CN110129324A - The extraction of gastric cancer serum excretion body marker hsa_circ_001477 a kind of and detection method and application - Google Patents
The extraction of gastric cancer serum excretion body marker hsa_circ_001477 a kind of and detection method and application Download PDFInfo
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- CN110129324A CN110129324A CN201910396660.9A CN201910396660A CN110129324A CN 110129324 A CN110129324 A CN 110129324A CN 201910396660 A CN201910396660 A CN 201910396660A CN 110129324 A CN110129324 A CN 110129324A
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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
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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Abstract
The invention belongs to field of biotechnology and molecular diagnostic techniques field, the extraction and detection method and application of a kind of gastric cancer serum excretion body marker hsa_circ_001477 are disclosed.Serum excretion body circRNA extracting method provided by the invention and kit easily and accurately can extract and detect excretion body circRNA from blood sample, be conducive to the analysis and application in laboratory and clinical serum excretion body circRNA；The has_circ_001477 detection method and kit of offer can quickly detect a variety of circRNA including has_circ_001477；Cancer diagnosis reagent box quickly detects has_circ_001477；It is experimentally confirmed, serum excretion body hsa_circ_001477 can become the novel Noninvasive marker of diagnosing gastric cancer.The present invention is early diagnosis provider just feasible marker and the detection method of gastric cancer, and also being applied to tumor markers to analyze more serum excretion body circRNA provides help.
Description
Technical field
The invention belongs to field of biotechnology and molecular diagnostic techniques field more particularly to a kind of gastric cancer serum excretion body marks
The extraction of will object hsa_circ_001477 and detection method and application.
Background technique
Currently, the prior art commonly used in the trade is such that
Gastric cancer is incidence and the higher malignant tumour of the death rate.Making a definite diagnosis for gastric cancer relies on tissue biopsy at present, has bright
Most patient has been middle and advanced stage when showing traumatic, and making a definite diagnosis, and prognosis is poor.Using operation excision and chemicotherapy as primary hand in treatment
Section, chemicotherapy has very macrolesion to human normal function, although chemotherapy is sometimes significant in efficacy, but timeliness is short, has drug
Side effect and drug resistance.Therefore, it is badly in need of establishing a kind of convenience, minimally invasive, sensitive and special diagnosis is especially early diagnosed and supervised
The means of survey.
Excretion body is that one kind is naturally present in the human body fluids such as blood, and diameter can be by most cells for 30-100nm's
The small film bubble of the double films of the lipid of secretion.Protein containing cell-specific, lipid and nucleic acid in vesica can exist as signaling molecule
It is played an important role in physiology and pathologic process.Analysis shows that RNA is the important side of intercellular signal transmitting in excretion body
Formula, can be used as the Molecular biomarkers and therapy target of the diseases such as tumour, and lipid two film construction can protect signal in vesica
Molecule caused by external environment and enzyme etc. from degrading, and energy specific enrichment disease associated molecule.Further, since blood and institute
Organized organ contact, can carry the largely real-time effective informations in relation to body, and sample mode is simple, traumatic small, cost
Cheap, sample is convenient for collecting and storing, and therefore, explore blood excretion body RNA is just becoming as the disease molecules such as tumour marker
The work hot spot of scientific research personnel.
CircRNA is a kind of novel non-coding RNA with virus covalently closed circular structure.Since its type and quantity is rich
Rich, stability and conservative it is high, in various histocytes and human body fluid it is widely distributed, there is disease specific and tissue
With developing stage specificity, the biological modulated of a variety of physiology and pathologic process such as proliferation, apoptosis, migration and invasion of tumour is participated in
Control, circRNA, which is expected to become, has promising New Type of Diseases molecular marker.Have analysis shows that circRNA stablizes in excretion body
And it is abundant, it is the ideal marker of diagnosing tumor.But point for circRNA in excretion body as diagnosing tumor marker at present
Analysis only has a small number of reports, carries out expression pattern analysis to circRNA using high throughput sequencing technologies mostly, diagnoses for early gastric caacer
Serum excretion body molecular marker there is not been reported.In addition, the extraction and detection method to serum excretion body circRNA rarely have
Report extracts in detection cell, tissue or blood merely with trizol method joint RT-qPCR and Northern Blot
circRNA。
In conclusion problem of the existing technology is:
(1) only have a small number of reports as the analysis of diagnosing tumor marker for circRNA in excretion body at present, mostly benefit
Expression pattern analysis is carried out to tissue circRNA with high throughput sequencing technologies, can be used for the serum excretion body of early gastric caacer diagnosis
CircRNA molecule is unclear.
