CN114369643A - Cell free DNA detection kit, detection system and operation method - Google Patents
Cell free DNA detection kit, detection system and operation method Download PDFInfo
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Abstract
The invention relates to the technical field of intelligent detection of tumors, in particular to a cell free DNA detection kit, a detection system and an operation method, wherein the detection kit comprises a padlock DNA probe, T4 ligase, phi29 DNA polymerase and deoxyribonucleoside triphosphate; or a DNAzyme hydrogel formed from cell free DNA, padlock DNA probes, T4 ligase, phi29 DNA polymerase and deoxyribonucleoside triphosphates; the sequence of the padlock DNA probe is shown as SEQ ID NO. 1. The invention realizes the field detection and analysis of cell free DNA without large-scale instruments, and has the advantages of convenient carrying, low cost, simple operation and the like.
Description
Technical Field
The invention relates to the technical field of intelligent tumor detection, in particular to a cell free DNA detection kit, a cell free DNA detection system and an operation method.
Background
In the past decades, DNA hydrogels have been widely used in the fields of drug release, tissue engineering, environmental analysis, biosensing, etc. due to their precise programmability, stability and biocompatibility, and in particular, pure DNA hydrogels consisting of pure DNA have attracted much attention. Pure DNA hydrogels are typically constructed by self-assembly or enzymatic amplification of DNA fragments, and thus have the advantage of being easier to synthesize than DNA hybridization hydrogels.
Cell-free DNA (cfDNA) is a typical DNA fragment, and has attracted much attention as a noninvasive diagnostic biomarker. In addition, cell-free DNA plays an important role in biomedical diagnostics, such as early diagnosis of cancer, assessment of tumor stage, selection of effective treatment regimens, monitoring prognosis, etc. Polymerase Chain Reaction (PCR) based methods such as droplet digital PCR (ddpcr), asymmetric PCR, emulsion digital PCR and reverse transcription quantitative PCR (RT-qPCR) are considered gold standards for cell free DNA detection. In addition, in recent years, detection methods such as electrochemistry, beads, emulsion, amplification and magnetic platforms, and Amplification Refractory Mutation Systems (ARMS) have been established to improve the performance of cell-free DNA analysis. Although these testing methods are promising, they still require reliance on sophisticated instrumentation and do not meet the needs of portable testing, so that they are not more widely used. Therefore, it is necessary to develop a visual cell-free DNA detection platform without instrument for rural areas with relatively scarce resources.
So far, a smart phone is widely applied to the fields of biomedicine, biological analysis, environmental monitoring and the like as an optical quantitative instrument by virtue of the functions of powerful imaging hardware, a built-in sensing system, advanced software design and the like.
Disclosure of Invention
In view of the above, the present invention provides a cell-free DNA detection kit, a detection system and an operation method, which can detect cell-free DNA in human serum quickly, conveniently and sensitively.
Based on the above purpose, the invention provides a cell free DNA detection kit, which comprises a padlock DNA probe, T4 ligase, phi29 DNA polymerase and deoxyribonucleoside triphosphate; or a DNAzyme hydrogel formed from cell free DNA, padlock DNA probes, T4 ligase, phi29 DNA polymerase and deoxyribonucleoside triphosphates; the sequence of the padlock DNA probe is shown as SEQ ID NO. 1.
The sequence of the padlock DNA probe shown in SEQ ID NO.1 is:
5’-phosphorylated-TTAGAGAGTATTTTTTTTTTTCCCCCTTTTTTCAGTGATT-3’
as an alternative embodiment, the preparation method of the DNAzyme hydrogel comprises the steps of:
mixing and incubating target cell free DNA, a padlock DNA probe and a T4 ligase buffer solution, and cooling to room temperature to obtain a treatment solution A;
step two, adding T4 ligase into the treatment solution A, culturing, adding phi29 DNA polymerase buffer solution, deoxyribonucleoside triphosphate and phi29 DNA polymerase, incubating, and heating at 50-60 ℃ for 25-35min to obtain treatment solution B;
and step three, adding a mixed solution of 90-100 mu M of heme and 20-25mM of phosphate buffer solution into the treatment solution B, standing for 5-7h at the temperature of 3-6 ℃, and then washing the heme-loaded DNA hydrogel by using the phosphate buffer solution to obtain the DNAzyme hydrogel with a microporous structure.
Preferably, the incubation temperature in the first step is 85-100 ℃ and the incubation time is 8-12 min.
Preferably, the culturing method in the second step is culturing at 16 ℃ for 8-12h, and then heating at 65 ℃ for 8-12 min.
