CN107604069A - People's JAK2 V617F mutation detection kits and method based on Taqman MGB probes - Google Patents
People's JAK2 V617F mutation detection kits and method based on Taqman MGB probes Download PDFInfo
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Abstract
The invention discloses AK2 V617F mutation fluorescent quantificationally PCR detecting kits and detection method, include the primer as shown in SEQ ID NO.7 SEQ ID NO.8 for people JAK2 V617F and the probe as shown in SEQ ID NO.13 SEQ ID NO.14.Detecting system after the probe and primer that the present invention designs, and optimization, there is extraordinary specificity and sensitivity.The present invention can not effectively expand the wild TaqMan MGB probes of wild allele and allele to the specific sealing process in wild allele site to suppress wild amplified allele jointly using allelic mutation primer, so as to establish with high sensitivity and specific JAK2 V617F mutation rate detection methods.
Description
Technical field
The invention belongs to biology field, is related to medical science and biotechnology, relates particularly to one kind and is based on
The people's JAK2 V617F mutation detection kits and method of Taqman-MGB probes.
Background technology
Bone marrow proliferative tumour (myelo-proliferative neoplasm/myelodysplastic/
Myeloprolifer-ative disorder, MPN/MDS/MPD) it is a kind of inertia tumour, it is white mainly to include chronic granulocyte
Blood disease (CML), polycythemia vera (PV), primary thrombocytosis (ET), and primary myelofibrosis
(PMF), CNL (CNL), chronic acidophil leukecythemia/high acidophil syndrome (CEL/HES)
Deng.Clinically the MPD of meaning is usually PV, IMF and the ET for reflecting pluripotential hemopoietic stem cell conversion at present.MPD falls ill without feature
And incubation period is grown, and is common in 40-70 year, year, mild case accounted for 20% during diagnosis.Because bone marrow proliferative diseases patient lacks allusion quotation
The clinical symptoms of type, in addition to CML, the diagnosis of other hypotypes depends on morphologic inspection in MPD for a long time, this
Tend not to make a definite diagnosis MPD type, and then patient can not obtain the effective treatment.
JAK2 is by one paid high attention in JAK (one of non-receptor type tyrosine protein kinase, Janus kinases) family
Member, it is distributed widely in body cell kytoplasm, participates in the signal transduction of hematopoiesis and the system such as immune.Multinomial research confirmation, PV,
JAK2 kinases V617F point mutation in the diseases such as ET, PMF be present.The mutational site DNA sequence dna the 1849th bit base, by chest
Gland pyrimidine instead of original guanine (G-T), causes the phenylalanine of encoding proteins the 617th to be replaced by valine, causes
The tyrosine kinase activity of the gene code increases.JAK2 V617F are the mutation for betiding stem cell levels, the world in 2008
Health organization incorporates JAK2 V617F mutation PV, PT and PMF diagnostic criteria.Therefore JAK2 V617F detection into
For the main molecules mechanism of causing a disease and diagnostic markers of the bone marrow proliferative such as PV, ET tumour (MPN).
At present, be mainly to PV and ET treatment method rapid bloodletting (remove excessive blood platelet or reduce blood volume) and
Drug therapy (radionuclide phosphorus, hydroxycarbamide or busulfan etc.).PMF treatment method is mainly suited the medicine to the illness and supportive treatment, such as
Splenectomy or rocalcitrol (1,25 (OH)2D3) treatment fibrosis etc..Histology knot of the JAK2 V617F mutation with reference to bone marrow biopsy
Fruit, foundation can be provided for ET and PMF diagnosis and MPD treatments.In addition, prognosis of the JAK2 mutation on MPD patient also has certain influence.
In ET and PV patients, JAK2 V617F express high, poor prognosis, and in PMF patient, JAK2 V617F expression is low, in advance
It is poor afterwards.
