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CN104131064A - In-situ hybridization detection kit of miR-378 in early stage of pathologic evolution of gastric cancer, detection method and application - Google Patents

In-situ hybridization detection kit of miR-378 in early stage of pathologic evolution of gastric cancer, detection method and application Download PDF

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CN104131064A
CN104131064A CN201310159214.9A CN201310159214A CN104131064A CN 104131064 A CN104131064 A CN 104131064A CN 201310159214 A CN201310159214 A CN 201310159214A CN 104131064 A CN104131064 A CN 104131064A
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hybridization
cancer
mir
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test kit
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裘建英
张云福
张玉丽
裘霖
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JIAXING RUIKANG BIOLOGICAL TECHNOLOGY Co Ltd
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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
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    • 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
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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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

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Abstract

The invention discloses an in-situ hybridization detection kit which includes a hybridization probe and a marker. The invention also discloses a method for detecting change of expression of a miR-378 gene, which is closely relative to pathologic evolution in an early stage of gastric cancer cancerization, through the kit in a manner of in-situ hybridization detection. The method includes following steps: (1) on the condition that the hybridization probe and a target sequence can form a stable hybridization complex, contacting a to-be-detected miR-378 in a substrate with the hybridization probe to form the hybridization complex; and (2) detecting the hybridization complex. By means of the kit and the method, an expression quantity of the miR-378 can be detected on a miRNA level. Compared with image medicine and a clinical biochemical detection index in the prior art, The kit and the method can be applied in an earlier stage and can achieve a true RNA-level screening operation during the early stage of cancerization. Meanwhile, the detection method is simple and convenient, is low in cost and is suitable for being popularized in hospitals in counties or districts.

Description

Hybridization in situ detection kit and detection method and the application of cancer of the stomach Pathologic miR-378 in early stage
Technical field
The present invention relates to field of biological detection, more particularly, relate to change with cancer of the stomach Pathologic rna expression the correlation detection technology of (Pathologic process).
Background technology
The data providing according to domestic and international authoritative institution, the newly-increased number of the annual cancer of China has reached 3,120,000, death toll nearly 2,600,000, patient surpasses 7,000,000, the annual newly-increased cancer patients 8,000,000 in the whole world, death toll approaches 8,000,000, and patient approximately has more than 8,400 ten thousand people, to the above number of the year two thousand twenty, will double, this is one group of fearful numeral.Cancer diagnosis and treatment cost is more and more higher, by cancer patients's year medical expense 200,000, (poor area may be higher, developed regions may exceed 200,000 far away), more than 700 ten thousand patients, annual cost is 1.4 trillion Renminbi, deduction cost 35% approximately 400,000,000,000, approximately has 1,000,000,000,000 Renminbi to consume in vain every year.And cancer patients is most of can be dead soon after treatment.Therefore, existing clinical cancer diagnosis and treatment pattern must change, and innovative point of the present invention is to accomplish in advance preventative examination, then gets involved in time preventative regulation and control and prophylactic treatment, accomplishes preventiveing treatment of disease of gene level cancer.
The inventor is in the middle discovery that studies for a long period of time, and causing the major reason that cancer mortality is not fallen is to accomplish real early diagnosis.According to existing clinical medicine image (B ultrasonic, CT, Magnetic resonance imaging etc.) and with other biochemistry (cancer antigen, carcinomebryonic antigen, carbohydrate hormone, the cytolemma factor, nuclear factor, cell streaming technology) index, carry out diagnosing cancer, all that tumour forms rear diagnosis, the former will learn in a organized way and change or existing occupying lesion, the latter's major part be tumour form rear secreted, discharge or the marker of tumour.The clinical idea of tradition thinks that occupancy cancer piece is the diagnosis that belongs to early-stage cancer under 2 centimeters, this concept is worth conscientiously discussing, it is rigorous not that 2 centimeters of early stage these of following cancer piece genus define science, from cytology angle, analyze, the lump of 1 centimeter approximately has 100,000,000 tumour cells, its three-dimensional cell stack number of the lump of 2 centimeters is far above 200,000,000 tumour cells, from canceration, to mono-clonal cancer cells, produce and form the cancer piece of 2 centimeters early stage, its Pathologic process is quite long, it may be 5 years or 10 years, or even 10 years above (except special case), what be difficult to confirm is in this Pathologic process, lump is the unique spot of cancer and independent focus, possible cancer cells moves to other tissue or organ growth already.Clinical study confirms already, and once form lump, other cancer cells moves to other position clonal growth by different approaches, once after excision primary tumor, other organ recurrence kitchen range or multiple cancer piece kitchen range successively form.Therefore, with 2 centimeters of following cancerous swelling piece sizes, whether define in early days clinically, rigorous not (some case, when finding primary lesion, find metastatic lesion simultaneously, not in the content of our statement), be at this moment diagnosis in late period and treatment of late stage in fact, this is the true cause that causes cancer mortality not fallen.
