WO2024098470A1 - Nucleic acid absolute quantification detection system and nucleic acid absolute quantification method - Google Patents

Abstract

Disclosed herein are a nucleic acid absolute quantification detection system and a nucleic acid absolute quantification method. The nucleic acid absolute quantification detection system comprises: four-arm polyethylene glycol acrylate, thiol-polyethylene glycol-thiol, a primer of a target nucleic acid molecule, and a nucleic acid amplification reaction reagent, wherein the mass ratio of the four-arm polyethylene glycol acrylate to the thiol-polyethylene glycol-thiol is (1-2):1. According to the present application, a unique hydrogel nucleic acid amplification reaction system is designed, the inherent porous structure of the hydrogel is utilized to uniformly confine the target nucleic acid molecules in a sample to be detected in pores, and amplification and fluorescence imaging are performed on the whole system; finally, according to the number of fluorescent bright spots in the hydrogel, the specific initial copy number of the target nucleic acid molecules in the sample is calculated in combination with the Poisson distribution theorem, thereby achieving absolute quantification, eliminating the need for an additional physical structure and the creation of a standard curve, and thus significantly reducing the cost and simplifying the process.

Description

A nucleic acid absolute quantitative detection system and a nucleic acid absolute quantitative method Technical Field

The present application belongs to the technical field of molecular biology and relates to a nucleic acid absolute quantitative detection system and a nucleic acid absolute quantitative method.

Background technique

Nucleic acids include DNA and RNA, which encode genetic information and act as storage media that allow genetic material to be passed between generations. Nucleic acid amplification and quantification has always been one of the core technologies in the field of molecular biology, and has been applied to molecular sequencing, gene expression analysis, gene mutation research, early molecular diagnosis of diseases, single nucleotide polymorphisms and drug screening, and plays an important role in research fields.

At present, the most widely used nucleic acid quantification technology is real-time fluorescence quantitative polymerase chain reaction (qPCR). This method observes the amplification of the template in real time according to the added fluorescent molecular probe signal during detection. The detection result is output in the form of a linear amplification signal, and the starting template copy number of the unknown sample is estimated based on the amplification curve of the known standard sample. Therefore, qPCR technology is a relative nucleic acid quantitative method, and its sensitivity and accuracy are limited. In recent years, digital polymerase chain reaction (dPCR) technology has developed rapidly. This technology separates single nucleic acid molecules in a single compartment for PCR reaction to identify the presence of target molecules. At present, dPCR technology mainly includes microfluidic chip array reaction chamber or droplet digital analysis technology and emulsion droplet digital analysis technology. The dPCR technology based on microfluidic devices and chips has limited scalability and low detection throughput. Emulsion droplet digital analysis technology uses the method of emulsion sealing magnetic beads to provide a higher throughput dPCR technology. However, this technology still has many defects, such as when the template and magnetic beads are not separated in the same droplet, the target template cannot be detected, the polymerase inhibitors in the DNA extract will affect the efficiency of the amplification reaction, and the complexity of the workflow and thermal cycle amplification.

In summary, how to provide a method for absolute nucleic acid quantification with high throughput, stable technology and simple operation is one of the problems that urgently need to be solved in the field of molecular biology.

Summary of the invention

The present application provides a nucleic acid absolute quantitative detection system and a nucleic acid absolute quantitative method. The present application designs a new nucleic acid absolute quantitative detection system, which can quickly and accurately realize nucleic acid absolute quantification without the need for a standard curve, and has simple operation and low cost.

In the first aspect, the present application provides a nucleic acid absolute quantitative detection system, which contains: four-arm polyethylene glycol acrylate, bis-thiol polyethylene glycol, primers of target nucleic acid molecules and reagents for nucleic acid amplification reaction, wherein the mass ratio of the four-arm polyethylene glycol acrylate and the bis-thiol polyethylene glycol is (1-2):1, including but not limited to 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 16:11, 1.5:1, 1.7:1, 1.8:1 or 2:1, preferably 16:11.

