CN109251964B - Circulating microRNAs detection kit, method for specifically detecting circulating microRNAs and application - Google Patents
Circulating microRNAs detection kit, method for specifically detecting circulating microRNAs and application Download PDFInfo
- Publication number
- CN109251964B CN109251964B CN201811360587.1A CN201811360587A CN109251964B CN 109251964 B CN109251964 B CN 109251964B CN 201811360587 A CN201811360587 A CN 201811360587A CN 109251964 B CN109251964 B CN 109251964B
- Authority
- CN
- China
- Prior art keywords
- dna
- hairpin
- hairpin structure
- circulating micrornas
- detection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/6844—Nucleic acid amplification reactions
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biology, and relates to a circulating microRNAs detection kit, a method for specifically detecting circulating microRNAs and application of the circulating microRNAs detection kit. The kit comprises: hairpin structure DNA with a cohesive end, a part of which is complementarily paired with at least a part of the target microRNA; double-stranded specific nuclease and reaction buffer; a circular DNA probe, a portion of which is complementary paired with a portion of hairpin DNA; optionally one of the following components: a SYBR Gold fluorescent nucleic acid dye; a DNA polymerase; DNA polymerase reaction buffer; 4 × dNTPs. The invention adopts double-chain specificity nuclease mediated target circulation reaction and rolling circle amplification reaction to carry out specificity detection on the circulating miRNAs, can reduce the detection background and play a role in bidirectional signal amplification, and has the characteristics of simple operation, rapid detection, high sensitivity, good specificity, low cost and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a circulating microRNAs detection kit, a method for specifically detecting circulating microRNAs, and application of the circulating microRNAs detection kit and the method in circulating microRNAs detection.
Background
Circulating microRNAs (miRNAs) are miRNAs with a length of about 19-23nt, endogenous, non-coding, and in an extracellular free state in body fluids such as serum, plasma or urine, which regulate the expression of genes at the translation level by complementarily binding to the 3 'noncoding region (3' UTR) of their target mRNA molecules, and thus, miRNAs are widely involved in various physiological processes of organisms, such as ontogeny, cell proliferation, differentiation and apoptosis, regulation of metabolism and stress response, and even disease occurrence, including tumor formation. miRNAs also participate in important biological program regulation of tumor cells, indirectly play a role of cancer promotion genes and cancer suppressor genes and play a crucial role in the occurrence and development of tumors, so the miRNAs are regarded as important potential biomarkers and targets for early diagnosis, prognosis and treatment of tumors.
Circulating miRNAs have the characteristics of short sequence, high homology, low content and extremely easy degradation, so that an efficient and specific miRNAs detection method is urgently needed in the aspects of biomedical research and early clinical diagnosis.
The traditional detection methods of miRNAs mainly comprise Northern blot, microarray and real-time fluorescence quantitative PCR (real-time PCR), and the detection methods not only need to use large and expensive instruments and equipment and have complex operation, but also have low sensitivity and poor specificity, so the detection methods are difficult to popularize.
Therefore, there is a need to develop a new method for detecting circulating microRNAs.
Disclosure of Invention
The invention aims to provide a circulating microRNAs detection kit, a method for specifically detecting circulating microRNAs and application. Under the condition based on SYBR Gold fluorescent nucleic acid dye, the target circulation reaction mediated by double-chain specific nuclease and the rolling circle amplification reaction are adopted to carry out specificity detection on the circulating miRNAs, so that the detection background can be reduced, the bidirectional signal amplification effect can be realized, and the method has the characteristics of simplicity in operation, rapidness in detection, high sensitivity, good specificity, low cost and the like.
In order to achieve the above object, a first aspect of the present invention provides a circulating microRNAs detection kit, comprising the following components:
(1) hairpin DNA having a sticky end, a portion of the hairpin DNA being complementary paired to at least a portion of the microRNA of interest; the complementary pairing enables hairpin structure DNA to be unfolded and form miRNA-DNA hybrid with target microRNA;
(2) double-chain specific nuclease and double-chain specific nuclease reaction buffer solution;
(3) a circular DNA probe, a portion of which is complementary paired to a portion of hairpin DNA;
(4) optionally one of the following components:
(4-1) SYBR Gold fluorescent nucleic acid dye;
(4-2) a DNA polymerase;
(4-3) a DNA polymerase reaction buffer;
(4-4)4×dNTPs。
the components in the kit of the present invention may be provided individually, or part of the components may be provided in the form of a mixture, as required.
