CN113801923A - A digital rolling circle amplification detection method that can be directly used for serum marker detection - Google Patents
A digital rolling circle amplification detection method that can be directly used for serum marker detection Download PDFInfo
- Publication number
- CN113801923A CN113801923A CN202111222855.5A CN202111222855A CN113801923A CN 113801923 A CN113801923 A CN 113801923A CN 202111222855 A CN202111222855 A CN 202111222855A CN 113801923 A CN113801923 A CN 113801923A
- Authority
- CN
- China
- Prior art keywords
- rolling circle
- serum
- circle amplification
- detection method
- digital
- 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.)
- Pending
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
-
- 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
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及一种可直接用于血清标志物检测的数字滚环扩增检测方法，将滚环扩增体系与血清混合后加入数字PCR芯片，通过优化血清稀释比例，生成粒径均一，单分散性良好的微液滴，血清标志物按照泊松分布分配进微液滴中，微液滴作为独立的反应室，在反应室中进行滚环扩增，可直接检测血清标志物的拷贝数，该技术有别于传统的滚环扩增检测方法，能够对待测物质进行绝对定量，因此提高了滚环扩增的灵敏度，并且可直接用于血清标志物的检测。
The invention relates to a digital rolling circle amplification detection method which can be directly used for the detection of serum markers. The rolling circle amplification system is mixed with serum and then added to a digital PCR chip. Serum markers are distributed into the droplets according to Poisson distribution, and the droplets are used as independent reaction chambers. Rolling circle amplification is performed in the reaction chamber, and the copy number of serum markers can be directly detected. Different from the traditional rolling circle amplification detection method, the technology can perform absolute quantification of the substance to be tested, thus improving the sensitivity of rolling circle amplification, and can be directly used for the detection of serum markers.
Description
Technical Field
The invention belongs to the field of biochemical detection and molecular diagnosis, and particularly relates to a digital rolling circle amplification detection method capable of being directly used for serum marker detection.
Background
The isothermal amplification reaction has single temperature and is not limited by thermal cycling, so that the method is simpler in actual operation and equipment requirements, and can realize portable rapid detection of the marker. Among them, Rolling Circle Amplification (RCA) is the earliest developed and the most widely used isothermal amplification detection method so far. The template for rolling circle amplification is circular DNA, and after the circular DNA is combined with a primer, the circular DNA can be amplified to form thousands of sequences complementary to the circular DNA under the action of Polymerase with strand displacement properties, such as phi29 Polymerase (phi 29 DNA Polymerase), Phage T7, Sequence and the like. The equipment required by rolling circle amplification is simple, the specificity is high, the flux is large, the reaction temperature is mild, and the structure and the function of the biomolecule can not be damaged, so the method has wide application prospect in the aspects of whole genome detection, single nucleotide polymorphism analysis, immunoassay, microarray solid phase detection and the like. However, the amplification efficiency of rolling circle amplification is limited, and the traditional linear rolling circle amplification has limitations on signal amplification, so that the detection sensitivity is unsatisfactory, and other signal amplification mechanisms, such as fluorescent quantum dots, nano-electrode composite materials and the like, are often combined for joint detection, which greatly increases the preparation time of the materials at the early stage and the complexity of operation, reduces the stability and repeatability of the method, and is not suitable for clinical application.
Disclosure of Invention
In order to solve the problems that the traditional linear rolling circle amplification detection method has low efficiency and cannot quantify substances to be detected with low concentration in a complex body fluid environment, the invention provides a digital rolling circle amplification detection method which can be directly used for detecting serum markers.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a digital rolling circle amplification detection method directly used for serum marker detection comprises the following steps:
(1) adding the rolling ring amplification system and the diluted serum into a digital PCR chip, adding droplet generation oil matched with the digital PCR into a sample adding hole corresponding to the chip, and placing the chip into a digital PCR instrument to generate micro droplets;
(2) carrying out isothermal incubation by taking the micro-droplets as an independent reaction chamber, and carrying out roller amplification;
(3) the positive reaction units are counted by a chip scanner, and the copy number of the serum marker is detected.
