CN112961908A - Visual detection method of annular RNA in extracellular vesicle - Google Patents
Visual detection method of annular RNA in extracellular vesicle Download PDFInfo
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- CN112961908A CN112961908A CN202110339760.5A CN202110339760A CN112961908A CN 112961908 A CN112961908 A CN 112961908A CN 202110339760 A CN202110339760 A CN 202110339760A CN 112961908 A CN112961908 A CN 112961908A
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Abstract
The invention discloses a visual detection method of annular RNA in an extracellular vesicle, S1, cracking, separating and purifying the extracellular vesicle to obtain RNA to be detected; s2, dividing the RNA into a group to be detected and a control group, and adding a certain amount of RNA to be detected into the total RNA of the group to be detected respectively; s3, synthesizing DNA by using the RNA to be detected as a template under the action of reverse transcriptase for the group to be detected and the control group respectively; s4, the group to be detected and the control group respectively realize rolling circle amplification replication under the conditions of proper temperature and pH value of an amplification substrate, a DNA template to be detected, DNA polymerase and a plurality of DNA primers; s5, carrying out visual detection on the amplified product, and respectively detecting whether the to-be-detected group and the control group contain the circular RNA and the content of the circular RNA. According to the invention, reverse transcription-rolling circle amplification reaction is utilized, an amplification substrate is an A base containing a biotin label, and an amplification product can be detected by using a streptavidin colloidal gold test strip, so that whether the extracellular vesicles to be detected contain cyclic RNA or not and the content of the cyclic RNA are detected.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a visual detection method of annular RNA in an extracellular vesicle.
Background
The circular RNA is a small molecular non-coding RNA widely existing in animals and plants, is mainly formed by splicing linear RNA through reverse shearing, and a specific base sequence formed after splicing is a key sequence for distinguishing the circular RNA from the linear RNA. In recent years, with the continuous innovation of high-throughput sequencing and nucleic acid molecular techniques, more and more research data show that circular RNA plays an important role in growth, development, metabolism, apoptosis, aging, and the like of the body.
Studies have demonstrated that there are over 1000 circular rnas (circrnas) in human blood exosomes and these circrnas can be used as potential markers of cancer. Where hsa _ circ _0036627 is a potential marker for pancreatic cancer. Compared with other non-coding RNAs, the circular RNAs are used as molecular markers for diagnosis, risk stratification and prognosis of cardiovascular diseases, pancreatic cancers and the like and are closely related to special biological characteristics of the circular RNAs. The specific biological properties include: 1) the circular RNA molecule lacks 3 'end cap and 5' end poly A tail structure, can resist the degradation of RNA enzyme in blood and environment, and has longer half-life; 2) the circular RNA molecule has obvious tissue and cell specificity in different tissue organs and cell types according to the type and expression abundance. However, the current methods for detecting circular RNA in blood at home and abroad mainly involve multiple links such as RNA extraction, purification, reverse transcription and the like. However, the detection of the circular RNA is not satisfactory for visualization, so that the clinical application of the circular RNA is greatly limited.
Therefore, there is an urgent need in the art to develop a method for detecting circular RNA in an extracellular vesicle, which has high sensitivity, high accuracy and visual detection, and aims to solve the problems of the prior art that the sensitivity for detecting circular RNA in an extracellular vesicle is not high and the visual detection cannot be performed.
Disclosure of Invention
The invention overcomes the defects of the prior art, provides a visual detection method of the annular RNA in the extracellular vesicle, and aims to solve the problems that the sensitivity of the detection of the annular RNA in the extracellular vesicle is not high and the visual detection cannot be realized in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that: the visual detection method of the annular RNA in the extracellular vesicles is characterized by comprising the following steps:
s1, RNA extraction: cracking, separating and purifying the extracellular vesicles to obtain RNA to be detected, and performing quality control on the RNA to be detected;
s2, grouping: grouping into a group to be detected and a control group, and adding a certain amount of RNA to be detected into the total RNA of the group to be detected respectively, so that the quality of the total RNA in the group to be detected and the quality of the total RNA in the control group are kept consistent;
s3, reverse transcription: respectively synthesizing DNA (deoxyribonucleic acid) by using RNA (ribonucleic acid) to be detected as a template under the action of reverse transcriptase in the to-be-detected group and the control group, and inactivating enzyme after the reaction is finished;
s4, rolling ring amplification: the group to be detected and the control group respectively realize rolling circle amplification replication under the conditions of proper temperature and pH value of an amplification substrate, a DNA template to be detected, DNA polymerase and a plurality of DNA primers;
s5, detection: and performing visual detection on the amplified product, and respectively detecting whether the to-be-detected group and the control group contain the circular RNA and the content of the circular RNA.
In a preferred embodiment of the present invention, the amplification substrate contains at least biotin-labeled A base.
In a preferred embodiment of the present invention, the test strip of colloidal gold is used to perform a color test reaction on the amplified products in the test group and the control group, and whether the control group contains the circular RNA is determined according to whether the control group has color development.
In a preferred embodiment of the present invention, the content of the circular RNA in the control group is determined by the degree of color development in the color reaction.
In a preferred embodiment of the present invention, in S5, the concentration of the RNA to be detected in the group to be detected is measured by using a solid fluorescence quantitative analyzer for quantification; the fluorescence intensity increases in the presence of the circular RNA, and increases as the concentration of the circular RNA increases.
In a preferred embodiment of the present invention, in S5, the amplicons in the test group and the control group can be sequenced, and the sequence ratio can be used to determine whether the test group contains circular RNA. And (3) comparing the sequences of the amplificates in the test group and the control group by detecting, and determining whether the sequences can form a circular structure mutually or not to determine whether the RNA to be tested is circular RNA or not.
In a preferred embodiment of the present invention, the sample to be tested for the extracellular vesicles is derived from blood, pleural effusion or peritoneal effusion.
In a preferred embodiment of the present invention, in S2, the mass ratio of the test RNA to the total RNA in the test group is 1: 100-120.
In a preferred embodiment of the present invention, in S4, the DNA primers are at least two pairs in forward and reverse directions.
In a preferred embodiment of the invention, each pair of primers is greater than 400bp in length.
In a preferred embodiment of the present invention, in S4, the conditions of the rolling circle amplification reaction are as follows: incubating for 1.5-3h at 30-37 ℃; the inactivation conditions are as follows: inactivating at 60-70 deg.C for 10-20 min.
In a preferred embodiment of the present invention, the test strip is a streptavidin colloidal gold test strip.
The invention solves the defects in the background technology, and has the following beneficial effects:
(1) the invention provides a visual detection method of annular RNA in an extracellular vesicle, which comprises the steps of RNA extraction, grouping, reverse transcription, rolling circle amplification and visual detection, and abandons a plurality of links such as traditional annular RNA concentration determination, quantitative PCR detection and the like, and is quicker and more convenient than the traditional detection method.
(2) According to the invention, reverse transcription-rolling circle amplification reaction (RT-RCA) is utilized, an amplification substrate in the invention is an A base containing a biotin label, so that an amplification product can be detected by using a streptavidin colloidal gold test strip, and the detection of whether the extracellular vesicles to be detected contain the cyclic RNA and the content of the cyclic RNA is realized.
(3) The invention provides a group to be detected and a control group, strictly controls the quality of total RNA in the group to be detected and the control group, adds RNA to be detected into the group to be detected, and performs rolling circle amplification and visual detection together with the total RNA in the control group. Therefore, the problem that no blank RNA reference control exists is solved, and the purpose of detecting the expression quantity of the circular RNA in the extracellular vesicle is realized.
(4) In the invention, the RNA to be detected is reversely transcribed into the DNA, so that the detection of the circular RNA is converted into the research on the DNA, and the subsequent work is facilitated.
(5) The invention is suitable for early screening of diseases and evaluation of disease severity, and can guide clinical diagnosis and treatment.
(6) The invention is a relatively broad-spectrum invention, and based on the biological characteristics of circular RNA tissue and cell specific expression, the method has higher specificity for diagnosing certain special diseases.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts;
fig. 1 is a flow chart of a preferred embodiment of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described herein, and therefore the scope of the present invention is not limited by the specific embodiments disclosed below.
In the description of the present application, it is to be understood that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in the orientation or positional relationship indicated in the drawings for convenience in describing the present application and for simplicity in description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed in a particular orientation, and be operated in a particular manner, and are not to be considered limiting of the scope of the present application. Furthermore, the terms "first," "second," and the like are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first," "second," etc. may explicitly or implicitly include one or more of that feature. In the description of the invention, the meaning of "a plurality" is two or more unless otherwise specified.
In the description of the present application, it is to be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present application can be understood by those of ordinary skill in the art through specific situations.
As shown in fig. 1, it is a flow chart of the method for visually detecting circular RNA in extracellular vesicle provided by the present invention. The method comprises the steps of RNA extraction, grouping, reverse transcription, rolling circle amplification and visual detection, and a plurality of links such as traditional circular RNA concentration determination, quantitative PCR detection and the like are abandoned, so that the method is quicker and more convenient than the traditional detection method. The visual detection method mainly comprises the following steps:
s1, RNA extraction: cracking, separating and purifying the extracellular vesicles to obtain RNA to be detected, and performing quality control on the RNA to be detected;
s2, grouping: grouping into a group to be detected and a control group, and adding a certain amount of RNA to be detected into the total RNA of the group to be detected respectively, so that the quality of the total RNA in the group to be detected and the quality of the total RNA in the control group are kept consistent;
s3, reverse transcription: respectively synthesizing DNA (deoxyribonucleic acid) by using RNA (ribonucleic acid) to be detected as a template under the action of reverse transcriptase in the to-be-detected group and the control group, and inactivating enzyme after the reaction is finished;
s4, rolling ring amplification: the group to be detected and the control group respectively realize rolling circle amplification replication under the conditions of proper temperature and pH value of an amplification substrate, a DNA template to be detected, DNA polymerase and a plurality of DNA primers; wherein the amplification substrate at least contains biotin-labeled A base; the conditions of the rolling ring amplification reaction are as follows: incubating for 1.5-3h at 30-37 ℃; the inactivation conditions are as follows: inactivating at 60-70 deg.C for 10-20 min;
s5, detection: and performing visual detection on the amplified product, and respectively detecting whether the to-be-detected group and the control group contain the circular RNA and the content of the circular RNA.
According to the invention, the colloidal gold test strip is used for carrying out color development experimental reaction on the amplification products in the group to be detected and the control group respectively, and whether the control group contains the cyclic RNA is judged according to whether the control group has color development. The content of the circular RNA in the control group was determined by the degree of color development in the color reaction. Wherein the test strip is a streptavidin colloidal gold test strip.
The invention can selectively add fluorescent dye during amplification, and calculate the content of the circular RNA in unit volume of blood according to fluorescence intensity. Therefore, the invention can also use a solid fluorescence quantitative instrument for quantification to measure the concentration of the RNA to be measured of the group to be measured; the fluorescence intensity increases in the presence of the circular RNA, and increases as the concentration of the circular RNA increases.
In the present invention, in S1, the extracellular vesicles are lysed, separated, and purified to obtain RNA to be detected, and an alcoholic organic solvent is added and washed with an alcoholic solution, wherein the alcoholic organic solvent includes but is not limited to at least one of methanol, ethanol, propanol, isopropanol, propylene glycol, glycerol, and isoamyl alcohol. Washing is performed using an alcohol solution including, but not limited to, at least one of methanol, ethanol, propanol, isopropanol, propylene glycol, glycerol, and isoamyl alcohol. In addition, in the invention S1, centrifugal treatment is required, the centrifugal acceleration of the high-speed centrifugal treatment is 10000g, and the duration is 30-60 min.
The invention can also sequence the amplimers in the middle group to be detected and the control group, and judge whether the group to be detected contains the circular RNA or not through the sequence ratio. And (3) comparing the sequences of the amplificates in the test group and the control group by detecting, and determining whether the sequences can form a circular structure mutually or not to determine whether the RNA to be tested is circular RNA or not.
The sample to be tested for the extracellular vesicles of the invention includes, but is not limited to, cell culture supernatant, blood, pleural effusion, peritoneal effusion, tissue mass, interstitial fluid, tissue leachate, serum, plasma, whole blood, interstitial fluid, lymph fluid, cerebrospinal fluid, aqueous humor, pleural fluid, ascites, synovial fluid, gastrointestinal secretion, digestive gland secretion, and cell culture supernatant.
In the present invention, in S2, the mass ratio of the test RNA to the total RNA in the test group is 1: 100-120. In S4, the DNA primers are at least two pairs in forward and reverse directions, and the length of each pair of primers is more than 400 bp.
In the invention, the RNA to be detected is reversely transcribed into the DNA, so that the detection of the circular RNA is converted into the research on the DNA, and the subsequent work is facilitated.
The invention provides a group to be detected and a control group, strictly controls the quality of total RNA in the group to be detected and the control group, adds RNA to be detected into the group to be detected, and performs rolling circle amplification and visual detection together with the total RNA in the control group. Therefore, the problem that no blank RNA reference control exists is solved, and the purpose of detecting the expression quantity of the circular RNA in the extracellular vesicle is realized.
The existing circular RNA detection method is still based on the traditional Northern Blot and depends on transfer and hybridization. The main disadvantages of this method are long working procedure, complicated operation, time consuming, large sample amount required, limited sensitivity, and the need for labeling of the probe with radioactive or alkaline phosphatase, which causes environmental pollution and waste of resources. In contrast, the present invention detects circular RNA by rolling circle amplification. Amplification in this manner is only a simple circular amplification and the newly synthesized product is always attached to the target or solid support, resulting in a specific local signal.
