CN103571923A - Detection probes and detection liquid phase chip for BIM gene deletion mutation - Google Patents
Detection probes and detection liquid phase chip for BIM gene deletion mutation Download PDFInfo
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Abstract
The present invention discloses a detection liquid phase chip and detection probes for BIM gene deletion mutation. The detection liquid phase chip comprises: BIM gene deletion mutation wild-type targeted detection probe coated microspheres and BIM gene deletion mutation mutant-type targeted detection probe coated microspheres, wherein the wild-type detection probe is SEQ ID NO.1 or SEQ ID NO.2, and the mutant-type detection probe is SEQ ID NO.3 or SEQ ID NO.4; and biotin labeled amplification primers. According to the present invention, the prepared BIM gene mutation detection liquid phase chip has a good signal-to-noise ratio, no cross-reaction basically exist among the designed detection probes, the amplification primers and the amplification product, and parallel detection on the wild-type sequence and the mutant-type sequence in the same reaction system can be achieved.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, concrete a kind of BIM deletion mutant detection probes and the liquid-phase chip of relating to.
Background technology
BIM gene is present on No. 2 karyomit(e)s of people, is a kind of B cell leukemia/lymphoma 2(BCL2 gene) the short apoptosis member of protein family.One of BIM genetic expression comprises BH3(BCL2-homology domain3) albumen of structural domain, by with the Bcl2-l1 of Bcl2 family, BCL-XL, BCL2A1, the combine inhibition tumor cell growth of some somatotrophic albumen such as MCL1, or and promote protein B AX, the BAK1 of the apoptosis promotion apoptosis of tumor cells that combines.BH3 structural domain is the key function territory that BIM gene plays a role, and research finds that this functional domain and TKIs play a role closely related.For example in the nonsmall-cell lung cancer (NSCLC) of TKIs medicine treated with gefitinib, need the normal expression of BIM gene, and BH3-similar medicine can obviously strengthen its result for the treatment of.The human tumor cell that a lot of kinase expressions of the mankind cause extremely can be by suppressing the expression of BIM gene or keeping the growth vigor of self by MAPK1 phosphorylation modification BIM albumen.In nearly all mankind's tumour, the activation of BIM gene expresses that for TKIs class medicine, to play a role be necessary, and the expression that suppresses BIM gene can cause TKIs resistance.Show that BIM gene plays a significant role in TKIs medicine plays a role process.
At present, BIM detection method of gene mutation mainly contains: Illumina optical fiber superbead chip technology, Affymetrix6.0 chip technology.Although Illumina optical fiber superbead chip technology is the high throughput testing system of highly sensitive and accuracy, but level of automation is low, manual operations is many, be difficult to meet the needs of practical application, high-throughout Affymetrix SNP6.0 chip technology comparative maturity, yet this chip technology in low-density clinical diagnosis cake core improper, be difficult to expansion in same reaction system and detect relevant SNP or the Tag SNP of numerous biological characters.In addition, Affymetrix SNP6.0 chip is mainly more intense on chip of expression spectrum, and species are more, on SNP chip relatively a little less than, and detect expensively, can not meet actual needs.
The BIM transgenation sudden change of target detect of the present invention, this sequence (SEQ IDNO.16: lack the sequence after sudden change) is since the 169th bit base, there are 2903 bit bases that deletion mutantion has occurred, in frame, sequence is disappearance region, those skilled in the art use routine techniques means, and the based composition according to before and after disappearance region just can easily obtain the based composition in this disappearance region in gene pool, therefore, the content of the BIM deletion mutant of target detect of the present invention is fully disclosed.