(2) extraction and detection method of serum excretion body circRNA are rarely reported, combine RT- merely with trizol method
CircRNA in qPCR and Northern Blot extraction detection cell, tissue or blood, this method are applied to outside micro serum
It secretes body circRNA extraction and is difficult to ensure its concentration, purity and ease-to-operate.
Solve the difficulty of above-mentioned technical problem: currently, being badly in need of developing a kind of extraction of simplicity and detecting serum excretion body
CircRNA method not only can guarantee that the concentration of extraction and purity were preferable, but also can guarantee the accuracy of detection.Above-mentioned technology is solved to ask
The meaning of topic: can diagnose for early gastric caacer and provide new potential excretion body circRNA marker and clinical labororatory's detection method,
Also help can be provided to excavate and analyzing more serum excretion body circRNA applied to tumor markers.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of gastric cancer serum excretion body marker hsa_circ_
001477 extraction and detection method and application.
The invention is realized in this way a kind of gastric cancer serum excretion body marker, outside a kind of serum for diagnosing gastric cancer
Body marker hsa_circ_001477 is secreted, sequence is as shown in SEQ ID NO:1.
Another object of the present invention is to provide the extractions of gastric cancer serum excretion body marker hsa_circ_001477 a kind of
Method, it is a variety of including has_circ_001477 for extracting using the extracts kit of serum excretion body circRNA
Serum excretion body circRNA.
Further, serum excretion body circRNA extracts kit includes:
(1) reagent needed for extracting serum excretion body: ExoQuickTMExosome Precipitation Solution；
(2) reagent needed for extracting excretion body circRNA: chloroform, dehydrated alcohol, miRNeasy Serum/Plasma Kit.
Further, the extracts kit of serum excretion body circRNA, can extract includes hsa_circ_ in serum excretion body
A variety of circRNA including 001477.
Another object of the present invention is to provide the detections of gastric cancer serum excretion body marker hsa_circ_001477 a kind of
Method utilizes cancer diagnosis reagent box, the method for the detection and identification for serum excretion body hsa_circ_001477.
Further, cancer diagnosis reagent box includes:
(1) reagent needed for reverse transcription: HiScript 1st Strand cDNA Synthesis Kit；
(2) reagent needed for quantitative fluorescent PCR: AceQ qPCR SYBR GREEN Master Mix, hsa_circ_
001477 upstream and downstream primer, β-actin upstream and downstream primer, sequence such as SEQ ID NO:2 and 3 and SEQ ID NO:4.
Further, cancer diagnosis reagent box can measure serum excretion body hsa_circ_001477 content.
Further, cancer diagnosis reagent box, the PCR primer containing detection hsa_circ_001477 content, primer sequence is such as
Shown in SEQ ID NO:2 and 3.
Further, cancer diagnosis reagent box also can detect other circRNA using other specificity circRNA primers.
Another object of the present invention is to provide detection circRNA marker reagents of expression quantity in serum excretion body to exist
Prepare the application in diagnosing gastric cancer preparation.
In conclusion advantages of the present invention and good effect are as follows:
Present invention firstly discovers that in serum excretion body there are has_circ_001477 and its for diagnosing gastric cancer valence
Value.The serum excretion body circRNA extracting method and kit of offer, can from blood sample easily and accurately extract and
Excretion body circRNA is detected, the analysis and application in laboratory and clinical serum excretion body circRNA are conducive to.The has_ of offer
Circ_001477 detection method and kit can be used for quickly detecting a variety of circRNA including has_circ_001477.
The present invention is early diagnosis provider just feasible marker and the detection method of gastric cancer, is also the more serum excretion bodies of analysis
CircRNA is applied to tumor markers and provides help.
First discovery of the present invention is used for the new excretion body circRNA of diagnosing gastric cancer, and proposition excretion body circRNA first is extracted
And detection method.
Detailed description of the invention
Fig. 1 is that Nanodrop instrument provided in an embodiment of the present invention detects the concentration of separated serum excretion body RNA and pure
Spend schematic diagram.
Fig. 2 is the expansion of real-time fluorescence quantitative PCR detection hsa_circ_1477 and β-actin provided in an embodiment of the present invention
Increase curve and melting curve figure.
Fig. 3 is PCR primer agarose electrophoresis provided in an embodiment of the present invention and sequencing identification schematic diagram.