The principle of the invention is shown in figure 1, and the target microRNA is taken as miR-21 as an example. The detection system comprises two steps of reaction, when the object miR-21 to be detected exists, the target miR-21 is firstly combined with the hairpin structure DNA and unfolded to form a miRNA-DNA hybrid which can be recognized and sheared by the double-strand specific nuclease, and after the double-strand specific nuclease shears the DNA part in the hybrid, the target miR-21 is released to enter the cycle of the first step of reaction. The remaining cohesive end of the hairpin DNA is complementary to the circular DNA, so that rolling circle amplification is performed by the action of DNA polymerase and dNTP, and the product is linear single-stranded DNA having a large number of repeated sequences (completely complementary to the circular DNA). And finally adding SYBR Gold fluorescent nucleic acid dye into the amplification system, and judging the content of the target miRNA through the strength-to-weakness comparison of fluorescent signals. When the object miR-21 to be detected does not exist, the hairpin structure DNA cannot be opened, so that the subsequent two-step reaction cannot be carried out, and the detection signal is extremely weak.
A double-strand specific nuclease (DSN) is a commercial heat-resistant nuclease which can selectively degrade DNA in double-stranded DNA (10-12bp) and DNA-RNA hybrid (15bp), but has no degradation effect on single-stranded DNA, single-stranded RNA and double-stranded RNA. Moreover, the enzyme can distinguish perfectly matched and incompletely matched DNA hybrids, and has high specificity.
Rolling Circle Amplification (RCA) is a detection method for signal amplification developed by taking advantage of the rolling circle amplification mechanism of circular DNA molecules in nature. RCA is a constant temperature enzymatic reaction, using single-stranded circular DNA/RNA as a template at a constant temperature, and under the action of specific DNA/RNA polymerase, a short single-stranded DNA/RNA primer is amplified to grow single-stranded DNA/RNA, and the product of RCA contains hundreds of thousands of tandem repeat sequences complementary with the circular template, and can be used for detecting trace biomarkers.
SYBR Gold Dye is a low-toxicity and stable commercialized fluorescent nucleic acid Dye, the self fluorescence is weak, but after the Dye is combined with nucleic acid (single-stranded DNA, single-stranded RNA, double-stranded DNA and double-stranded RNA), the fluorescent signal can be obviously enhanced (1000 times), so that the detection background can be obviously reduced.
According to the present invention, hairpin DNA of various structures based on the above principle can achieve this effect. Preferably, a part of the hairpin DNA near the long cohesive end is complementarily paired with at least a part of the microRNA of interest; more preferably, a part of the hairpin DNA starting from the long sticky end is perfectly complementary paired with the microRNA of interest; further preferably, the number of bases of a part of the long cohesive end of the hairpin structure DNA which are complementarily paired with at least a part of the target microRNA is 15-24.
Correspondingly, a part of the circular DNA probe is complementarily paired with a part of the short cohesive end of hairpin structure DNA.
The concept of "long cohesive end" and "short cohesive end" as described in the present invention can be understood by those skilled in the art to refer to a protruding end and an un-protruding end of the cohesive ends, respectively.
According to a specific embodiment of the invention, the target microRNA is miR-21, and has a nucleotide sequence shown in SEQ ID NO: 1; the hairpin structure DNA has the nucleotide sequence shown in SEQ ID NO: 2; the circular DNA probe has a nucleotide sequence shown as SEQ ID NO: 3, and (b) is shown in the specification.
According to the invention, the hairpin DNA is preferably provided in the form of a hairpin DNA buffer at a concentration of 1nM to 100 nM; such as Tris-HCl buffer (100mM NaCl,5mM MgCl)2，pH7.4)。
Preferably, the circular DNA probe is provided in the form of a rolling circle amplification reaction solution of the circular DNA probe at a concentration of 1nM to 100 nM. The rolling circle amplification reaction solution may be any of various conventional reaction solutions suitable for rolling circle amplification reactions.