SEQ ID NO.16BIM deletion mutant
GTTGGTAGAGTTATCAATTAGGAAACCCAGTACAGAGTCTATTATAATTTAGATTGTACCTCATGATGAAGGCTAACTCAACAAACCCATCAGAACAGACACTGGAACAAAATGACATTTCTAAATACCATCCAGCTCTGTCTTCATAGGCTTCAGTGAGGTAAATCA
CTGTTCTCCATAGAGGCTGTGCCATTTTACATTCCCACCAACAGGGCACAAGGGTTCCAGTTTCTCCACATACTTACCAACACTTTTTTTTTTTTTTTTTTAACAGTAGTCATCCTAGAGGATATAGGTGATCTTTCACTGTGCTTTGGATTTATATTTACTGGCTTAGATTTGTATGGCCACCACCATAGTCAAGATACAGAACAACTCAACCACAAGGATTTCTCATGATACCTTTTTATAGCCACAGCCACCTCTCTCCCTCTTCCTTGAGCATTTTGTCATATGGTCATTGGTGATTAAA
Summary of the invention
One of object of the present invention is to provide a kind of BIM deletion mutant and detects liquid-phase chip, and this liquid-phase chip can be used for BIM deletion mutant and detects.
Realize above-mentioned purpose technical scheme as follows.
Deletion mutant detects a liquid-phase chip, includes
A, be coated with the microballoon for wild-type and the saltant type detection probes of BIM deletion mutant respectively, described wild-type detection probes is SEQ ID NO.1 or SEQ ID NO.2, and described saltant type detection probes is SEQ ID NO.3 or SEQ ID NO.4;
B, for amplify need to detect, with primer biotin labeled, that contain the genotypic target sequence of BIM deletion mutantion.
In an embodiment, described amplimer is therein: SEQ ID NO.5-SEQ ID NO.7.
In an embodiment, described amplimer biotin labeling is positioned at the 5 ' end of SEQ ID NO.6 and SEQ ID NO.7 therein.
In an embodiment, in the middle of detection probes is connected with microballoon, be also provided with spacerarm sequence therein, more preferably, described spacerarm sequence is 20-50 T.
Another object of the present invention is to provide the probe detecting for BIM deletion mutant.
Concrete technical scheme is as follows:
The probe detecting for BIM deletion mutant, described probe is: for detection probes SEQ ID NO.1 or the SEQ ID NO.2 of wild-type, and for probe SEQ ID NO.3 or the SEQ ID NO.4 of the detection of saltant type.
Major advantage of the present invention is:
1. the identical rate of the detected result of BIM gene mutation detection liquid-phase chip provided by the present invention and sequencing is up to 100%.And detect the needed time well below conventional sequencing technologies, realistic especially application needs.Prepared BIM gene mutation detection liquid-phase chip has extraordinary signal-noise ratio, and between designed detection probes and amplimer, amplified production, substantially there is not cross reaction, in same reaction system, realize the parallel detection of wild-type and saltant type sequence.
2. the present invention, by the design experiences of the long-term accumulation of contriver and a large amount of experimental implementation, has chosen optimum 2 groups from numerous detection probes.The detection probes of the present invention design can the sensitive Sudden change region of identifying specifically target detect, accurately distinguishes the genotype of various types; In same reaction system, between different detection probes, substantially there is not cross reaction between detection probes and the pcr amplification product of non-target detect, detection specificity is good, and cross reacting rate is lower than 3%.
3. detection method step of the present invention is simple, can complete the synchronous detection of wild-type and saltant type in single job, greatly improves Detection accuracy, has embodied accurate while qualitative and quantitative analysis feature.
4. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, the poor defect of repeatability of detected result, the fluorescent signal value of detection improves greatly, thereby the sensitivity detecting is further enhanced, signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1BIM deletion mutant detects liquid-phase chip, mainly includes:
One, detection probes
For wild-type and the saltant type of the BIM deletion mutant of target detect of the present invention, design detection probes, as shown in the table:
The detection probes sequence of table 1BIM deletion mutant
4 kinds of microballoons selecting, purchased from U.S. Luminex company, are coated in (one probe is corresponding to a kind of microballoon) on microballoon by detection probes.Between detection probes and microballoon, be connected with the spacerarm sequence of 20-50 T, before every is detected sequence, add the spacerarm sequence of the preceding paragraph 20-50 T, detection probes sequence is synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.By synthetic detection probes sterilizing ddH
2o is made into the stock solution of 100nmol/ml.