JAK2 V617F abrupt climatic changes use more and use PCR- RFLPs (restriction more
Fragment length polymorphism, RFLP), cut model and flashlight model detector (amplification
Refractory mutation system, ARMS) or direct Sequencing analyze the methods of, but the above two exist false positive (or
Sensitivity is relatively low), no standard measure detection, the latter's detection is more accurate, but time-consuming cost, in MPD follow-ups and Molecular remission judge
The limited popularization and application for being unsuitable for clinical diagnosis of effect.
The content of the invention
It is an object of the invention to provide it is a kind of with good specificity and sensitivity based on Taqman-MGB probes
People's JAK2 V617F mutation detection kits, the kit has the high advantage of detection resolution.
To achieve the above object, the technical solution adopted by the present invention is as follows：
People's JAK2 V617F mutation detection kits based on Taqman-MGB probes, include for people JAK2 V617F
The primer as shown in SEQ ID NO.7-SEQ ID NO.8 and the probe as shown in SEQ ID NO.13-SEQ ID NO.14；
5 ' ends of the probe are marked with fluorophor, and 3 ' ends are marked with quenching group.
Concentration of the SEQ ID NO.13-SEQ ID NO.14 in detection architecture is 200nM/ml；Ratio is SEQ ID
NO.14：SEQ ID NO.13 amount ratio 1:2.So that the Ct differences for detecting two Air conduct measurements during homozygous sample are sufficiently large,
Ct differences when detecting heterozygous sample are sufficiently small.
In one of the embodiments, the kit also includes buffer solution, magnesium ion, dNTP, Taq enzyme.
In one of the embodiments, the fluorophor that the probe 5 ' is held is one kind in FAM, VIC or HEX, and 3 ' hold
Quenching group be NFQ.
It is a further object to provide a kind of people's JAK2 V617F abrupt climatic changes based on Taqman-MGB probes
Method.
Realize that above-mentioned purpose technical scheme is as follows.
People's JAK2 V617F mutation detection methods based on Taqman-MGB probes, comprise the following steps：
(1) DNA of template to be measured is extracted；
(2) reaction solution is prepared, magnesium ion, dNTP, above-mentioned primer and probe, Taq enzyme are added in buffer solution；
(3) template DNA to be measured that rapid (1) obtains is added separately in the PCR reaction solutions, mixed in fluorescent PCR pipe
After closing uniformly centrifugation, it is put into quantitative fluorescent PCR instrument and carries out PCR reactions；
(4) genotype of detection site is determined by data collected by quantitative real time PCR Instrument.
In one of the embodiments, the quantitative fluorescent PCR reaction condition used for：50 DEG C of 2min, 95 DEG C of 15min, 94
DEG C 30s, 55 DEG C of 30s, 72 DEG C of 30s, after 40 circulations, 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s, 60 DEG C of 15s, then from 60 DEG C
Slowly it is warmed to 95 DEG C.
In one of the embodiments, detect in reaction system, SEQ ID NO.14 and SEQ ID NO.14 concentration ratio
For for 1:1.8-2.2.Consider from the integrated cost and Detection results for making kit, most preferably SEQ ID NO.14 and SEQ
ID NO.14 concentration ratio is 1:2.
In one of the embodiments, SEQ ID NO.14 detect final concentration of 10nM/ml -15nM/ in reaction system
ml。
After quantitative fluorescent PCR EP (end of program), corresponding amplification curve diagram is obtained, observes mesh in specific PCR reaction system
Detection fluorescence signal whether form amplification curve, it is more representated by the specific reaction system to have the then DNA sample to be checked
The positive of state property type.
Main advantages of the present invention are：
It is wild that 1. the present invention utilizes allelic mutation primer effectively to expand wild allele and allele
TaqMan-MGB probes suppress wild amplified allele jointly to the specific sealing process in wild allele site, from
And establish with high sensitivity and specific JAK2 V617F mutation rate detection methods, moreover, the kit also has inspection
Survey the advantage of high resolution.