The invention also provides a cell-free DNA detection system which comprises a DNAzyme detection platform, a cell-free DNA detection kit arranged on the detection platform, a smart phone, an image processing module and a fitting linear curve processing module, wherein pictures shot by the smart phone are led into the image processing module, and the fitting linear curve processing module performs linear fitting on digital information processed by the image processing module.
The construction and detection method of the detection platform comprises the following steps:
(1) preparation of a detection platform and qualitative detection of cell free DNA: as shown in fig. 1 a: cell-free DNA was probed with a padlock DNA probe at T4 ligase, phi29 DNA polymerase and deoxyribonucleoside triphosphatesUnder the action of phosphoric acid, DNAzyme hydrogel is formed through rolling circle amplification, G tetrad in the DNAzyme hydrogel is one of deoxyribozymes and catalyzes H2O2Simultaneously adding colorless 2, 2-azo-bis (3-ethylbenzthiazoline-6-sulfonic Acid) (ABTS)2-) Catalysis to green oxidation product ABTS-Generating visible signals to realize qualitative detection of cell free DNA;
(2) quantitative detection of cell free DNA: as shown in fig. 1b, the reaction system is placed in a 3D printing detection kit (the kit entity is as shown in fig. 1c), an image is recorded by a smart phone, a corresponding light absorption value is obtained by analyzing a signal by software in the smart phone, and then a quantitative relationship is established to realize quantitative detection of cell free DNA, the process is as shown in fig. 1D. The invention realizes the field detection and analysis of cell free DNA without large-scale instruments, and has the advantages of convenient carrying, low cost, simple operation and the like.
Preferably, the cell free DNA detection kit is a 3D printing kit made by a 3D printer, and a centrifuge tube, a light source and an optical filter used for being placed in front of a camera of a smart phone are arranged in the 3D printing kit.
Preferably, the 3D printing kit has a size of 150 mm × 75 mm × 30 mm, and the light source has a size of 70 mm × 30 mm; the size of the filter is 12mm × 12 mm.
Preferably, the fitting linear curve processing module is origin software.
The invention also provides an operation method of the cell-free DNA detection system, which comprises the following steps:
s1, preparing DNAzyme hydrogel;
s2, adding 1-2 mu M ABTS and 3-5 mu M H into the prepared DNAzyme hydrogel2O2Carrying out catalytic reaction, then putting the DNAzyme hydrogel subjected to catalytic reaction into a 3D printing kit, and taking pictures of different colors of corresponding solutions under different cell free DNA concentrations by using a smart phone under the illumination condition;
s3, importing the pictures shot by the smart phone into an image processing module to convert the color intensity of the images into digital information;
s4, fitting a linear standard curve by origin software, wherein the abscissa of the obtained curve is the logarithm of the cell free DNA concentration, and the ordinate is the intensity corresponding to the color of the image.
The mechanism of the invention is as follows:
the carrier of the kit is hydrogel for target response, cell free DNA takes E542K as a target to firstly trigger rolling circle amplification to generate macroscopic DNAzyme hydrogel, and the hydrogel is easy to observe by naked eyes and convenient for qualitative detection. In addition, DNAzyme hydrogel has a horseradish peroxidase (HRP) catalytic function, and colorless 2, 2-azo-bis (3-ethylbenzothiazoline-6-sulfonic Acid) (ABTS)2-) Catalysis to green oxidation product ABTS-The intelligent cell phone can collect and analyze images in the kit and establish a relation curve to realize quantitative detection of cell free DNA.
The invention has the beneficial effects that:
in the invention, the model target point of the cell free DNA detection is E542K, and E542K is a hot point mutation of PIK3CA gene and is widely existed in breast cancer, gastric cancer, lung cancer and brain cancer.
The DNAzyme hydrogel and the cell free DNA detection kit designed by the invention can be used for detecting cell free DNA in human serum, the platform has good sensitivity and recovery rate, and can be used for field test.
Drawings
In order to more clearly illustrate one or more embodiments or prior art solutions of the present specification, the drawings that are needed in the description of the embodiments or prior art will be briefly described below, and it is obvious that the drawings in the following description are only one or more embodiments of the present specification, and that other drawings may be obtained by those skilled in the art without inventive effort from these drawings.