Along with molecular biotechnology is day by day perfect, functional genomics, the deep expansion of the research such as cancer gene group, in order to seek more early stage diagnosing cancer, treatment cancer and preventing cancer, makes great progress.So far, we likely do more accurate early screening and diagnosis on the one-level functional transcription product (mRNA level or microRNA) of gene, front in canceration early stage or cancer cells formation (mono-clonal), just can accomplish early prediction and examination.
The death toll of annual cancer of the stomach surpasses 1,000,000,75% Patients with Gastric Cancer in Asia in the world, and China has accounted for significant proportion.And M & M is also rising.In experimental study, find miR-378 great expression in patients with gastric cancer, and just have obviously and increase in cancer of the stomach early expression amount.
The present invention selects many group clinical samples (Patients with Gastric Cancer, high risk population, normal control) to adopt nucleic acid hybridization in situ technology and ImmunohistochemistryMethods Methods to detect analysis to the early warning of miR-378 and cancer of the stomach.The inventor finds that miR-378 has obvious expression amount to change patients with gastric cancer, cancer high risk population's (chronic atrophic gastritis and stomach have Leptospira), normal control people, has very important clinical diagnosis meaning using miR-378 in earlier stage as the pathological change of early screening cancer of the stomach under study for action.The Preventive early warning of doing in after cancer of the stomach canceration examination in early stage and curing gastric cancer also has very important clinical meaning.
Contriver is in long-term research, drawn a kind of new concept, the clinical diagnosis and treatment pattern of cancer and other clinical major disease must change, can not only stop and control now oneself disease (diagnosis and treatment after morbidity), accomplish preventative diagnosis and treatment, accomplish to preventive treatment of disease, only in this way could reduce the M & M of major disease, reduce social cost and medical treatment cost.Therefore, contriver, in the rna level kit for screening and medicine of development and production major disease, innovates theoretical and technical.Particularly screen clinical samples (normal population, cancer high risk population, tumour patient), broken through the consistency research and development thinking of healthy tissues and tumor tissues comparison, find and develop the rna level becoming before cancer, develop closely related with cancer early gene physiopathology, and the extremely important target of clinical meaning (disease gene), tumour is clinically formed to the preventative diagnosis and treatment that rear diagnosis and treatment pattern becomes tumour, striven for time and the space of tumor diagnosis and treatment, reach preventing cancer.
At present, someone has applied for that miR-378 express spectra detects patent, use be the analytical technologies such as chip, RT-PCR, sample is serum.The present invention adopts hybridization in situ technique and groupization immunization method to detect the detection technique of miR-378 expression amount, adopts peripheral blood leucocyte sample or histocyte sample, and similar reagents box and screening method have no report.
The inventor is in the requirement for novelty invention, designed (patients with gastric cancer, high risk population, normal control) different pieces of information example group, with hybridization in situ technique, detect, result shows above Patients with Gastric Cancer miR-378 high expression level, high risk population has and expresses in various degree 6-18%, and normal control is all zero to express.Show that miR-378 is the important symbol thing of cancer of the stomach canceration examination in early stage.