2. the primer and probe designed by the present invention, there is the advantages of high specificity, high sensitivity.Moreover, particularly, this
The dosage that designed probe is found in invention has an impact to testing result, then by many experiments, is finally determined that the present invention is logical
Cross to the 2 kinds of type concentration and probe concentrations and ratio in fluorescent PCR system, so that detecting the Ct of two Air conduct measurements during homozygous sample
Difference is sufficiently large, and Ct differences when detecting heterozygous sample are sufficiently small.Eventually finding optimal concentration and probe concentration ratio i.e. concentration is
During 200nM/ml, ratio W：M=1：2 or so, so as to realize high detection resolution ratio.
3. the JAK2 V617F mutation detection kits of design of the present invention, have and greatly shorten experimental period, sample
This, high flux, it is reproducible the advantages of, therefore this method has higher use value in clinical diagnosis and Treatment monitoring,
Detection cycle can effectively be shortened, effectively aid in MPD patient's pathological diagnosis, offer effectively reference is probed into for clinical therapeutic efficacy
Foundation.
Brief description of the drawings
Accompanying drawing 1 is B1 sample JAK2 gene V617F site kit fluorescence quantitative PCR detection figures；
Accompanying drawing 2 is B4 sample JAK2 gene V617F site kit fluorescence quantitative PCR detection figures；
Accompanying drawing 3 is B8 sample JAK2 gene V617F site kit fluorescence quantitative PCR detection figures；
Accompanying drawing 4 is in embodiment 3：JAK2 V617F primer screening results；
Accompanying drawing 5 is in embodiment 3：SEQ ID NO.13 probes and the comparison of SEQ ID NO.15 probe in detecting results；
Accompanying drawing 6 is in embodiment 3：SEQ ID NO.14 probes and the comparison of SEQ ID NO.16 probe in detecting results；
Accompanying drawing 7 is in embodiment 4：The sensitivity results of JAK2 V617F mutation detection kits detection；
Accompanying drawing 8 is in embodiment 5：Detected for homozygous mutation, the probe ratiometric result schematic diagram of experimental group 8；
Accompanying drawing 9 is in embodiment 5：Detected for homozygous mutation, the probe ratiometric result schematic diagram of experimental group 14；
Accompanying drawing 10 is in embodiment 5：Detected for heterozygous mutant, wild type and saltant type probe ratio are 1:1 probe
Ratiometric result schematic diagram；
Accompanying drawing 11 is in embodiment 5：Detected for heterozygous mutant, wild type and saltant type probe ratio are 1:2 ratio
Result schematic diagram.
Embodiment
The present invention is described in further detail below in conjunction with the embodiment given by accompanying drawing.
Embodiment 1
People's JAK2 V617F mutation detection kits based on Taqman-MGB probes described in the present embodiment, in kit
Polymorphism-specificity PCR reaction solutions including this site of V617F, wherein PCR reaction solutions contain PCR and react necessary buffering
It is also corresponding to include following specific detection primers and probe outside the materials such as liquid, magnesium ion, dNTP, Taq enzyme：
The JAK2 gene extension primer sequences of table 1 and probe
The fluorophor that the probe 5 ' is held is one kind in FAM, VIC or HEX, and the fluorophor in the present embodiment is
FAM and HEX, 3 ' ends are MGB.
In use, according to following procedure and step：
(1) extraction of DNA profiling
Recommend the tissue DNA that commercialized kit extracts people to be measured；
(2) PCR reaction systems configuration optimization
The reaction detection system of each PCR amplifications is 20 μ l, and obtained mixing is added after detected sample into reaction tube
Each component final concentration and its content are as follows in thing：
(3) fluorescent PCR detects
By the PCR system configured in fluorescent PCR pipe be well mixed centrifugation after, be put into quantitative fluorescent PCR instrument according to
Specific temperature cycles and signal acquisition program carry out fluorescence quantitative PCR detection, the quantitative fluorescent PCR reaction condition used for；
50 DEG C of 2min, 95 DEG C of 15min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, after 40 circulations, 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C
15s, 60 DEG C of 15s, then slowly it is warmed to 95 DEG C from 60 DEG C.