In a second aspect of the invention, a method for specific detection of circulating microRNAs is provided, comprising the steps of:
1) mixing the substance to be detected with the hairpin structure DNA, adding double-strand specific nuclease, and carrying out enzymolysis reaction;
2) adding a circular DNA probe into an enzymolysis reaction product to carry out rolling circle amplification;
3) detecting the rolling circle amplification product;
wherein the content of the first and second substances,
the hairpin structure DNA has a cohesive end, and a part of the hairpin structure DNA is complementary and matched with at least one part of the target microRNA; the complementary pairing enables hairpin structure DNA to be unfolded and form miRNA-DNA hybrid with target microRNA;
a portion of the circular DNA probe is complementary paired with a portion of hairpin DNA.
According to the present invention, preferably, the rolling circle amplification product is detected using a SYBR Gold fluorescent nucleic acid dye.
According to the present invention, hairpin DNA of various structures based on the above principle can achieve this effect. Preferably, a part of the hairpin DNA near the long cohesive end is complementarily paired with at least a part of the microRNA of interest; more preferably, a part of the hairpin DNA starting from the long sticky end is perfectly complementary paired with the microRNA of interest; further preferably, the number of bases of a part of the long cohesive end of the hairpin structure DNA which are complementarily paired with at least a part of the target microRNA is 15-24.
Correspondingly, a part of the circular DNA probe is complementarily paired with a part of the short cohesive end of hairpin structure DNA.
In the present invention, the enzymatic reaction in step 1) can be performed under conventional conditions of double-strand specific nuclease reaction, such as incubation at 50-60 ℃ for 15-60 minutes.
In the present invention, in step 2), the rolling circle amplification conditions may be reaction conditions conventional in the art, for example, incubation at 20-40 ℃ for 60-120 minutes.
In a third aspect, the invention provides the use of the above-described kit for the detection of circulating microRNAs and/or the above-described method for the detection of circulating microRNAs.
The miRNAs are detected by using the kit and the method of the invention, no chemical modification, no complex reaction process and no expensive instrument are needed, the miRNAs can be recycled in the detection system, the operation is simple and convenient, the detection time is short, the cost is low, the sensitivity is high, the specificity is good, and the clinical application prospect is good.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 is a schematic diagram of the detection principle of the present invention.
FIG. 2 shows the results of 20% native polyacrylamide gel electrophoresis of the first step reaction product.
FIG. 3 shows the results of 0.4% agarose gel electrophoresis of the reaction products of the entire detection system.
FIGS. 4A and 4B show fluorescence spectra (A) and fluorescence intensities (B) in the presence of different concentrations of the target miR-21. In FIG. 4A, the spectral lines from top to bottom represent the control group, and the concentrations of miR-21 are 1fM, 10fM, 100fM, 1pM, 10pM, 100pM, and 1nM in this order.
FIG. 5 shows the results of the assay specific for the detection system of the present invention.
FIGS. 6A and 6B show the results of the detection system of the present invention for detecting miR-21 in breast cancer cells MDA-MB-231 and T47D, wherein the lines represent concentrations of 0, 1 ng/. mu.L, 2 ng/. mu.L, 5 ng/. mu.L and 10 ng/. mu.L from top to bottom.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
The invention is further illustrated by the following examples. The conditions not specified in the examples were carried out according to the conditions of the conventional molecular biology procedures or the conditions recommended by the manufacturer, and various conventional reagents were commercially available.
Kit composition
4a) Detection solution 1: 20mM Tris-HCl buffer (100mM NaCl,5mM MgCl)2pH 7.4) (hairpin structure-containing DNA probe HP: 1nM to 100nM), Duplex-specific nuclease (DSN) 0.1U/. mu.L (containing 10 XDSN master buffer).
4b) Detection solution 2: DNA circular probe 1nM-100nM, Phi29DNA polymerase 0.5U/. mu.L (including RCA reaction solution), dNTP: 0.5 mM.
4c) SYBR Gold fluorescent nucleic acid dyes.