The present application designs a nucleic acid absolute quantitative detection system, which utilizes a hydrogel system composed of two monomers, four-arm polyethylene glycol acrylate (4Arm-PEG-AC) and bis-thiol polyethylene glycol (SH-PEG-SH). The system can spontaneously polymerize into a gel at room temperature without the need for initiation, thereby avoiding affecting the nucleic acid amplification reaction system. In addition, by controlling the mass ratio of the two, the speed and efficiency of nucleic acid amplification are further improved and the fluorescence diffusion is improved, thereby achieving rapid and accurate quantitative detection.

Preferably, the weight average molecular weight (Mw) of the four-arm polyethylene glycol acrylate is 5000-20000, including but not limited to 5100, 5500, 6000, 8000, 10000, 12000, 15000, 18000 or 19000.

Preferably, the weight average molecular weight of the bis-thiol polyethylene glycol is 1000-10000, including but not limited to 1200, 1500, 1600, 2000, 3000, 5000, 6000, 8000 or 9000.

In the present application, by controlling the weight average molecular weight of four-arm polyethylene glycol acrylate and dithiol polyethylene glycol in the detection system, the shape and size of the fluorescent bright spot can be further controlled to further facilitate detection and analysis.

Preferably, in the nucleic acid absolute quantitative detection system, the weight average molecular weight of the four-arm polyethylene glycol acrylate is 10,000 and the weight average molecular weight of the bis-thiol polyethylene glycol is 3,400.

Preferably, the target nucleic acid molecule includes any one or a combination of at least two of the Escherichia coli 23S ribosomal gene, the cytokeratin 19 gene or the HPV gene.

It is understood that common nucleic acid amplification methods in the art are applicable to the present application.

Preferably, the nucleic acid amplification reaction may include any one of loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), polymerase chain amplification (PCR) or rolling circle amplification (RCA).

It will be understood that the common nucleic acid amplification reaction reagents in the field are suitable for the present application, such as LAMP reaction reagents WarmStart LAMP 2×Master Mix (Biolabs) and LAMP Fluorescent Dye (Biolabs).

It can be understood that by designing primers according to different target nucleic acid molecules, it is possible to detect any target nucleic acid molecule.

Preferably, the nucleic acid sequence of the forward external primer of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.1.

Preferably, the nucleic acid sequence of the forward inner primer of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.2.

Preferably, the nucleic acid sequence of the reverse external primer of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.3.

Preferably, the nucleic acid sequence of the reverse inner primer of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.4.

Preferably, the nucleic acid sequence of the forward loop guide of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.5.

Preferably, the nucleic acid sequence of the reverse loop guide of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.6.

SEQ ID NO.1(F3):5’-GGCGTTAAGTTGCAGGGTAT-3’.

SEQ ID NO.2(FIP):

5’-CGGTTCGGTCCTCCAGTTAGTGTTTTCCCGAAACCCGGTGATCT-3’.

SEQ ID NO.3(B3):5’-TCACGAGGCGCTACCTAA-3’.

SEQ ID NO.4(BIP):

5’-TAGCGGATGACTTGTGGCTGGTTTTTCGGGGAGAACCAGCTATC-3’.

SEQ ID NO.5 (LoopF): 5’-ACCTTCAACCTGCCCATG-3’.

SEQ ID NO.6 (LoopB): 5’-GTGAAAGGCCAATCAAACC-3’.

Preferably, the nucleic acid sequence of the primer of the cytokeratin 19 gene includes the sequence shown in SEQ ID NO.7.

SEQ ID NO.7(Primer):CCTGTTCCGTTCTTCCTTCA.

Preferably, the nucleic acid sequence of the forward primer of the HPV gene includes the sequence shown in SEQ ID NO.8.

Preferably, the nucleic acid sequence of the reverse primer of the HPV gene includes the sequence shown in SEQ ID NO.9.

SEQ ID NO.8(FP):5’-CTCTTTGGCTGCCTAGTGAG-3’.

SEQ ID NO.9(RP):5’-GCGTGCAACATATTCATCCG-3’.

In a second aspect, the present application provides the use of the nucleic acid absolute quantification detection system described in the first aspect in the preparation of a nucleic acid absolute quantification kit.

In a third aspect, the present application provides a nucleic acid absolute quantification kit, which contains four-arm polyethylene glycol acrylate, bis-thiol polyethylene glycol, primers of target nucleic acid molecules, and reagents for nucleic acid amplification reactions.

In the nucleic acid absolute quantification kit of the present application, four-arm polyethylene glycol acrylate and dithiol polyethylene glycol can spontaneously polymerize into a hydrogel at room temperature, thereby being able to load and separate the target nucleic acid molecules to be tested, so that the target nucleic acid molecules are amplified at the initial position and the products do not diffuse, forming amplified fluorescent spots with distinct shapes, thereby realizing quantitative detection.

In a fourth aspect, the present application provides a method for absolute quantification of nucleic acids, the method comprising:

Prepare the nucleic acid absolute quantitative detection system described in the first aspect, perform nucleic acid amplification reaction, perform fluorescence imaging of the reaction product, calculate the number of fluorescent bright spots, and calculate the copy number of the target nucleic acid molecule based on the number of fluorescent bright spots combined with the Poisson distribution theorem.

In the present application, a hydrogel nucleic acid amplification reaction system is constructed, the inherent porous structure of the hydrogel is used to uniformly confine the target nucleic acid molecules in the sample to be tested in the pores, and the entire system is amplified and fluorescently imaged. Finally, based on the number of fluorescent bright spots in the hydrogel and combined with the Poisson distribution theorem, the specific starting copy number of the target nucleic acid molecules in the sample is calculated to achieve absolute quantification without the need for additional physical structures and the preparation of standard curves, which significantly reduces costs and simplifies the process.

Preferably, the nucleic acid amplification reaction comprises any one of a loop-mediated isothermal amplification reaction, a recombinase polymerase amplification reaction, a polymerase chain amplification reaction or a rolling circle amplification reaction.

It can be understood that the common fluorescence analysis methods in the field are applicable to the present application. For example, the software hAmplicon_count-master can be selected to count the amplification points and quantify the fluorescence signals. The quantification of the target nucleic acid can be achieved through the Poisson distribution formula, and the concentration of the target nucleic acid in the original sample can be further calculated, thereby achieving absolute quantification of the nucleic acid without the need for a standard curve.

Poisson distribution can calculate the probability Pr(n) that each microcompartment in the hydrogel contains n copies of the target, that is, if the average number of target copies in each microcompartment is C, then:

Enter n = 0 to get the probability that the corresponding micro-segment is empty for a given C value:

Pr(0)＝e ^-C

For a large number of microcompartments, the observed proportion of empty microcompartments (E) is an unbiased estimate of Pr(0), so:

E＝e ^-C

C＝-ln(E)

C is the number of copies per microcompartment. To convert it to copies per microliter, divide it by the volume of the microcompartment:

Combining the above two equations, we can get:

By definition, there are:

Where N _neg is the number of negative microcompartments and N is the total number of droplets.

Combining the above two equations, we can get:

或者C＝ln(N)-ln(N _neg)

Or C = ln(N) - ln(N _neg )

Concentration refers to the concentration of target nucleic acid molecules in the original sample.

Compared with the prior art, this application has the following beneficial effects:

The present application designs a unique hydrogel nucleic acid amplification reaction system, which utilizes the inherent porous structure of the hydrogel to uniformly confine the target nucleic acid molecules in the sample to be tested in the pores, and performs amplification and fluorescence imaging on the entire system. Finally, based on the number of fluorescent bright spots in the hydrogel and combined with the Poisson distribution theorem, the specific starting copy number of the target nucleic acid molecules in the sample is calculated to achieve absolute quantification without the need for additional physical structures or the preparation of standard curves, significantly reducing costs and simplifying the process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1A is a comparison of the shapes and sizes of fluorescent bright spots in hydrogels of monomers with different molecular weights.

Figure 1B is a comparison of the shapes and sizes of fluorescent bright spots in hydrogels of monomers with different molecular weights.

FIG2 is a diagram showing amplification points and diffusion in hydrogels with different monomer mass ratios.

Figure 3 is a diagram of the optical path of a light sheet fluorescence microscope.

Figure 4 is a real picture of a light sheet fluorescence microscope.

FIG5 is a schematic diagram of slicing a hydrogel after amplification using a laser sheet in a light sheet fluorescence microscope.

FIG. 6 is a diagram showing the nucleic acid amplification point counting results.

FIG. 7 is a diagram showing the copy number detection results in Example 1.

FIG8 is a graph showing the consistency analysis results of the hydrogel LAMP and ddPCR results in Example 1.

Figure 9 is a graph showing the consistency analysis results of the hydrogel RCA and ddPCR results in Example 2.

Figure 10 is a graph showing the consistency analysis results of the hydrogel PCR and ddPCR results in Example 3.

Detailed ways

To further illustrate the technical means and effects adopted by the present application, the present application is further described below in conjunction with the embodiments and drawings. It is understood that the specific implementation methods described herein are only used to explain the present application, rather than to limit the present application.

If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

Conventional nucleic acid extraction methods in the field are applicable to the present application. For example, in a specific embodiment, a sample containing the nucleic acid to be tested is collected, and the cells or bacteria in the sample are lysed using a nucleic acid extraction kit to expose the nucleic acid to the solution. Taking an E. coli bacterial liquid sample as an example, to quantify the E. coli 23S ribosomal DNA, 10 μL of the sample is mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies), placed at 65°C for 6 minutes, and then placed at 95°C for 4 minutes, the sample DNA is extracted, and diluted 10 times for later use.

In order to achieve quantitative analysis of target nucleic acid molecules, the present application designs primers for the target region.

Taking the LAMP amplification of the Escherichia coli 23S gene as an example, but not limited to LAMP, the present application is also applicable to other nucleic acid amplification reactions of other target nucleic acids such as PCR, RPA, RCA, etc.

The sequences and concentrations of the primers are as follows:

23S forward outer primer SEQ ID NO.1 (F3, 0.2μM): 5’-GGCGTTAAGTTGCAGGGTAT-3’.

23S reverse external primer SEQ ID NO.3 (B3, 0.2μM): 5’-TCACGAGGCGCTACCTAA-3’.

23S forward inner primer SEQ ID NO.2 (FIP, 1.6 μM):

5’-CGGTTCGGTCCTCCAGTTAGTGTTTTCCCGAAACCCGGTGATCT-3’.

23S reverse inner primer SEQ ID NO.4 (BIP, 1.6 μM):

5’-TAGCGGATGACTTGTGGCTGGTTTTTCGGGGAGAACCAGCTATC-3’.

23S forward loop primer SEQ ID NO.5 (LoopF, 0.4μM): 5’-ACCTTCAACCTGCCCATG-3’.

23S reverse loop primer SEQ ID NO.6 (LoopB, 0.4μM): 5’-GTGAAAGGCCAATCAAACC-3’.

In this application, taking LAMP as an example, the hydrogel nucleic acid amplification reaction system contains: WarmStart LAMP 2X Master Mix (Biolabs), primer mixture, LAMP Fluorescent Dye (Biolabs), 4Arm-PEG-AC, SH-PEG-SH, 10-fold diluted test sample DNA and sterile water.

Gelation of the reaction system: After the prepared system is mixed, it is sealed in a 250μL centrifuge tube and can be gelled within 5 minutes at room temperature.

Nucleic acid amplification: transfer the centrifuge tube to a thermal amplification instrument for amplification (LAMP reaction conditions are 65°C amplification for 25 minutes).

Result interpretation: After amplification, a light sheet fluorescence microscope was used to scan and detect the fluorescence signal, and the software hAmplicon_count-master was used to count the fluorescent spots.

Commonly used hydrogel monomers such as ultraviolet crosslinking, thermal crosslinking, and catalyst crosslinking, such as hydroxyethyl methacrylate (HEMA) and polyethylene glycol diacrylate (PEGDA), can be polymerized under the action of initiator HMPP and ultraviolet light, or polymerized under the action of initiator APS and high temperature. These polymerization conditions may damage the nucleic acid amplification system, affect the amplification efficiency and fluorescence intensity, and although amplification can be performed, nucleic acids will be degraded during ultraviolet polymerization, resulting in quantitative results that are smaller than expected. Such temperature-responsive gelling hydrogels as agarose and gelatin are affected by temperature because the sol and gel states are affected by temperature, and the nucleic acid amplification process also requires specific temperature conditions, which is not conducive to establishing a stable amplification system based on hydrogels. Therefore, the present application first needs to establish a mild hydrogel polymerization condition. Attempt to amplify in a milder agarose gel, and no fluorescent spots can be observed in the results. The polymer with thiol group can spontaneously polymerize into gel at room temperature without initiation, and will not affect the nucleic acid amplification reaction system. Therefore, the hydrogel system composed of two monomers, four-arm polyethylene glycol acrylate (4Arm-PEG-AC) and double thiol polyethylene glycol (SH-PEG-SH), was selected to form a gel. Nine different hydrogels were formed by polymerizing 4Arm-PEG-AC with different molecular weights (Mw: 5000, 10000 and 20000) and SH-PEG-SH with different molecular weights (Mw: 1000, 3400 and 10000). The shape and size of the fluorescent bright spots in the hydrogel were observed. The results are shown in Figures 1A and 1B. According to the results, the hydrogel system composed of two monomers, 4Arm-PEG-AC, Mw: 10000 and SH-PEG-SH, Mw: 3400, can be selected to form a gel.

The ratio of the above two monomers plays a decisive role in the hydrogel gelation speed, porosity, pore size, etc., and thus affects the speed and efficiency of nucleic acid amplification and fluorescence diffusion. The hydrogel LAMP system with different monomer ratios was gelled and amplified. The best monomer ratio was selected by observing the fluorescence intensity and fluorescence diffusion of the amplification point after amplification. Figure 2 shows the amplification images with monomer ratios (4Arm-PEG-AC: SH-PEG-SH) of 2:1, 16:11 and 1:1, respectively. It can be seen that the diffusion is the least when the monomer ratio is 16:11, so the mass ratio of 4Arm-PEG-AC: SH-PEG-SH can be selected as 16:11.

In this application, a microscope can be built according to the optical path of the light sheet fluorescence microscope. In the illumination optical path (Figure 3), the laser is first collimated into a Gaussian beam with a diameter of about 3.5 mm, and then passes through a sandwich structure composed of an aspheric lens (f = 8 mm) and two cylindrical lenses (CL1 and CL2, f = 20 mm and 12.7 mm) to convert the circular beam into an ellipse with a short optical path. The beam expansion ratios in the z-axis and y-axis directions are × 0.4 and × 1.6, respectively, forming an elliptical beam with a size of 5.6 × 1.4 mm. There is an orthogonally placed adjustable mechanical slit (0-8 mm) behind the sandwich structure to further cut off the beam and adjust the height and thickness of the laser sheet. The light sheet fluorescence microscope is shown in Figure 4. After the laser passes through the illumination cylindrical lens (CL3, f = 50 mm), it finally produces a large and wide laser sheet, which optically separates the hydrogel layer by layer. In the detection light path, a ×2 infinity-corrected wide-field detection path (Leica Plan Fluor 4×/0.13 objective lens + Thorlabs TTL100 tube lens) was constructed, which is perpendicular to the illumination light path to collect fluorescence signals. The amplified sample centrifuge tube is mounted on the motorized translation stage through a tube clamp. Within a few seconds required for signal readout, the centrifuge tube and the hydrogel quickly pass through the scanning laser sheet along the z axis. The schematic diagram of slicing the amplified hydrogel with the laser sheet in the light sheet fluorescence microscope is shown in Figure 5. The high-speed camera (Phantom Miro C320) continuously records images from the laser sheet plane at a high speed of up to hundreds of frames per second.

In this application, python+OpenCV is used to write the counting software hAmplicon_count-master. The result data after the sample scan is in video format. After importing the video into the software, obtain the length, width, frame rate and encoding format of the video. Create a new video according to the length, width, frame rate and encoding format of the imported video as the output result of the software. Read each frame of the imported video one by one, convert it into a grayscale image, and use the Hough circle transform to detect the fluorescent amplification points in the image after removing the noise by median blur. The basic idea of the Hough circle transform is to assume that every non-zero pixel on the image may be a potential point on a circle, generate a cumulative coordinate plane by voting, and set a cumulative weight to locate the circle. The equation of the circle in the Cartesian coordinate system is:

(xa) ² +(yb) ² = r ²

Where (a, b) is the center of the circle and r is the radius, it can also be expressed as:

x＝a+rcos(θ)

y＝b+rsin(θ)

Therefore, in the three-dimensional coordinate system composed of abr, a point can uniquely determine a circle. In the Cartesian xy coordinate system, all circles passing through a certain point are mapped to a cone in the abr coordinate system. The circle equations of all points on the same circle in the xy coordinate system are the same, and they are mapped to the same point in the abr coordinate system, so in the abr coordinate system, the point should have the total number of pixels of the circle intersecting with the curve. By judging the number of intersections (cumulative) of each point in abr, points greater than a certain threshold are considered to be circles. After Hough circle detection, the detection result circle is drawn into the original image, and at the same time, it is judged and counted whether the circle comes from the same amplification point as the circle in the previous frame. Finally, the counting result is drawn to the original image, synchronized with the original video, and written into the new video for output.

In the present application, LAMP is taken as an example, but it is not limited to LAMP. The present method is also applicable to other nucleic acid amplification reactions such as PCR, RPA, RCA, etc.

(1) The 25 μL hydrogel nucleic acid amplification reaction system was as follows: 12.5 μL WarmStart LAMP 2X Master Mix (Biolabs), 2.5 μL primer mixture, 0.5 μL LAMP Fluorescent Dye (Biolabs), 1.92 mg 4Arm-PEG-AC, 1.32 mg SH-PEG-SH, 2.5 μL 10-fold diluted sample DNA, and 2 μL sterile water.

(2) Gelation of the reaction system: After the prepared system is mixed evenly, it is sealed in a 250 μL centrifuge tube and can be gelled within 5 minutes at room temperature.

(3) Nucleic acid amplification: The centrifuge tube was transferred to a thermal amplification instrument for amplification (LAMP reaction conditions were 65°C for 25 min).

(4) Result interpretation: After amplification, a light-sheet fluorescence microscope was used to scan and detect the fluorescence signal, and the software hAmplicon_count-master was used to count the fluorescent spots. Each fluorescent spot represented a target, and the number of fluorescent spots was calculated to quantify the target.

In the present application, according to the three-dimensional fluorescent nucleic acid amplification point signals obtained by scanning with a light-sheet fluorescence microscope, the software hAmplicon_count-master is used to count the amplification points and quantify the fluorescence signals. The Poisson distribution formula can be used to quantify the target nucleic acid, and the concentration of the target nucleic acid in the original sample can be further calculated, thereby achieving absolute quantification of the nucleic acid without the need for a standard curve.

Poisson distribution can calculate the probability Pr(n) that each microcompartment in the hydrogel contains n copies of the target, that is, if the average number of target copies in each microcompartment is C, then:

Enter n = 0 to get the probability that the corresponding micro-segment is empty for a given C value:

Pr(0)＝e ^-C

For a large number of microcompartments, the observed proportion of empty microcompartments (E) is an unbiased estimate of Pr(0), so:

E＝e ^-C

C＝-ln(E)

C is the number of copies per microcompartment. To convert it to copies per microliter, divide it by the volume of the microcompartment:

Combining the above two equations, we can get:

By definition, there are:

Where N _neg is the number of negative microcompartments and N is the total number of droplets.

Combining the above two equations, we can get:

或者C＝ln(N)-ln(N _neg)

Or C = ln(N) - ln(N _neg )

Concentration refers to the concentration of target nucleic acid in the original sample.

Specifically, the present application uses the Escherichia coli 23S ribosomal gene, cytokeratin 19 (CK19) gene and HPV gene as examples to verify the nucleic acid absolute quantitative detection system and method of the present application.

Example 1

This example uses the hydrogel LAMP system to quantitatively analyze the Escherichia coli 23S ribosomal gene.

Primers (SEQ ID NO.1 to SEQ ID NO.6) were designed for the 23S ribosomal gene of Escherichia coli. 10 μL of Escherichia coli culture medium (ATCC: 25922) was mixed evenly with 90 μL of DNA Extraction Solution 1.0 (Biosearch Technologies), placed at 65°C for 6 minutes, and then placed at 95°C for 4 minutes to extract sample DNA. The 25μL reaction system of hydrogel LAMP is: 12.5μL WarmStart LAMP 2X Master Mix (NEB), 2.5μL 10× primer mixture, 0.5μL LAMP Fluorescent Dye (NEB), 1.92mg 4Arm-PEG-AC (Mw: 10000), 1.32mg SH-PEG-SH (Mw: 3400), 2.5μL 10-fold diluted test sample DNA, 2μL sterile water, prepare a system containing different copy numbers of the test sample DNA (copy number 1 to 13, named CN1 to CN13 respectively), and use the system without adding the test sample DNA as the control (NTC). After the prepared system is mixed, it is sealed in a 250μL centrifuge tube and placed at room temperature for 5 minutes to gel. After gelation, the hydrogel is placed in an amplification instrument for amplification (LAMP reaction conditions are 65℃ amplification for 25 minutes). After amplification, the sample was mounted on the electric translation stage through a tube clamp, and the fluorescence signal was detected using a light sheet fluorescence microscope. The fluorescence amplification points were counted using the software hAmplicon_count-master. Each system was repeated once. For example, the quantitative results of one slice are shown in Figure 6. As shown in Figure 7, it is shown that the method of the present application can accurately detect samples with copy numbers ranging from a single copy (CN1) to 13 copies (CN13) with high resolution. In addition, the experimental results were compared with the results of the analysis of the same samples using the Bio-Red ddPCR system, which showed excellent consistency and an intraclass correlation coefficient of 0.996 was obtained, as shown in Figure 8.

Example 2

This example uses a hydrogel RCA system to quantitatively analyze the cytokeratin 19 (CK19) gene.

Primers were designed for the CK19 gene (SEQ ID NO.7). The 25 μL reaction system of hydrogel RCA was: 2.5 μL of 10× phi29 DNA Polymerase Reaction Buffer (NEB), 0.25 μL of recombinant albumin, 5 μL of dNTPs, 2.5 μL of 10× primer mixture, 1 μL of phi29 DNA Polymerase (NEB), 1.92 mg 4Arm-PEG-AC (Mw: 10000), 1.32 mg SH-PEG-SH (Mw: 3400), 2.5 μL of the sample DNA to be tested, 0.25 μL of Sybr Green dye, and then sterile water was added to make up to 25 μL. After the prepared system was mixed, it was sealed in a 250 μL centrifuge tube and placed at room temperature for 5 minutes to gel. After gelation, the hydrogel was placed in an amplification instrument for amplification (RCA reaction conditions were 30°C for 120 minutes). After amplification, the sample was mounted on the electric translation stage through a tube clamp, and the fluorescence signal was detected using a light sheet fluorescence microscope. The software hAmplicon_count-master was used to count the fluorescence amplification points. The experimental results were compared with the results of the same sample analyzed by the Bio-Red ddPCR system, which showed excellent consistency and obtained an intraclass correlation coefficient of 0.994, as shown in Figure 9.

Example 3

This example uses a hydrogel PCR system to quantitatively analyze HPV DNA.

Primers were designed for HPV DNA (SEQ ID NO.8 to SEQ ID NO.9). The 25 μL reaction system for hydrogel PCR was: 12.5 μL PrimeSTAR Max Premix (2×), 2.5 μL 10× primer mixture, 0.5 μL Eva Green dye, 1.92 mg 4Arm-PEG-AC (Mw: 10000), 1.32 mg SH-PEG-SH (Mw: 3400), 2.5 μL DNA of the sample to be tested, and then sterile water was added to make up to 25 μL. After the prepared system was mixed, it was sealed in a 250 μL centrifuge tube and placed at room temperature for 5 minutes to gel. After gelation, the hydrogel was placed in an amplification instrument for amplification (PCR reaction conditions were 98°C for 20 seconds and 68°C for 1 minute). After amplification, the sample was mounted on the electric translation stage through a tube clamp, and the fluorescence signal was detected using a light sheet fluorescence microscope, and the fluorescence amplification points were counted using the software hAmplicon_count-master. The experimental results were compared with the results of the same samples analyzed using the Bio-Red ddPCR system, which showed excellent consistency and an intraclass correlation coefficient of 0.996, as shown in Figure 10.

In summary, the present application designs a unique hydrogel nucleic acid amplification reaction system, which utilizes the inherent porous structure of the hydrogel to uniformly confine the target nucleic acid molecules in the sample to be tested in the pores, and performs amplification and fluorescence imaging on the entire system. Finally, based on the number of fluorescent bright spots in the hydrogel and combined with the Poisson distribution theorem, the specific starting copy number of the target nucleic acid molecules in the sample is calculated to achieve absolute quantification without the need for additional physical structures or the preparation of standard curves, significantly reducing costs and simplifying the process.

The applicant declares that the present application uses the above-mentioned embodiments to illustrate the detailed methods of the present application, but the present application is not limited to the above-mentioned detailed methods, that is, it does not mean that the present application must rely on the above-mentioned detailed methods to be implemented. The technicians in the relevant technical field should understand that any improvement to the present application, the equivalent replacement of the raw materials of the product of the present application, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present application.

Claims (10)

A nucleic acid absolute quantitative detection system, comprising: four-arm polyethylene glycol acrylate, bis-thiol polyethylene glycol, primers of target nucleic acid molecules and reagents for nucleic acid amplification reaction; Wherein, the mass ratio of the four-arm polyethylene glycol acrylate to the dithiocarbyl polyethylene glycol is (1-2):1. The nucleic acid absolute quantitative detection system according to claim 1, wherein the weight average molecular weight of the four-arm polyethylene glycol acrylate is 5000 to 20000; Preferably, the weight average molecular weight of the bis-mercapto polyethylene glycol is 1,000 to 10,000. According to the nucleic acid absolute quantitative detection system according to claim 1 or 2, wherein the target nucleic acid molecule includes any one or a combination of at least two of the Escherichia coli 23S ribosomal gene, the cytokeratin 19 gene or the HPV gene. The nucleic acid absolute quantitative detection system according to any one of claims 1 to 3, wherein the nucleic acid amplification reaction comprises any one of a loop-mediated isothermal amplification reaction, a recombinase polymerase amplification reaction, a polymerase chain amplification reaction or a rolling circle amplification reaction. The nucleic acid absolute quantitative detection system according to claim 3, wherein the nucleic acid sequence of the forward external primer of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.1; Preferably, the nucleic acid sequence of the forward inner primer of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.2; Preferably, the nucleic acid sequence of the reverse external primer of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.3; Preferably, the nucleic acid sequence of the reverse inner primer of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.4; Preferably, the nucleic acid sequence of the forward loop guide of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.5; Preferably, the nucleic acid sequence of the reverse loop guide of the Escherichia coli 23S ribosomal gene includes the sequence shown in SEQ ID NO.6. According to the nucleic acid absolute quantitative detection system according to claim 3, the nucleic acid sequence of the primer of the cytokeratin 19 gene includes the sequence shown in SEQ ID NO.7. The nucleic acid absolute quantitative detection system according to claim 3, wherein the nucleic acid sequence of the forward primer of the HPV gene includes the sequence shown in SEQ ID NO.8; Preferably, the nucleic acid sequence of the reverse primer of the HPV gene includes the sequence shown in SEQ ID NO.9. Use of the nucleic acid absolute quantitative detection system according to any one of claims 1 to 7 in the preparation of a nucleic acid absolute quantitative kit. A method for absolute quantification of nucleic acid, comprising: Prepare the nucleic acid absolute quantitative detection system according to any one of claims 1 to 7, perform nucleic acid amplification reaction, perform fluorescence imaging of the reaction product, calculate the number of fluorescent bright spots, and calculate the copy number of the target nucleic acid molecule based on the number of fluorescent bright spots combined with the Poisson distribution theorem. The method for absolute quantification of nucleic acid according to claim 9, wherein the nucleic acid amplification reaction comprises any one of a loop-mediated isothermal amplification reaction, a recombinase polymerase amplification reaction, a polymerase chain amplification reaction or a rolling circle amplification reaction.

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