In the prior art, a method for detecting circular RNA in blood mainly relates to multiple links of RNA extraction, purification, reverse transcription and the like. However, the detection of the circular RNA is not satisfactory for visualization, so that the clinical application of the circular RNA is greatly limited. Therefore, the invention is improved on the basis of the prior art, reverse transcription-rolling circle amplification reaction (RT-RCA) is utilized in the invention, Rolling Circle Amplification (RCA) is a high-efficiency isothermal enzymatic nucleic acid amplification technology, and the method is widely applied to detection of DNA, RNA and protein due to simple operation, strong universality, high specificity and high sensitivity. The miRNA can be used as a connecting template of a padlock probe chain and also can be used as a primer for isothermal amplification, so that a novel detection way is provided for the detection of the miRNA. The amplification substrate is A base containing biotin labels, so that an amplification product can be detected by using a streptavidin colloidal gold test strip, and whether the extracellular vesicles to be detected contain the cyclic RNA or not and the content of the cyclic RNA are detected. The invention is suitable for early screening of diseases and evaluation of disease severity, and can guide clinical diagnosis and treatment. The invention is a relatively broad-spectrum invention, and based on the biological characteristics of circular RNA tissue and cell specific expression, the method has higher specificity for diagnosing certain special diseases.
In light of the foregoing description of the preferred embodiment of the present invention, it is to be understood that various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.
Claims (10)
1. The visual detection method of the annular RNA in the extracellular vesicles is characterized by comprising the following steps:
s1, RNA extraction: cracking, separating and purifying the extracellular vesicles to obtain RNA to be detected, and performing quality control on the RNA to be detected;
s2, grouping: grouping into a group to be detected and a control group, and adding a certain amount of RNA to be detected into the total RNA of the group to be detected respectively, so that the quality of the total RNA in the group to be detected and the quality of the total RNA in the control group are kept consistent;
s3, reverse transcription: respectively synthesizing DNA (deoxyribonucleic acid) by using RNA (ribonucleic acid) to be detected as a template under the action of reverse transcriptase in the to-be-detected group and the control group, and inactivating enzyme after the reaction is finished;
s4, rolling ring amplification: the group to be detected and the control group respectively realize rolling circle amplification replication under the conditions of proper temperature and pH value of an amplification substrate, a DNA template to be detected, DNA polymerase and a plurality of DNA primers;
s5, detection: and performing visual detection on the amplified product, and respectively detecting whether the to-be-detected group and the control group contain the circular RNA and the content of the circular RNA.
2. The method for visually detecting a circular RNA inside an extracellular vesicle according to claim 1, wherein: the amplification substrate contains at least biotin-labeled A base.
3. The method for visually detecting a circular RNA inside an extracellular vesicle according to claim 1, wherein: and (3) performing color development experimental reaction on the amplification products in the to-be-detected group and the control group by using a colloidal gold test strip, and judging whether the control group contains the cyclic RNA or not according to whether the control group develops color or not.
4. The method for visually detecting a circular RNA inside an extracellular vesicle according to claim 3, wherein: the content of the circular RNA in the control group was determined by the degree of color development in the color reaction.
5. The method for visually detecting a circular RNA inside an extracellular vesicle according to claim 1, wherein: in the step S5, quantifying by using a solid fluorescence quantifier, and measuring the concentration of the RNA to be detected of the group to be detected; the fluorescence intensity increases in the presence of the circular RNA, and increases as the concentration of the circular RNA increases.
6. The method for visually detecting a circular RNA inside an extracellular vesicle according to claim 1, wherein: in S5, the amplicons in the test and control groups can be sequenced, and the presence or absence of circular RNA in the test group can be determined by the sequence ratio.
7. The method for visually detecting a circular RNA inside an extracellular vesicle according to claim 1, wherein: the sample to be detected of the extracellular vesicles is derived from blood, pleural effusion or peritoneal effusion.
8. The method for visually detecting a circular RNA inside an extracellular vesicle according to claim 1, wherein: in S2, the mass ratio of the test RNA to the total RNA in the test group is 1: 100-120.
9. The method for visually detecting a circular RNA inside an extracellular vesicle according to claim 1, wherein: in S4, the DNA primers are at least two pairs in forward and reverse directions.
10. The method for visually detecting a circular RNA inside an extracellular vesicle according to claim 1, wherein: in S4, the conditions of the rolling circle amplification reaction are: incubating for 1.5-3h at 30-37 ℃; the inactivation conditions are as follows: inactivating at 60-70 deg.C for 10-20 min.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113564230A (en) * | 2021-07-27 | 2021-10-29 | 华侨大学 | A kind of circular RNA in situ detection method |
| CN115058488A (en) * | 2022-07-27 | 2022-09-16 | 苏州爱宝德生物科技有限公司 | Annular RNA visual detection method based on exosome extraction in peripheral blood |
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2021
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113564230A (en) * | 2021-07-27 | 2021-10-29 | 华侨大学 | A kind of circular RNA in situ detection method |
| CN113564230B (en) * | 2021-07-27 | 2023-10-31 | 华侨大学 | In-situ detection method for circular RNA |
| CN115058488A (en) * | 2022-07-27 | 2022-09-16 | 苏州爱宝德生物科技有限公司 | Annular RNA visual detection method based on exosome extraction in peripheral blood |
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