CN106906299A - People's JAK2V617F gene mutation detection kits - Google Patents
People's JAK2V617F gene mutation detection kits Download PDFInfo
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Abstract
The invention provides a kind of people JAK2V617F gene mutation detection kits, including A boxes and B boxes, A boxes include magnetic bead BC, lysate BL, cleaning solution BW1 and eluent CE；B boxes include premixed liquid I, premixed liquid II, positive criteria product, negative standards' product, ultra-pure water, the Proteinase K Solution of 15mg/mL, DNA Loading Buffer and DNA Marker.The kit of detection people's JAK2V617F gene mutations that the present invention is provided simplifies the operating procedure of experiment by the extraction of gene and abrupt climatic change organic combination a to kit, improves the efficiency of detection.
Description
Technical field
The present invention relates to gene engineering technology field, and in particular to a kind of people JAK2V617F gene mutation detection kits.
Background technology
Polycythemia vera (PV) is a kind of Clonal disease of candidate stem cell, clinical aobvious with red blood cell number and capacity
With the characteristics of work increases, there is the performance caused by sanguine temperament and Hyperviscosity syndrome, often with splenomegaly.PV insidious onsets, make slow progress, evening
Phase can occur various conversions.The incidence of disease of this disease in crowd is about 2.8/10 ten thousand, more typically in male (about 1.4: 1),
Average age during diagnosis is 60 years old, and scope is 15 to 90 years old, but children are rare, less than 40 years old when 5% patient falls ill.
Polycythemia vera belongs to bone marrow proliferative diseases category, is the one kind in typical marrow proliferating tumor.Bone
Marrow proliferating tumor (Myeloproliferative neoplasms, MPNs) refers to that myeloid tissue is continued to multiply and caused
Group disease, often showing as the one of candidate stem cell is or polyphyly malignant hyperplasia.The difference of the cell line according to hyperplasia, MPNs
Typical MPNs and atypia MPNs can be divided into again.Typical MPNs include polycythemia vera (Polycythemia vera,
PV), primary thrombocytosis (Essential thrombocythemia, ET), PMF (Primary
Myelofibrosis, PMF) and chronic myelogenous leukemia (Chronic myeloid leukemia, CML), relation between them
Closely, can often convert with similar Clinical symptoms and mutually or merge presence.
Early in eighties of last century sixties, just clearly, i.e., there is Philadelphia dyeing in the pathogenesis of CML in CML patient's body
Body, therefore CML is also known as Philadelphia Chromosome Positive MPNs.At present, domestic and international various big hospital is also exactly based on whether detection patient deposits
The diagnosis of CML is carried out in Philadelphia chromosome.Until in April, 2005, scientists are in Philadelphia chromosome negative MPNs pathogenesis
Research aspect achieve substantial breakthrough.Result of study shows：Nearly 97% PV patient, about 50% ET patient and 50%
There is the mutation of JAK2V617F, subsequent JAK2V617F transgene mouse models and bone marrow transplanted mice in the PMF patient of left and right
It is to cause the main cause of MPNs that model demonstrates JAK2V617F mutation, discloses the molecular mechanism of MPNs morbidities.
At present, the diagnosis for MPNs, domestic overwhelming majority hospital is still mainly examined using conventional diagnostic method, i.e. basis
In conjunction with secondary diagnosis index, (leucocyte increases severed finger mark (red blood cell volume, arterial oxygen saturation, spleen enlargement situation etc.)
Many, neutrophil alkaline phosphatase activity, serum VB12 contents etc.) diagnosed, checkup item is various, costly.And
And traditional diagnostic method can not often be made a definite diagnosis and cause mistaken diagnosis, delay the treatment of patient, to sufferers themselves and its family members with
Great pain and burden are come.There is sufficient scientific basis due to making a definite diagnosis MPNs by detecting JAK2V617F, 2008
Year, the World Health Organization (WHO) will detect a main diagnosis indexs of the JAK2V617F as PV, ET and PMF.
In sum, the present invention intends providing a kind of kit for detecting people's JAK2V617F gene mutations, makes MPNs diseases,
Especially the diagnosis of PV diseases is more accurate, convenient and quick.
