CN107699622A - A kind of disheveled protein 1（DVL1）Gene G136D mutation detection kits - Google Patents

A kind of disheveled protein 1（DVL1）Gene G136D mutation detection kits Download PDF

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CN107699622A
CN107699622A CN201711161143.0A CN201711161143A CN107699622A CN 107699622 A CN107699622 A CN 107699622A CN 201711161143 A CN201711161143 A CN 201711161143A CN 107699622 A CN107699622 A CN 107699622A
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dvl1
nucleic acid
peptide nucleic
pna
primer
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唐景峰
陈兴珍
周策凡
胡苗
钱学红
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Hubei University of Technology
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Abstract

The invention discloses a kind of disheveled protein 1（DVL1）Gene G136D mutation detection kits, belong to biotechnology and clinical molecular diagnosis technical field.Kit of the present invention includes being used for the primer pair of specific detection DVL1 genes G136D mutation（SEQ ID NO.1、2）With peptide nucleic acid fluorescence probe（SEQ ID NO.3）, for blocking the peptide nucleic acid probe of wild type DVL1 gene magnifications（SEQ ID NO.4）, include detecting the primer pair and peptide nucleic acid fluorescence probe of internal reference（SEQ ID NO.5‑7）, positive reference product, negative reference product and PCR reagent etc..The present invention can quickly have found DVL1 gene mutations situation in Wnt signal paths from transcriptional level, and for screening anticancer medicine, new targeted drug is inquired into, basic scientific research provides instrument.

Description

A kind of disheveled protein 1 (DVL1) gene G136D mutation detection kits
Technical field
The present invention relates to biotechnology and clinical molecular diagnosis technical field, and in particular to a kind of disheveled protein 1 (DVL1) Gene G136D mutation detection kits.
Background technology
Wnt signal paths are widely present in invertebrate and vertebrate, are that one kind is high during spore The conservative signal path of degree.Wnt signals are in the early development of animal embryo, orga- nogenesis, regeneration and other physiology courses In, there is vital effect.If the key protein in this signal paths is undergone mutation or unconventionality expression, cause letter Number abnormal activation, it is possible to the generation of induced cancer.Wnt signal paths include classical Wnt signal paths and non-classical Wnt Signal path, in classical path is Wnt- β-catenin signal paths, the Wnt factors pass through on active cell film Suppress phosphorylation and the degraded of endocellular liberation β-catenin albumen after Frizzle/LRP5/6 cooperative expert systems, in cytoplasm The core that β-catenin albumen occurs is shifted after the rise of β-catenin protein levels, causes β-catenin albumen in nucleus Rise, in karyon β-catenin albumen can combine Pygo2, Bcl-9 and FoxM1 albumen jointly with TCF/LEF-1 transcriptions because Sub-family forms complex and activates the transcriptional activation of Wnt signal path downstream target genes.
DVL1 albumen is one of β-catenin upstream important members in Wnt signal paths.Its main function is to enable to GSK3 β phosphorylations, and then promote APC (adenomatous polyposis coli), GSK3 β, CK1 complexs degraded, promote That enters β-catenin in cytoplasm enters core.Increasing research is it has been found that DVL1 albumen is in many tumours at present Now different degrees of mutation.
The core element regulatory mechanism of research core signal path at present, and expression of some important members in cell Level, have become a kind of key means for the treatment of tumour, although research of the Wnt signal paths in cancer in recent years, and Research of the DVL1 albumen as its mutation level of Wnt signal paths important member, have become research and development tumour medicine and be badly in need of The major issue of solution, especially in fluffy and disorderly specific (Dishevelled specific domain, aa90-247) domain The mutation of middle generation, to the very important effect of DVL1 protein exhibits function ons, such as detected in colorectal cancer cell G136D mutation etc..