Fig. 4 is that real-time fluorescence quantitative PCR provided in an embodiment of the present invention compares patients with gastric cancer and healthy person serum excretion body
Hsa_circ_001477 differential expression schematic diagram.
Fig. 5 is that serum excretion body hsa_circ_001477 expression provided in an embodiment of the present invention faces with patients with gastric cancer
Relation schematic diagram between bed pathological factor.
Fig. 6 is ROC curve analysis serum excretion body hsa_circ_001477 provided in an embodiment of the present invention to diagnosing gastric cancer
Specificity and sensitivity schematic diagram.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
In order to solve existing technical problem, the present invention provides one kind to extract and detect from serum excretion body
The method and use this method of circRNA extracts application of the detection circRNA in diagnosing gastric cancer.
Gastric cancer serum excretion body marker provided in an embodiment of the present invention, a kind of serum excretion body mark for diagnosing gastric cancer
Will object hsa_circ_001477, sequence is as shown in SEQ ID NO:1.
The extracting method of gastric cancer serum excretion body marker hsa_circ_001477 provided in an embodiment of the present invention utilizes
The extracts kit of serum excretion body circRNA, for extracting a variety of serum excretion bodies including has_circ_001477
circRNA。
Serum excretion body circRNA extracts kit provided in an embodiment of the present invention includes:
(1) reagent needed for extracting serum excretion body: ExoQuickTMExosome Precipitation Solution, purchase
From SBI company, article No. EXOQ5A；
(2) reagent needed for extracting excretion body circRNA: chloroform, dehydrated alcohol, miRNeasy Serum/Plasma Kit
Purchased from QIAGNE and article No. 217184.
The extracts kit of serum excretion body circRNA provided in an embodiment of the present invention, can extract and wrap in serum excretion body
Include a variety of circRNA including hsa_circ_001477.
The detection method of gastric cancer serum excretion body marker hsa_circ_001477 provided in an embodiment of the present invention utilizes
Cancer diagnosis reagent box, the method for the detection and identification for serum excretion body hsa_circ_001477.
Cancer diagnosis reagent box provided in an embodiment of the present invention includes:
(1) reagent needed for reverse transcription: HiScript 1st Strand cDNA Synthesis Kit, it is public purchased from Vazyme
Department, article No. R111-02；
(2) reagent needed for quantitative fluorescent PCR: AceQ qPCR SYBR GREEN Master Mix is purchased from Vazyme company
And article No. Q111-02；
Hsa_circ_001477 upstream and downstream primer,β- actin upstream and downstream primer is synthesized, sequence by Invitrogen company
Such as SEQ ID NO:2 and 3 and SEQ ID NO:4.
Cancer diagnosis reagent box provided in an embodiment of the present invention can measure serum excretion body hsa_circ_001477 content.
Cancer diagnosis reagent box provided in an embodiment of the present invention, the PCR containing detection hsa_circ_001477 content draw
Object, primer sequence is as shown in SEQ ID NO:2 and 3.
Cancer diagnosis reagent box provided in an embodiment of the present invention also can detect it using other specificity circRNA primers
He is circRNA.
Detection circRNA marker reagent of expression quantity in serum excretion body provided in an embodiment of the present invention is preparing stomach
Application in cancer diagnostic preparation.
Application principle of the invention is further described in specific embodiment below；
If not specializing.Chemical reagent used in embodiment is conventional commercial reagent, technology used in example
The conventional means that means are well known to those skilled in the art.
Embodiment 1；The extraction of serum excretion body circRNA
(1) acquisition and preparation of serum
1) with blood plasma separation gel anticoagulant tube acquire ulnar vein blood 5ml, after blood sampling immediately gentle inversion 5 times to mix well；
2) after being stored at room temperature 30min, serum and blood clot quilt are made with horizontal centrifuge room temperature 3000r/min centrifugation 15min
Separation gel separates completely；
3) upper serum is shifted with micropipettor and dispenses into 1.5ml import EP pipe (300 μ l/ pipe)；- 80 DEG C of guarantors
Spare or 4 DEG C of placements are deposited to use immediately.
(2) serum excretion body precipitating and extraction
1) serum sample is thawed from -80 DEG C of 4 DEG C of taking-up juxtapositions；With 4 DEG C, it is broken that 3000g is centrifuged 15min removal residual cells
Piece etc.；
2) 63 μ l ExoQuick are added in micropipettor transfer 250 μ l of supernatant to new import EP pipeTM
Exosome Precipitation Solution, soft piping and druming mix；4 DEG C of standing 30min are set sufficiently to precipitate excretion body；
3) with 4 DEG C, sediment is centrifuged to tube bottom by 1500g centrifugal mixture 30min；Supernatant is removed, then with 4 DEG C,
1500g is centrifuged 5min and removes residual liquid, avoids touching tube bottom precipitating；
4) tube bottom excretion body precipitating is resuspended with 50-250ul sterilizing 1xPBS, 4 DEG C of placement 10min to dissolve excretion body, and to
It further uses.
(3) excretion body circRNA is extracted
1) 600 μ l QIAzol lysates are added in above-mentioned excretion body, be sufficiently mixed by inversion, and be stored at room temperature 5min；Add
Enter the chloroform equal with excretion body volume, be acutely vortexed concussion 15s, and places and be stored at room temperature balance 2-3min；
2) 4 DEG C of centrifuge are set, 12000g is centrifuged 15min；Supernatant is drawn into newly imported EP pipe with micropipettor, is added
1.5 times of volume dehydrated alcohols, are mixed by inversion；
3) 700 μ l mixed liquors are first pipetted in the splitter for being cased with collecting pipe outside, 4 DEG C, 12000g, 15s centrifugation are abandoned and collected
Waste liquid in pipe, then splitter is added in residual mixed liquor, repeat aforesaid operations；
4) 700 μ l RWT solution are added into splitter, 4 DEG C, waste liquid in collecting pipe is abandoned in 12000g, 15s centrifugation；
5) 700 μ l RPE solution are added into splitter again, 4 DEG C, waste liquid in collecting pipe is abandoned in 12000g, 15s centrifugation；
6) 80% ethyl alcohol that 500 μ l are prepared with no RNase enzyme water is added into splitter, 4 DEG C, 12000g, 2min are centrifuged,
Abandon collecting pipe；
7) above-mentioned splitter is put into mating new collecting pipe, 4 DEG C, 12000g, 5min, abandons collecting pipe；Splitter is put
Enter in mating newly imported EP pipe, 14 μ l are added without RNase enzyme water among micropipettor alignment splitter, stand 3-5min,
To be sufficiently humidified so as to and dissolve RNA；
8) with 4 DEG C of centrifuge, the circRNA for extracting and isolating is collected by centrifugation in 12000g, 5min；- 80 DEG C save or set 4
DEG C to use immediately.
(4) RNA concentration and purity detecting
Nanodrop instrument detects the concentration and purity of separated serum excretion body RNA, as a result as shown in Figure 1.
(contain as shown in Figure 1, Nanodrop instrument provided in an embodiment of the present invention detects separated serum excretion body RNA
CircRNA concentration and purity schematic diagram).
Embodiment 2；The detection and identification of serum excretion body has_circ_001477
(1) preparation of cDNA:
Reaction product can be placed in 4 DEG C and be immediately available for PCR reaction, or places in -20 DEG C of half a year and use, or -80 DEG C of preservations are kept away
Exempt from multigelation.
CDNA can specifically take the circumstances into consideration to dilute 2-5 times with RNase free ddH2O, or be directly used in PCR reaction system.
(2) real-time fluorescence quantitative PCR:
In Bio-Rad CFX96TMReal-time fluorescence quantitative PCR reaction is carried out in instrument.
PCR product identification: the amplification curve and melting curve of qPCR as the result is shown, as shown in Figure 2；Agarose gel electrophoresis
Analysis, as shown in Figure 3；The building of T-A cloned plasmids and sequencing, as shown in Figure 3.
As shown in Fig. 2, real-time fluorescence quantitative PCR provided in an embodiment of the present invention detects hsa_circ_1477 and β-actin
Amplification curve and melting curve figure.
As shown in figure 3, PCR primer agarose electrophoresis provided in an embodiment of the present invention and sequencing identification schematic diagram.
Embodiment 3；Gastric cancer serum excretion body has_circ_001477 is worth as diagnosis marker
(1) present invention receives what 35 December in January, 2017-went to a doctor in the attached First People's Hospital's gastrointestinal surgery of Jiangsu University
Patients with Gastric Cancer collects its age, gender, tumor size, position, differentiation degree, lymphatic metastasis, far-end transfer, blood vessel and mind
Through basic documents such as involvement, TNM stage, histological classification, CA199, acquires its preoperative ulnar vein blood and detect serum excretion body
Has_circ_001477 is horizontal.Same period physical examination of healthy population 35 is included in simultaneously by age to be matched with gender.Two groups of samples are same
Phase collects, sample, dispense, preservation condition it is consistent, carry out serum excretion body has_circ_001477 content detection and point
Analysis.
(2) data are analyzed: this experimental data uses the analysis method of relative quantification, withβ- actin is reference gene (primer
Sequence is as shown in SEQ ID NO:4 and 5), with △ CT=CThsa_circ_001477-CT β -actinIt indicates each sample testing number value, compares
Difference organizes other difference, and is analyzed with GraphPad Prism and SPSS16.0.
(3) result:
As shown in figure 4, patients with gastric cancer provided in an embodiment of the present invention and healthy person serum excretion body has_circ_001477
Expression compares.
As shown in figure 5, serum excretion body has_circ_001477 expression provided in an embodiment of the present invention and gastric cancer are suffered from
Relationship between person's clinical pathological factors.
As shown in fig. 6, ROC curve provided in an embodiment of the present invention analyzes serum excretion body hsa_circ_001477 (ex-
Circ1477) to the specificity of diagnosing gastric cancer and sensitivity.
Serum excretion body hsa_circ_001477 (ex-circ1477) provided by the invention is used as diagnosing gastric cancer marker
And its application.The present invention provides the extracts kits of serum excretion body circRNA a kind of, can facilitate from blood sample,
Accurately extract excretion body circRNA.The present invention also provides a kind of cancer diagnosis reagent box, are drawn using specific circRNA
Object can be used for quickly detecting has_circ_001477.It is experimentally confirmed, serum excretion body hsa_circ_001477 can be with
Novel Noninvasive marker as diagnosing gastric cancer.The present invention be gastric cancer early diagnosis provider just feasible marker and
Detection method also provides help to study more serum excretion body circRNA applied to tumor markers.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
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<120>extraction of gastric cancer serum excretion body marker hsa_circ_001477 a kind of and detection method and application
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Claims (10)
1. a kind of gastric cancer serum excretion body marker, which is characterized in that the gastric cancer serum excretion body marker is a kind of use
It is hsa_circ_001477 in the serum excretion body circRNA marker of diagnosing gastric cancer, sequence is as shown in SEQ ID NO:1.
2. a kind of extracting method of gastric cancer serum excretion body marker as described in claim 1, which is characterized in that the gastric cancer
The extracting method of serum excretion body marker utilizes the extracts kit of serum excretion body circRNA, and extracting includes has_circ_
A variety of serum excretion body circRNA including 001477.
3. the extracting method of gastric cancer serum excretion body marker as claimed in claim 2, which is characterized in that the serum excretion
Body circRNA extracts kit includes:
(1) reagent needed for extracting serum excretion body: ExoQuickTMExosome Precipitation Solution；
(2) reagent needed for extracting excretion body circRNA: chloroform, dehydrated alcohol, miRNeasy Serum/Plasma Kit.
4. the extracting method of gastric cancer serum excretion body marker as claimed in claim 2, which is characterized in that the serum excretion
The extracts kit of body circRNA extracts a variety of circRNA in serum excretion body including hsa_circ_001477.
5. a kind of detection method of gastric cancer serum excretion body marker as described in claim 1, which is characterized in that the gastric cancer blood
The detection method of clear excretion body marker, the inspection using cancer diagnosis reagent box, for serum excretion body hsa_circ_001477
The method surveyed and identified.
6. the detection method of gastric cancer serum excretion body marker as claimed in claim 5, which is characterized in that the diagnosing gastric cancer
Kit includes:
(1) reagent needed for reverse transcription: HiScript 1st Strand cDNA Synthesis Kit；
(2) reagent needed for quantitative fluorescent PCR: on AceQ qPCR SYBR GREEN Master Mix, hsa_circ_001477
Downstream primer, β-actin upstream and downstream primer, sequence such as SEQ ID NO:2 and SEQ ID NO:3 and SEQ ID NO:4.
7. the detection method of gastric cancer serum excretion body marker as claimed in claim 5, which is characterized in that the diagnosing gastric cancer
Kit can measure serum excretion body hsa_circ_001477 content.
8. the detection method of the gastric cancer serum excretion body marker as described in right wants 5, which is characterized in that the diagnosing gastric cancer examination
Agent box, the PCR primer containing detection hsa_circ_001477 content, primer sequence such as SEQ ID NO:2 and SEQ ID NO:3
It is shown.
9. a kind of application of gastric cancer serum excretion body marker as described in claim 1 in diagnosing gastric cancer, which is characterized in that
Application in gastric cancer uses other specificity circRNA primers, also can detect other circRNA.