The content of the invention
The kit of detection people's JAK2V617F gene mutations that the present invention is provided is organic by the extraction of gene and abrupt climatic change
It is integrated into a kit, simplifies the operating procedure of experiment, improves the efficiency of experiment.
The technical scheme that the present invention takes is as follows：
People's JAK2V617F gene mutation detection kits, including A boxes and B boxes, A boxes include magnetic bead BC, lysate BL, wash
Wash liquid BW1 and eluent CE；B boxes include premixed liquid I, premixed liquid II, positive criteria product, negative standards' product, ultra-pure water, 15mg/
The Proteinase K Solution of mL, DNALoading Buffer and DNA Marker.
Preferably, the premixed liquid I preparation processes include：By 20 μM of JAK3 primers (shown in SEQ ID NO.2) and 20 μ
The JAK4 primers (shown in SEQ ID NO.3) of M mix in equal volume, then by mix primer and 2 × Taq PCR MIX Loading
Dye-free by volume 2: 55 ratio mix obtain final product.
Preferably, the premixed liquid II preparation processes include：According to final concentration of 5 μM of J2f primers (SEQ ID NO.4 institutes
Show), 5 μM of J2r primers (shown in SEQ ID NO.5), 10 μM of J2nf primers (shown in SEQ ID NO.6) and 40 μM of J2mr draw
The amount of thing (shown in SEQ ID NO.1) prepares nest-type PRC primer, then by nest-type PRC primer and 2 × Taq PCR MIX
Loading Dye-free by volume 2: 55 proportions, mixing obtains final product.
Preferably, the Proteinase K Solution is that Proteinase K is dissolved in into Tris-HCl, CaCl2With the mixing of glycerine three
Obtained in solution；The Tris-HCl concentration is 10mM, pH value 7.5, CaCl2Concentration is 20mM, and qualities of glycerin fraction is 50%.
Preferably, negative standards' product preparation process includes：The μ L of pKS-JAK2 plasmids 1 are added to 100 μ L E.coli
In DH5 α competent cell solution, it is well mixed, 42 DEG C of water-bath heat shock 90s, ice bath is cooled to 0 DEG C, adds 800 μ L LB liquid
Culture medium, 37 DEG C of slight oscillatory recovery 1h take 100 μ L cultures and apply the LB solid plates containing 100 μ g/mL Amp, 37 DEG C of cultures
12~14 hours；Single bacterium colony shaken overnight culture is chosen, DNA is extracted, DNA 10ng/ μ L is diluted to ultra-pure water, i.e.,
It is negative standards' product.
Preferably, the preparation process of the positive criteria product includes：The μ L of pKS-JAK2V617F plasmids 1 are added to 100 μ L
In E.coli DH5 α competent cell solution, it is well mixed, 42 DEG C of water-bath heat shock 90s, ice bath is cooled to 0 DEG C, adds 800 μ L
LB fluid nutrient mediums, 37 DEG C of slight oscillatory recovery 1h take 100 μ l cultures and apply the LB solid plates containing 100 μ g/mL Amp, 37
DEG C culture 12~14 hours；Single bacterium colony shaken overnight culture is chosen, plasmid is extracted, DNA is diluted to 10ng/ μ with ultra-pure water
L, and by 1：9 volume ratio is well mixed with negative standards' product and obtains final product positive criteria product.
Preferably, the amount of each sample is in the kit：
Seminar of the present invention is using ApoE gene (Allele-specific PCR, ASP) and DNA sequence dna point
Analysis method, analysis have detected the JAK2V617F gene mutations from the blood sample of Sino-Japanese Party Hospital, Jilin Univ.'s random collecting
Situation.Ended for the end of the year 2005, in 3935 non-MPNs patients, detect that 39 patients have JAK2V617F gene mutations, sun
The incidences of disease of the MPNs in crowd such as property rate nearly 1%, significantly larger than PV.And blood routine result shows, these positive patients are equal
The not up to diagnostic criteria of MPNs, but statistical analysis show, the leucocyte and platelet counts of Positive Populations apparently higher than
Population with Negative.Next, seminar has carried out the sieve of JAK2V617F gene mutations to 2613 Patients with Cardiovascular/Cerebrovascular Diseases again
Look into, find JAK2V617F positive gene mutations 44, positive rate is 1.68%.This imply that JAK2V617F genes not only with blood
The anormogenesis of liquid cell is relevant, or even to be also possible to other major diseases with the mankind directly related.Therefore, JAK2V617F genes
Mutation detection kit can be not only used for MPNs, particularly for the diagnosis of PV, and in major diseases such as cardiovascular and cerebrovascular diseases
Early diagnosis, auxiliary diagnosis and ahead of time prevention etc. aspect can play a significant role, with wide market application foreground.
The beneficial effects of the present invention are：
1) this kit integrates extracting genome DNA and detection in Gene Mutation, increased the continuity of operation, reduces
Due to extracting the unstability of the genomic DNA quality for obtaining using distinct methods, the success rate of detection is significantly increased；
2) test limit of this kit is up to 1%, therefore, for MPNs, early diagnosis especially for PV diseases and
Treatment has important directive significance；
3) this kit is easy to operate, time-consuming short, and whole process detection only needs 3 hours, according to operation instructions, ordinary skill
Personnel are operable, and result judgement easily, it is accurate, as long as occur with compare the band that is consistent by draw a conclusion；
4) equipment is simple needed for this kit, it is only necessary to common PCR instrument and imaging device, wider using scope
It is general, it is adaptable to which that medical condition is undeveloped or low developed area；
5) this kit Cleaning Principle clearly, enhances the science of MPNs diagnosis, can effectively help doctor to carry out really
Examine；
6) testing cost reduction, patient need not do excessive, unnecessary auxiliary examination, as long as doing some routine inspections again
Made a definite diagnosis by carrying out this detection in Gene Mutation, be alleviated or avoided because making a definite diagnosis the secondary injury brought to patient body, while
Alleviate the financial burden of patient.
Brief description of the drawings
Fig. 1 is kit test limit experimental result of the present invention；
Fig. 2 is kit specificity experiments result of the present invention；
Fig. 3 is kit of the present invention repeatability experimental result.
Specific embodiment
The embodiment of technical solution of the present invention will be described in detail below.Following examples are only used for clearer
Ground explanation technical scheme, therefore example is only used as, and can not be limited the scope of the invention with this.
Following reagents are purchase commodity, and its manufacturer is：
2 × Taq PCR MIX Loading Dye-free, purchased from GenStar；6 × DNA loading buffer, purchase
From GenStar；2K Plus DNA Marker, purchased from TransGen；Proteinase K, purchased from Roche；Primer JAK3 and JAK4, purchase
From Invitrogen；Magnetic bead BC, lysate BL, cleaning solution BW1, eluent CE are purchased from the Changchun will limited public affairs of high biotechnology
Department.
JAK2 genes Genbank accession number 3717, gene mutation position is that the G on 93526 sports T (i.e. V617
Point).The gene fragment order (including V617 sites) that JAK2 genes are 93253 to 93774 is as shown in SEQ ID NO.7.
Embodiment 1
People's JAK2V617F gene mutation detection kits, including A boxes and B boxes, A box reagents have：Magnetic bead BC, lysate BL,
Cleaning solution BW1, eluent CE；B box reagents have：MIX I, MIX II, positive criteria product, negative standards' product, ultra-pure water, protease
K、DNA Loading Buffer、DNA Marker.The specification of the kit has but is not limited to 20 times, 50 times and 100 times, each examination
Agent loading amount is shown in Table 1.
The reagent loading amount of table 1
It is prepared by negative standards' product：The μ L of pKS-JAK2 plasmids 1 are added to 100 μ LE.coli DH5 α competent cell solution
In, it is well mixed, 42 DEG C of water-bath heat shock 90s, ice bath is cooled to 0 DEG C, adds 800 μ L LB fluid nutrient mediums, 37 DEG C of slight oscillatories
(＜ 200rpm) recovery 1h, takes 100 μ L cultures and applies LB solid plates (containing 100 μ g/mL Amp), and 37 DEG C of cultures 12~14 are small
When；Single bacterium colony shaken overnight culture is chosen, DNA is extracted.DNA is diluted to 10ng/ μ L with ultra-pure water, as negative mark
Quasi- product.