FIG. 1a is a schematic diagram of DNAzyme hydrogel platform for detecting cell-free DNA (cfDNA); b is a schematic diagram of the internal structure of the 3D printing kit; c is a 3D printing kit photo in real detection; d is a flow of smartphone analysis;
in FIG. 2, a is color intensity of a sample solution analyzed based on smartphone DNAzyme hydrogel, wherein I is cell free DNA and locked DNA probe added with heme and ABTS/H after rolling circle amplification2O2(ii) a II is when no cell free DNA exists, adding heme and ABTS/H after the padlock DNA probe is subjected to rolling circle amplification reaction2O2(ii) a III is heme and ABTS/H2O2(ii) a IV is ABTS/H2O2(ii) a b is the corresponding absorbance value curve of the sample solution and corresponds to a, cell free DNA, heme, ABTS and H2O2At concentrations of 30nM, 100. mu.M, 2mM and 4mM, respectively; in the c, the left side is a photograph of deoxyribozyme hydrogel in water, the middle part is a photograph of 20 XSYBR Green I dyed DNAzyme hydrogel in the sun, and the right side is a photograph under an ultraviolet lamp; d is an SEM image of DNAzyme hydrogel at a scale of 1 μm;
in fig. 3, a is an optical image of a sample collected by a smart phone; b is a calibration curve of the color intensity of the sample analyzed by the smart phone and the concentration of cell free DNA; c is a linear relation between the color intensity and the logarithm of the concentration of the free DNA of the cells, and the range is from 0.1pM to 1500 nM;
in FIG. 4, a is the selectivity of the DNAzyme detection platform based on the smartphone for cell free DNA detection; b is the anti-interference capability of a DNAzyme detection platform based on a smart phone on cell free DNA detection, the sample and the concentration detected in a and b are respectively the cell free DNA concentration of 30nM, the non-cell free DNA of 300nM, all interference substrates of 300nM, and a blank sample is 20mM phosphate buffer solution (pH 7.4); c is the stability of the DNAzyme platform based on the smart phone at different temperatures; d is the stability of the DNAzyme platform based on the smart phone in different storage time;
in FIG. 5, a is a schematic diagram illustrating the analysis of a biopsy sample; b is the result of E542K quantification in the plasma of gastric cancer patients and healthy individuals by using a DNAzyme hydrogel platform based on a smart phone; c is the quantitative result of conventional droplet digital PCR (ddPCR).
Detailed Description
To make the objects, technical solutions and advantages of the present disclosure more apparent, the present disclosure is further described in detail below with reference to specific embodiments.
It is to be noted that unless otherwise defined, technical or scientific terms used in one or more embodiments of the present specification should have the ordinary meaning as understood by those of ordinary skill in the art to which this disclosure belongs. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items.
The preparation method of the DNA hydrogel comprises the following steps:
(1) mixing 0.5-0.8 mu M of cell free DNA and 0.2-0.5 mu M of padlock DNA probe in T4 ligase buffer solution, incubating at 85-100 ℃ for 8-12min, and cooling to room temperature.
(2) Adding 10-15U/. mu.L of T4 ligase into the cooling solution, culturing at 16 ℃ for 8-12h, heating at 65 ℃ for 8-12min, and adding phi29 DNA polymerase buffer solution.
(3) 8-12mM deoxyribonucleoside triphosphate and 1-5U/. mu.L phi29 DNA polymerase are incubated for 20-24 h at 30 ℃, and then heated for 25-35min at 50-60 ℃.
(4) After the operation is finished, adding a mixed solution of 90-100 mu M of heme and 20-25mM of phosphate buffer (pH 7.4), placing the mixed solution at 3-6 ℃ for 5-7h, and finally washing the heme-loaded DNA hydrogel twice by using the phosphate buffer, so as to obtain the three-dimensional macroscopic DNA hydrogel with the microporous structure. The following is a detailed description by specific examples.
Example 1
The preparation method of the DNAzyme hydrogel comprises the following steps:
(1) mu.M cell free DNA was mixed with 0.3. mu.M padlock DNA probe in T4 ligation buffer solution enzyme, incubated at 90 ℃ for 10min, and then cooled to room temperature.
(2) 10U/. mu.L of T4 ligase was added to the cooled solution, incubated at 16 ℃ for 10 hours, heated at 65 ℃ for 10 minutes and added with phi29 DNA polymerase buffer.
(3)10mM deoxyribonucleoside triphosphate and 2U/. mu.L phi29 DNA polymerase were incubated at 30 ℃ for 24h, followed by heating at 55 ℃ for 30 min.