The invention mixes the rolling ring amplification system with serum and adds the mixture into a digital PCR chip, generates micro-droplets with uniform quantity and particle size and good monodispersity by optimizing the dilution ratio of the serum, distributes serum markers into the micro-droplets according to Poisson distribution, takes the micro-droplets as an independent reaction chamber, carries out rolling ring amplification in the reaction chamber, and can directly detect the copy number of the serum markers.
As a modification of the present invention, in step (1), the serum markers in the serum are distributed into microdroplets according to a poisson distribution.
As an improvement of the invention, in the step (1), the dilution multiple of the serum is 50-1250 times.
As an improvement of the invention, in the step (1), the dilution multiple of the serum is 200-300 times.
As a modification of the invention, in the step (1), the serum is diluted by 250 times.
As an improvement of the invention, in the step (2), the temperature of the isothermal incubation is 25-37 ℃, and the time of the isothermal incubation is 30-90 min.
As an improvement of the invention, the temperature of the isothermal incubation is 30 ℃, and the time of the isothermal incubation is 45 min.
The invention has the beneficial effects that:
the invention combines a rolling circle amplification detection method with a digital micro-droplet technology to establish a digital rolling circle amplification detection method, thousands of micro-droplets are generated by utilizing a commercial digital PCR instrument to serve as independent reaction chambers, serum markers are distributed into the micro-droplets according to Poisson distribution, rolling circle amplification is carried out in the reaction chambers, and positive reaction units are counted by a commercial chip scanner after the amplification is finished.
The method does not depend on a control sample and a standard curve, can be used for accurate absolute quantitative detection, has higher sensitivity and accuracy compared with the traditional rolling circle amplification detection method, and has great advantages in extremely low concentration of substances to be detected and complex body fluid environments. The technology improves the sensitivity of rolling circle amplification by absolutely quantifying the substance to be detected, and can be directly used for detecting serum markers. In addition, compared with combined detection combined with other signal amplification mechanisms, the commercialized digital PCR instrument has higher stability and repeatability, has lower requirements on the background and the proficiency of operators, and is more suitable for clinic.
Drawings
FIG. 1 is a fluorescence imaging diagram of micro-droplets under different serum dilution multiples, wherein A-D dilution multiples are respectively 10 times, 50 times, 250 times and 1250 times in sequence, the left column is a positive group, and the right column is a control group;
FIG. 2 shows the detection results of the fluorescence signals of the microdroplets under different isothermal incubation times, wherein A is the detection result of the fluorescence signals of the microdroplets, and B is the number of the microdroplets;
fig. 3 is the positive group of micro-droplet fluorescence confocal imaging under the optimal experimental conditions, wherein the left image is a bright field, the middle image is a dark field, and the right image is a bright field and dark field superposition image.
Detailed Description
The present invention will be further illustrated with reference to the accompanying drawings and specific embodiments, which are to be understood as merely illustrative of the invention and not as limiting the scope of the invention.
A digital rolling ring amplification detection method capable of being directly used for serum marker detection is characterized in that a rolling ring amplification system and serum are mixed and then added into a digital PCR chip, serum dilution proportion is optimized, micro-droplets with uniform particle size and good monodispersity are generated, serum markers are distributed into the micro-droplets according to Poisson distribution, the micro-droplets are used as independent reaction chambers, rolling ring amplification is carried out in the reaction chambers, and the copy number of the serum markers can be directly detected.
The method comprises the following specific steps:
(1) adding the rolling circle amplification system and the diluted serum into a digital PCR chip to generate micro-droplets;
(2) carrying out isothermal incubation by taking the micro-droplets as an independent reaction chamber, and carrying out roller amplification;
(3) the positive reaction units are counted by a chip scanner, and the copy number of the serum marker is detected.
The invention is further described with reference to specific examples below:
example 1
Adding the rolling circle amplification system and the serum diluted by different times into a digital PCR chip to generate micro-droplets; the detection target here was mucin 1 (MUC 1).
Taking 1 mu L of diluted 10, 50, 250 and 1250 times serum respectively, adding a rolling circle amplification system (0.5 mu L of probe, 1 mu L of circular template, 1 mu L of second primer (2 mu M), 2 mu L of 10 XPhi 29 DNA polymerase buffer solution, 1 mu L of Phi29 polymerase (10U/mu L) and 1 mu L of dNTP (10 mM)), adding DEPC processing water to complement to 20 mu L, adding the solution into a sample tank of a digital PCR chip after uniformly mixing, adding 75 mu L of droplet generation oil matched with digital PCR into a sample adding hole corresponding to the chip, and placing the chip into a digital PCR instrument to generate micro-droplets.
Wherein the probe sequence is as follows: 5'-ATGTACTGCATGCACACCACTTCAACTATGCAGTACAT-3', the probe can bind with mucin 1 and bind with mucin 1 to the circular template, and acts as a primer for rolling circle amplification, and when the substance to be detected is not present, the probe cannot bind with the circular template, and the rolling circle amplification cannot be carried out.
The sequence of the circular template is as follows:
5’-ACTACGCGACTCTACTAGTTCTATCATTCTCATATGTACTGCATCTCGTTTGGTGGACCTGAATCATGT-3’。
the second primer sequence is: 5'-TACTAGTTCTATCATTCTCATA-3' are provided.
The fluorescence imaging of the micro-droplets is carried out under the condition of different serum dilution multiples, as shown in figure 1, the left side is a positive group (diluted serum of a gastric cancer patient), the right side is a control group (normal human serum diluted by the same proportion), the dilution multiples are 10, 50, 250 and 1250 in sequence from top to bottom, the result shows that under different serum dilution multiples, the fluorescence difference of the positive droplets and the negative droplets is different, the low-concentration dilution group has the effect that the probe stability is influenced by the components in the serum due to higher serum proportion, so that the low-concentration dilution group is combined with an annular template and the rolling ring amplification is started under the condition that a substance to be detected does not exist, so that the droplets of the experimental group and the control group emit strong fluorescence, while the highest-concentration dilution group has no fluorescence due to too low concentration of the substance to be detected, and the experimental group and the control group have obvious fluorescence, and the fluorescence signals of the droplets of the experimental group and the control group have obvious difference in 50-fold dilution and 1250-fold dilution, since there are individual droplets of larger size in the 50-fold dilution, the 250-fold dilution was finally selected as the optimum experimental condition in view of droplet uniformity.
Example 2
(1) Adding the rolling circle amplification system and the serum diluted by 250 times into a digital PCR chip to generate micro-droplets (same as the example 1);
(2) carrying out isothermal incubation for 0-90min at 30 ℃ by taking the micro-droplets as an independent reaction chamber, and carrying out roller amplification;
(3) the positive reaction units are counted by a chip scanner, and the copy number of the serum marker is detected.
The result of detecting the fluorescence signal of the micro-droplets with isothermal incubation time of 0, 30, 45, 60, 75, and 90min is shown in fig. 2, and the result shows that as the incubation time increases, the number of positive micro-droplets (i.e. micro-droplets above the dotted line) increases, and the total number of micro-droplets decreases, because the commercial digital PCR instrument is developed for PCR, the reaction temperature and the reaction buffer are different from the rolling circle amplification, and in addition, serum also has a certain influence on the reaction system, so the longer the reaction time, the more unstable the system of (W/O), and since the number of micro-droplets is crucial to the detection method, the fluorescence signal intensity and the total number of micro-droplets are considered, and the incubation time of 45min is selected as the optimal time.
Fig. 3 is fluorescence confocal imaging of positive micro-droplets under the optimal experimental conditions, which is a superposition graph of a bright field, a dark field and a bright field and a dark field from left to right in sequence, and the results show that the particle size distribution of the micro-droplets formed after the experimental conditions are optimized is uniform, and the micro-droplets containing the markers are obviously compared with the micro-droplets not containing the markers under the dark field.
It should be noted that the above-mentioned contents only illustrate the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and it is obvious to those skilled in the art that several modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations fall within the protection scope of the claims of the present invention.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111222855.5A CN113801923A (en) | 2021-10-20 | 2021-10-20 | A digital rolling circle amplification detection method that can be directly used for serum marker detection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111222855.5A CN113801923A (en) | 2021-10-20 | 2021-10-20 | A digital rolling circle amplification detection method that can be directly used for serum marker detection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113801923A true CN113801923A (en) | 2021-12-17 |
Family
ID=78937797
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111222855.5A Pending CN113801923A (en) | 2021-10-20 | 2021-10-20 | A digital rolling circle amplification detection method that can be directly used for serum marker detection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113801923A (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101529252A (en) * | 2006-05-04 | 2009-09-09 | 英国开放大学 | Aptamers to recombinant MUC1 |
| WO2015048173A2 (en) * | 2013-09-24 | 2015-04-02 | The Regents Of The University Of California | Encapsulated sensors and sensing systems for bioassays and diagnostics and methods for making and using them |
| WO2015083002A2 (en) * | 2013-12-02 | 2015-06-11 | Vanadis Diagnostics | Multiplex detection of nucleic acids |
| CN106133211A (en) * | 2014-01-14 | 2016-11-16 | 爱普济德生物技术有限公司 | Nucleic acid reagent for carrying out hypersensitive Digital Detecting and quantitative purposes to target molecule |
| WO2018031691A1 (en) * | 2016-08-10 | 2018-02-15 | The Regents Of The University Of California | Combined multiple-displacement amplification and pcr in an emulsion microdroplet |
| CN108251512A (en) * | 2018-01-02 | 2018-07-06 | 苏州锐讯生物科技有限公司 | A kind of double-aqueous phase system and its application |
| CN108285931A (en) * | 2018-03-30 | 2018-07-17 | 武汉大学 | A kind of the droplet type digital pcr method and kit of clinical detection HBV cccDNA |
| US20190388894A1 (en) * | 2018-06-21 | 2019-12-26 | Sharp Life Science (Eu) Limited | Ewod system and methods to increase dynamic range for digital nucleic acid amplification |
| CN111394433A (en) * | 2020-04-10 | 2020-07-10 | 南京鼓楼医院 | Extracellular vesicle detection technology based on aptamer hairpin-triggered hyperbranched rolling circle amplification |
-
2021
- 2021-10-20 CN CN202111222855.5A patent/CN113801923A/en active Pending
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101529252A (en) * | 2006-05-04 | 2009-09-09 | 英国开放大学 | Aptamers to recombinant MUC1 |
| WO2015048173A2 (en) * | 2013-09-24 | 2015-04-02 | The Regents Of The University Of California | Encapsulated sensors and sensing systems for bioassays and diagnostics and methods for making and using them |
| CN105764490A (en) * | 2013-09-24 | 2016-07-13 | 加利福尼亚大学董事会 | Encapsulated sensors and sensing systems for bioassays and diagnostics and methods of making and using same |
| WO2015083002A2 (en) * | 2013-12-02 | 2015-06-11 | Vanadis Diagnostics | Multiplex detection of nucleic acids |
| CN105934523A (en) * | 2013-12-02 | 2016-09-07 | 瓦纳迪斯诊断公司 | Multiplex detection of nucleic acids |
| CN106133211A (en) * | 2014-01-14 | 2016-11-16 | 爱普济德生物技术有限公司 | Nucleic acid reagent for carrying out hypersensitive Digital Detecting and quantitative purposes to target molecule |
| WO2018031691A1 (en) * | 2016-08-10 | 2018-02-15 | The Regents Of The University Of California | Combined multiple-displacement amplification and pcr in an emulsion microdroplet |
| CN110088290A (en) * | 2016-08-10 | 2019-08-02 | 加利福尼亚大学董事会 | Multiple displacement amplification and PCR are combined in lotion droplet |
| CN108251512A (en) * | 2018-01-02 | 2018-07-06 | 苏州锐讯生物科技有限公司 | A kind of double-aqueous phase system and its application |
| CN108285931A (en) * | 2018-03-30 | 2018-07-17 | 武汉大学 | A kind of the droplet type digital pcr method and kit of clinical detection HBV cccDNA |
| US20190388894A1 (en) * | 2018-06-21 | 2019-12-26 | Sharp Life Science (Eu) Limited | Ewod system and methods to increase dynamic range for digital nucleic acid amplification |
| CN110628878A (en) * | 2018-06-21 | 2019-12-31 | 夏普生命科学（欧洲）有限公司 | EWOD system and method for increasing dynamic range of digital nucleic acid amplification |
| CN111394433A (en) * | 2020-04-10 | 2020-07-10 | 南京鼓楼医院 | Extracellular vesicle detection technology based on aptamer hairpin-triggered hyperbranched rolling circle amplification |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Garg et al. | Recent advances in loop-mediated isothermal amplification (LAMP) for rapid and efficient detection of pathogens | |
| Kaushik et al. | Droplet microfluidics for high‐sensitivity and high‐throughput detection and screening of disease biomarkers | |
| US9157116B2 (en) | Combinatorial amplification and detection of nucleic acids | |
| Ning et al. | A self-digitization chip integrated with hydration layer for low-cost and robust digital PCR | |
| CN107326085B (en) | Encoding and decoding of signals in multiplexed biochemical assays | |
| Khodakov et al. | An oligonucleotide microarray for multiplex real-time PCR identification of HIV-1, HBV, and HCV | |
| JPH11505126A (en) | Wide dynamic range nucleic acid detection using primer series populations | |
| US9347097B2 (en) | Arrangement for determining the concentration of nucleic acids | |
| Peng et al. | RT-RPA-assisted CRISPR/Cas12a for rapid and multiplex detection of respiratory infectious viruses based on centrifugal microfluidics | |
| Ding et al. | Monolithic, 3D-printed lab-on-disc platform for multiplexed molecular detection of SARS-CoV-2 | |
| Li et al. | All‐in‐one OsciDrop digital PCR system for automated and highly multiplexed molecular diagnostics | |
| CN102015996A (en) | System for biological pathogen detection and use thereof | |
| JP2001269196A (en) | Quantitative method for nucleic acid in test object and method for counting number of molecule of nucleic acid in test object | |
| Gulinaizhaer et al. | Isothermal nucleic acid amplification technology in HIV detection | |
| Chen et al. | Smartphone-assisted fluorescence-based detection of sunrise-type smart amplification process and a 3D-printed ultraviolet light-emitting diode device for the diagnosis of tuberculosis | |
| JP2023541767A (en) | Composition for determining false positives using a specific artificial nucleotide sequence and method for determining false positives using the same | |
| Mu et al. | Microfluidic-based approaches for COVID-19 diagnosis | |
| Jiang et al. | Single-molecule RNA capture-assisted droplet digital loop-mediated isothermal amplification for ultrasensitive and rapid detection of infectious pathogens | |
| CN107760763A (en) | A kind of stem-loop primer for stem-loop primer-rolling circle amplification reaction and the application of stem-loop primer-rolling circle amplification | |
| Cao et al. | Development of a cost-efficient micro-detection slide system for the detection of multiple shrimp pathogens | |
| Liu et al. | An amplification-free digital droplet assay for influenza A viral RNA based on CRISPR/Cas13a | |
| Wang et al. | Recent developments in isothermal amplification technology for rapid detection of SARS-CoV-2 | |
| WO2021202815A1 (en) | Colorimetric and multiplexed isothermal rna-and antibody-based assay for sars-cov-2 and other viral diagnostics and cell analysis | |
| CN113801923A (en) | A digital rolling circle amplification detection method that can be directly used for serum marker detection | |
| Bel Hadj Ali et al. | Rapid detection of SARS-CoV-2 RNA using a one-step fast multiplex RT-PCR coupled to lateral flow immunoassay |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20211217 |