It is prepared by positive criteria product：The μ L of pKS-JAK2V617F plasmids 1 are added to 100 μ L E.coli DH5 α competence thin
In cell lysis liquid, it is well mixed, 42 DEG C of water-bath heat shock 90s, ice bath is cooled to 0 DEG C, adds 800 μ L LB fluid nutrient mediums, 37 DEG C light
Micro oscillation (＜ 200rpm) recovery 1h, takes 100 μ l cultures and applies LB solid plates (containing 100 μ g/mL Amp), and 37 DEG C of cultures 12~
14 hours；Single bacterium colony shaken overnight culture is chosen, plasmid is extracted, DNA is diluted to 10ng/ μ L with ultra-pure water, and by 1: 9
Ratio is well mixed with negative standards' product obtained above and obtains final product positive criteria product.
Determine the homogeneity of standard items using the method for DNA sequencing, negative standards' product sequencing result be should be 100% open country
Raw type JAK2, positive criteria product sequencing result should be 100% JAK2V617F saltant types.The preparation of MIX I：Take primer JAK3
(20 μM, SEQ ID NO.2) and JAK4 (20 μM, SEQ ID NO.3) are prepared by mixing into JAK3/4 in equal volume, by JAK3/4 and 2
× Taq PCR MIX Loading Dye-free are mixed with 2: 55 ratio and are obtained final product MIX I.
The preparation of MIX II：According to final concentration of 5 μM of J2f primers (SEQ ID NO.4), 5 μM of J2r primers (SEQ ID
NO.5), 10 μM of J2nf primers (SEQ ID NO.6) and 40 μM of J2mr primers (SEQ ID NO.1) prepare nest-type PRC primer
MIX 60, by MIX 60 and 2 × Taq PCR MIX Loading Dye-free with 2: 55 proportions, mixing obtains final product MIX
II。
The preparation of Proteinase K Solution：A certain amount of Proteinase K is weighed, 10mM Tris-HCl, 20mM of pH 7.5 is dissolved in
CaCl2In the storage buffer of 50% glycerine, the concentration for being made Proteinase K is the Proteinase K Solution of 15mg/mL.
It should be noted that MIX I, MIX II, positive criteria product, negative standards' product, ultra-pure water, Proteinase K, DNA
Loading Buffer, DNA Marker, eluent CE should be as clear as crystal liquid, without precipitation, no suspended substance, without floccule.
Lysate BL, cleaning solution BW1 if any solid separate out, should be placed in 50 DEG C of warm bath, solid dissolving and fully mixing after
As clear as crystal liquid is should be, without precipitation, no suspended substance, without floccule.
Embodiment 2
The method that kit detects JAK2V617F gene mutations, it is specific as follows：
1st, the extraction of poba gene group DNA
1) 20 μ L Proteinase Ks are taken, is added in 1.5mL centrifuge tubes, notice that Proteinase K will be added to centrifugation bottom of the tube.By whole blood
After sample is fully mixed, take in 200 μ L addition centrifuge tubes, add 200 μ L lysates.Vortex oscillation is mixed, 70 DEG C of digestion 15
Minute, period can vortex oscillation 1 time.
It should be noted that please ensure that Proteinase K, sample and lysate are sequentially added into, should not be in lysate directly
Add Proteinase K.In addition, whole blood sample can be preserved for a long time in less than -20 DEG C, such as in 2~8 DEG C of preservations, the holding time please don't surpass
Spend 10 days.If the whole blood sample holding time is long, can be by proper extension digestion time (such as 20 minutes) improving extraction
Effect.
2) it is incubated and terminates rear brief centrifugation, 480 μ L absolute ethyl alcohols and 60 μ L magnetic beads are then added in each sample, is vortexed
Vibration is mixed carries out DNA absorption for 10 minutes after being stored at room temperature, and period can vortex oscillation 2~3 times.After room temperature absorption, vortex shakes
Swing mixing, centrifuge tube be placed on magnetic frame, magnetic 1 minute or to solution clarification.Carefully centrifugation liquid in pipe is inhaled completely
Abandon, be careful not to touch magnetic bead.
It should be noted that magnetic bead is drawn at once after mixing need to be fully vibrated before, but the continuous oscillation time should not surpass
30 seconds are spent, repeatedly can be vibrated in short-term.Magnetic bead can be in storage on magnetic frame before and after.Conditions permit is such as tested, is stored at room temperature
Also the upset 10 minutes (50rpm) on upset well distributing rocker can be changed to carries out DNA absorption.In experimentation in addition to magnetic step, its
Should not be placed in centrifuge tube on magnetic frame by its step.Centrifuge tube is placed on magnetic frame before magnetic every time answer vortex oscillation equal
It is even.Suction is abandoned step and be should be noted that and should not touch magnetic bead.
3) add 700 μ L to add the cleaning solution BW1 of ethanol, cover centrifuge tube lid, vortex oscillation is mixed 20 seconds, put back to
On magnetic frame.In the case where centrifuge tube is without departing from magnetic frame, magnetic frame is overturned repeatedly, centrifuge tube lid is cleaned, 1 point of magnetic
Clock or to solution clarification.The liquid in pipe abandon lid is first inhaled, is then inhaled and is abandoned all liq.
4) add 800 μ L to add the cleaning solution BW2 of ethanol, washed 1 time according to the method for step 3.
5) ethanol solution of 800 μ L volume fractions 80% is added, is washed 1 time according to the method for step 3.
6) centrifuge tube is placed on magnetic frame, room temperature is uncapped and dried 5~10 minutes.
It should be noted that please noting observation magnetic bead drying regime in drying process, such as magnetic bead can still flow or table
Face is shinny, shows that magnetic bead is not dried fully, need to continue drying, to be dried matt to magnetic bead surfaces or magnetic bead color is become by brown
It is light brown, shows that magnetic bead has been dried fully.Note to make magnetic bead overdrying (magnetic bead color being changed into pale yellow from brown
Color), overdrying can influence the elution efficiency of DNA.
7) 50~100 μ L eluents are added, vortex oscillation is mixed after 70 DEG C of warm bath 10 minutes.Vibration is vortexed again for mix
Magnetic bead, supernatant is drawn after 1 minute in magnetic on magnetic frame, carries out subsequent experimental, or stand-by in -20 DEG C of preservations.
It is noted that being careful not to touch magnetic bead during imbibition, to draw at a slow speed, magnetic bead should not be taken out of.If draw
There is magnetic bead to take out of, liquid should be blown back and be drawn again into centrifuge tube.
2nd, PCR detections JAK2V617F mutation
1) first run PCR
Draw blood genomic DNA, positive reference substance, each 1 μ L of negative standards' product, are added to equipped with premixed liquid MIX respectively
In the PCR pipe of I, performing PCR is entered according to following reaction condition：94℃3min；94 DEG C of 30s, 61 DEG C of 20s, 72 DEG C of 30s, totally 35 are followed
Ring；72℃5min；4 DEG C store for future use；
2) nest-type PRC
The μ L of first run PCR primer 1 are drawn, is added in the PCR pipe equipped with premixed liquid MIX II, entered according to following reaction condition
Performing PCR：94℃3min；94 DEG C of 30s, 60 DEG C of 20s, 72 DEG C of 30s, totally 30 circulations；72℃5min.
PCR primer 120V voltages in 3% Ago-Gel carry out electrophoresis 30 minutes, observe result.
3rd, result judgement：
If sample P CR product bands are consistent with positive reference substance, you can judge that the sample is mutated as JAK2V617F positive；
If sample P CR product bands are consistent with negative standards' product, you can judge that the sample is normal specimens.
Embodiment 3
The determination of kit test limit
Experimental procedure is as follows：
1) the μ L of negative standards' product 99 mix with the μ L of positive criteria product 1 in taking kit, are configured to JAK2V617F mutant DNAs
It is 1% sample；
2) 9.6 μ L ultra-pure waters are drawn with pipettor and 11.4 μ L MIX I is added in PCR pipe, be subsequently adding 1 μ L mutation
Rate is 1% sample, is well mixed, parallel to prepare 10 parts；
3) 9.6 μ L ultra-pure waters are drawn with pipettor and 11.4 μ L MIX I is added in PCR pipe, be subsequently adding 1 μ L ultrapure
Water, is well mixed, as blank；
4) PCR pipe is placed in PCR instrument, performing PCR is entered according to following reaction condition：94 DEG C of predegeneration 2min；Major cycle：94
DEG C denaturation 20s, 61 DEG C annealing 20s, 72 DEG C extension 30s；After 30 circulations, 72 DEG C of extension 5min；
5) by step 4) in the first run PCR primer ultra-pure water that obtains dilute 15 times, the head for having diluted is drawn with pipettor
Wheel each 1 μ L (including blank) of product, are added separately in the PCR pipe equipped with 11.4 μ L MIX II and 9.6 μ L ultra-pure waters,
It is well mixed；
6) PCR pipe is placed in PCR instrument, performing PCR is entered according to following reaction condition：94 DEG C of predegeneration 1min；Major cycle：94
DEG C denaturation 20s, 62 DEG C annealing 20s, 72 DEG C extension 30s；After 25 circulations, 72 DEG C of extension 5min；
7) agarose of 1.8g is weighed, adds 7.5mL ultra-pure waters and 65mL 1xTAE to be configured to 3% Ago-Gel；
8) the DNA Marker of 3 μ L and step 6 are separately added into the loading hole of Ago-Gel) in obtain 11
PCR primer, electrophoresis observes result after 30 minutes.
Result is as shown in figure 1, in figure, blank sample refers to control samples of the first run PCR with ultra-pure water as template；M samples
Refer to DNA standard molecular weights；1-10 samples are 10 parallel samples with the DNA containing 1%JAK2V617F as template.If
There are P bands and can determine that the detection of this kit is limited to 1% in electrophoresis result.Final to determine, the test limit of kit can reach
Having 1% JAK2 genes that V617F mutation occur in 1%, i.e. sample can just detect.
Embodiment 4
The specific detection of kit
Experimental procedure is as follows：
1) first run PCR
The μ L of JAK2V617F mutation positive patient poba gene groups DNA 1 are drawn, the PCR equipped with premixed liquid MIX I is added to
Guan Zhong, performing PCR is entered according to following reaction condition：94 DEG C of predegeneration 3min；94 DEG C of denaturation 30s, 61 DEG C of annealing 20s, 72 DEG C of extensions
30s, totally 35 circulations；72 DEG C of extension 5min.
2) nest-type PRC
The μ L of first run PCR primer 1 are drawn, is added in the PCR pipe equipped with premixed liquid MIX II, entered according to following reaction condition
Performing PCR：94℃3min；94 DEG C of predegenerations 30s, 60 DEG C of annealing 20s, 72 DEG C of extension 30s, totally 30 circulations；72 DEG C of extension 5min.
Wherein 1,2, No. 3 samples are the periphery of the JAK2V617F mutation positive patients obtained from No.1 Hospital of Jilin Univ.
Blood sample；No. 4 samples are the Normal human peripheral's blood sample from infection from hospital；Mouse：Mouse peripheral blood sample；Ultra-pure water：Electrical conductivity
It is the pure water of 18.2M Ω .cm.
From figure 2 it can be seen that any band does not occur in the ultrapure water sample without any species DNA, this examination is illustrated
It must be DNA rather than the other materials as ultra-pure water class that agent box has material specificity, the i.e. template of PCR.Due to this kit
The primer for using be according to human genome DNA's sequences Design, therefore, all samples from people source are (regardless of whether in the presence of prominent
Become) and corresponding band has been amplified after negative standards' product enter performing PCR, and the sample from mouse source does not obtain any
Band, illustrates that design of primers is successful, and kit is with species specificity.
Embodiment 5
Kit repeatability detection
Take No.1 Hospital of Jilin Univ.'s acquisition is defined as JAK2V617F mutation sun with the detection method of quantitative fluorescent PCR
The blood sample of property patient is used as positive reference product, coincidence rate：4 parts of positive reference product are carried out using kit of the present invention
Determine, all positives of testing result.
Mouse peripheral blood genomic DNA and ultra-pure water are taken as negative reference product, coincidence rate：Using reagent of the present invention
Box detects that testing result does not have any band to 2 parts of negative reference product, as a result all feminine genders.
2 the JAK2V617F positives and feminine gender DNA reference materials of concentration level are taken respectively, use same batch kit pair
Positive and each duplicate detection of negative sample 10 times, testing result is shown in Fig. 3, and each row sample is as follows in A figures in Fig. 3：M：DNA
Marker；1-10：Parallel 10 of positive sample (0.2ng/ μ L) after dilution；11-20：Positive sample (5ng/ μ L) after dilution
Parallel 10.Each row sample of B figures is as follows in Fig. 3：M：DNA Marker；1-10：Negative sample (0.1ng/ μ L) after dilution is put down
Row 10；11-20：Parallel 10 of negative sample (3.7ng/ μ L) after dilution.Result understands that this kit has as shown in Figure 3
There is good assay reproducibility.
Finally it should be noted that：Various embodiments above is merely illustrative of the technical solution of the present invention, rather than its limitations；To the greatest extent
Pipe has been described in detail with reference to foregoing embodiments to the present invention, it will be understood by those within the art that：Its according to
The technical scheme described in foregoing embodiments can so be modified, or which part or all technical characteristic are entered
Row equivalent；And these modifications or replacement, the essence of appropriate technical solution is departed from various embodiments of the present invention technology
The scope of scheme, it all should cover in the middle of the scope of claim of the invention and specification.
The ＞ applicants of ＜ 110：Sharp gill bio tech ltd of Jilin Province
The ＞ people's JAK2V617F gene mutation detection kits of ＜ 120
〈160〉7
〈210〉1
〈211〉21
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ J2mr primers of ＜ 223
〈400〉1
cttactctcg tctccacaga a 21
〈210〉2
〈211〉26
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ JAK3 primers of ＜ 223
〈400〉2
ttccaggctt acacaggggt ttcctc 26
〈210〉3
〈211〉26
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ JAK4 primers of ＜ 223
〈400〉3
ctataatact ctattgtttg ggcatt 26
〈210〉4
〈211〉25
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ J2f primers of ＜ 223
〈400〉4
gttgatggca gttgcaggtc catat 25
〈210〉5
〈211〉19
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ J2r primers of ＜ 223
〈400〉5
tttaacttca ttgctttcc 19
〈210〉6
〈211〉23
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ J2nf primers of ＜ 223
〈400〉6
ggttttaaat tatggagtat gtg 23
〈210〉7
〈211〉522
〈212〉DNA
The ＞ homo sapiens of ＜ 213（HOMO SAPIENS）
〈220〉
The gene fragment order (including V617 sites) of the ＞ JAK2 genes 93253 to 93774 of ＜ 223
〈400〉7
ttccaggctt acacaggggt ttcctcagaa cgttgatggc agttgcaggt ccatataaag 60
ggaccaaagc acattgtatc ctcatctata gtcatgctga aagtaggaga aagtgcatct 120
ttattatggc agagagaatt ttctgaacta tttatggaca acagtcaaac aacaattctt 180
tgtacttttt tttttcctta gtctttcttt gaagcagcaa gtatgatgag caagctttct 240
cacaagcatt tggttttaaa ttatggagta tgtgtctgtg gagacgagag taagtaaaac 300
tacaggcttt ctaatgcctt tctcagagca tctgtttttg tttatataga aaattcagtt 360
tcaggatcac agctaggtgt cagtgtaaac tataatttaa caggagttaa gtatttttga 420
aactgaaaac actgtaggac tattcagtta tatcttgtga aaaaggaaag caatgaagtt 480
aaaagtagaa ggttacaatg cccaaacaat agagtattat ag 522
Claims (7)
1. people JAK2V617F gene mutation detection kits, it is characterised in that including A boxes and B boxes, A boxes include magnetic bead BC, split
Solution liquid BL, cleaning solution BW1 and eluent CE；B boxes include premixed liquid I, premixed liquid II, positive criteria product, negative standards' product, ultrapure
Water, the Proteinase K Solution of 15mg/mL, DNA Loading Buffer and DNA Marker.