Peptide nucleic acid (peptide nucleic acids, PNA) is a kind of DNA for substituting sugared phosphate backbone with polypeptide backbone Analog.It is on the basis of the first generation, second generation antisense agent, is built by Computer Design and final artificial synthesized Third generation antisense agent, it is a kind of brand-new DNA analogs, i.e., is substituted with the peptide chain acid amides 2- aminoethylglycines key of neutrality Pentose phosphate diester linkage skeleton in DNA, remaining is identical with DNA, PNA can pass through Watson-Crick base pairings Form identifies and combines DNA or RNA sequence, forms stable double-spiral structure.Because PNA is not negatively charged, with DNA and RNA Between be not present electrostatic repulsion, thus the stability and specificity that combine all greatly improve；Different between DNA or DNA, RNA Hybridization, PNA and DNA or RNA hybridization are hardly influenceed by hybridization system salinity, remote with the hybridization ability of DNA or RNA molecule Better than DNA/DNA or DNA/RNA, very high hybridization stability, excellent distinguished sequence recognition capability are shown, not by nuclease And protease hydrolytic.
The present invention is using the PNA oligomers of distinguished sequence as probe.Because PNA is a kind of class of artificial synthesized nucleic acid Like thing, and it is achirality, uncharged molecule, avoids when oligonucleotides is combined with its target gene because electric charge is mutually exclusive Caused hybridization unstability, with reference to being not easy to be influenceed by hybridization solution ionic strength, so as to show extremely strong heterosis, hybrid vigor, Substantially increase detection sensitivity.
The content of the invention
The purpose of the present invention is the signaling molecule DVL1 bases for how to determine core in Wnt signal paths in the prior art Because of catastrophe, and how to explain the difficulty of the horizontal change of mutation of the core element DVL1 in tumour cell, there is provided one The kit of DVL1 gene mutations in kind detection Wnt signal paths.
The peptide nucleic acid PNA fluorescence probes that kit provided by the invention includes detecting DVL1 gene mutations (are used for special Property detection DVL1 genes G136D mutation peptide nucleic acid fluorescence probe), by combining the PCR method of peptide nucleic acid PNA fluorescence probes, Can more sensitively detect the mutation that DVL1 genes in cell occur, and for study Wnt signal paths provide it is most direct Evidence；Also include in the kit：Detect one section of peptide with DVL1 gene wild type Complementary hybridizations needed for DVL1 gene mutations The nucleic acid PNA probe peptide nucleic acid probe of wild type DVL1 gene magnifications (be used for block), for qualitative detection DVL1 gene mutations Primer.
The principle of the present invention is by building the one section peptide nucleic acid PNA sequence complementary with DVL1 genes wild type, when peptide core When sour PNA sequences are combined with DVL1 gene complementations, any mutation can cause PNA/DNA to produce mispairing, so that dissolving temperature Degree changes.And then specific peptide nucleic acid PNA probe and primer pair DVL1 mutation are designed according to DVL1 genic mutation types Type gene carries out fluorescent quantitative PCR, after the PCR amplifications of a wheel, you can DVL1 genic mutation types are entered with wild type Row makes a distinction.
For achieving the above object, the present invention uses following technical scheme：
A kind of disheveled protein 1 (DVL1) gene G136D mutation detection kits, it includes following primer and probe：
Forward primer DVL1-F for specific detection DVL1 genes G136D mutation：5'- cccagggcacggacagccac-3'；
Reverse primer DVL1-R for specific detection DVL1 genes G136D mutation：5'- cgtgctgccatcctcgtc-3'；
Peptide nucleic acid fluorescence probe DVL1-PNA-P for specific detection DVL1 genes G136D mutation：5'- fluorescence reports Group-cgagacagacacggagtccatgg- quenching groups -3'；
For blocking the peptide nucleic acid probe DVL1-PNA of wild type DVL1 gene magnifications：5'- cgagacaggcacggagtccatgg-3'。
Further, described kit also includes：
Detect the forward primer DVL1-F' of internal reference：5'-cccaaatgtggccagc-3'；
Detect the reverse primer DVL1-R' of internal reference：5'-cctcctcgcggttcc-3'；
Detect the peptide nucleic acid fluorescence probe DVL1-PNA-P' of internal reference：5'- fluorescent reporter groups- Ggtcagtcaccggcgggagc- quenching groups -3'.
Further, described kit also includes PCR reaction reagents.The reaction reagent of the PCR includes：DEPC water, Archaeal dna polymerase, dNTPs, PCR Buffer, RNASIN, reverse transcriptase, the solution (MgCl of oligo (dT) and the ion containing Mg2) Deng.Described archaeal dna polymerase is preferably with archaeal dna polymerase exo-acting 5' → 3'.