Embodiment 2
The embodiment of the present invention is mutated inspection according to the people JAK2 V617F based on Taqman-MGB probes as described in Example 1
Test agent box, parting detection is carried out to tissue DNA sample respectively, comprised the following steps：
(1) extraction of DNA profiling
Recommend the tissue DNA that commercialized kit extracts people to be measured；
(2) PCR reaction detections system configurations
19 μ L V617F PCR reaction solutions are taken into a PCR reaction tube, and a step (1) is added into PCR reaction tubes
The μ L of DNA 1 of middle extraction, each sample repeat three pipes, specifically as described in Example 1；
(3) fluorescent PCR detects
By the PCR system configured in fluorescent PCR pipe be well mixed centrifugation after, be put into quantitative fluorescent PCR instrument according to
Specific temperature cycles and signal acquisition program carry out fluorescence quantitative PCR detection, the quantitative fluorescent PCR reaction condition used for；
50 DEG C of 2min, 95 DEG C of 15min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, after 40 circulations, 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C
15s, 60 DEG C of 15s, then slowly it is warmed to 95 DEG C from 60 DEG C.
(4) Genotyping interpretation
The genotype of detection site is determined by data collected by quantitative real time PCR Instrument.Quantitative fluorescent PCR EP (end of program)
Afterwards, the amplification curve diagram in corresponding site is obtained, observes whether purpose detection fluorescence signal in specific PCR reaction system forms expansion
Increase curve, then the DNA sample to be checked is the positive of the polymorphism type representated by the specific reaction system.
Experimental result：
The people's JAK2 V617F abrupt climatic change result parting interpretations of table 2
The qPCR partings testing result of above-mentioned all detection samples is by DNA sequencing checking, sequencing result and the present invention
Method testing result is consistent；Prove the result of the inventive method detection accurately and reliably.
Accompanying drawing 1 is B1 sample JAK2 gene V617F site kit fluorescence quantitative PCR detection figures, and detection figure shows it
There is wild-type probe fluorescence signal to form amplification curve, then judge the sample for JAK2 V617F wild-type homozygotes.
Accompanying drawing 2 is B4 sample JAK2 gene V617F site kit fluorescence quantitative PCR detection figures, and detection figure shows that it is wild
Raw type probe and saltant type fluorescence probe signal are respectively formed amplification curve, then judge the sample for JAK2 V617F heterozygous mutations
Son.
Accompanying drawing 3 is B8 sample JAK2 gene V617F site kit fluorescence quantitative PCR detection figures, and detection figure shows it
There is wild-type probe fluorescence signal to form amplification curve, then judge the sample for the homozygous mutons of JAK2 V617F.
The contrast of the selection of the people's JAK2 V617F mutation detection specific primer sequences of embodiment 3
By taking the common gene mutations site of people's JAK2 gene mutations as an example, specific primer sequence is designed, with the mutation
The complementary series forward or backwards of target sequence is template where site, for the wild type and saltant type in V617F mutational sites
It is (or big to design in specific probe sequence, including the embodiment of the present invention 1 preferable specific primer and probe sequence and 2
In 2) alternative specific primer and probe sequence, as shown in Table 3, 4.
The primer sequence of table 3
The specific probe sequence of table 4
The present invention first screens a series of primers and probe in development, then final through PCR conditions and system optimization
Screen that a group-specific is most strong, the optimal primer and probe of amplification efficiency.
Detection method according to embodiment 2 is detected.
Primer combines：V617F-1 is SEQ ID NO.1 and SEQ ID NO.2；V617F-2 is SEQ ID NO.3 and SEQ
IDNO.4；V617F-3 is SEQ ID NO.5 and SEQ ID NO.6；V617F-4 is SEQ ID NO.7 and SEQ ID NO.8；
V617F-5 is SEQ ID NO.9 and SEQ ID NO.10；V617F-6 is SEQ ID NO.11 and SEQ ID NO.12.
Fig. 4 is JAK2 V617F primer screening results, and V617F-4 combination amplification efficiencies are best.