Hybridization in situ technique (in situ hybridization) is that molecular biology and Cytochemical Technique are combined, and the nucleic acid molecule of mark of take is probe, in the technology of histocyte in situ detection specific nucleic acid molecule.Its principle is the nucleic acid strand (being probe) that makes to contain distinguished sequence, passes through mark, under optimum conditions with histocyte in complementary nucleic acid strand be that target nucleic acid is hybridized, with radioautograph or immunocytochemistry, label probe is surveyed again, thereby shown special DNA or RNA molecule at cell in-situ.
The probe of in situ hybridization is the molecule of known array or sequence the unknown but known nucleic acid molecule (though indefinite this molecule full sequence of molecule, but known its for what target molecule), the kind of probe can be divided into again DNA probe, cDNA probe, cRNA probe and synthetic oligonucleotide probe by properties of nucleic acids difference.For the ease of spike, probe must, with certain means mark in addition, be beneficial to later detection.Conventional marker comprises radionuclide and the large class of non-radioactive marker two.Conventional isotopic label has 3h, 35s, 125i and 32p.The advantages such as susceptibility is high although isotopic label has, back end is comparatively clear, because radio isotope all can damage human and environment, have the trend being replaced by heterotope recently.At present the most frequently used in heterotope marker have three kinds of vitamin H, digoxin and fluoresceins.The method that detects these markers is all extremely sensitive.
According to probe used and the difference that will detect nucleic acid, can be divided into DNA-DNA again, RNA-DNA, RNA-RNA hybridization.No matter but the hybridization of any form, all must be through five large processes, histiocytic fixing, prehybridization, hybridization, flushing and demonstration.The present invention adopts the Crossing system of RNA-RNA, synthetic probe (RNA) and the target RNA detecting are the principles that adopts base complementrity (hybridization is complementary), simultaneously through long-time research with observe, start and termination place residue on not impact of the result detecting.
In view of the diagnosis of cancer of the stomach clinically (gastroscope, medical imaging and biochemical indicator thing are all the diagnosis after tumour forms) is at present diagnosis in late period, treatment is also treatment of late stage, is also the treatment pattern that causes mortality ratio not fallen.Original intention of the present invention is to want to improve at present the diagnosis and treatment pattern of major disease clinically, from treating the disease affected, become preventative preventiveing treatment of disease, reach preventative diagnosis and treatment, current medical imaging means and numerous biochemical marker cannot be detected and become rna level before cancer and quantize change technology, do the technological breakthrough of novelty, provide cancer the front rna level examination technology that becomes.Made to have had clinically a front technology (tumour does not have before formation) that becomes the real early screening of rna level of new cancer, for the diagnosis and treatment of clinical cancer of the stomach are raced against time and space.
In sum, first object of the present invention is to provide a kind of hybridization in situ detection kit, and it comprises in situ hybridization detection probes and marker.Secondly, the present invention shifts the relevant in situ hybridization detection method of early warning after also will providing mentioned reagent box for the examination in early stage of cancer of the stomach Pathologic and treatment.
Summary of the invention
For realizing object of the present invention, technical scheme of the present invention is as follows:
First the present invention provides a kind of hybridization in situ detection kit, and it comprises hybridization probe and marker, and wherein, described hybridization probe has respectively sequence number shown in sequence table SEQ ID: NR_029870, nucleotide sequence length 66bp.
A preferred version of test kit of the present invention is that described marker is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, fluorescein, enzyme and nano material.
A preferred version of test kit of the present invention is also to comprise hybridization solution.
A preferred version of test kit of the present invention is also to comprise toughener.
A preferred version of test kit of the present invention is also to comprise developer.
Cancer of the stomach Pathologic of the present invention miR-378 kit for screening in early stage using value is, to the examination in early stage of cancer of the stomach Pathologic, and early warning occurs for Preventive, diffusion after canceration or after treatment, further coordinates clinical treatment.
The present invention also provides a kind of detection method of miR-378 in situ hybridization, comprises the following steps:
(1) described hybridization probe and target sequence can form under the condition of stablizing hybridization complex in the above, RNA to be measured in substrate contacted to formation hybridization complex with hybridization probe; With
(2) detect described hybridization complex.
Detection method of the present invention, wherein preferably, the condition that hybridization complex is stablized in described forming is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour.
Detection method of the present invention, wherein preferably, described substrate is selected people's blood leucocyte sample or other organ-tissue cell specimen.More preferably, described blood preparation or other organ-tissue cell specimen are from the high risk population of patients with gastric cancer, cancer of the stomach, healthy normal population.
Detection kit of the present invention is to adopt nucleic acid hybridization technique and groupization immunization method to combine, take miR-378 as detected object, synthesising probing needle is miR-378 hybridization complementary sequence, and the substrate of detection is the expression amount of blood of human body sample white corpuscle or histiocytic miR-378.The display packing of hybridization in situ technique can provide sxemiquantitative or the quantitative expression deciding degree of miR-378.The expression amount of judging above mRNA according to immunohistochemical methods colour developing after hybridization, normal people miR-378 zero expresses, and does not develop the color; The low expression of high risk population, a small amount of cell dyeing; Patients with Gastric Cancer high expression level, a large amount of cell dyeings.
The component of diagnostic kit of the present invention is by hybridization probe, hybridization solution, developer, the compositions such as synergistic agent.The nucleic acid hybridization principle of this test kit is that molecular biology insider all knows, and concrete operation step is under sample disposal, prehybridization, hybridization, immunohistochemical staining, mirror, to carry out quantitative analysis, report the test, and wherein the concrete steps of hybridization comprise:
1). sample to be measured is put into reactive tank;
2). instrument discards liquid automatically, automatically adds Digestive system;
3). instrument discards liquid automatically, automatically rear fixing;
4). instrument discards liquid automatically, automatically prehybridization (42 ℃);
5). instrument discards liquid automatically, automatically cleans;
6). instrument discards liquid automatically, automatically hybridization (42 ℃);
7). instrument discards liquid automatically, automatically cleans;
8). instrument discards liquid automatically, and automatic and DIG antibody is cultivated (room temperature);
9). instrument discards liquid automatically, automatically cleans colour developing;
10). take out mounting microscopy.
The scheme of a preferred embodiment of the present invention is: with miR-378 synthetic digoxigenin labeled for nucleic acid probe (cDNA of digoxigenin labeled, RNA and oligonucleotide probe, not only probe has a biotin labeling advantage, also overcome biotin labeled probe and in crossover process, organized the shortcomings such as the dry sorrow of Endogenous Biotin in position), this hybridization probe and the leukocytic RNA nucleic acid to be measured of blood of human body are hybridized, by the method for immunohistochemical methods, develop the color again, existence and location at light Microscopic observation RNA, according to the cell count of dyeing, the expression amount of judgement object RNA.
The inventive method is current conventional nucleic acid hybridization in situ technology, the method is by the miR-378 expression amount in detection substrate cell, be used for determining the cancer of the stomach Pathologic RNA variable quantity in early stage, early warning cancer of the stomach whether occur and patients with gastric cancer treatment after the whether prediction of Preventive.Because miR-378 is zero expression in normal people, if expression amount increases, the risk getting a cancer of the stomach is described, illustrate that cancer of the stomach change occurs, or the postoperative Preventive of Patients with Gastric Cancer, thereby the diagnostic message of acquisition cancer of the stomach.
A test kit can many person-portions be used or person-portion use.
The present invention has following beneficial effect:
Clinical meaning of the present invention is to follow the tracks of more in early days the variation that detects miR-378 expression amount in cancer of the stomach pathology evolution process, and early warning cancer of the stomach occurs, development trend.Diagnostic kit of the present invention is with other detects cancer of the stomach mark clinically, and medical imaging inspection has and showing difference.The present invention can examination cancer of the stomach Pathologic the miR-378 unconventionality expression in early stage, before the recurrence of occupancy carninomatosis kitchen range is not found in medical imaging inspection, before cancer biochemical indicator does not produce extremely, also before not forming tumor focus, can accomplish early the information acquisition of miR-378 abnormal expression, give real early warning of clinical patients with gastric cancer, so just likely implement early screening, early prevention, the early treatment of cancer, likely from source, thoroughly effect a radical cure cancer of the stomach foul disease.
In addition, feature highly sensitive, high specificity that test kit provided by the invention has, meanwhile, detection method of the present invention is convenient and simple for operation, and Neng Municipal Hospitals are widely used and promoted.
The simple declaration of accompanying drawing
Fig. 1 is that illustration is implemented in miR-378 in situ hybridization of the present invention;
Fig. 2 is that in the embodiment of the present invention, Patients with Gastric Cancer miR-378 expresses picture;
Fig. 3 is that high risk population miR-378 expresses picture;
Fig. 4 is that in the embodiment of the present invention, normal people miR-378 expresses picture.
Embodiment:
Below in conjunction with embodiment, be described more specifically content of the present invention.Should be appreciated that, the following examples are non-limiting content of the present invention for explanation, and any pro forma change or accommodation will fall into protection scope of the present invention.
Embodiment 1
The in situ hybridization test kit of preparing the present embodiment according to ordinary method, this test kit comprises hybridization probe, marker, the specification sheets with miR-378 design, wherein:
The probe mark thing of the present embodiment is selected digoxin.
Test kit hybridization solution forms:
Reagent preparation working concentration
1). by 10 * damping fluid I with tri-distilled water by being diluted to 1 * damping fluid I at 1: 10;
2). by 20 * damping fluid II with tri-distilled water by being diluted to 2 * damping fluid II at 1: 10;
By being diluted to 0.2 * damping fluid II at 1: 100; By being diluted to 0.1 * damping fluid II at 1: 200;
3). by 10 * damping fluid III with tri-distilled water by being diluted to 1 * damping fluid III at 1: 10;
4) .10 * damping fluid IV with tri-distilled water by 1: 10 be diluted to * damping fluid IV (get 1#, 2#, each 10mL of 3#, add water to 100mL both can).
Embodiment 2
The implementation process of application nucleic acid hybridization in situ detection method to each group blood preparation miR-378 expression amount:
1). get two of samples to be measured;
2). in glass jar, add Digestive system (Digestive system 100 μ L add 1 * damping fluid I99.9ml, are working concentration) 50m1,37 ℃ of water-bath preheating 10min, put 16 slides into, process 12min, then use 1 * damping fluid I to wash 5min for 37 ℃;
3). the protection liquid with 0.2% (protection liquid 1ml adds 1 * damping fluid I, and 99ml is working concentration) is washed 10min, and tri-distilled water is washed 5mi n (above process is all carried out at glass jar), takes out slide, allows its seasoning;
4). slide is put into moisture preservation box, add prehybridization solution 25 μ L/ sheets (being added in the place of cell), covered, covers tightly moisture preservation box, is placed in 42 ℃ of constant water bath box more than 3h;
5). take out slide, discard cover glass, slide is put into glass jar, the ethanol with 70%, 90%, 95% is respectively washed 2min, takes out seasoning;
6). slide is put into moisture preservation box, and one adds just hybridization solution 25 μ L/ sheets, and another adds antisense hybridization solution 25 μ L/ sheets, and covered, covers tightly moisture preservation box, is placed on 16-24h in 42 ℃ of constant water bath box;
7). take out slide, discard cover glass, slide is put into glass jar:
In 42 ℃ of constant water bath box, with 2 * damping fluid II, wash twice, each 15min;
In 42 ℃ of constant water bath box, with 0.2 * damping fluid II, wash once each 15min;
In 42 ℃ of constant water bath box, with 0.1 * damping fluid II, wash twice, each 15min;
8). with 1 * damping fluid, III washes 30s, takes out slide, seasoning;
9). slide is put into moisture preservation box, add 0.5% confining liquid (1ml confining liquid adds 5ml1 * damping fluid III), 100 μ L/ sheets, cover tightly moisture preservation box, at room temperature act on 30min.(this step does not need to add cover glass);
10). take out slide, with 1 * damping fluid, III washes 30s, seasoning;
11). slide is put into moisture preservation box, add X-AP antibody and (get a pipe alkaline phosphatase enzyme antibody, add wherein 1.8ml1 * damping fluid III) 100 μ L/ sheets, cover tightly moisture preservation box and at room temperature act on 30min, time can not be long, otherwise can produce false positive (this step does not need to add cover glass);
12). take out slide, with 1 * damping fluid III, wash 3 times, each 15min;
13). with 1 * damping fluid, IV washes 2min, adds developer (developer A73.3 μ L, developer B157.5 μ L is added in 30mL1 * damping fluid IV, mixes), and room temperature lucifuge 16h is to more than 18h;
14). with tri-distilled water, wash 5min, seasoning, (add with glycerine 10% 1 * damping fluid I mix) mounting microscopy.
Nucleic acid hybridization in situ detection method of the present invention is by object miR-378 digoxigenin labeled, become RNA nucleic acid probe, the RNA nucleic acid to be measured of probe and human leukocytes is hybridized, by the method for immunohistochemical methods, develop the color again, therefore in existence and the location of light Microscopic observation RNA, according to the cell count of dyeing, the expression amount of judgement object RNA.
20 of Patients with Gastric Cancer, 20 of high risk population's (chronic atrophic gastritis and stomach have leptospiral infection), 20 of Normal groups.Take out all people's to be checked peripheral blood 3-5 milliliter (separated white corpuscle) and do in situ hybridization.Result represents, all patients with gastric cancer miR-378 expression amounts are very high, and cell dyeing is dark; High risk population expresses slightly and reduces, decimal dyeing; Normal group miR-378 zero expresses, and cell does not dye, and concrete outcome is shown in Fig. 2, Fig. 3, Fig. 4.

Claims (10)

1. a hybridization in situ detection kit, comprises hybridization probe and marker, it is characterized in that, described hybridization probe has respectively sequence total length 66bp shown in sequence table SEQ ID NO:NR_029870.
2. test kit as claimed in claim 1, is characterized in that, described marker is selected from radioactive substance, chemoluminescence or substance that show color, vitamin H, metal king crab, fluorescein, enzyme and nano material.
3. test kit as claimed in claim 1, is characterized in that, this test kit also comprises hybridization solution.
4. test kit as claimed in claim 1, is characterized in that, this test kit also comprises synergistic agent.
5. test kit as claimed in claim 1, is characterized in that, this test kit also comprises developer.
6. an AE1 gene hybridization in situ detection method, is characterized in that, the method comprises the following steps:
(1) at hybridization probe claimed in claim 1 and target sequence, can form under the condition of stablizing hybridization complex, RNA to be measured in substrate is contacted with hybridization probe, form hybridization complex; With
(2) detect described hybridization complex.
7. detection method as claimed in claim 6, is characterized in that, the condition that hybridization complex is stablized in described forming is: the temperature of nucleic acid hybridization is 42 ℃; The time of nucleic acid hybridization is 16-24 hour.
8. detection method as claimed in claim 6, is characterized in that, described substrate choosing is human peripheral blood white corpuscle sample.
9. detection method as claimed in claim 6, is characterized in that, described blood preparation (cancer, high risk population, normal people's sample).
10. detection method as claimed in claim 9, is characterized in that, described in comprise high risk population's cancer of the stomach clinically Pathologic process in early stage, and the recurrence of various cancer after treatment and shift Pathologic process.
CN201310159214.9A 2013-05-03 2013-05-03 In-situ hybridization detection kit of miR-378 in early stage of pathologic evolution of gastric cancer, detection method and application Pending CN104131064A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109957610A (en) * 2017-12-14 2019-07-02 安徽普元生物科技股份有限公司 MicroRNA 378 (MIR378) nucleic acid quantitative determination reagent kit (PCR- fluorescence probe method)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109957610A (en) * 2017-12-14 2019-07-02 安徽普元生物科技股份有限公司 MicroRNA 378 (MIR378) nucleic acid quantitative determination reagent kit (PCR- fluorescence probe method)

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