Described spacerarm is for for by detection probes and microsphere surface is spaced apart or detection probes is placed in to the sequence of hydrophilic environments.By the spacerarm sequence of suitable length is set between detection probes and microballoon, can reduce sterically hinderedly, improve the efficiency of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly(dT), oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n >=3), as (CH2) 12, (CH2) 18 etc.In addition, if there is poly(dA) disturb, can also use poly(TTG) as spacerarm.Spacerarm of the present invention is preferably 20-50 T, and the coated process of microballoon is as follows:
Get respectively 5 * 10
6the carboxylated microballoon of individual above-mentioned numbering (purchased from Luminex company) is suspended in the MES solution of 50ul0.1mol/L (pH4.5), adds the synthetic detection probes molecule (100nmol/ml) of 10ul.The EDC(N-(3-Dimethylaminopropyl-N-ethylcarbodiimide of preparation 10ng/ml) (purchased from Pierce Chemical company) working fluid.Toward the EDC working fluid that adds 2.5ul in microballoon suspension, constant-temperature incubation 30 minutes, then add the EDC working fluid of 2.5ul, then constant-temperature incubation 30 minutes.After reaction finishes, the Tween-20 with 0.02% washs once, then washs once with 0.1% SDS liquid.The Tris-EDTA solution [10mmol/L Tris(pH8.0)] that the microballoon that is coated with detection probes after washing is resuspended in to 100ul, in 1mmol/L EDTA, 2-8 ℃ keeps in Dark Place.
Two, amplify the primer that contains target sequence
According to the sequence of BIM deletion mutant, form situation, respectively for the feature of wild-type sequence and saltant type sequence, design 2 pairs of amplimers to (in Table 2), for the SEQ ID NO.5 of wild-type and SEQ ID NO.6, for SEQ ID NO.5 and the SEQ ID NO.7 of saltant type, wherein, 5 ' a kind of end in forward primer (Forward) or reverse primer (Reverse-W and Reverse-M) is with biotin labeling.
While there is wild-type sequence in genome, amplimer Forward and Reverse-W amplify the product of 189bp, while there is saltant type sequence in genome, amplimer Forward and Reverse-M amplify the product of 143bp, while there is two kinds of types in genome, amplify 189bp and 143bp product simultaneously simultaneously.
Table 2 amplifies the primer of wild-type and saltant type target sequence
| SEQ IDNO. | Primer title | Sequence 5 ' → 3 ' |
| 5 | Forward | ACCTCATGATGAAGGCTAACT |
| 6 | Reverse-W | TCACAGGTGTGCCCAACCT |
| 7 | Reverse-M | AATGTAAAATGGCACAGCCTC |
All primers are synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with respectively the stock solution of 100pmol/mL with 10mmol/L Tris Buffer.
The detection of BIM deletion mutant liquid-phase chip described in embodiment 2 utilization embodiment 1 to sample
The formula of described various solution is as follows:
MES damping fluid (pH5.0) formula (250ml) of 50mM:
2 * Tm hybridization buffer
After filtration, be stored in 4 ℃.
ExoSAP-IT test kit is purchased from U.S. USB company.
Biotin labeled dCTP is purchased from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
Methods involving with reference to < < molecular cloning > > about DNA extraction, obtains DNA to be detected.
Two, the pcr amplification of testing sample
For BIM deletion mutant, design two pairs of primers, and use 5 ' of biotin labeling reverse primer (SEQ ID NO.6 and SEQ ID NO.7) to hold, amplify the target sequence with biotin labeled wild-type and saltant type, product size is respectively 189bp and 143bp, and primer sequence (SEQ ID NO.5-7) is shown in shown in above-mentioned table 2.While there is wild-type sequence in genome, amplimer Forward and Reverse-W amplify the product of 189bp, while there is saltant type sequence in genome, amplimer Forward and Reverse-M amplify the product of 143bp, while there is two kinds of types in genome, amplify 189bp and 143bp product simultaneously simultaneously.
