CN114369643A - Cell-free DNA detection kit, detection system and operation method - Google Patents

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

Cell-free DNA detection kit, detection system and operation method

technical field

The invention relates to the technical field of intelligent tumor detection, in particular to a cell-free DNA detection kit, a detection system and an operation method.

Background technique

In the past few decades, DNA hydrogels have been widely used in drug release, tissue engineering, environmental analysis, biosensing and other fields due to their precise programmability, stability and biocompatibility, especially Pure DNA hydrogels composed of pure DNA have attracted extensive attention. The construction of pure DNA hydrogels is generally through self-assembly or enzymatic amplification of DNA fragments, so pure DNA hydrogels have the advantage of being easier to synthesize than DNA hybrid hydrogels.

Cell-free DNA (cfDNA), a typical DNA fragment, has attracted widespread attention as a non-invasive diagnostic biomarker. In addition, cell-free DNA plays an important role in biomedical diagnosis, such as early diagnosis of cancer, assessment of tumor stage, selection of effective treatment options, monitoring of 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 the gold standard for cell-free DNA detection. In addition, detection methods such as electrochemistry, beading, emulsion, amplification and magnetic platforms, and Amplification Refractory Mutation System (ARMS) have been established in recent years to improve the performance of cell-free DNA analysis. Although these test methods are promising, they still rely on sophisticated instruments and cannot meet the needs of portable detection, so that they cannot be used more widely. Therefore, it is very necessary to develop an instrument-free and visualized cell-free DNA detection platform for rural areas with relatively scarce resources.

So far, smartphones, as optical quantitative instruments, have been widely used in biomedicine, biological analysis, environmental monitoring, and other fields by virtue of their powerful imaging hardware, built-in sensing systems, and advanced software design.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide a cell-free DNA detection kit, detection system and operation method, which can quickly, conveniently and sensitively detect cell-free DNA in human serum.

Based on the above purpose, the present invention provides a cell-free DNA detection kit, comprising a padlock DNA probe, T4 ligase, phi29 DNA polymerase and deoxyribonucleoside triphosphate; or a cell-free DNA, padlock DNA probe A DNAzyme hydrogel formed by needle, T4 ligase, phi29 DNA polymerase and deoxyribonucleoside triphosphate; the sequence of the padlock DNA probe is shown in SEQ ID NO.1.

The sequence of the padlock DNA probe shown in SEQ ID NO.1 is:

5’-phosphorylated-TTAGAGAGTATTTTTTTTTTTCCCCCTTTTTCAGTGATT-3’

As a kind of optional embodiment, the preparation method of described DNAzyme hydrogel comprises the steps:

Step 1. After mixing and incubating the free DNA of the target cell with the lock-type DNA probe and the T4 ligase buffer solution, it is cooled to room temperature to obtain the A treatment solution;

Step 2: Add T4 ligase to the A treatment solution, after culturing, adding phi29 DNA polymerase buffer, deoxyribonucleoside triphosphate and phi29 DNA polymerase for incubation, and then heating at 50-60 ° C for 25-35min, get B treatment solution;

Step 3. Add a mixture of 90-100μM heme and 20-25mM phosphate buffer to the treatment solution B, and place it at 3-6°C for 5-7h, and then use phosphate buffer to hydrocoagulate the heme-loaded DNA. The gel is washed to obtain a DNAzyme hydrogel with a microporous structure.

Preferably, the incubation temperature in the first step is 85-100° C., and the time is 8-12 min.

Preferably, the method for culturing in the second step is culturing at 16° C. for 8-12 hours, and then heating at 65° C. for 8-12 minutes.

The present invention also provides a cell-free DNA detection system, comprising a DNAzyme detection platform, a cell-free DNA detection kit set on the detection platform, a smart phone, an image processing module and a fitting linear curve processing module. The image is imported into the image processing module, and the fitting linear curve processing module performs linear fitting on the digital information processed by the image processing module.