(4) After the operation is completed, adding a mixed solution of 100 μ M heme and 20mM phosphate buffer (pH 7.4), standing at 4 ℃ for 6h, and finally washing the heme-loaded DNA hydrogel twice with phosphate buffer to obtain a three-dimensional macroscopic DNAzyme hydrogel with a microporous structure, wherein SEM images at a 1 μ M scale are shown in fig. 2d, and color intensity and corresponding absorbance value curves of the obtained DNAzyme hydrogel analysis sample solution are shown in fig. 2a and fig. 2 b; the selectivity of the DNAzyme detection platform consisting of the three-dimensional macroscopic DNAzyme hydrogel to cell free DNA detection, the stability of the anti-interference level at different temperatures and storage times are shown in FIG. 4.
Smart phone image acquisition
Add 2. mu.M ABTS and 4. mu. M H2O2And (3) carrying out catalytic reaction, putting hydrogel into a 3D printing kit, taking pictures of different colors of corresponding solutions in the kit under different cell free DNA concentrations by using a smart phone under the condition that a common light source provides light, and observing that the colors of different kits obtained on the pictures gradually become dark along with the increase of the cell free DNA concentration, wherein the result is shown in figure 3 a.
Analysis of images by software
(1) The corresponding pictures under different cell free DNA concentrations are acquired, the picture shot by the smart phone is guided into an Image J program, and the Image intensity of the Image of the kit is converted into digital information by clicking Analyze → Gels → Plot lanes → Analyze → Measure;
(2) a linear standard curve was fitted by origin software, and the abscissa of the curve obtained as shown in FIG. 3c is the logarithm of the cell-free DNA concentration, and the ordinate is the intensity corresponding to the color of the kit.
Detection of cell-free DNA in real samples (detailed procedure in FIG. 5a)
Human serum samples were first diluted 1:10 and then tested by adding cell free DNA at concentrations of 5pM, 10pM and 50 pM. The concentration of cell free DNA in human serum is obtained by detection by using the detection platform developed by the invention, and the recovery rate of the cell free DNA is calculated, and the data is shown in Table 1. The detection result is consistent with the addition concentration. The Relative Standard Deviation (RSD) of all three groups of samples was less than 7%. The sample recovery rate is 96.8-104.2%, and the analysis requirement is met. Based on the expected results obtained from the spiked serum sample experiments, we tested the measurement performance of E542K directly in the plasma of gastric cancer patients as a real application for cancer diagnosis. The patient group (30.7237.14 ng/mL, replicate analysis n-4) and healthy group (6.227.39 ng/mL) were also significantly differentiated, the quantification results are shown in fig. 5b, and the method is shown to be sufficiently accurate and reproducible in actual sample detection compared to the traditional droplet digital pcr (ddpcr) quantification results in fig. 5 c.
TABLE 1
Those of ordinary skill in the art will understand that: the discussion of any embodiment above is meant to be exemplary only, and is not intended to intimate that the scope of the disclosure, including the claims, is limited to these examples; within the spirit of the present disclosure, features from the above embodiments or from different embodiments may also be combined, steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present description as described above, which are not provided in detail for the sake of brevity.
It is intended that the one or more embodiments of the present specification embrace all such alternatives, modifications and variations as fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalents, improvements, and the like that may be made within the spirit and principles of the invention are intended to be included within the scope of the disclosure.
Sequence listing
<110> university of teacher's university of Anqing
<120> cell free DNA detection kit, detection system and operation method
<130> 1
<141> 2022-01-14
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
ttagagagta tttttttttt tccccctttt ttcagtgatt 40
Claims (10)
1. A kit for detecting cell free DNA is characterized by comprising a padlock DNA probe, T4 ligase, phi29 DNA polymerase and deoxyribonucleoside triphosphate; or a DNAzyme hydrogel formed from cell free DNA, padlock DNA probes, T4 ligase, phi29 DNA polymerase and deoxyribonucleoside triphosphates; the sequence of the padlock DNA probe is shown as SEQ ID NO. 1.
2. The kit for detecting cell-free DNA according to claim 1, wherein the DNAzyme hydrogel is prepared by a method comprising the steps of:
mixing and incubating target cell free DNA, a padlock DNA probe and a T4 ligase buffer solution, and cooling to room temperature to obtain a treatment solution A;
adding T4 ligase into the treatment solution A, culturing, adding phi29 DNA polymerase buffer solution, deoxyribonucleoside triphosphate for incubation and phi29 DNA polymerase, and heating at 50-60 ℃ for 25-35min to obtain treatment solution B;
and step three, adding a mixed solution of 90-100 mu M of heme and 20-25mM of phosphate buffer solution into the treatment solution B, placing for 5-7h at the temperature of 3-6 ℃, and then washing the heme-loaded DNAzyme hydrogel by using the phosphate buffer solution to obtain the DNAzyme hydrogel with a microporous structure.