First prepare multiple PCR primer working fluid: respectively get respectively the primer stock solution 100ul of SEQ ID NO.5-7 in 1.5ml Eppendorf tube, mix and be multiple PCR primer working fluid.Multi-PRC reaction system is as follows:
PCR reaction conditions
Four, hybridization system
1. according to the detection probes of design, select corresponding 2 kinds of microballoons that are coated with detection probes, wherein, for wild-type, select No. 1 coated microballoon of SEQ ID NO.1 detection probes, for saltant type, select No. 3 coated microballoons of SEQ ID NO.3 detection probes, between detection probes and microballoon, be provided with the spacerarm sequence of 30T, every kind of microballoon concentration is 2.5 * 10
5individual/ml;
2. get respectively the microballoon of every kind of numbering of 1ul in the Eppendorf tube of 1.5ml;
3. microballoon is in >=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in the Tm hybridization buffer of 100ul, and vortex mixes;
5. get the above-mentioned microballoon suspension of 48ul in the 96 corresponding holes of hole filter plate;
6. get the amplified production of 2ul in corresponding hole, control wells adds the ddH of 2ul
2o; ;
7. with masking foil, encase 96 orifice plates with lucifuge, 95 ℃ of 3min, 52 ℃ of 15min are hatched hybridization;
8. adding 25ul concentration is the SA-PE (SA-PE) of 20ug/ml; Hatch 5min for 12.52 ℃, on Luminex instrument, detect.
Five, result detects and data analysis
Reaction after product detects by Luminex serial analysis instrument.Detected result is as shown in table 3.
Fluorescent value (MFI) and data processing are had to following requirement:
1. each site need have at least an allelotrope MFI to be greater than 300 and be greater than 10 * PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI(NET MFI be less than 0 with 0 expression);
3. meet the data of above two conditions, by following formula, calculate sudden change ratio:
Sudden change ratio=saltant type NET MFI ÷ (saltant type NET MFI+ wild-type NET MFI)
4. the sudden change ratio definite threshold (cut-off value) to each detection site rule of thumb, to divide wild-type homozygote, heterozygote and saltant type homozygote.
Use present method to detect 20 increments BIM deletion mutant originally, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Arranging of threshold value (cut-off value) is as follows: sudden change ratio range is considered as wild-type homozygote at 0%-20%; 30%-70% is considered as heterozygote; 80%-100% is considered as anomaly homozygote.With sequencing, detect with detected result of the present invention and compare, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments BIM genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.Visible BIM deletion mutant provided by the present invention detects the sudden change situation that liquid-phase chip can detect BIM gene exactly, and result is reliable and stable.
Table 3 sample B IM deletion mutant detected result and Polymorphism Analysis
BIM detection in Gene Mutation detection probes in embodiment 3 embodiment 1
One, the design that prepared by liquid-phase chip
Use in embodiment 1 respectively for 2 wild-type detection probes (SEQ ID NO.1 and SEQ ID NO.2) of BIM deletion mutant and the detection probes (SEQ ID NO.3 and SEQ ID NO.4) of saltant type, form different detection system, with more different wild-type probe and saltant type probe combinations, whether obtain consistent detection effect, concrete probe combinations is as shown in table 4, detection probes is coated with microballoon (being provided with the spacerarm sequence of 30T between detection probes and microballoon), amplimer (5 ' end of biotin labeling reverse primer (SEQ ID NO.6 and SEQ ID NO.7)), detection method is like described in embodiment 1 and embodiment 2.
Table 4 hybridization probe sequence
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 21-40 by testing process described in embodiment 3 and method, and detected result is as follows:
Table 5 sample B IM Gene A 145G detected result and Polymorphism Analysis
Table 6 Analysis of test results
From the present embodiment, liquid-phase chip technology scheme of the present invention is selected the arbitrary combination in the detection probes (SEQ ID NO.3 or SEQ ID NO.4) of wild-type detection probes (SEQ ID NO.1 or SEQ ID NO.2) in table 1 and saltant type, all can realize the specific detection of saltant type, and the detection effect of agreeing.