Further, each component concentration in specific detection in PCR reaction systems is as follows：The final concentration of archaeal dna polymerase For 0.01U/ μ L~0.05U/ μ L, dNTPs final concentration of 0.2~0.6mM, PCR Buffer final concentration of 1 ×, RNASIN Final concentration of 40U/ μ L~60U/ μ L, M-MLV reverse transcriptases final concentration of 200U/ μ L~320U/ μ L, Mg ions (MgCl2) final concentration of 1.5~5.0mM, final concentration of 0.05~0.9 μM of forward primer, reverse primer it is final concentration of 0.05~0.9 μM, 0.05~0.9 μM of oligo (dT), final concentration of 0.05~0.9 μM of peptide nucleic acid probe, peptide nucleic acid fluorescence is visited Final concentration of 0.05~0.9 μM of pin.
Further, described kit also includes positive reference substance, positive reference product and negative reference product.Described sun Property reference substance be containing the extron G136D of DVL1 genes the 4th mutation DNA and contain the exon sequence of DVL1 genes the 5th DNA mixture, concentration 3ng/ μ L.Described positive reference product are to be mutated containing the extron G136D of DVL1 genes the 4th DNA, negative reference product be containing DNA wild the extron G136 of DVL1 genes the 4th, concentration is 3ng/ μ L. In the detection, positive reference substance CT values are located between positive reference product CT values and negative reference product CT values.
The present invention successfully works out by lot of experiments, research and analysis and can be used in quick, sensitive, effective detection The kit of DVL1 genes G136D mutation.Mankind's DVL1 gene G136D catastrophes can be detected using this kit.
The step of kit provided by the invention is not included to testing sample processing and template extraction, but for from formaldehyde The piece segment DNA that the sample and fresh tissue sample of fixed FFPE are obtained still has the expansion same with cell sample DNA Increasing and detectability.
Compared with prior art, the advantages and positive effects of the present invention are：DVL1 genes be β in Wnt signal paths- Catenin albumen upstream plays the gene of critical function, and the invention provides directly detect DVL1 genes in Wnt signal paths The reagent of abrupt climatic change, can be by quantitative real-time PCR in transcriptional level quick detection by means of the detection reagent Go out the mutation of DVL1 genes, and unlike common real-time fluorescence quantitative PCR, the peptide nucleic acid PNA fluorescence probes that we use Sensitiveer, the present invention is the screening of cancer therapy drug, and the Mechanism Study of new targeted drug both provides very strong instrument.Behaviour Make simply, to be once loaded, react start to finish from PCR, carried out always in the reaction system of closing, not only reduce pollution Probability, and reduce result and the probability of deviation occur.As a result interpretation is clearly directly perceived.
In a word, the kit experimental implementation letter of disheveled protein 1 (DVL1) gene G136D abrupt climatic changes of the present invention Single, low-cost, as a result repeated, sensitiveness is good, is study the tumour related drugs mechanism of action and basic scientific research one Kind important means.
Brief description of the drawings
Fig. 1 is peptide nucleic acid mass spectrogram described in the embodiment of the present invention；
Fig. 2 is G136D saltant types described in the embodiment of the present invention and reference wild-type product PCR amplification figures, it is upper in figure, in, Lower three bars of lines respectively represent the amplification curve of the bit codon mutation type reference materials of DVL1 the 136th, sample and reference wild-type product.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with embodiment and accompanying drawing to this hair Bright embodiment is described in further detail.
Embodiment 1
The present embodiment is illustrated by colorectal cancer cell, but the present invention is not limited to colorectal cancer cell.
SW-480 used in the present embodiment (Human colorectal carcinoma SW-480 cell lines) is purchased from U.S. ATCC, and culture cell is used RPMI-1640 culture mediums and 10% hyclone be purchased from handsome company (invitrogen), other reagents are mainly purchased from treasured Bioengineering (Dalian) Co., Ltd.
First, primed probe designs
(1) select one section of sequence containing 136 amino acids codons in DVL1mRNA as target sequence design primer and Probe.