TABLE 1 nucleic acid (containing DNA and miRNAs) sequences for use in the present inventiona
aUnderlined bases in the hairpin DNA indicate complementary sequences that will form the complementary region (stem region) of the hairpin probe. Bases in hairpin DNA that are bolded represent complementary regions of the target miR-21. The bold bases in the circular DNA probe indicate complementary regions of hairpin DNA. The underlined bases in M1, M2 and M3 indicate the mutation site of miR-21.
Procedure for the preparation of the
(a) And (3) fully and uniformly mixing the miRNA-containing solution with the detection solution 1, and then incubating at 55 ℃ for 30 minutes.
(b) The detection solution 2 was added to the above reaction solution (a), and incubated at 30 ℃ for 90 minutes.
(c) Adding SYBR Gold fluorescent nucleic acid dye into the reaction solution (b), and reading a fluorescent signal by using a microplate reader.
Test example 1
The product of the first reaction (i.e., the product obtained in step (a)) was subjected to 20% native polyacrylamide gel electrophoresis, and the results are shown in FIG. 2. When the detection system only contains miR-21(1) and hairpin structure DNA (2), the reaction of the first step can not be started, and when the detection system contains miR-21 and double-strand specific nuclease (3) or hairpin structure DNA and double-strand specific nuclease (4), the double-strand specific nuclease has no degradation effect on the target miR-21 and hairpin structure DNA. When the detection system contains target miR-21 and hairpin structure DNA (5), only partial reaction of the first step can be started. Only when hairpin structure DNA, double-strand specific nuclease and a target miR-21 exist in the detection system simultaneously (6), the reaction of the first step can be completely started. These experimental phenomena are consistent with the detection principle shown in fig. 1, and the feasibility of the technical scheme is powerfully proved.
Test example 2
The reaction product of the whole detection system (i.e., the product obtained in step (c)) was subjected to 0.4% agarose gel electrophoresis, and the results are shown in FIG. 3. When the detection system does not have the target miR-21, the system cannot perform the target circulation reaction and the rolling circle amplification reaction (1) mediated by the double-strand specific nuclease, and when the detection system contains the target miR-21, the target circulation reaction and the rolling circle amplification reaction (2) mediated by the double-strand specific nuclease can be performed, so that the phenomenon is consistent with the detection principle of the system, and the feasibility of the technical scheme is further proved.
Test example 3
The test case is used for testing the sensitivity, the specificity and the application in complex biological samples of the detection system. As shown in FIG. 4, as the concentration of the target miR-21 is increased, the corresponding fluorescence intensity is gradually increased. The fluorescence intensity of lgCmiR-21 at concentrations of 1 to 4 can be plotted as a standard calibration curve (B), and the linear regression equation is that the fluorescence intensity (λ ex 485nm, λ em 544nm) is 297.5+36.21 × lgCmiR-21 (linear correlation coefficient R)20.991), the detection limit of the detection system on miR-21 is 1fM, which shows that the method has extremely high sensitivity.
Test example 4
This test example serves to illustrate the specificity of the detection system of the present invention.
As shown in FIG. 5, the relative fluorescence intensities of the target miR-21-containing samples are 3 times, 6.6 times and 11.4 times of the relative fluorescence intensities of 1, 2 and 3 base mismatches miR-21, respectively. The experimental result proves that the technical scheme has extremely high specificity.
Test example 5
The test example is used to illustrate that the detection system is used to detect miR-21 in breast cancer cells MDA-MB-231 and T47D.
Total miRNAs in breast cancer cells MDA-MB-231 and T47D are extracted, miR-21 in the total miRNAs with different concentrations is detected by the method, as shown in figure 6, the method can detect miRNA in the cells and has a certain clinical application prospect.
Test example 6
The test example is used for illustrating the evaluation of the detection effect of the detection system when the detection system is applied to actual detection samples. The results are shown in table 2, when the detection system contains 10% of normal human serum, the detection result is not affected, and the result shows that the technical scheme can detect miRNAs in complex biological samples (serum), and has very good application prospect and practical value.