The selection of embodiment 4BIM detection in Gene Mutation detection probes
One, the design that prepared by liquid-phase chip (selection of wild-type and saltant type specific primer sequence)
The complementary sequence forward or backwards of this BIM deletion mutant place target sequence of take is template, respectively for wild-type and saltant type design detection probes, comprise preferred 2 detection probes and 4 alternative detection probes in the embodiment of the present invention 1, as shown in table 7.Detection probes is coated with microballoon (being provided with the spacerarm sequence of 30T between detection probes and microballoon), amplimer (5 ' end of biotin labeling reverse primer (SEQ ID NO.6 and SEQ ID NO.7)), detection method like described in embodiment 1 and embodiment 2.
Table 7 hybridization probe sequence
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects sample 41-60 by testing process described in embodiment 4 and method, and detected result is as follows:
One of table 8 sample B IM deletion mutant Analysis of test results
Two of table 9 sample B IM deletion mutant Analysis of test results
From the present embodiment, when detection probes is selected embodiment 1, effect better (signal to noise ratio is better), referring to the present embodiment test group 5 and test group 6.Other is different from different detection probes sequences described in embodiment 1, that derive from the complementary sequence forward or backwards of target detect deletion mutantion place sequence, and it detects effect and all can not show a candle to SEQ ID NO.1-SEQ IDNO.4.
Embodiment 5, the experiment of BIM gene mutation detection liquid-phase chip detection sensitivity
In order to detect the detection sensitivity of this liquid-phase chip, design detects the ability of mutant DNA in BIM wild-type DNA, investigates the minimum ratio of the mutant DNA that this liquid-phase chip system can detect.It is as shown in table 10 that the copy number of wild-type DNA, mutant DNA in sample is respectively organized in this experiment.The plasmid mixture of take in table 10 detects (be configured to cumulative volume as the plasmid mixture of 200 μ L, get 10 μ L loadings, establish respectively 3 holes and repeat experiment) as template, establishes 1 negative control, the sensitivity that sudden change detects according to Analysis of test results.Detection probes is coated with microballoon (being provided with the spacerarm sequence of 30T between detection probes and microballoon), amplimer (5 ' end of biotin labeling reverse primer (SEQ ID NO.6 and SEQ ID NO.7)), detection method like described in embodiment 1 and embodiment 2, wherein, detection probes combination is in Table 11.
Table 10BIM gene mutation detection liquid-phase chip sensitivity detected result
Table 11 hybridization probe sequence
| Wild-type probe | Saltant type probe | Experimental group |
| SEQ ID NO.1 | SEQ ID NO.3 | Group11 |
| SEQ ID NO.2 | SEQ ID NO.4 | Group12 |
Two, sample detection
The liquid-phase chip that adopts above-mentioned design to prepare, detects the plasmid mixture of sequence number 1-10 by testing process described in embodiment 5 and method, and detected result is as follows:
The experiment of table 12BIM deletion mutant detection sensitivity
The result detecting shows: adopt BIM gene mutation detection liquid-phase chip of the present invention, 1% saltant type plasmid can be detected in sample, detection sensitivity is at least 1%.Simultaneously; in detected result, can find out; in sequence number 1 plasmid mixture, only there is wild plasmid; in the plasmid mixture of sequence number 8, only there is saltant type plasmid; and all there is not cross reaction in wild-type and saltant type detection probes in liquid-phase chip of the present invention; energy specific recognition target detect plasmid, has good specificity.
The above embodiment has only expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (6)
1. BIM deletion mutant detects a liquid-phase chip, it is characterized in that, includes
A, be coated with the microballoon for wild-type and the saltant type detection probes of BIM deletion mutant respectively, described wild-type detection probes is SEQ ID NO.1 or SEQ ID NO.2, and described saltant type detection probes is SEQ ID NO.3 or SEQ ID NO.4;
B, for amplify need to detect, with primer biotin labeled, that contain the genotypic target sequence of BIM deletion mutantion.
