CN105603069A - Bim (Bcl-2 interacting mediator of cell death) gene deletion fluorescent quantitative PCR (polymerase chain reaction) detection primer, probe and detection reagent kit - Google Patents
Bim (Bcl-2 interacting mediator of cell death) gene deletion fluorescent quantitative PCR (polymerase chain reaction) detection primer, probe and detection reagent kit Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention discloses a Bim (Bcl-2 interacting mediator of cell death) gene deletion fluorescent quantitative PCR (polymerase chain reaction) detection primer, a probe and a detection reagent kit, and belongs to the field of molecular biological detection. The Bim gene deletion fluorescent quantitative PCR detection primer and the probe comprise forward primers 5'-CAACAAACCCATCAGAACAGACAC-3', reverse primers 5'-ACAGCCTCTATGGAGAACAGTGATT-3 and fluorescent probes 5'-FAM-CAGACACTGGAACAAA-MGB-3'. The detection reagent kit comprises the Bim gene deletion fluorescent quantitative PCR detection primer, the probe, premixed liquid of PCR liquid and positive control samples A. The Bim gene deletion fluorescent quantitative PCR detection primer, the probe and the detection reagent kit have the advantages that Bim gene deletion mutation can be detected by the aid of the Bim gene deletion fluorescent quantitative PCR detection primer, the probe and the detection reagent kit, and the Bim gene deletion fluorescent quantitative PCR detection primer, the probe and the detection reagent kit are simple, convenient and feasible and are high in accuracy and sensitivity and particularly suitable for detecting clinical samples; Bim deletion mutation can be detected, and accordingly the Bim gene deletion fluorescent quantitative PCR detection primer, the probe and the detection reagent kit can bring convenience for guiding individual treatment on bodies.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of Bim gene delection fluorescence quantitative PCR detection primer andProbe, detection kit.
Background technology
At present, lung cancer case fatality rate in the developed countries such as America and Europe and China's malignant tumour is the highest, and its incidence of disease is gone up year by yearRise, wherein NSCLC accounts for 80%～85%. In recent years, the targeted therapy of lung cancer makes substantial progress, the target of multiclass high-efficiency low-toxicityCome out one after another to medicine, and be applied to clinically, mainly contain the targeted therapy taking EGFR as target spot, the target taking Angiogenesis as target spotTo treatment, NMPI (TIMP), the targeted therapy taking method Geordie transferase as target spot, the few core of lung cancer antisenseThuja acid targeted therapy and lung cancer target vaccine etc.; Gefitinib is the representative drugs of targeted therapy, is signal transduction pathway junketPropylhomoserin inhibitors of kinases. In addition, the gene therapy taking adenovirus as carrier has a wide range of applications in clinical. At present manyCount and studies confirm that, the EGFR-TKI such as Gefitinib can be by increasing Bim quantity inducing cell endogenous apoptosis in cell. Bim is heavyGroup Adenovirus Transfection has inducing action to EGFR sudden change NSCLC Apoptosis, and mechanism is for raising Bim albumen table in cellReach; This gene therapy that is NSCLC provides foundation, but about Bim TIF grinding aspect therapy of tumorStudy carefully very few.
Bim, as important apoptosis regulatory protein, is not only eliminating autoimmunity, maintains the stable of the interior environment of hematopoietic cellIn play important regulating action, and have powerful to pathology damage (as neurodegeneration, tumour, epilepsy etc.) cellKilling ability. The research of cells involved apoptosis is recognized people, and cell death inducing can be used as the new way for the treatment of tumour.Bim is the more popular a kind of apoptosis regulatory protein of Recent study, studies have reported that the chromosome of the mankind Bim assignment of genes gene mappingRegion is positioned at the chromosomal region of a lot of tumours, and is mainly the tumour in hemopoietic system source. The disappearance of Bim gene can causeThe generation of kinds of tumors, mostly wherein is hematopoietic system cancer, thereby proves that forcefully Bim is a kind of TIF. KingDeng finding at normal proliferative stage of endometrium, atypical hyperplasia endometrium, endometrium with immunohistochemical method researchIn this process of cancer, the expression of Bim albumen reduces gradually. The pro-apoptotic function of Bim, becomes in therapy of tumorA desirable target.