Table 2 detection of miR-21(n ═ 3) in the presence of 10% normal human serum
| Sample (I) | Actual addition value (pM) | Mean calculated value (pM)b | Average recovery (%)c | Relative standard deviation (%)d |
| 1 | 10 | 12.25 | 122.57 | 4.29 |
| 2 | 50 | 56.41 | 56.41 | 2.26 |
| 3 | 100 | 117.23 | 117.23 | 0.88 |
bdValues obtained in three independent experiments
cAverage recovery rate (%) < 100 × (C)Mean calculated value/CActual value of addition)
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
<110> Shenzhen Shenkang pharmaceutical science and technology Limited
<120> cyclic microRNAs detection kit, method for specifically detecting cyclic microRNAs and application
<130> 1800234
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> Homo sapiens
<400> 1
uagcuuauca gacugauguu ga 22
<210> 2
<211> 42
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcaacatcag tctgataagc taccatgtgt agaatgctta tc 42
<210> 3
<211> 54
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
agcacaatct acacatcaaa gcccatacta caacaactac aacattgata gata 54
<210> 4
<211> 22
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
uagcuuauca gaccgauguu ga 22
<210> 5
<211> 22
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
uagcauauca gaccgauguu ga 22
<210> 6
<211> 22
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
uagcaaauca gaccgauguu ga 22
Claims (7)
1. A circulating microRNAs detection kit is characterized by comprising the following components:
(1) hairpin structure DNA with a cohesive end, wherein a part of the hairpin structure DNA beginning from the long cohesive end is completely complementary and paired with the target microRNA; the complementary pairing enables hairpin structure DNA to be unfolded and form miRNA-DNA hybrid with target microRNA;
(2) double-chain specific nuclease and double-chain specific nuclease reaction buffer solution;
(3) a circular DNA probe, wherein a part of the circular DNA probe is complementarily paired with a part of the short cohesive end of hairpin structure DNA;
(4) optionally one of the following components:
(4-1) SYBR Gold fluorescent nucleic acid dye;
(4-2) a DNA polymerase;
(4-3) a DNA polymerase reaction buffer;
(4-4)4×dNTPs。
2. the circulating microRNAs detection kit of claim 1, wherein the number of bases that a part of long sticky end of hairpin DNA is complementary paired with at least a part of target microRNA is 15-24.
3. The circulating microRNAs detection kit of claim 1, wherein said hairpin DNA is provided in the form of hairpin DNA buffer at a concentration of 1nM to 100 nM;
the circular DNA probe is provided in the form of a rolling circle amplification reaction solution of the circular DNA probe, and the concentration of the circular DNA probe is 1nM-100 nM.
4. The circulating microRNAs detection kit of claim 1, wherein said target microRNA has an amino acid sequence as set forth in SEQ ID NO: 1; the hairpin structure DNA has the nucleotide sequence shown in SEQ ID NO: 2; the circular DNA probe has a nucleotide sequence shown as SEQ ID NO: 3, and (b) is shown in the specification.
5. A method for specifically detecting circulating microRNAs, comprising the steps of:
1) mixing the substance to be detected with the hairpin structure DNA, adding double-strand specific nuclease, and carrying out enzymolysis reaction;
2) adding a circular DNA probe into an enzymolysis reaction product to carry out rolling circle amplification;
3) detecting the rolling circle amplification product;
wherein the content of the first and second substances,
the hairpin structure DNA has a cohesive end, and a part of the hairpin structure DNA beginning from the long cohesive end is completely complementary and paired with the target microRNA; the complementary pairing enables hairpin structure DNA to be unfolded and form miRNA-DNA hybrid with target microRNA;
a portion of the circular DNA probe is complementary paired with a portion of the short sticky end of hairpin DNA.
6. The method of claim 5, wherein the rolling circle amplification product is detected using a SYBR Gold fluorescent nucleic acid dye.