10. a kind of diagnosing gastric cancer preparation comprising gastric cancer serum excretion body marker described in claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910396660.9A CN110129324A (en) | 2019-05-14 | 2019-05-14 | The extraction of gastric cancer serum excretion body marker hsa_circ_001477 a kind of and detection method and application |
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|---|---|---|---|
| CN201910396660.9A CN110129324A (en) | 2019-05-14 | 2019-05-14 | The extraction of gastric cancer serum excretion body marker hsa_circ_001477 a kind of and detection method and application |
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| Publication Number | Publication Date |
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| CN110129324A true CN110129324A (en) | 2019-08-16 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111540469A (en) * | 2020-05-29 | 2020-08-14 | 杭州广科安德生物科技有限公司 | Method for constructing mathematical model for in-vitro detection of gastric cancer and application thereof |
| CN111549142A (en) * | 2020-06-19 | 2020-08-18 | 江苏华美健晟生物科技有限公司 | Primer and kit for detecting serum exosome circ-KCNQ1 and application of primer and kit in gastric cancer liver metastasis |
| CN111549141A (en) * | 2020-06-19 | 2020-08-18 | 江苏华美健晟生物科技有限公司 | Primer and kit for detecting peripheral blood exosome circ-PPCDC (circulating-positive-negative-positive |
| CN111808956A (en) * | 2020-06-19 | 2020-10-23 | 镇江市第一人民医院 | Primer and kit of serum exosome RP11-470P21-2 and application of primer and kit in diagnosis and treatment of gastric cancer liver metastasis |
| CN113999909A (en) * | 2021-11-17 | 2022-02-01 | 江苏大学 | Serum exosome marker for gastric cancer diagnosis, application thereof, amplification primer pair and diagnosis kit |
| CN114606321A (en) * | 2022-04-15 | 2022-06-10 | 张家港澳洋医院有限公司 | Gastric cancer plasma exosome circRNA marker and kit and application thereof |
| CN115747333A (en) * | 2022-11-24 | 2023-03-07 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Tumor marker detection kit, detection and analysis system and application thereof |
-
2019
- 2019-05-14 CN CN201910396660.9A patent/CN110129324A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| RONG LI 等: "Exosomal sorting of circRNA promotes cancer progression and serves as a novel biomarker for gastric cancer", 《JOURNAL OF EXTRACELLULAR VESICLES》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111540469A (en) * | 2020-05-29 | 2020-08-14 | 杭州广科安德生物科技有限公司 | Method for constructing mathematical model for in-vitro detection of gastric cancer and application thereof |
| CN111549142A (en) * | 2020-06-19 | 2020-08-18 | 江苏华美健晟生物科技有限公司 | Primer and kit for detecting serum exosome circ-KCNQ1 and application of primer and kit in gastric cancer liver metastasis |
| CN111549141A (en) * | 2020-06-19 | 2020-08-18 | 江苏华美健晟生物科技有限公司 | Primer and kit for detecting peripheral blood exosome circ-PPCDC (circulating-positive-negative-positive |
| CN111808956A (en) * | 2020-06-19 | 2020-10-23 | 镇江市第一人民医院 | Primer and kit of serum exosome RP11-470P21-2 and application of primer and kit in diagnosis and treatment of gastric cancer liver metastasis |
| CN111549142B (en) * | 2020-06-19 | 2022-12-20 | 江苏华美健晟生物科技有限公司 | Primer and kit for detecting serum exosome circ-KCNQ1 and application of primer and kit in gastric cancer liver metastasis |
| CN113999909A (en) * | 2021-11-17 | 2022-02-01 | 江苏大学 | Serum exosome marker for gastric cancer diagnosis, application thereof, amplification primer pair and diagnosis kit |
| CN113999909B (en) * | 2021-11-17 | 2024-03-29 | 江苏大学 | Serum exosome marker for gastric cancer diagnosis and application thereof, amplification primer pair and diagnosis kit |
| CN114606321A (en) * | 2022-04-15 | 2022-06-10 | 张家港澳洋医院有限公司 | Gastric cancer plasma exosome circRNA marker and kit and application thereof |
| CN115747333A (en) * | 2022-11-24 | 2023-03-07 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Tumor marker detection kit, detection and analysis system and application thereof |
| CN115747333B (en) * | 2022-11-24 | 2024-02-20 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Tumor marker detection kit, detection analysis system and application thereof |
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