2. people JAK2V617F gene mutation detection kits according to claim 1, it is characterised in that the premixed liquid I
Preparation process includes：By 20 μM of JAK3 primers (shown in SEQ ID NO.2) and 20 μM of JAK4 primers (SEQ ID NO.3 institutes
Show) isometric mixing, then by mix primer and 2 × Taq PCR MIX Loading Dye-free by volume 2:55 ratio
Example is mixed and obtained final product.
3. people JAK2V617F gene mutation detection kits according to claim 1, it is characterised in that the premixed liquid
II preparation process includes：According to final concentration of 5 μM of J2f primers (shown in SEQ ID NO.4), 5 μM of J2r primers (SEQ ID
Shown in NO.5), 10 μM of J2nf primers (shown in SEQ ID NO.6) and 40 μM of amounts of J2mr primers (shown in SEQ ID NO.1)
Nest-type PRC primer is prepared, then by nest-type PRC primer and 2 × Taq PCR MIX Loading Dye-free by volume 2:
55 proportions, mixing is obtained final product.
4. people JAK2V617F gene mutation detection kits according to claim 1, it is characterised in that the Proteinase K
Solution is that Proteinase K is dissolved in into Tris-HCl, CaCl2Obtained with the mixed solution of glycerine three；The Tris-HCl is dense
It is 10mM to spend, pH value 7.5, CaCl2Concentration is 20mM, and qualities of glycerin fraction is 50%.
5. people JAK2V617F gene mutation detection kits according to claim 1, it is characterised in that the negative mark
Quasi- product preparation process includes：The μ L of pKS-JAK2 plasmids 1 are added in 100 μ L E.coli DH5 α competent cell solution, are mixed
Close uniform, 42 DEG C of water-bath heat shock 90s, ice bath is cooled to 0 DEG C, add 800 μ L LB fluid nutrient mediums, 37 DEG C of slight oscillatory recoveries
1h, takes 100 μ L cultures and applies the LB solid plates containing 100 μ g/mL Amp, and 37 DEG C are cultivated 12~14 hours；Choose single bacterium colony vibration
Incubated overnight, extracts DNA, and DNA is diluted into 10ng/ μ L, as negative standards' product with ultra-pure water.
6. people JAK2V617F gene mutation detection kits according to claim 1, it is characterised in that the positive mark
The preparation process of quasi- product includes：The μ L of pKS-JAK2V617F plasmids 1 are added to 100 μ L E.coli DH5 α competent cells molten
In liquid, it is well mixed, 42 DEG C of water-bath heat shock 90s, ice bath is cooled to 0 DEG C, adds 800 μ L LB fluid nutrient mediums, 37 DEG C is slightly shaken
Recovery 1h is swung, 100 μ l cultures is taken and is applied the LB solid plates containing 100 μ g/mL Amp, 37 DEG C are cultivated 12~14 hours；Choose single bacterium
The shaken overnight that falls culture, extracts plasmid, and DNA is diluted into 10ng/ μ L with ultra-pure water, and by 1：9 volume ratio and feminine gender
Standard items are well mixed to obtain final product positive criteria product.
7. people JAK2V617F gene mutation detection kits according to claim 1, it is characterised in that the kit
The amount of middle each sample is：
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710251443.1A CN106906299A (en) | 2017-04-18 | 2017-04-18 | People's JAK2V617F gene mutation detection kits |
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| CN201710251443.1A CN106906299A (en) | 2017-04-18 | 2017-04-18 | People's JAK2V617F gene mutation detection kits |
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| CN106906299A true CN106906299A (en) | 2017-06-30 |
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| CN101918588A (en) * | 2007-11-08 | 2010-12-15 | 奎斯特诊断投资公司 | The JAK2 sudden change |
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| CN104818340A (en) * | 2015-05-29 | 2015-08-05 | 沈阳优吉诺生物科技有限公司 | Primers and kit for detecting JAK2 gene V617F site polymorphism, and PCR (polymerase chain reaction) method thereof |
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| CN101054412A (en) * | 2006-04-12 | 2007-10-17 | 辽宁省计划生育科学研究院 | Pathogenic gene for far end arthrosis bend, detection method and application thereof |
| CN101918588A (en) * | 2007-11-08 | 2010-12-15 | 奎斯特诊断投资公司 | The JAK2 sudden change |
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Application publication date: 20170630 |