The construction and detection method of the detection platform are as follows:

(1) Preparation of the detection platform and qualitative detection of cell-free DNA: As shown in Figure 1a: cell-free DNA is treated with a padlock DNA probe as a template under the action of T4 ligase, phi29 DNA polymerase and deoxyribonucleoside triphosphate. DNAzyme hydrogel was formed by rolling circle amplification. The G-quadruplex in DNAzyme hydrogel is a kind of deoxyribozyme, which catalyzes H ₂ O ₂ and converts colorless 2,2-azo-bis(3- Ethylbenzothiazoline-6-sulfonic acid) (ABTS ^2- ) catalyzes the green oxidation product ABTS- ^to generate visible signal to realize the qualitative detection of cell-free DNA;

(2) Quantitative detection of cell-free DNA: As shown in Figure 1b, put the reaction system into the 3D printing detection kit (the actual kit is shown in Figure 1c), record the image through the smartphone, and analyze the signal through the software in the mobile phone. The corresponding absorbance value, and then establish a quantitative relationship to achieve quantitative detection of cell-free DNA, the process is shown in Figure 1d. The invention realizes the on-site detection and analysis of cell-free DNA without needing large-scale instruments, and has the advantages of convenient portability, 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 the 3D printing kit is provided with a centrifuge tube, a light source and a filter for placing in front of a smartphone camera.

Preferably, the size of the 3D printing kit is 150 mm×75 mm×30 mm, the size of the light source is 70 mm×30 mm, and the size of the filter is 12 mm×12 mm.

Preferably, the fitting linear curve processing module is origin software.

The present invention also provides an operation method of the cell-free DNA detection system, comprising the following steps:

S1, prepare DNAzyme hydrogel;

S2. Add 1-2 μM ABTS and 3-5 μM H ₂ O ₂ to the prepared DNAzyme hydrogel for catalytic reaction, and then put the catalytically reacted DNAzyme hydrogel into the 3D printing kit. Under the condition of light, Use a smartphone to take pictures to record the pictures of different colors of the corresponding solutions under different cell-free DNA concentrations;

S3. Import the picture taken by the smartphone into the image processing module to convert the color intensity of the image into digital information;

S4. Fitting a linear standard curve by origin software, the abscissa in 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 present invention:

The carrier of the kit is a target-responsive hydrogel. The cell-free DNA first triggers rolling circle amplification with E542K as the target to generate a macroscopic DNAzyme hydrogel. The hydrogel is easy to observe with the naked eye and facilitate qualitative detection. In addition, DNAzyme hydrogel has horseradish peroxidase (HRP) catalytic function, which converts colorless 2,2-azo-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS ^2- ) catalyzes the green oxidation product ABTS ^- to generate a visible signal, the smart phone can collect and analyze the images in the kit, and establish a relationship curve to realize the quantitative detection of cell-free DNA. The invention can carry out on-site detection and analysis of cell-free DNA, It has the advantages of convenient portability, low cost and simple operation.

Beneficial effects of the present invention:

In the present invention, the model target for cell-free DNA detection is E542K, which is a hot spot mutation of the PIK3CA gene and widely exists in breast cancer, gastric cancer, lung cancer and brain cancer.

The DNAzyme hydrogel and cell-free DNA detection kit designed in the present invention can be used to detect cell-free DNA in human serum. The platform has good sensitivity and recovery rate, and can be tested on-site. The method is based on a portable smart phone. The availability of the platform provides a new and promising diagnostic tool for resource-constrained fields, with simple operation, short detection time, equipment cost savings, and no need for large instrumentation.

Description of drawings

In order to more clearly illustrate one or more embodiments of the present specification or the technical solutions in the prior art, the following briefly introduces the accompanying drawings used in the description of the embodiments or the prior art. Obviously, in the following description The accompanying drawings are only one or more embodiments of the present specification, and for those of ordinary skill in the art, other drawings can also be obtained from these drawings without any creative effort.