3. The kit for detecting cell-free DNA according to claim 2, wherein the concentration of the padlock DNA probe is 0.2-0.5. mu.M, the content of T4 ligase is 10-15U/. mu.L, the content of deoxyribonucleoside triphosphate is 8-12mM, and the content of phi29 DNA polymerase is 1-5U/. mu.L.
4. The kit for detecting cell-free DNA according to claim 2, wherein the incubation temperature in the first step is 85-100 ℃ and the incubation time is 8-12 min.
5. The kit for detecting cell-free DNA according to claim 2, wherein the culturing in the second step is carried out by culturing at 16 ℃ for 8-12 hours and then heating at 65 ℃ for 8-12 min.
6. The kit for detecting cell free DNA according to claim 1, wherein the detection limit of the kit on the target cell free DNA is 0.042pM, and the linear range of a detection platform is 0.1pM-1500 nM.
7. A cell free DNA detection system is characterized by comprising a DNAzyme detection platform, a cell free DNA detection kit arranged on the detection platform, a smart phone, an image processing module and a fitting linear curve processing module, wherein pictures shot by the smart phone are led into the image processing module, and the fitting linear curve processing module performs linear fitting on digital information processed by the image processing module.
8. The cell-free DNA detection system according to claim 7, wherein the cell-free DNA detection kit is a 3D printing kit manufactured by a 3D printer, and a centrifuge tube, a light source and a filter used for being placed in front of a camera of a smart phone are arranged in the 3D printing kit.
9. The system for detecting cell-free DNA according to claim 8, wherein the 3D printing kit has a size of 150 mm x 75 mm x 30 mm, and the light source has a size of 70 mm x 30 mm; the size of the optical filter is 12mm multiplied by 12 mm; the fitting linear curve processing module is origin software.
10. The method for operating a cell-free DNA detection system according to any one of claims 7 to 9, comprising the steps of:
s1, preparing DNA hydrogel;
s2, adding 1-2 mu M ABTS and 3-5 mu M H into the prepared DNA hydrogel2O2Carrying out catalytic reaction, then putting the DNA hydrogel after catalytic reaction into a 3D printing kit, and taking pictures of different colors of corresponding solutions under different cell free DNA concentrations by using a smart phone under the illumination condition;
s3, importing the pictures shot by the smart phone into an image processing module to convert the color intensity of the images into digital information;
s4, fitting a linear standard curve by origin software, wherein the abscissa of the obtained curve is the logarithm of the cell free DNA concentration, and the ordinate is the intensity corresponding to the color of the image.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557459A (en) * | 2017-09-29 | 2018-01-09 | 沈阳药科大学 | A kind of method that DNA hydrogels and DNAzyme detections SNP is used in combination |
| CN112063691A (en) * | 2020-09-18 | 2020-12-11 | 湖北医药学院 | Method for detecting single-chain target nucleic acid sequence based on G4-heme DNase system |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107557459A (en) * | 2017-09-29 | 2018-01-09 | 沈阳药科大学 | A kind of method that DNA hydrogels and DNAzyme detections SNP is used in combination |
| CN112063691A (en) * | 2020-09-18 | 2020-12-11 | 湖北医药学院 | Method for detecting single-chain target nucleic acid sequence based on G4-heme DNase system |
Non-Patent Citations (5)
| Title |
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| SHAOWEI LIU ET AL.: ""Smartphone-Based Pure DNAzyme Hydrogel Platform for Visible and Portable Colorimetric Detection of Cell-Free DNA"", 《ACS SENS.》, vol. 7, pages 658 - 665 * |
| XIAOXIA MAO ET AL.: "" Fabrication of DNAzyme-functionalized hydrogel and its application for visible detection of circulating tumor DNA"", 《SENSORS AND ACTUATORS B: CHEMICAL》, vol. 285, pages 385 - 390 * |
| XIAOXIA MAO ET AL.: ""Fabrication of DNAzyme-functionalized hydrogel and its application for visible detection of circulating tumor DNA"", 《SENSORS AND ACTUATORS B: CHEMICAL》, vol. 285, pages 385 - 390 * |
| ZHANMIN LIU ET AL.: ""Amplified visual detection of microRNA-378 through a T4 DNA ligase-mediated circular template specific to target and target-triggering rolling circle amplification"", 《ANAL.METHODS》, vol. 11, pages 2082 - 2088 * |
| 毛晓霞: ""新型DNA水凝胶的制备及其在生化分析中的应用"", 《中国优秀硕士学位论文全文数据库 工程科技I辑》, no. 2, pages 1 - 134 * |
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