2. BIM deletion mutant according to claim 1 detects liquid-phase chip, it is characterized in that, the base sequence of described amplimer is: SEQ ID NO.5-SEQ ID NO.7.
3. BIM deletion mutant according to claim 2 detects liquid-phase chip, it is characterized in that, the biotin labeling of described amplimer is positioned at the 5 ' end of SEQ ID NO.5, or the biotin labeling of described amplimer is positioned at the 5 ' end of SEQ ID NO.6 and SEQ ID NO.7.
4. according to the BIM deletion mutant described in claims 1 to 3 any one, detect liquid-phase chip, it is characterized in that, in the middle of detection probes is connected with microballoon, be also provided with spacerarm sequence.
5. BIM deletion mutant according to claim 4 detects liquid-phase chip, it is characterized in that, described spacerarm sequence is 20-50 T.
6. the probe detecting for BIM deletion mutant, is characterized in that, described probe is: for detection probes SEQ ID NO.1 or the SEQ ID NO.2 of wild-type, and for detection probes SEQ ID NO.3 or the SEQ ID NO.4 of saltant type.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073552A (en) * | 2013-04-16 | 2014-10-01 | 张以哲 | BIM (Bcl-2 Interacting Mediator of Cell Death) gene mutation detection method and kit |
| CN104450924A (en) * | 2014-12-16 | 2015-03-25 | 广东省人民医院 | Method and kit for detecting drug-resistance and toxic and side effects related gene polymorphism in lung cancer treatment |
| CN105603069A (en) * | 2016-01-20 | 2016-05-25 | 安徽达健医学科技有限公司 | Bim (Bcl-2 interacting mediator of cell death) gene deletion fluorescent quantitative PCR (polymerase chain reaction) detection primer, probe and detection reagent kit |
| 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 |
| CN114606318A (en) * | 2022-03-25 | 2022-06-10 | 杭州瑞普基因科技有限公司 | Application of probe set in detecting BIM gene deletion polymorphism |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399899A (en) * | 2011-12-09 | 2012-04-04 | 邵棠 | Liquid phase chip method and kit for detecting polymorphism of cytochrome P450 1A2 (CYP1A2) genes |
-
2012
- 2012-07-18 CN CN201210250293.XA patent/CN103571923B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399899A (en) * | 2011-12-09 | 2012-04-04 | 邵棠 | Liquid phase chip method and kit for detecting polymorphism of cytochrome P450 1A2 (CYP1A2) genes |
Non-Patent Citations (1)
| Title |
|---|
| NQ KP ET AL.: "A common BIM deletion polymorphism mediates intrinsic resistance and inferior responses to tyrosine kinase inhibitors in cancer", 《NAT MED.》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073552A (en) * | 2013-04-16 | 2014-10-01 | 张以哲 | BIM (Bcl-2 Interacting Mediator of Cell Death) gene mutation detection method and kit |
| CN104450924A (en) * | 2014-12-16 | 2015-03-25 | 广东省人民医院 | Method and kit for detecting drug-resistance and toxic and side effects related gene polymorphism in lung cancer treatment |
| CN105603069A (en) * | 2016-01-20 | 2016-05-25 | 安徽达健医学科技有限公司 | Bim (Bcl-2 interacting mediator of cell death) gene deletion fluorescent quantitative PCR (polymerase chain reaction) detection primer, probe and detection reagent kit |
| 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 |
| CN114606318A (en) * | 2022-03-25 | 2022-06-10 | 杭州瑞普基因科技有限公司 | Application of probe set in detecting BIM gene deletion polymorphism |
| CN114606318B (en) * | 2022-03-25 | 2023-12-01 | 杭州瑞普基因科技有限公司 | Application of probe set in detecting BIM gene deletion polymorphism |
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