CN105506035A - Method for preparing RNA or DNA probe by taking multiple DNA fragments as formwork - Google Patents
Method for preparing RNA or DNA probe by taking multiple DNA fragments as formwork Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12P19/28—N-glycosides
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- 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
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
Abstract
The invention relates to a method for preparing an RNA or DNA probe by taking multiple DNA fragments as a formwork. The method comprises the following steps of screening a plurality of target sequences from in vitro transcription gene specificity cDNA or particular gene DNA sequences in genomes, and designing one or more pairs of primers for each target sequence, wherein PCR amplification products of the primers are multiple DNA fragments, and the multiple DNA fragments can include all of the target sequences; taking the multiple DNA fragments as the formwork to perform the following reaction: adding RNA polymerase and UTP with biotin labeling for the reaction, adding the UTP into the products, and then performing purification to obtain the RNA probe; or adding DNA polymerase and dUTP with biotin or luminescence labelling for the reaction, adding the dUTP to the products, and then performing purification to obtain the DNA probe. According to the method disclosed by the invention, the multiple DNA fragments are used as the formwork for the first time; aimed at the target sequences to be detected, the pairs of the primers are designed, a promoter or the RNA polymerase is added on a 5' end of a backward primer, and through the combination of the sequences, the finally obtained RNA probe can totally cover the target sequences.
Description
Technical field
The invention belongs to diagnostic nucleic acid field, be specifically related to method and the application thereof of a kind of RNA or DNA probe mark.
Background technology
Fluorescence in situ hybridization technique (Flu or escenceinsituhybridization, FISH) be a kind of important nonradioactivein situhybridization technology, the target DNA that the specific nucleic acid probe of fluorochrome label is corresponding in cell or RNA molecule are hybridized, if detected karyomit(e) target DNA and nucleic acid probe used are homologous complementaries, the two is through sex change-annealing-renaturation, because DNA molecular is linearly arrange along the karyomit(e) longitudinal axis on chromosome, thus probe can directly and karyomit(e) carry out hybridizing thus specific gene located on chromosome.By at fluorescence microscopy Microscopic observation fluorescent signal, with determine with specific probe hybridization after combine region of DNA territory or the location of RNA molecule in karyomit(e) or other organoids of fluorescent probe, DNA to be measured is carried out qualitative, quantitatively or relative positioning analysis.
Fluorescence in situ hybridization technique (FISH) has lot of advantages, is mainly reflected in: (1), without the need to labelled with radioisotope, security is higher; (2) experimental period of FISH is short, and probe steady is strong; (3) pass through repeatedly immuno-chemical reaction, its hybridization signal is amplified, improve sensitivity, the sensitivity of detection is hybridized close to isotope probe; (4) resolving power of FISH is up to 3-20Mb; (5) FISH can mark different probes with different modifying nucleic acid molecule, namely polychromatic probe can be prepared into, therefore in same section, the location of several probe can be observed simultaneously, directly obtain their relevant position and order, accelerate the research of the function assignment of genes gene mapping and biological genome.
FISH technology is widely applied in the field such as detection, antenatal diagnosis, mammalian chromosome Study on Evolution of the assignment of genes gene mapping, gene mapping, gene amplification and disappearance, particularly in diagnosing tumor, FISH technology is widely used in the early diagnosis of the solid tumors such as mammary cancer, bladder cancer, lung cancer, lymphoma, examination of curative effect, several aspect such as individualized treatment and Index for diagnosis.Research shows, the expression of some genes and the generation of tumour closely related.
Fluorescence in situ hybridization (FISH) is generally that application DNA probe detects, and DNA probe can detect DNA sample (genomic dna), also can detect RNA sample (mRNA or ncRNA).DNA probe good stability, is easy to preserve.Rna probe is generally used for and detects RNA sample (mRNA or ncRNA).So the advantage of rna probe is at single-stranded probe, hybridization efficiency is high, and crossbred comparatively DNA probe stablize.In fluorescence in situ hybridization, can follow according to respective probe advantage and detect target select.
Summary of the invention
The object of this invention is to provide a kind of take multiple DNA fragment as the method for Template preparation RNA or DNA probe, the method can prepare high specificity, the RNA that highly sensitive and stability is strong or DNA probe effectively in a large number, with the demand of satisfied production FISH lesion detection test kit.
Of the present invention take multiple DNA fragment as the method for Template preparation RNA or DNA probe, comprise the following steps: A. filters out multiple target sequence from specific gene DNA sequence dna the gene specific cDNA or genome of in-vitro transcription, at least one pair of primer is designed for each target sequence, the pcr amplification product of described primer is multiple DNA fragment, and described multiple DNA fragment can comprise all target sequences; B. with described multiple DNA fragment for template, carry out following reaction: a. adds RNA polymerase and reacts with biotin labeled UTP, UTP is mixed product, obtains rna probe after purifying; Or b. adds archaeal dna polymerase and reacts with the dUTP of vitamin H or fluorescent labelling, dUTP is mixed product, obtains DNA probe after purifying.
The RNA polymerase that probe mark is conventional comprises: t7 rna polymerase, SP6RNA polysaccharase, T3RNA polysaccharase and TrcRNA polysaccharase.
The archaeal dna polymerase that probe mark is conventional comprises: DNA polymerase i, archaeal dna polymerase Klenow fragment, Taq DNA polymerase and other are for the archaeal dna polymerase in PCR.
According to the further feature preparing the method for RNA or DNA probe of the present invention, in described steps A, described screening comprises: search specific mRNA to be detected, or searches specific gene DNA sequence dna in genome, gets rid of other non-specific sequences; Then, screen 2-50 intronless and tumor-necrosis factor glycoproteins particular sequence region, homology region sequence can be selected as required, as target sequence.
According to the further feature preparing the method for RNA or DNA probe of the present invention, in described steps A, described design of primers comprises: at least one pair of primer is respectively designed in selected each region, makes amplified production length be 100-1000bp.
According to the further feature preparing the method for RNA or DNA probe of the present invention, described RNA polymerase is t7 rna polymerase, and the 5' end of primer adds T7 promoter sequence.
According to the further feature preparing the method for RNA or DNA probe of the present invention, described RNA polymerase is SP6RNA polysaccharase, and the 5' end of primer adds SP6 promoter sequence respectively.
According to the further feature preparing the method for RNA or DNA probe of the present invention, described RNA polymerase is T3RNA polysaccharase, and the 5' end of primer adds T3 promoter sequence respectively.
According to the further feature preparing the method for RNA or DNA probe of the present invention, described RNA polymerase is TrcRNA polysaccharase, and the 5' end of primer adds Trc promoter sequence respectively.
T7 promoter sequence: 5 '-TAATACGACTCACTATAG-3 '
SP6 promoter sequence: 5 '-CATACGATTTAGGTGACACTATA-3 '
T3 promoter sequence: 5 '-AATTAACCCTCACTAAAG-3 '
Trc promoter sequence: 5 '-TGTTGACAATTAATCATCCGGCTCGTATAAT-3 '
Above-mentioned four kinds of RNA polymerase and corresponding promotor be combined as design of the same type, can use be replaced.
Invention further provides the test kit of the cytokine that a kind of rapid detection cancer is correlated with.
The test kit of the cytokine that rapid detection cancer of the present invention is correlated with comprises RNA prepared by method of the present invention or DNA probe.
The present invention is template with cDNA sequence, utilizes bioinformatics technique, the template sequence as probe mark that squama is elected by comparing with gene library.Multiple DNA fragment refers to the cDNA sequence removing to contain and replace full-length gene with the DNA fragmentation of 2 or more, these multiple sequence dna fragments for certain gene can not only fully demonstrate specificity and the representativeness of this gene, and some tumor-necrosis factor glycoproteinss or homologous sequence can be avoided to strengthen the specificity of probe targetedly.
The present invention uses multiple DNA fragment as template first, designs multipair primer for target sequence to be detected, and adds promotor or RNA polymerase binding sequence at reverse primer 5 ' end, all covers all target sequences to enable the rna probe finally obtained.Probe preparation method of the present invention simplifies probe preparation steps, significantly shortens whole experimental period, the more important thing is, probe specificity and stability are stronger, and reduce background signal, sensitivity is higher.On this basis, the invention provides the novelty teabag of kinds of tumors first visit rapid detection aspect and relevant blanket test kit.
The method of the RNA of preparation of the present invention or DNA probe can prepare high specificity, the RNA that highly sensitive and stability is strong or DNA probe effectively in a large number.Rna probe for diagnosing tumor has more advantage than DNA probe, such as: avoid double chain DNA probe possibility of easy renaturation between two chains in hybridization, improve the susceptibility of hybridization; The crossbred formed between RNA and RNA is more stable than DNA RNA hybrid; And the crossbred formed between RNA-RNA is not by the impact of RNA enzyme, and therefore available RNA ferment treatment after hybridization, removes unconjugated probe with wash-out, can reduce background signal, therefore have the advantage that background is low.Therefore, the rna probe utilizing the method for the invention to prepare is conducive to related reagent and the test kit of researching and developing and prepare easy storage and transport, in particular for high-quality probe and the related kit of Diagnosis and Treat kinds of tumors.
Traditional probes label methods is directly using cDNA as mark template.When preparation detects the probe of mRNA, be generally application full length cDNA clone plasmid or PCR primer as template, utilize RNA polymerase or archaeal dna polymerase to prepare RNA or DNA probe.But most gene cDNA, at more than 1000bp, can be unfavorable for because probe length is oversize entering cell and hybridization as directly carried out hybridization, and can produce unspecific background signal.RNA or the DNA probe of preparation generally need alkaline lysis or DNA enzymatic process, make probe cleavage become the fragment of below 1000bp.But, RNA alkaline lysis or DNA enzymatic treating processes and degree wayward, therefore probe mass is difficult to ensure.
The advantage of method of the present invention is: adopt many DNA fragmentations comprising described aim sequence can be prepared the rna probe of 100-500nt ideal length by single step reaction, and can be directly used in next step cross experiment.Probe preparation process does not need the steps such as alkaline lysis, just can obtain the good probe of length homogeneity, is the fragment of cross experiment ideal length, therefore strengthens probe specificity, reduces background signal interference, and saves the great many of experiments time, improve test efficiency.
In DNA and RNA hybridization, the ideal length of probe is 100-500bp (MichaelR.GreenandJosephSambrook, MolecularCloning:ALab or at or yManual.ColdSpringHarb or Lab or at or yPress; 4thedition (June15,2012)).If probe is too short, hybrid specificities is not high, and hybridization is unstable; If probe is oversize, it is not easily through cell, and hybridization efficiency reduces, and easily produces unspecific background signal.
The method of existing RNA or DNA probe mark is all difficult to effectively the probe length marked be controlled within the scope of this.Therefore, prior art must go to degrade the probe that marked to reach above-mentioned ideal length by alkaline lysis (rna probe) or DNA enzymatic (DNA probe).
PCR reaction accurately can control the length of product, but not high by the efficiency of the direct label probe of PCR, and much all effectively can not be incorporated in product by the dUTP of mark for the enzyme of PCR reaction and go.PCR reaction can not be used for labeled rna probe.
The present invention utilizes PCR to react the feature that accurately can control product length, label probe is removed as template with the DNA fragmentation that it prepares multiple ideal lengths, just effectively control the probe length of mark like this in DNA profiling level, the RNA of preparation or DNA probe are directly used in hybridization without RNA alkaline lysis or DNA enzymatic treating processes.In addition, with the DNA fragmentation of multiple ideal lengths as template, specific target gene sequence can also effectively be covered according to the needs of testing goal.Add RNA polymerase binding sequence at each prime end simultaneously, make the DNA fragmentation of multiple ideal lengths in same labeled reactant, can prepare rna probe as template simultaneously.
Following table 1 compares routine and prepares the method for probe and the similarities and differences of method of the present invention.
Method of the present invention is conducive to screening and prepares high specific probe, and can eliminate the impact of non-coding sequence and other non-specific sequences.The design of multiple DNA fragment is more targeted to detection sequence, not only eliminates some non-coding sequences and other non-specific sequences, the specificity of probe also can be made greatly to improve.
Method of the present invention is conducive to the different shear-forms distinguishing homology sequence and RNA.The design of multiple DNA fragment of the present invention can be determined to add or delete some homology sequences according to detected object, also only can comprise or does not comprise different shear-form and reach the object of detection specificity target sequence.
When designing primer in the present invention, add promoter sequence or RNA polymerase binding sequence at reverse primer 5' end, can guarantee that each fragment can be transcribed into probe, design multipair primer for target sequence, can ensure that probe covers all special regions of target gene.
Accompanying drawing explanation
The schema of Fig. 1 to be of the present invention with multiple DNA fragment be method of Template preparation rna probe, showing with multiple DNA fragment is the rna probe labeling process of template.
Fig. 2 is the experimental result of rna probe in FISH adopting the method for the invention to prepare.
The schema of Fig. 3 to be of the present invention with multiple DNA fragment be method of Template preparation DNA probe, showing with multiple DNA fragment is template, prepares DNA probe with random primer labelling (RandomPriming).
Embodiment
Embodiment one: take multiple DNA fragment as Template preparation rna probe
Of the present invention is that the method for Template preparation rna probe is see Fig. 1 with multiple DNA fragment.
Below for Her2 (ErbB-2) gene RNA probe, illustrate and how to adopt aforesaid method to prepare biotin labeled Her2RNA probe.
One, the design of PCR primer:
(1) search Her2 gene mRNA sequence, get rid of other non-specific RNA sequence.
Such as, in GenBank (http://www.ncbi.nlm.nih.gov/genbank/), search Her2mRNA sequence, get rid of other non-specific RNA sequence with NCBIBLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
(2) screen 2-50 intronless and tumor-necrosis factor glycoproteins particular sequence region, homology region sequence can be selected as required, as target sequence.
(3) at least one pair of primer is respectively designed for each target sequence, make amplified production length be 100-1000bp.Design of primers is as follows:
Primer involved by upper table is only citing, and those skilled in the art principle design according to the present invention can go out more suitable primer.
(4) as required T7 promotor or other RNA polymerase binding sequences are added in the 5' end of reverse primer, to guarantee PCR primer and object fragment complementation and to comprise whole target sequence (seeing the above table middle lowercase).
Two, mark the preparation of template: by PCR from cDNA plasmid template (from GeneCopoeiacDNA plasmid storehouse) amplification with the object fragment of T7 promotor and Her2DNA template.
Three, the preparation of rna probe:
Preparation 10 × NTPs, in the NTPs of preparation, make the concentration of ATP, CTP, GTP be 10mM, the concentration of TTP is 5mM.
In an EP pipe without RNase, add following composition:
Above-mentioned reaction operates in without the environment of RNase.
Mixing reaction solution, reacts 2-3 hour at 37 DEG C.
Add 1 μ LDNaseI and 5 μ LDNaseI damping fluids, after mixing at 37 DEG C 15 minutes, to remove template DNA.
Add 2 μ L0.5MEDTA (pH8.0), react 10min at 65 DEG C with termination reaction.
Get the product electrophoresis detection of 2-3 μ L, damping fluid is with 1 × MOPS (3-morpholine propanesulfonic acid).
Add 100 μ LDEPC water (diethylpyrocarbonate), 150 μ L phenol and 200 μ L chloroforms are in EP pipe, and concussion mixes, the centrifugal 10min of 10000rpm.
Supernatant liquor is transferred in new EP pipe, adds isopyknic Virahol, and after mixing leaves standstill 10min, the centrifugal 10min of 12000rpm, removes supernatant.
After the ethanol wash precipitation of 75%, add DEPC water dissolution, survey production concentration and electrophoresis detection, be stored in-20 DEG C.
Thus, obtaining take multiple DNA fragment as the rna probe of Template preparation.
The biotin labeled Her2RNA probe obtained by aforesaid method is used for the expression level detecting human breast cancer cell line Bcap-37 (purchased from ATCC) Her2mRNA.
At fluorescence microscopy Microscopic observation, experimental result as shown in Figure 2.Zuo Tu: with DAPI and 4', 6-diamidino-2-phenylindone (4', 6-diamidino-2-phenylindole) dyeing, blue-fluorescence showed cell core.Middle figure: hybridize with described probe and object mRNA, the distribution of red fluorescence display human breast cancer cell line Bcap-37 Her2mRNA and expression level.Right figure is the composograph of left figure and middle figure, intuitively showed cell core and Her2mRNA relative position.
Embodiment two: take multiple DNA fragment as Template preparation DNA probe
Of the present invention is that the method for Template preparation DNA probe is see Fig. 3 with multiple DNA fragment.
One, the design of PCR primer:
(1) search specific mRNA to be detected, or search specific gene DNA sequence dna in genome, get rid of other non-specific RNA sequence.
(2) screen 2-50 intronless and tumor-necrosis factor glycoproteins particular sequence region, homology region sequence can be selected as required.
(3) at least one pair of primer is respectively designed in each region selected by, makes amplified production length be 100-1000bp.
In a particular embodiment, random primer (Randomprimer) can be designed, the oligonucleotide fragment of namely random length 6 Nucleotide.They can combine (annealing) with the random complementation of the double-stranded DNA of sex change, to provide 3 ' hydroxyl terminal, without under archaeal dna polymerase large fragment (as the Klenow large fragment) effect of 5 ' → 3 ' 5 prime excision enzyme activity, add Nucleotide one by one until next primer place at 3 ' C-terminal of primer.There are institute's likely binding sequences in 6 mixture of ribonucleotides, the combination of primer and template occurs in a kind of random mode, can the total length of uniform fold target patch segment DNA so mark.
Two, the preparation of template is marked: by PCR from cDNA plasmid template (from the GeneCopoeiacDNA plasmid storehouse) object that increases fragment.
Three, the preparation of DNA probe:
Preparation 10 × dNTPs, wherein, the concentration of dATP, dCTP, GTP is 2mM, and the concentration of dTTP is 1mM.
In an EP, add following composition:
Mixing reaction solution, reacts 5h at 37 DEG C.
Add 2 μ L0.5MEDTA (PH8.0), react 10min at 65 DEG C with termination reaction.
Get the product electrophoresis detection of 2-3 μ L, damping fluid 1 × TAE.
Add 100 μ L water, 150 μ L phenol and 200 μ L chloroforms are in EP pipe, and concussion mixes, the centrifugal 10min of 10000rpm.
Supernatant liquor is transferred in new EP pipe, adds isopyknic Virahol, and after mixing leaves standstill 10min, the centrifugal 10min of 12000rpm, removes supernatant.
After the ethanol wash precipitation of 75%, be dissolved in water, survey production concentration and electrophoresis detection, be stored in-20 DEG C.
Thus, obtaining take multiple DNA fragment as the DNA probe of Template preparation.
Claims (9)
1. be a method for Template preparation RNA or DNA probe with multiple DNA fragment, it is characterized in that, comprise the following steps:
A. from specific gene DNA sequence dna the gene specific cDNA or genome of in-vitro transcription, multiple target sequence is filtered out, at least one pair of primer is designed for each target sequence, the pcr amplification product of described primer is multiple DNA fragment, and described multiple DNA fragment can comprise all target sequences;
B. with described multiple DNA fragment for template, carry out following reaction:
A. add RNA polymerase and react with biotin labeled UTP, UTP being mixed product, after purifying, obtains rna probe; Or
B. add archaeal dna polymerase and react with the dUTP of vitamin H or fluorescent labelling, dUTP being mixed product, after purifying, obtains DNA probe.
2. the method preparing RNA or DNA probe according to claim 1, is characterized in that, described RNA polymerase is t7 rna polymerase, and the 5' end of primer adds T7 promoter sequence.
3. the method preparing RNA or DNA probe according to claim 1, is characterized in that, described RNA polymerase is SP6RNA polysaccharase, and the 5' end of primer adds SP6 promoter sequence respectively.
4. the method preparing RNA or DNA probe according to claim 1, is characterized in that, described RNA polymerase is T3RNA polysaccharase, and the 5' end of primer adds T3 promoter sequence respectively.
5. the method preparing RNA or DNA probe according to claim 1, is characterized in that, described RNA polymerase is TrcRNA polysaccharase, and the 5' end of primer adds Trc promoter sequence respectively.
6. the method preparing RNA or DNA probe according to claim 1, is characterized in that, described archaeal dna polymerase is selected from: DNA polymerase i, archaeal dna polymerase Klenow fragment, Taq DNA polymerase and other are for the archaeal dna polymerase in PCR.
7. the method preparing RNA or DNA probe according to claim 1, it is characterized in that: in described steps A, described screening comprises: search specific mRNA to be detected, or searches specific gene DNA sequence dna in genome, gets rid of other non-specific sequences; Then, screen 2-50 intronless and tumor-necrosis factor glycoproteins particular sequence region, homology region sequence can be selected as required, as target sequence.
8. the method preparing RNA or DNA probe according to claim 2, it is characterized in that: in described steps A, described design of primers comprises: at least one pair of primer is respectively designed in selected each region, makes amplified production length be 100-1000bp.
9. the rapid detection cancer test kit of cytokine of being correlated with, is characterized in that: comprise the rna probe prepared by method according to claim 1 or DNA probe.
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| WO2017133608A1 (en) * | 2016-02-05 | 2017-08-10 | 广州复能基因有限公司 | Method for preparing rna or dna probe with multiple dna fragments as templates |
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| WO2017133608A1 (en) * | 2016-02-05 | 2017-08-10 | 广州复能基因有限公司 | Method for preparing rna or dna probe with multiple dna fragments as templates |
| CN114891787A (en) * | 2022-05-09 | 2022-08-12 | 珠海圣美生物诊断技术有限公司 | Random probe, preparation method and application |
| WO2023216938A1 (en) * | 2022-05-09 | 2023-11-16 | 珠海圣美生物诊断技术有限公司 | Random probe, preparation method therefor and use thereof |
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