Expanded with V617F-4 primers, the comparison of different probe under the same conditions (as shown in Fig. 5~6).According to embodiment
Detection method described in 2 is detected.
Fig. 5：SEQ ID NO.14 probes, wild type have amplification curve, and saltant type does not have, and parting is clear and definite；SEQ ID
NO.16, wild type have amplification curve, but indefinite compared with SEQ ID NO.14 probe partings；Illustrate that JAK2 V617F wild types are visited
Pin SEQ IDNO.14 specificity is better than SEQ ID NO.16.
Fig. 6：SEQ ID NO.13 probes, saltant type have amplification curve, and wild type does not have, and parting is clear and definite；SEQ ID
NO.15 probes, wild type and saltant type have amplification, indefinite compared with SEQ ID NO.13 probe partings；Illustrate JAK2 V617F
Saltant type probe SEQ ID NO.13 specificity is better than SEQ ID NO.15.
It can be seen that the primer as shown in SEQ ID NO.7-SEQ ID NO.8 of the present invention for JAK2 V617F
With the probe as shown in SEQID NO.13-SEQ ID NO.14, have amplification efficiency optimal, detect the most strong advantage of specificity.
The sensitivity experiment of embodiment 4JAK2 V617F mutation detection kits detection
Healthy human peripheral blood genomic DNA is extracted, 30ng/ μ l are calibrated to by ultraviolet specrophotometer.People is red white
Blood disease cell line (HEL, purchased from Chinese Academy of Sciences's cell bank) extracts genomic DNA conduct after cultivating to Exponential growth stage
The pure and mild mutation standard items of JAK2V617F.Because clone's number of JAK2 V617F in each hel cell strain can be more than more than 2.Cause
This is copied when configuring mutation rate standard items according to the JAK2 V617F allele in healthy human gene group DNA (30ng/ μ l)
Number, using the wild plasmid standards of JAK2 V617F of structure as standard curve, by quantitative fluorescent PCR, by hel cell strain base
Because group DNA copy number is calibrated, to ensure the accuracy of JAK2 V617F mutation rates in standard items.With Healthy People 30ng/ μ
L genomic DNAs are as dilution matrix, by the hel cell pnca gene group DNA after calibration and 30ng/ μ l healthy volunteer's genomes
DNA presses 1：10 dilution proportions, it is configured to 100%-0.1% JAK2 V617F mutation standard items.According to the kit of embodiment 1
And the detection method described in embodiment 2 is detected.
Experimental result is as shown in fig. 7, respectively with 100%, 10%, 1%, 0.1%, 0% (i.e. wild plasmid standard)
JAK2 V617F mutation standard items carry out carrying out real-time fluorescence quantitative PCR amplification under the same conditions as template, are sent out through experiment
Existing, 0.1% JAK2V617F mutation standard items have obvious amplification curve, wild plasmid standard does not have under test conditions
Have, illustrate that this method sensitivity is higher, there is good detection application value.
Embodiment 5
Inventor has found in the actual design kit, because wild type and saltant type probe are in same reaction system
In, it is designed to probe different concentration and ratio experimental result is produced a very large impact, therefore to the concentration ratio of probe
Example configuration has carried out substantial amounts of research.By repeated screening, finally determine first to use all experiments for same homozygous sample
Group setting is tested, and further verification experimental verification is carried out for heterozygous sample, it can thus be seen that present invention determine that most
Excellent concentration and probe concentration proportional arrangement, Detection results are good.With reference to such as following table, experimental group 1-20 is set.
The experimental group 1-20 of table 5 wild type and the concentration and usage ratio of saltant type probe
(1) extraction of DNA profiling
Recommend the tissue DNA that commercialized kit extracts people to be measured；
(2) PCR reaction systems configuration optimization
The reaction detection system of each PCR amplifications is 20 μ l, and obtained mixing is added after detected sample into reaction tube
Each component final concentration and its content are as follows in thing：
(3) fluorescent PCR detects
By the PCR system configured in fluorescent PCR pipe be well mixed centrifugation after, be put into quantitative fluorescent PCR instrument according to
Specific temperature cycles and signal acquisition program carry out fluorescence quantitative PCR detection, the quantitative fluorescent PCR reaction condition used for；
50 DEG C of 2min, 95 DEG C of 15min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, after 40 circulations, 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C
15s, 60 DEG C of 15s, then slowly it is warmed to 95 DEG C from 60 DEG C.