7. The method according to claim 5, wherein the number of bases that a part of the long sticky end of the hairpin DNA is complementary-paired with at least a part of the microRNA of interest is 15-24.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810380776 | 2018-04-25 | ||
| CN2018103807769 | 2018-04-25 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109251964A CN109251964A (en) | 2019-01-22 |
| CN109251964B true CN109251964B (en) | 2021-09-14 |
Family
ID=65043426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811360587.1A Active CN109251964B (en) | 2018-04-25 | 2018-11-15 | Circulating microRNAs detection kit, method for specifically detecting circulating microRNAs and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109251964B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706225B (en) * | 2019-03-05 | 2021-11-02 | 山东大学 | Electrochemical detection method of palladium nanoparticles to microRNA based on rolling circle amplification mediation |
| CN110004214B (en) * | 2019-04-11 | 2023-01-31 | 合肥工业大学 | Method for detecting microRNA-21 by double DNA machine |
| CN112941154B (en) * | 2021-02-21 | 2022-09-09 | 中南大学 | Molecular beacon probe, application of molecular beacon probe in preparation of circBART2.2 detection preparation and kit |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555838B (en) * | 2013-10-31 | 2016-09-14 | 深圳先进技术研究院 | A kind of miRNA detection probe based on rolling circle amplification reaction, detection method and test kit |
| CN104593520B (en) * | 2015-02-28 | 2016-10-05 | 上海赛安生物医药科技有限公司 | A kind of pulmonary carcinoma miRNA detection kit and the application of miRNA thereof |
| CN106967794B (en) * | 2017-03-14 | 2020-04-14 | 清华大学深圳研究生院 | Kit and method for detecting miRNA (micro ribonucleic acid) by bidirectional signal amplification |
| CN106947811B (en) * | 2017-03-17 | 2020-03-31 | 中国科学技术大学 | Method, probe set and kit for detecting miRNAs-21 |
-
2018
- 2018-11-15 CN CN201811360587.1A patent/CN109251964B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN109251964A (en) | 2019-01-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Siddika et al. | Bringing MicroRNAs to light: methods for MicroRNA quantification and visualization in live cells | |
| CN103210092B (en) | The quantitative PCR of the oligonucleotide mediated reverse transcription of stem-ring of modifying and base intervals restriction | |
| CN113604564A (en) | Method for detecting exosome-associated microRNA (ribonucleic acid) molecules | |
| US9169520B2 (en) | Cancer marker, method for evaluation of cancer by using the cancer marker, and evaluation reagent | |
| Zhang et al. | An ultrasensitive label-free electrochemical biosensor for microRNA-21 detection based on a 2′-O-methyl modified DNAzyme and duplex-specific nuclease assisted target recycling | |
| Li et al. | A strategy for co-analysis of microRNAs and DNA | |
| CN106967794B (en) | Kit and method for detecting miRNA (micro ribonucleic acid) by bidirectional signal amplification | |
| CN109251964B (en) | Circulating microRNAs detection kit, method for specifically detecting circulating microRNAs and application | |
| CN107130024B (en) | Method for detecting microRNA based on helicase-dependent DNA isothermal amplification technology | |
| CN111020031A (en) | Method for detecting tumor gene mutation by combining sequence specific blocker with specific PCR (polymerase chain reaction) program | |
| WO2023025259A1 (en) | Method and kit for detecting microrna | |
| JP7392048B2 (en) | Analysis method and kit | |
| Fan et al. | Label-free fluorescence detection of circulating microRNAs based on duplex-specific nuclease-assisted target recycling coupled with rolling circle amplification | |
| Chi et al. | CRISPR-Cas14a-integrated strand displacement amplification for rapid and isothermal detection of cholangiocarcinoma associated circulating microRNAs | |
| CN105463110B (en) | A method of MicroRNA is detected using two-step amplification method | |
| Van Etten et al. | A guide to design and optimization of reporter assays for 3′ untranslated region mediated regulation of mammalian messenger RNAs | |
| CN107385037B (en) | MiRNA indirect real-time fluorescence quantitative PCR detection method | |
| Huang et al. | Target-induced multiple palindrome-mediated strand displacement amplification of Scarecrow-shaped DNA nanoprobe for ultrasensitive detection of MicroRNA | |
| CN103352070B (en) | ROS1 fusion gene screening method | |
| CN115895857A (en) | PCR chip and method for detecting micro RNA in blood sample | |
| CN114196733A (en) | Telomere G quadruplex DNA and thioflavin T mediated fluorescence biosensor and application thereof in lncRNA detection | |
| CN102899390A (en) | Small cell lung cancer markers and their detection | |
| CN113322306A (en) | Biosensor combining exponential amplification reaction and CRISPR-Cas12a as well as detection method and application thereof | |
| JP7346533B2 (en) | Analysis method and kit | |
| EP4079850A1 (en) | Analytical method and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |