2+free) 2 μ l;
Mg
2+damping fluid (2.5mM) 2.5 μ l;
DNTP mixed solution (2.5mM) 1.6 μ l;
Primer 1 (0.5 μ M) 1 μ l;
Primer 2 (10 μ M) 1 μ l;
SYTO-9 fluorescence dye (purchased from Invitrogen company) (50 μ M) 0.8 μ l; With
Deionized water 10 μ l.
Pcr amplification step is: 95 ℃ of 2min of denaturation, 70 thermal cyclings (95 ℃ 1 second, 55 ℃ 1 second, 72 ℃ 1 second); With liquation instrument, carry out liquation (55 ℃-90 ℃, 0.2 ℃/Step), generate fluorescence-melting temperature (Tm) derivative curve (dF/dT).
As shown in Figure 1, detected sample is wild-type sequence to result, has lower Tm (63 ℃); Detected sample is saltant type sequence, in liquation process, will produce higher Tm (70 ℃); And detected sample is the biased sample that contains a small amount of saltant type, between wild-type and the fusion zone of saltant type, produce respectively the corresponding peak that melts, and the form of melting curve changes the content with saltant type, thereby can carry out semi-quantitative analysis, can distinguish in biased sample, whether to contain the wherein shared content of saltant type sequence of saltant type sequence and preresearch estimates.
Claims (9)
1. for the method for the V617F sudden change of the detection JAK2 gene of non-medical diagnosis on disease object, it is characterized in that, it comprises:
(1) detected sample is carried out to pcr amplification, use therein primer pair sequence is:
5’-TGAAGCAGCAAGTATGATGAG-3’，
5 '-GGGAGTATGTGTCTGTGGAGACTGACACCTAGCTGTGATCCTG-3 '; With
(2) with liquation instrument, carry out liquation;
The step of described pcr amplification is: 95 ℃ of 2min of denaturation, 70 thermal cyclings, wherein each circulation be 95 ℃ 1 second, 55 ℃ 1 second, 72 ℃ 1 second.
2. by method claimed in claim 1, it is characterized in that, wherein detected sample is the biased sample from Different Individual.
3. by method claimed in claim 2, it is characterized in that, wherein in biased sample, the JAK2 gene content of V617F saltant type is no more than 20%.
4. by method claimed in claim 1, it is characterized in that, wherein the system of pcr amplification also comprises SYTO-9 fluorescence dye.
5. by method claimed in claim 1, it is characterized in that, wherein liquation is to generate fluorescence-melting temperature (Tm) derivative curve.
6. primer pair, its sequence is:
5’-TGAAGCAGCAAGTATGATGAG-3’，
5’-GGGAGTATGTGTCTGTGGAGACTGACACCTAGCTGTGATCCTG-3’。
7. for detection of the test kit of the V617F sudden change of JAK2 gene, it is characterized in that, it comprises primer pair claimed in claim 6.
8. by test kit claimed in claim 7, it is characterized in that, it also comprises SYTO-9 fluorescence dye.
9. the application of the test kit described in primer pair claimed in claim 6 and/or claim 7 or 8 in the test article of the arbitrary described method for the preparation of claim 1～5.
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| CN104818340B (en) * | 2015-05-29 | 2018-06-26 | 沈阳优吉诺生物科技有限公司 | Detect primer, kit and its PCR method of JAK2 gene V617F loci polymorphisms |
| CN105441573A (en) * | 2016-01-11 | 2016-03-30 | 武汉海吉力生物科技有限公司 | Primer, probe and kit for detecting mutation of human JAK2 gene V617F |
| CN106086204B (en) * | 2016-07-14 | 2017-08-04 | 上海金域医学检验所有限公司 | The primer and its method of a kind of detection JAK2 V617F gene mutations |
| CN107841536A (en) * | 2017-10-24 | 2018-03-27 | 复旦大学附属华山医院 | A kind of Primer composition and its kit and detection method of detection JAK2 V617F gene mutations |
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| CN101622363A (en) * | 2007-07-13 | 2010-01-06 | 爱科来株式会社 | Probe for detecting mutation in JAK2 gene and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101072870A (en) * | 2004-10-27 | 2007-11-14 | 巴黎国立医院（Ah-Hp） | Identification of a JAK2 mutation in polycythemia vera |
| CN101622363A (en) * | 2007-07-13 | 2010-01-06 | 爱科来株式会社 | Probe for detecting mutation in JAK2 gene and use thereof |
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| Title |
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