In Figure 1, a is a schematic diagram of the detection of cell-free DNA (cfDNA) by the DNAzyme hydrogel platform; b is a schematic diagram of the internal structure of the 3D printing kit; c is the photo of the 3D printing kit in the actual detection; d is the process of smartphone analysis ;

In Figure 2, a is the color intensity of the sample solution based on smartphone DNAzyme hydrogel analysis, where I is the addition of heme and ABTS/H ₂ O ₂ after cell-free DNA and padlock DNA probe rolling circle amplification; II is no In the presence of cell-free DNA, heme and ABTS/H ₂ O ₂ are added to the padlock DNA probe after rolling circle amplification reaction; III is heme and ABTS/H ₂ O ₂ ; IV is ABTS/H ₂ O ₂ ; b is the corresponding absorbance value curve of the above sample solution, and corresponds to a. The concentrations of cell-free DNA, heme, ABTS and H ₂ O ₂ are 30 nM, 100 μM, 2 mM and 4 mM, respectively; the left side of c is the deoxyribozyme hydrogel The photo of the glue in water, the middle is the photo of 20×SYBR Green I stained DNAzyme hydrogel under sunlight, the right is the photo under UV light; d is the SEM image of DNAzyme hydrogel at 1 μm scale;

In Figure 3, a is the optical image of the sample collected by the smartphone; b is the calibration curve of the color intensity of the sample analyzed by the smartphone and the cell-free DNA concentration; c is the linear relationship between the color intensity and the logarithm of the cell-free DNA concentration, ranging from 0.1pM～1500nM;

In Figure 4, a is the selectivity of the smartphone-based DNAzyme detection platform for cell-free DNA detection; b is the anti-interference ability of the smartphone-based DNAzyme detection platform for the detection of cell-free DNA. The samples and concentrations detected in a and b are respectively is cell-free DNA concentration of 30 nM, non-cell-free DNA of 300 nM and all interfering substrates at 300 nM, blank is 20 mM phosphate buffer (pH=7.4); c is the stability of the smartphone-based DNAzyme platform at different temperatures d is the stability of the smartphone-based DNAzyme platform under different storage times;

In Figure 5, a is a schematic diagram of human biopsy sampling analysis; b is the quantitative result of E542K in the plasma of gastric cancer patients and healthy individuals using a smartphone-based DNAzyme hydrogel platform; c is the quantitative result of traditional droplet digital PCR (ddPCR).

Detailed ways

In order to make the objectives, technical solutions and advantages of the present disclosure clearer, the present disclosure will be further described in detail below with reference to specific embodiments.

It should be noted that, unless otherwise defined, the technical or scientific terms used in one or more embodiments of the present specification shall have the usual meanings understood by those with ordinary skill in the art to which this disclosure belongs. "Comprises" or "comprising" and similar words mean that the elements or things appearing before the word encompass the elements or things recited after the word and their equivalents, but do not exclude other elements or things.

The preparation method of the DNA hydrogel involved in the present invention comprises the following steps:

(1) Mix 0.5-0.8 μM cell-free DNA and 0.2-0.5 μM padlock DNA probe in T4 ligase buffer solution, incubate at 85-100°C for 8-12 min, and then cool to room temperature.

(2) Add 10-15U/μL of T4 ligase to the cooling solution, incubate at 16°C for 8-12h, then heat at 65°C for 8-12min, and then add phi29 DNA polymerase buffer.

(3) 8～12mM deoxyribonucleoside triphosphate and 1～5U/μL phi29 DNA polymerase were incubated at 30℃ for 20～24h, and then heated at 50～60℃ for 25～35min.

(4) After the operation is completed, add a mixed solution of 90-100 μM heme and 20-25 mM phosphate buffer (pH=7.4) and place it at 3-6 °C for 5-7 hours. The hydrogel was washed twice to obtain a three-dimensional macroscopic DNA hydrogel with a microporous structure. The following is a detailed description through specific examples.