Target sequence：
cccagggcacggacagccacacagacctgcccccgcctcttgagcggacaggcggcatcggggactcccggcccccc tccttccacccaaatgtggccagcagccgtgacgggatggacaacgagacaggcacggagtccatggtcagtcaccg gcgggagcgtgcccgacgccggaaccgcgaggaggccgcccggaccaatgggcacccaaggggagaccgacggcggg atgtggggctgcccccagacagcgcgtccaccgccctcagcagcgagcttgagtccagcagctttgtggactcggac gaggatggcagcacg。
Primer and the probe for detecting DVL1G136D mutation are as follows：
DVL1-F：5'-cccagggcacggacagccac-3'；
DVL1-R：5'-cgtgctgccatcctcgtc-3'；
DVL1-PNA-P：5'-FAM-cgagacagacacggagtccatgg-BHQ-3'；
DVL1-PNA：5'-cgagacaggcacggagtccatgg-3'.
(2) region of above-mentioned target sequence (being free of 136 amino acids codons) is different from using the extrons of DVL1 the 4th Ginseng, the primer and probe for detecting internal reference are as follows：
DVL1-F'：5'-cccaaatgtggccagc-3'；
DVL1-R'：5'-cctcctcgcggttcc-3'；
DVL1-PNA-P'：5'FAM-ggtcagtcaccggcgggagc-BHQ3'.
More than：F：Forward, it is positive；DVL1-F/DVL1-F' represents the forward primer for detecting nucleic acid；
R：Reverse, reversely；DVL1-R/DVL1-R' represents the reverse primer for detecting nucleic acid；
P：Probe, fluorescence probe；P/P' represents the fluorescence probe for detecting nucleic acid, and the fluorescence probe is that TaqMan is glimmering Light probe.
2nd, the culture of Human colorectal carcinoma SW-480 cell lines and passage
1) cell culture
All cell lines use RPMI-1640 culture mediums (Invitrogen, Carlsbad, CA), 10% hyclone (Invitrogen, Carlsbad, CA), in 37 DEG C, 5%CO2Cultivated under environment.
2) passage
The nutrient solution in Tissue Culture Dish is suctioned out first by sterilized straw, PBS is added and cleans 2 times, toward carefully Appropriate trypsase is slowly added dropwise in born of the same parents, treats that cell rounding, adjustment angle cell add 3mL and contain 10% tire ox after can moving The DMEM culture mediums of serum, after gently blowing and beating repeatedly, micro- Microscopic observation cellular morphology simultaneously counts, according to cell in culture dish In the culture dish that content sterilizes appropriate passage to other, 5%CO is put into after adding 5mL DMEM culture mediums2Culture Case.
Cell count formula (individual/mL)：(4 big lattice TCS) × 104× extension rate/4.
3rd, the extraction of total serum IgE
1) outwell culture medium, after PBS, 1mL Trizol are directly injected into blake bottle (wherein cell 5 × 106Individual/ ML), suction is uniform repeatedly.
2) 0.2mL chloroform (for the 1/5 of Trizol cumulative volumes) is added in the centrifuge tube equipped with lysate, vibration is mixed 30 seconds, stand 5 minutes at room temperature.
3) 4 DEG C of 12000rpm is centrifuged 15 minutes, and split-phase is three layers.Upper strata：RNA (about the 60% of Trizol)；It is middle： DNA；Lower floor：Protein (phenol-chloroform).
4) careful Aspirate supernatant, it is transferred in another EP pipes.Supernatant volume caused by 1mL lysates is about 0.4~ 0.6mL.DNA and protein are contained in organic phase and intermediate layer, avoid touching.
5) supernatant adds about 0.5mL isopropanol, and vibration is mixed 30 seconds.Stand 10 minutes at room temperature.
6) 4 DEG C of 12000rpm is centrifuged 10 minutes.
7) RNA precipitate will be formed in the side at centrifugation bottom.Careful inhale abandons supernatant, pays attention to avoiding suction from abandoning RNA precipitate.
8) centrifuge tube adds 75% ethanol (1mL Trizol at least 1mL ethanol cleaning DNA) of 1mL precoolings, and vibration is mixed 30 seconds, vibrate precipitation, room temperature 12000rpm is centrifuged 1~2 minute.Inhale as far as possible and abandon supernatant, prevent RNA precipitate from losing Lose.Repeat above cleaning step once.
9) centrifuge tube is inverted on filter paper, dries RNA, but can not be completely dried (5~10 minutes).It is molten with the μ L of DEPC water 15 Solution precipitation, 55~60 DEG C are incubated 10~15 minutes.
4th, real-time fluorescence quantitative PCR
The total serum IgE 1-5 μ g of extraction are taken, add PCR reaction solutions, PCR reaction solutions include：Sterilized water, have 5' → 3' circumscribed Archaeal dna polymerase, dNTPs, 10 × PCR Buffer, RNASIN, M-MLV reverse transcriptase, oligo (dT), primer, the PNA of activity The solution of probe, PNA fluorescence probes and the ion containing Mg.Wherein, the DNA for having 5' → 3' exo-acting that concentration is 5U/ μ L gathers The μ L of synthase 0.3, concentration are the 10mmol/L μ L of 2 μ L, 10 × PCR Buffer of dNTPs 5, and concentration is 40U/ μ L RNASIN 0.6 μ L, concentration are the 200U/ μ L μ L of M-MLV reverse transcriptases 0.6, and concentration is 25mmol/L MgCl2The μ L of solution 5, concentration 10 μm ol/L positive, reverse trip each 1.2 μ L of primer, concentration are 10 μm of ol/L PNA probe, each 1.5 μ L additions of PNA fluorescence probes Sterilized water to volume is 50 μ L.Wherein, it can be Taq enzyme to have the exo-acting archaeal dna polymerases of 5' → 3'.
PCR is expanded：Each reaction tube is put into the reactive tank of quantitative fluorescent PCR instrument, setting mark fluorescent radical species, Sample ID and type, sample well is defined, and the amplification program that according to the form below provides enters performing PCR amplification：
Table 1 is pcr amplification reaction amplification program
Fluorescent value is read in the end of a period of the 3rd step of amplification program.
5th, data analysis judges：
Quantifying system CT values should meet 7≤CT value≤33 first, if the explanations of quantifying system FAM signal CT values ＜ 7 treat test sample This DNA sample concentrations are excessive；If CT values ＞ 33 illustrates that sample to be tested DNA sample concentrations are too low or degraded without CT values, Again processing sample to be tested DNA is verified again.If experiment meets that conditions above judges to detect the clear condition of mutation of sample again, select simultaneously Institute's sample sheet saltant type (positive) reference material corresponding with the pattern detection site and wild type (feminine gender) refer to sample wells, contrast three Hole PCR amplification curves (CTARepresent sample aperture CT values, CTWRepresent wild type CT values, CTMRepresent saltant type CT values)：
Work as CTW＜ CTA≤CTMWhen, show that the sample has mutation；
Work as CTW=CTAWhen, it is wild type to show the sample.
Fig. 1 is peptide nucleic acid mass spectrogram described in the embodiment of the present invention；
Fig. 2 is G136D saltant types described in the embodiment of the present invention and reference wild-type product PCR amplification figures, it is upper in figure, in, Lower three bars of lines respectively represent the amplification curve of the bit codon mutation type reference materials of DVL1 the 136th, sample and reference wild-type product, table Bright this presence of institute's sample mutation.
Above example is only used for illustrating technical scheme, rather than is limited, although with reference to foregoing reality Example is applied the present invention is described in detail, for the person of ordinary skill of the art, still can be to foregoing implementation Example described in technical scheme modify, or to which part technical characteristic carry out equivalent substitution, and these modification or Replace, the essence of appropriate technical solution is departed from the spirit and scope of claimed technical solution of the invention.
Sequence table
<110>Hubei University Of Technology
<120>A kind of disheveled protein 1（DVL1）Gene G136D mutation detection kits
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
cccagggcac ggacagccac 20
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
cgtgctgcca tcctcgtc 18
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
cgagacagac acggagtcca tgg 23
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
cgagacaggc acggagtcca tgg 23
<210> 5
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
cccaaatgtg gccagc 16
<210> 6
<211> 15
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
cctcctcgcg gttcc 15
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
ggtcagtcac cggcgggagc 20
<210> 8
<211> 323
<212> DNA
<213> Homo sapiens
<400> 8
cccagggcac ggacagccac acagacctgc ccccgcctct tgagcggaca ggcggcatcg 60
gggactcccg gcccccctcc ttccacccaa atgtggccag cagccgtgac gggatggaca 120
acgagacagg cacggagtcc atggtcagtc accggcggga gcgtgcccga cgccggaacc 180
gcgaggaggc cgcccggacc aatgggcacc caaggggaga ccgacggcgg gatgtggggc 240
tgcccccaga cagcgcgtcc accgccctca gcagcgagct tgagtccagc agctttgtgg 300
actcggacga ggatggcagc acg 323

Claims (8)

  1. A kind of 1. DVL1 genes G136D mutation detection kits, it is characterised in that：It is described including primer and peptide nucleic acid probe Primer includes the forward primer DVL1-F and reverse primer DVL1-R for being used for specific detection DVL1 genes G136D mutation, described Peptide nucleic acid probe include be used for specific detection DVL1 genes G136D mutation peptide nucleic acid fluorescence probe DVL1-PNA-P and use In the peptide nucleic acid probe DVL1-PNA for blocking wild type DVL1 gene magnifications, the primer, the sequence of peptide nucleic acid probe are as follows：
    DVL1-F：5'-cccagggcacggacagccac-3'；
    DVL1-R：5'-cgtgctgccatcctcgtc-3'；
    DVL1-PNA-P：5'- fluorescent reporter group-cgagacagacacggagtccatgg- quenching groups -3'；
    DVL1-PNA：5'-cgagacaggcacggagtccatgg-3'.
  2. 2. kit according to claim 1, it is characterised in that：Including PCR reagent.
  3. 3. kit according to claim 1 or 2, it is characterised in that：Including positive reference substance, positive reference product and feminine gender Reference material.
  4. 4. kit according to claim 3, it is characterised in that：Described positive reference substance is to contain DVL1 genes the 4th The mixture of the DNA and the DNA containing the exon sequence of DVL1 genes the 5th of extron G136D mutation.
  5. 5. kit according to claim 3, it is characterised in that：Described positive reference product are to contain DVL1 genes the 4th The DNA of extron G136D mutation, negative reference product are to contain DNA wild the extron G136 of DVL1 genes the 4th.
  6. 6. kit according to claim 1, it is characterised in that：Including detecting the forward primer DVL1-F' of internal reference and anti- To primer DVL1-R', and the peptide nucleic acid fluorescence probe DVL1-PNA-P' of detection internal reference, the primer, peptide nucleic acid fluorescence probe Sequence it is as follows：
    DVL1-F'：5'-cccaaatgtggccagc-3'；
    DVL1-R'：5'-cctcctcgcggttcc-3'；
    DVL1-PNA-P'：5'- fluorescent reporter group-ggtcagtcaccggcgggagc- quenching groups -3'.
  7. 7. the primer for DVL1 gene G136D abrupt climatic changes, it is characterised in that：Described primer includes forward primer DVL1-F It is as follows with reverse primer DVL1-R, sequence：
    DVL1-F：5'-cccagggcacggacagccac-3'；
    DVL1-R：5'-cgtgctgccatcctcgtc-3'.
  8. 8. the supporting peptide nucleic acid probe with the primer described in claim 7, it is characterised in that：Described peptide nucleic acid probe includes using In the peptide nucleic acid fluorescence probe DVL1-PNA-P of specific detection DVL1 genes G136D mutation and for blocking wild type DVL1 bases The peptide nucleic acid probe DVL1-PNA of gene-amplification, sequence are as follows：
    DVL1-PNA-P：5'- fluorescent reporter group-cgagacagacacggagtccatgg- quenching groups -3'；
    DVL1-PNA：5'-cgagacaggcacggagtccatgg-3'.
CN201711161143.0A 2017-11-20 2017-11-20 A kind of disheveled protein 1（DVL1）Gene G136D mutation detection kits Pending CN107699622A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103667492A (en) * 2013-12-13 2014-03-26 青岛大学医学院附属医院 WNT signal channel detecting reagent, PCR (polymerase chain reaction) detection method and application thereof
CN105624796A (en) * 2014-11-07 2016-06-01 天津华大基因科技有限公司 Chip and uses of chip in deafness related gene detection
CN105838796A (en) * 2016-04-28 2016-08-10 湖北工业大学 AXIN2 gene mutation detection reagent and application

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103667492A (en) * 2013-12-13 2014-03-26 青岛大学医学院附属医院 WNT signal channel detecting reagent, PCR (polymerase chain reaction) detection method and application thereof
CN105624796A (en) * 2014-11-07 2016-06-01 天津华大基因科技有限公司 Chip and uses of chip in deafness related gene detection
CN105838796A (en) * 2016-04-28 2016-08-10 湖北工业大学 AXIN2 gene mutation detection reagent and application

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Application publication date: 20180216