Bim (Bcl-2interactingmediatorofcelldeath) is the sub-family of BH3-only in Bcl-2 familyThe member of family, is a kind of important apoptosis regulatory protein, is that O ' connoretall found in 1998, mankind Bim gene positionIn 2q12 or 2q13, formed by 6 extrons and 3 intrones. The formation of Bim isomers is the selection by Bim Pre-mRNAProperty montage and producing. Because each isoforms mRNA has retained different exon sequences, thereby the each isomers of Bim is had notSame domain, pro-apoptosis bioactivity is also different. Find that at present Bim exists 11 kinds of isomers. Bim albumen belongs to Bcl-2Family, only containing the BH3 domain in 4 homeodomains of Bcl-2 family (BHl-4), and BH3 territory is considered to Apoptosis and risesThe necessary factor beginning, can and insert to line grain jointly by interactions such as the effect of antagonism anti-apoptosis factor or direct and BaxOn body film, cause that cytochrome c discharges and cell death inducing. Bim all has expression in multiple normal cell, comprising makingHaemocyte, epithelial cell, nerve cell and reproduction cell etc. Nearest research finds, Bim gene is as tumor suppressor gene, its tableReach disappearance and can impel the generation of kinds of tumors, comprise prostate cancer, breast cancer, carcinoma of endometrium etc. And Bim is also many anti-One of important medium of tumour medicine cell death inducing. The disappearance of Bim gene can cause the generation of kinds of tumors, mostly wherein isHematopoietic system cancer. Bim is at the non-small cell lung cancer cell of tyrosine kinase inhibitor induction epidermal growth factor receptor mutationsIn strain apoptotic process, play an important role, also relevant to the drug resistance of glucocorticoid to leukaemia, carry with Bim gene expressionBody transfection tumor cell likely becomes the new way of therapy of tumor.
The method that is usually used at present detecting Bim gene delection mainly contains: direct sequencing, FISH(florescenceinsituhybridization, FISH), immunohistochemistry (immunohistochemistry,IHC), RT-polymerase chain reaction (reversetranscription-polymerisechainreaction, RT-PCR). Although the result of direct sequencing has objectivity and specificity, it but exists, and sense cycle is long, operating process is multipleAssorted, easily contaminated, the shortcoming such as the interpretation of sequencing result is more complicated, time-consuming, and expense is higher, in addition, for detecting, heterogeneity is higherClinical tumor tissue samples be that direct sequencing false negative rate is high. FISH can be to chromosome or specific geneNumerical abnormality, diagnosis research is carried out in disappearance, transposition and the rearrangement of specific fragment, complicated but workload is large, efficiency is lower. Exempt fromThe histochemical testing cost of epidemic disease is lower, and the pathology department of most of hospital can carry out, but Bim gene is depended in IHC detectionExpression and specificity and the sensitiveness of corresponding antibodies, before not finding desirable antibody, the accuracy that IHC detects and heavyRenaturation is still difficult to ensure card. The general principle of RT-PCR is first through reverse transcriptase effect, under Auele Specific Primer exists, RNA is contraryBe transcribed into the CDNA of strand, then by PCR, CDNA increased, confirmed results after electrophoresis, has responsive special, the required tissue mass utmost pointThe advantage such as few, but the quality of RNA in tissue is had higher requirements, if the RNA degraded in tissue is serious, may affect finalTesting result, and time-consuming, recall rate is lower, is difficult to promote the wider application in routine work or laboratories. But existing inspectionThe existence of survey method cannot take into account simultaneously and be applicable to clinical sample, detection method is simple and easy to do, accuracy rate is high, highly sensitive, resultJudge succinctly shortcoming fast and accurately.
Real-Time Fluorescent Quantitative PCR Technique (rea1-timefluorescentquantitativePCR, FQ-PCR) inWithin 1996, released by AppliedBiosystems company of the U.S., it is a kind ofly in the time of pcr amplification, to add the same of pair of primersTime add a specific fluorescence probe, this probe is an oligonucleotides, two ends respectively report fluorophor of mark andA cancellation fluorophor. When probe is complete, the fluorescence signal of reporter group transmitting is quenched group and absorbs; While just beginning, visitPin is combined on the arbitrary strand of DNA; When pcr amplification, probe enzyme is cut degraded by 5 ' end-3 ' end 5 prime excision enzyme activity of Taq enzyme,Make to report that fluorophor separates with cancellation fluorophor, thereby fluorescence monitoring system can receive fluorescence signal, i.e. every amplification oneBar DNA chain, just has a fluorescence molecule to form, and accumulation and the PCR product of having realized fluorescence signal form Complete Synchronization. This technologyNot only realized to DNA profiling quantitatively, and there is highly sensitive, specificity and reliability is stronger, can realize multiple reaction,The features such as automaticity is high, nonstaining property, tool real-time and accuracy, be widely used at present molecular biology research andThe fields such as medical research, have no report but be applied to the detection primer of BIM deletion mutation and probe or kit.
In sum, in order to realize clinically the target for the treatment of lung cancer, in this area, need to develop simple and easy to do, accuratelyThe detection method that rate is high, highly sensitive and/kit, be especially applicable to clinical sample detect method and/kit, to useIn detecting BIM deletion mutation, be convenient to instruct the personalized treatment of body.
Summary of the invention
The problem existing for solving above-mentioned prior art, the invention provides a kind of based on fluorescence quantitative PCR detection Bim baseBecause of the detection primer of deletion mutation and probe, detection kit.
One of object of the present invention is to provide a kind of Bim gene delection fluorescence quantitative PCR detection primer and probe, and forward drawsThing sequence is that ForwardPrimer:5 '-CAACAAACCCATCAGAACAGACAC-3 ', reverse primer sequence areReversePrimer:5 '-ACAGCCTCTATGGAGAACAGTGATT-3 ', fluorescence probe sequence be Taqman-Probe:5 '-FAM-CAGACACTGGAACAAA-MGB-3 ', 5 of fluorescence probe ' end flag F AM fluorescence report group, 3 ' end mark MGB fluorescenceQuenching group.
The present invention is based on fluorescence quantifying PCR method, real-time fluorescence quantitative PCR is nucleic acid amplification, hybridization, spectrum analysis and realityTime detection technique combine, detect PCR product by means of fluorescence signal, having solved normal PCR technology can not be quantitatively and expansionIncrease the problem of product pollution, avoided the pollution in common quantitative PCR operating process, make it easy and simple to handle, quick, result standardReally, become the strong instrument of quantitative gene expression, had that sensitiveness and specificity are high, reproducible and quantitative scope is wideEtc. feature. Quantitatively testing requirement detection method sensitivity, special, accurate, accurate, reproducible, the range of linearity is wide, and at each baseBecause of indifference between type. Full automatic quantitative fluorescent PCR, because of its more excellent accuracy, accuracy and repeatability, becomesPromising quantitative measurement technology.
The principle of Bim gene delection fluorescence quantitative PCR detection is: adopt fluorescence quantifying PCR method to enter Bim gene delectionRow quantitatively detects. A pair of for the special design of conserved sequence TaqMan probe and the primer of Bim gene respectively, when running into Bim baseDuring because of disappearance variation specific gene sequence, the combination completely with it of TaqMan probe, when pcr amplification, fluorescence report groupSend fluorescence because enzymolysis separates, can detect in real time the accumulation of corresponding fluorescence signal, thereby realization detects Bim gene delectionObject.
First,, for the design of Bim gene delection fluorescence quantitative PCR detection primer, in prior art, quantitative fluorescent PCR drawsThe design of thing often needs to consider many factors, comprising: adopt conserved region sequence carry out design of primers, primer self or primer itBetween can not form complementation, amplified production and avoid forming the control (40%～60%) of secondary structure, G+C content, primer lengthControl (conventionally between 17-25 base), fluorescent quantitative PCR product length restriction (80-150bp, the longest 300bp) etc.
Inventor designs in the middle of primer process, on the basis that meets above-mentioned primary condition, further promotes by designThe specificity of primer and probe and amplification efficiency. Conventionally, primer is longer, the higher (nucleotide primer of every increase of its specificitySpecificity improves 4 times), but because primer is longer, its annealed combination forms stablizing for archaeal dna polymerase combination to target DNAThe speed of double-stranded template is less, and amplification efficiency is lower. Inventor is through too much group primer pair is than screening, and finishing screen is chosen the present inventionDescribed upstream primer and downstream primer, it possesses higher specificity and good amplification efficiency simultaneously; In addition inventor's design,Downstream primer is the combination of Bim deletion sequence (2903bp) upstream 5bp and deletion sequence (2903bp) downstream 20bp sequence, and it hasEffect avoided simple employing Bim deletion sequence downstream gene group sequence as primer amplification time genome fragment or mRNA causeThe problem of disturbing only comprises Bim deletion sequence (2903bp) upstream 5bp base in downstream primer simultaneously, can not produce 5bp baseThe amplification that causes of pairing, has improved specificity and the accuracy of fluorescent quantitative PCR greatly.
Especially, clinical sample variation, especially for sample paraffin embedded tissues, to the specificity of primer and probe and applicableProperty is had relatively high expectations. Detection primer of the present invention and probe adopt fluorescent quantitative PCR, have high specificity and accurateProperty.
The upstream primer Tm61.1 that the present invention obtains DEG C, G+C content are 45.8%, and Δ G is-41.7kcal/mol, underTm60.7 DEG C of primer of trip, G+C content are 44.0%, and Δ G is-43.2kcal/mol.
Secondly, for the design of Bim gene delection fluorescence quantitative PCR detection probe, in order further to increase fluorescent quantitationThe specificity of pcr amplification, the TaqMan-MGB probe that the present invention adopts, compares with traditional TaqMan-TAMRA probe, MGBProbe has the TM value of raising, makes the contraction in length of probe, and improve match and non-matching template between TM value difference different; Carry simultaneouslyHigh s/n ratio, is used in conjunction with specificity and primer, further improves the accuracy and the specificity that detect, makes experimental result more smartReally, resolution ratio is higher; More simplify experiment, MGB probe optimum experimental step is simple, and the stability of hybridization improves greatly, repeatabilityThe advantage such as raising greatly.
Inventor has also carried out selective optimization for fluorescence report group simultaneously, and existing fluorescence report group comprises 6-Fluoresceincarboxylic acid (6-carboxyfluorescein, FAM), chlordene-6-methyl fluorescein (Hexachloro-6-Methylfluorescein, HEX), VIC fluorescent dye, tetrachloro-6-Fluoresceincarboxylic acid (tetrachloro-6-Carboxyfluorescein, TET), carboxyl-X-rhodamine (Carboxy-x-rhodamine, ROX), 6-carboxyl tetramethyl sieveRed bright (6-carboxytetramethylrhodamine, TAMRA), sulphonyl rhodamine (Sulforhodamine101, TexasRed), 6-carboxyl-4 ', 5 '-bis-chloro-2 ', 7 '-dimethoxy fluorescein succinimide esters (6-Carboxy-4 ', 5 '-Dichloro-2 ', 7 '-dimethoxyfluorescein, JOE), Hua Jing 3 (cyanine3, Cy3), Hua Jing 3.5(cyanine3.5, Cy3.5), Hua Jing 5 (cyanine5, Cy5) and flower cyanines 5.5 (cyanine5.5, Cy5.5) etc. are multiple, inventionPeople, by the selection to probe 5 ' end reporter group, finds 5 ' end flag F AM fluorescence report group and MGB type probe, its cancellationIn process, FRET is better, and in quantitative fluorescent PCR process, fluorescence curve effect is more obvious.
Two ends provided by the invention all indicate the specificity fluorescent probe of fluorescence radiation group, in the time that probe is complete, and two basesGroup's distance on space structure is mutually close, 5 ' the end fluorescence that sends of reporter group because FRET (FRET) andBy 3 ' end quenching group cancellation, so there is no the variation of fluorescence signal in system. Once and the combination of it and template specificity, itsBinding site is between two primers, and along with the extension of primer, TaqDNA polymerase runs into and template phase in chain extension processIn conjunction with probe, its 5 '-3 ' 5 prime excision enzyme activity will cut off probe, fluorescence report group is away from fluorescent quenching group, like thisJust destroyed the FRET between two fluorophors, the fluorescence that reporter group discharges just can be built in instrumentInterior fluorescence photometer detects. PCR is every through a circulation, and fluorescence signal is also the same with object fragment, has a sync index to increaseLong process, the power of fluorescence signal just represented template DNA copy number number. Therefore the present invention not only can be used for simplyQualitative detection, also can be used as the quantitative detection of the concrete content of sample.
Another object of the present invention comprises the reagent that above-mentioned primer and probe detect for Bim deletion mutant in preparationApplication in box, chip or other detection reagent.
Another object of the present invention is to provide a kind of Bim gene delection fluorescent quantificationally PCR detecting kit, this kit bagDraw together above-mentioned detection primer and probe.
Preferably, kit of the present invention further comprises positive control sample A: comprise detected Bim gene delectionGenomic fragment DNA plasmid.
Preferred, the invention provides a kind of PCR kit for fluorescence quantitative that fast, accurately detects Bim gene delection, shouldKit comprises fluorescent quantitation reactant liquor premixed liquid (PCRMasterMix), fluorescent quantitation reactant liquor A, positive control sample A;
Fluorescent quantitation reactant liquor A: detection be Bim gene delection, reaction system comprises: detect the drawing of Bim gene delectionThing and probe sequence: forward primer sequence is ForwardPrimer:5 '-CAACAAACCCATCAGAACAGACAC-3 ', reversePrimer sequence is that ReversePrimer:5 '-ACAGCCTCTATGGAGAACAGTGATT-3 ', fluorescence probe sequence areTaqman-Probe:5 '-FAM-CAGACACTGGAACAAA-MGB-3 ', 5 ' end flag F AM fluorescence report base of fluorescence probeGroup, 3 ' end mark MGB fluorescent quenching group;
Positive control sample A: the genomic fragment DNA plasmid that comprises detected Bim gene delection.
The genomic fragment DNA plasmid of the Bim gene delection that detects
Another object of the present invention is to provide a kind of detection method of Bim deletion mutant, comprising:
From sample tissue, extract genomic DNA;
Utilize above-mentioned primer and probe to carry out quantitative fluorescent PCR reaction;
According to fluorescent quantitative PCR curve detection Bim deletion mutant.
Preferably, sample tissue is clinical tissue wax stone.
Preferably, the condition of quantitative fluorescent PCR is: reaction system is 25 μ L, the each 0.2 μ L of positive and negative primer, each 0.2 μ of probeL, sample template DNA2 μ L, 2 × TaqmanuniversalPCRMasterMix15 μ L, ddH2O7.4 μ L, PCR reactionCondition: 95 DEG C of denaturations 30 seconds; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, 45 circulations of amplified reaction.
It should be noted that, detection method of the present invention can not directly be diagnosed individual ill situation, does not belong to diseaseSick diagnostic method.
The present invention has following beneficial effect:
With existing detection method comparison, this technology adopts fluorescent quantitative PCR technique detection Bim gene delection to have following excellentGesture:
1. detection sensitivity is high, and specificity is good: the present invention has designed Auele Specific Primer for Bim gene conserved sequence respectivelyAnd fluorescence probe, improving detection sensitivity and specificity, false positive is low; The present invention is applicable to the detection of clinical tissue sample, veryTo can effectively detecting for paraffin embedded tissues.
2. linear relationship is good, can quantitatively detect, because the power of fluorescence signal and the logarithm of template amplification product are linearRelation, carries out quantitatively sample original template concentration by the detection of fluorescence signal, and error is little;
3. simple to operate, automaticity is high, the amplification of FQ-PCR technology to PCR product and detecting the in the situation that of stopped pipeOne step completes, and does not need to uncap, and cross pollution and contaminated environment chance are few, has therefore also just reduced the probability of result error;
4. result interpretation is clear and definite, directly perceived, can carry out quantitative analysis to result. The interpretation of fluorescence quantitative PCR method result: in territoryValue line has the sample of amplification curve to be positive sample above, and result interpretation is very simple, directly perceived;
5. the sample size detecting is large, once can detect at most 384 examples;
6. safety: in whole system, do not comprise poisonous and harmful substance, to operator and environment all without harm;
7. there is no post processing, need not hybridization, electrophoresis, take pictures.
Brief description of the drawings
The FQ-PCR figure of Figure 1B im gene delection
The sequencing result figure of Fig. 2 Bim gene delection
Detailed description of the invention
Design and the screening of embodiment mono-Bim gene delection fluorescence quantitative PCR detection primer and probe
According to conventional fluorescence quantification PCR primer design factor, design 20 groups of forward primers, reverse primer and fluorescence probes,According to primer screening design principle, screening obtains higher three groups of relative score, and part primer and probe sequence, referring to table 1, drawThing design factor comprises: 1, primer designs and has a specificity in nucleic acid conserved region; 2. amplified production avoids forming secondary knotStructure (free energy is less than 58.61KJ/mol); 3. primer length is generally between 17-25 base, and upstream and downstream primer can not differ tooGreatly; 4.G+C content between 40%～60%, 45-55% the best; 5. base is wanted random distribution, as far as possible evenly; 6. primer selfCan not there is the complementation of continuous 4 bases; 7. between primer, can not there is the complementation of continuous 4 bases; 8. primer 5 ' end can be modified;9.3 ' end can not be modified, and will avoid AT, and the region of GCrich, avoids T/C, A/G continuous structure (2-3); 10. primer3 ' end will be avoided the 3rd of codon; 11. primer global design free energys, the 5 ' end that distributes is greater than 3 ' end, and 3 ' end free energyBe less than well 9KJ/mol; 12. to be fluorescent quantitation product length 80-150bp best, and the longest is 300bp; 13. design of primers are avoidedDNA pollutes, preferably across extron connector area; The homology of 14. primers and non-specific amplification sequence be preferably less than 70% orThere are 8 complementary base homologies; 15. check the existence that has or not pseudogene; 16.TM value is between 58-62 degree.
The design of table 1Bim gene delection fluorescence quantitative PCR detection primer and probe and screening sequence
Adopting positive control sample A (the genomic fragment DNA plasmid that comprises detected Bim gene delection) to carry out fluorescence determinesAmount pcr amplification, wherein primer and probe groups two S type J curve effectJs are good, and amplification efficiency is higher, primer and probe groups three and drawThing and probe groups one increase not obvious. Therefore, choose primer and probe groups two is carried out clinical sample detection.
The detection of embodiment bis-Bim gene delection samples
In this embodiment, positive sample used is collected and from pathology department of Tumor Hospital Attached to Zhongshan Univ. Clinicopathologic Diagnosis isThe wax stone tissue of Bim gene delection, negative sample is the individual wax stone tissue of wild homozygote (BIM does not lack), wherein positive sampleThese 83 examples, negative sample 9 examples. From wax stone, extract genomic DNA for following experimental applications.
Probe and primer are:
ForwardPrimer：5’-CAACAAACCCATCAGAACAGACAC-3’、
ReversePrimer:5’-ACAGCCTCTATGGAGAACAGTGATT-3’、
Taqman-Probe:5’-FAM-CAGACACTGGAACAAA-MGB-3’；
Then optimizing reaction system is carried out the detection of FQ-PCR: reaction system is 25 μ L, each 0.2 μ L (20 μ of positive and negative primerΜ), the each 0.2 μ L of probe (20 μ Μ), sample template DNA2 μ L (100-300ng/ μ l), 2*TaqmanuniversalPCRMasterMix (purchased from applying biological company of the U.S.) 15 μ L, ddH2O7.4 μ L. PCR reaction condition: 95 DEG C of denaturation 30sec;And by 95 DEG C 5 seconds, 58 DEG C 33 seconds, 45 circulations of amplified reaction.
In fluorescent quantitation test, when sample need to be detected, also need to arrange sample reference material, to determine examination simultaneouslyThe validity (negative control does not detect) of testing as shown in Figure 1, is the fluorescent quantitative PCR song of Bim gene delection positive sampleLine; In addition, the sample in embodiment 2 is carried out to sequence verification, find to detect test by above-mentioned fluorescent quantitation in the present embodimentDetect Bim gene delection, as shown in Figure 2, can find out that by sequencing result corresponding gene occurs in this site really to be lackedLose, it is in full accord that itself and fluorescent quantitation detect the result of testing. Positive sample recall rate 100%, negative sample finds no expansionIncrease. Find to adopt Bim gene delection fluorescent quantificationally PCR detecting kit of the present invention can determine exactly missing gene with thisSudden change situation.
As from the foregoing: utilize mentioned reagent box to carry out FQ-PCR method and detect, be not only quick, efficient, sensitive, andThe interpretation of its result is very clear and definite, directly perceived, and result is also reliable, special.
By reference to the accompanying drawings the specific embodiment of the present invention is described although above-mentioned, not the present invention is protected to modelThe restriction of enclosing, one of ordinary skill in the art should be understood that, on the basis of technical scheme of the present invention, those skilled in the art are notNeed to pay various amendments that creative work can make or distortion still in protection scope of the present invention.
Claims (8)
1. Bim gene delection fluorescence quantitative PCR detection primer and a probe, forward primer sequence is ForwardPrimer:5 '-CAACAAACCCATCAGAACAGACAC-3 ', reverse primer sequence be ReversePrimer:5 '-ACAGCCTCTATGGAGAACAGTGATT-3 ', fluorescence probe sequence is Taqman-Probe:5 '-FAM-CAGACACTGGAACAAA-MGB-3 ', 5 of fluorescence probe ' end flag F AM fluorescence report group, 3 ' end mark MGB fluorescent quenchingGroup.
2. the kit, chip that described in claim 1, primer and probe detect for Bim deletion mutant in preparation or otherDetect the application in reagent.
3. a Bim gene delection fluorescent quantificationally PCR detecting kit, is characterized in that, this kit comprises claim 1State and detect primer and probe.
4. detection kit according to claim 3, is characterized in that, described kit further comprises positive control sampleA: the genomic fragment DNA plasmid that comprises detected Bim gene delection.
5. fast, accurately detect a PCR kit for fluorescence quantitative for Bim gene delection, this kit comprises that fluorescent quantitation is anti-Answer liquid premixed liquid, fluorescent quantitation reactant liquor A, positive control sample A;
Fluorescent quantitation reactant liquor A: detect primer and the probe sequence of Bim gene delection, forward primer sequence is ForwardPrimer:5 '-CAACAAACCCATCAGAACAGACAC-3 ', reverse primer sequence be ReversePrimer:5 '-ACAGCCTCTATGGAGAACAGTGATT-3 ', fluorescence probe sequence are Taqman-Probe:5 '-FAM-CAGACACTGGAACAAA-MGB-3 ', 5 of fluorescence probe ' end flag F AM fluorescence report group, 3 ' end mark MGB fluorescent quenchingGroup;
Positive control sample A: the genomic fragment DNA plasmid that comprises detected Bim gene delection.
6. a detection method for Bim deletion mutant, comprising:
From sample tissue, extract genomic DNA;
Utilize described in claim 1 described in primer and probe or claim 3-5 any one kit to carry out quantitative fluorescent PCR anti-Should;
According to fluorescent quantitative PCR curve detection Bim deletion mutant.
7. detection method according to claim 6, is characterized in that, sample tissue is clinical tissue wax stone.
8. according to the detection method described in claim 6 or 7, it is characterized in that, the condition of quantitative fluorescent PCR is: reaction systemBe 25 μ L, the each 0.2 μ L of positive and negative primer, the each 0.2 μ L of probe, sample template DNA2 μ L, 2 × TaqmanuniversalPCRMasterMix15μL,ddH2O7.4 μ L, PCR reaction condition: 95 DEG C of denaturations 30 seconds; And by 95 DEG C 5 seconds, 58 DEG C 33 seconds,45 circulations of amplified reaction.
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|---|---|---|---|---|
| CN108396066A (en) * | 2018-05-14 | 2018-08-14 | 广州医科大学附属肿瘤医院 | Primer, probe and the kit of BIM genetic polymorphism detections |
| CN112553326A (en) * | 2020-12-30 | 2021-03-26 | 昆明和合医学检验所有限公司 | Primer, probe and fluorescent PCR kit for detecting neonatal jaundice UGT1A1 genotype and GST gene deletion type |
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| CN108396066A (en) * | 2018-05-14 | 2018-08-14 | 广州医科大学附属肿瘤医院 | Primer, probe and the kit of BIM genetic polymorphism detections |
| CN112553326A (en) * | 2020-12-30 | 2021-03-26 | 昆明和合医学检验所有限公司 | Primer, probe and fluorescent PCR kit for detecting neonatal jaundice UGT1A1 genotype and GST gene deletion type |
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