Example 1

The preparation method of DNAzyme hydrogel comprises the following steps:

(1) 0.6 μM cell-free DNA and 0.3 μM padlock DNA probe were mixed in T4 ligation buffer solution enzyme, incubated at 90°C for 10 min, and then cooled to room temperature.

(2) 10U/μL of T4 ligase was added to the cooling solution, incubated at 16°C for 10h, heated at 65°C for 10min, and then added with phi29 DNA polymerase buffer.

(3) 10mM deoxyribonucleoside triphosphate and 2U/μL phi29 DNA polymerase were incubated at 30°C for 24h, and then heated at 55°C for 30min.

(4) After the operation is completed, add a mixed solution of 100 μM heme and 20 mM phosphate buffer (pH=7.4) and place it at 4°C for 6 hours. Finally, the heme-loaded DNA hydrogel is washed twice with phosphate buffer. That is, a three-dimensional macroscopic DNAzyme hydrogel with a microporous structure can be obtained. The SEM image of the 1 μm scale is shown in Figure 2d. The obtained DNAzyme hydrogel analysis sample solution color intensity and corresponding absorbance value curve are shown in Figure 2a and Figure 2b; Figure 4 shows the selectivity and anti-interference level of DNAzyme detection platform composed of three-dimensional macroscopic DNAzyme hydrogel for cell-free DNA detection at different temperatures and storage times.

Smartphone to acquire image

2μM ABTS and 4μM H ₂ O ₂ were added to catalyze the reaction, and the hydrogel was placed in the 3D printing kit. Under the condition of light provided by a common light source, take pictures with a smartphone to record the corresponding kits under different cell-free DNA concentrations. For pictures of different colors of the solution, it can be observed that with the increase of cell-free DNA concentration, the colors of different kits on the pictures gradually become darker, and the results are shown in Figure 3a.

Analysis of the image by the software

(1) The obtained pictures corresponding to different cell-free DNA concentrations are imported into the Image J program through the pictures taken by the smartphone, and click Analyze→Gels→Plot lanes→Analyze→Measure to convert the color intensity of the image of the kit to a number information;

(2) Fitting a linear standard curve by origin software, in the curve obtained as shown in Figure 3c, the abscissa 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 (the specific process is shown in Figure 5a)

Human serum samples were first diluted 1:10 and then assayed by addition of cell-free DNA at concentrations of 5pM, 10pM and 50pM. The detection platform developed by the present invention is used to detect the concentration of cell-free DNA in human serum and calculate the recovery rate of cell-free DNA. The data are shown in Table 1. The detection results were consistent with the added concentration. The relative standard deviation (RSD) of the three groups of samples were all less than 7%. The recovery rate of sample addition was 96.8%～104.2%, which met the analysis requirements. Based on the expected results obtained from experiments with spiked serum samples, we tested the measurement performance for the direct determination of E542K in the plasma of gastric cancer patients as a real application in cancer diagnosis. The patient group (30.72 ± 37.14 ng/mL, repeated analysis n = 4) and the healthy group (6.22 ± 7.39 ng/mL) could also be significantly differentiated, and the quantitative results are shown in Figure 5b, and are consistent with traditional droplet digital PCR (ddPCR) in Figure 5c Comparison of quantitative results shows that this method has sufficient accuracy and reproducibility in actual sample detection.

Table 1

Those of ordinary skill in the art should understand that the discussion of any of the above embodiments is only exemplary, and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples; under the spirit of the present disclosure, the above embodiments or Technical features in different embodiments may also be combined, steps may be implemented in any order, and there are many other variations of the different aspects of one or more embodiments of this specification as described above, which are not in detail for the sake of brevity supply.

The embodiment or embodiments of this specification are intended to cover all such alternatives, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included within the protection scope of the present disclosure.

sequence listing

<110> Anqing Normal University

<120> A 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

<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.

Images