(4) experimental result reads analysis
For homozygous testing this detection, carried out according to the concentration and probe concentration and ratio reaction result of experimental group 1-20 experimental groups
Analysis, represents result as shown in accompanying drawing 8-9, wherein, Fig. 9 is the result for the experiment that the concentration and probe concentration of experimental group 14 is carried out, specifically
Property it is good, and saltant type probe amplification fluorescent value is high, and amplification efficiency is strong.Fig. 8 is the experimental result that the concentration and probe concentration of experimental group 8 is carried out
(result of other experiments, similar to Fig. 8, the reason for because of length, not providing), its be not it is bad with specific amplification be exactly to expand
Energization power is poor, thus can primarily determine that the optium concentration of probe is 200nM/ml, while is evident that when wild and prominent
Modification concentration and probe concentration is wild type in 200nM/ml and system：The usage ratio of saltant type is 1：When 2, the fluorescence of amphitypy probe
It is worth higher and no cross reaction, the Ct differences of two Air conduct measurements are sufficiently large when detecting homozygous sample.
Wild type during in order to confirm that wild type and saltant type concentration and probe concentration are 200nM/ml：The optimal proportion of saltant type,
When being 200nM/ml to concentration, multigroup experimental group is set, it is 3 finally to determine ratio:1 to 1：Between 3, according to wild type and mutation
Type concentration and probe concentration is 3:1,2:1,1:1,1:2,1：3 are tested, it is found that when wild and saltant type concentration and probe concentration is
200nM/ml, and wild type in system：The ratio of saltant type is 1：Sufficiently small (the ginseng of Ct differences when heterozygous sample is detected when 2
See Figure 11), and other testing results are not as shown in figure 11, such as wild type and saltant type probe ratio are 1:1 probe ratio
Example result, referring to Figure 10, its fluorescent value is low, and amplification ability is not strong.The result figure of other experiments is similar with Figure 10.Reagent of the present invention
In box, when concentration is 200nM/ml, probe usage ratio is W：M=1:Detection results can be only achieved optimal when 2.
The repeated experiment of embodiment 5
Detected according to the detection method described in the kit and embodiment 2 of embodiment 1.By 0.1%, 1%, 10%
And replication is distinguished in 100%JAK2 V617F standard item groups, between group 10 times, aberration rate is shown in Table.Aberration rate maximum number in group
Value 4.235%, minimum 0.234%.Average 1.805%；Between-group variation rate maximum 3.742%, minimum 0.116% are average
1.722%.Experimental result fully shows that this kit has good stability in actual mechanical process.
The JAK2 V617F of table 5 detect replica test result
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope that this specification is recorded all is considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
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Claims (10)
1. people's JAK2V617F mutation detection kits based on Taqman-MGB probes, it is characterised in that include for people
JAK2V617F primer as shown in SEQ ID NO.7-SEQ ID NO.8 and such as SEQ ID NO.13-SEQ ID NO.14 institutes
The probe shown；5 ' ends of the probe are marked with fluorophor, and 3 ' ends are marked with quenching group.
2. people JAK2V617F mutation detection kits according to claim 1, it is characterised in that the kit also wraps
Buffer solution, magnesium ion, dNTP, Taq enzyme are included.
3. people JAK2V617F mutation detection kits according to claim 1, it is characterised in that what the probe 5 ' was held
Fluorophor is one kind in FAM, VIC or HEX, and the quenching group at 3 ' ends is NFQ.
4. people's JAK2V617F mutation detection kits according to claim any one of 1-3, it is characterised in that detecting
In reaction system, SEQ ID NO.14 and SEQ ID NO.14 concentration ratio is 1:1.8-2.2.
5. people JAK2V617F mutation detection kits according to claim 4, it is characterised in that in detection reaction system
In, SEQ ID NO.14 and SEQ ID NO.14 concentration ratio is 1:2.
6. people JAK2V617F mutation detection kits according to claim 5, it is characterised in that SEQ ID NO.14 are examined
Final concentration of 10nM/ml-the 15nM/ml surveyed in reaction system.
7. a kind of people's JAK2V617F mutation detection methods based on Taqman-MGB probes, it is characterised in that including following step
Suddenly：
(1) DNA of template to be measured is extracted；
(2) reaction system is prepared, magnesium ion, dNTP are added in buffer solution, for people JAK2V617F such as SEQ ID NO.7-
Primer shown in SEQ ID NO.8 and the probe as shown in SEQ ID NO.13-SEQ ID NO.14, Taq enzyme；
(3) template DNA to be measured that rapid (1) obtains is added separately in the PCR reaction solutions, mixed in fluorescent PCR pipe equal
After even centrifugation, it is put into quantitative fluorescent PCR instrument and carries out PCR reactions；
(4) genotype of detection site is determined by data collected by quantitative real time PCR Instrument.
8. people's JAK2V617F mutation detection methods according to claim 7 based on Taqman-MGB probes, its feature exist
In, the quantitative fluorescent PCR reaction condition used for：50 DEG C of 2min, 95 DEG C of 15min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 40
After individual circulation, 95 DEG C of 15s, 60 DEG C of 1min, 95 DEG C of 15s, 60 DEG C of 15s, then slowly 95 DEG C are warmed to from 60 DEG C.
9. people's JAK2V617F mutation detection kits according to claim any one of 7-8, it is characterised in that detecting
In reaction system, SEQ ID NO.14 and SEQ ID NO.14 concentration ratio is 1:1.8-2.2.
10. people JAK2V617F mutation detection kits according to claim 9, it is characterised in that SEQ ID NO.14 are examined
Final concentration of 10nM/ml-the 15nM/ml surveyed in reaction system.
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| CN201711015883.3A CN107604069A (en) | 2017-10-26 | 2017-10-26 | People's JAK2 V617F mutation detection kits and method based on Taqman MGB probes |
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| CN201711015883.3A CN107604069A (en) | 2017-10-26 | 2017-10-26 | People's JAK2 V617F mutation detection kits and method based on Taqman MGB probes |
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| CN110951860A (en) * | 2019-12-23 | 2020-04-03 | 济南金域医学检验中心有限公司 | Method for detecting JAK2V617F mutation rate and special primer and probe thereof |
| CN111471768A (en) * | 2020-04-15 | 2020-07-31 | 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 group and kit for detecting JAK2V617F and CA L R ninth exon gene mutation |
| CN114350767A (en) * | 2022-01-17 | 2022-04-15 | 武汉海希生物科技有限公司 | Primer and probe for detecting JAK2 gene V617F mutation, application thereof and kit |
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| CN102994613A (en) * | 2011-09-09 | 2013-03-27 | 苏州苏大赛尔免疫生物技术有限公司 | Jak2 gene mutation detection method and kit thereof |
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| CN1932038A (en) * | 2006-09-27 | 2007-03-21 | 北京大学人民医院 | Method of detecting JAK2V617F mutation and its special primer and TaqMan MGB probe |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110951860A (en) * | 2019-12-23 | 2020-04-03 | 济南金域医学检验中心有限公司 | Method for detecting JAK2V617F mutation rate and special primer and probe thereof |
| CN111471768A (en) * | 2020-04-15 | 2020-07-31 | 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 group and kit for detecting JAK2V617F and CA L R ninth exon gene mutation |
| 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 |
| CN114350767A (en) * | 2022-01-17 | 2022-04-15 | 武汉海希生物科技有限公司 | Primer and probe for detecting JAK2 gene V617F mutation, application thereof and kit |
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