Microfluidic chip and portable fluorescence detection system for nucleic acid detection

Abstract

The present application relates to a microfluidic chip for nucleic acid detection and a portable fluorescence detection system, the fluorescence detection system includes a laser light source, a first filter, a microfluidic chip, a second filter, the microfluidic chip is provided with a circular optical detection area for holding samples, the bottom of the optical detection area is provided with an optical detection area of ​​a layer of densely stacked microspheres, the laser light source is arranged below the optical detection area, the first filter is arranged between the laser light source and the optical detection area, and its transmission wavelength is the excitation wavelength of the sample; the second filter is arranged just above the optical detection area, and its transmission wavelength is the emission wavelength of the sample. The fluorescence detection system only uses a laser light source, two filters, and a microfluidic chip with a simple structure. It has a simple structure, is easy to carry, and has low cost. It can enhance the intensity of the excitation light through the microcavity effect and focusing effect of the microspheres, thereby improving the fluorescence signal of the sample and improving the sensitivity and detection limit of the fluorescence detection technology.

Description

Technical Field

[0001] The present application relates to the field of nucleic acid detection, and specifically to a microfluidic chip and a portable fluorescence detection system for nucleic acid detection. Background Art

[0002] The nucleic acid test of the new coronavirus generally requires centralized sampling and then sending the samples to the laboratory for unified testing, which prolongs the waiting time for nucleic acid test results. In order to speed up nucleic acid testing, it is very important to provide a sensitive, portable, and low-cost fluorescence detection system. Summary of the invention

[0003] In order to improve the sensitivity of fluorescent detection of nucleic acids, the present application provides a microfluidic chip that can increase the excitation light intensity by more than 2 times, and develops a low-cost portable fluorescence detection system based on the microfluidic chip. The fluorescence detection system can achieve sensitive and rapid detection of nucleic acids.

[0004] The technical solutions provided by this application are as follows:

[0005] In a first aspect, the present application provides a microfluidic chip, wherein the microfluidic chip is provided with an optical detection area, wherein the optical detection area is in a circular cake shape, and a layer of densely packed microspheres is provided at the bottom thereof.

[0006] Based on the above technical solution, the microspheres are polystyrene microspheres.

[0007] Based on the above technical solution, the diameter of the microspheres is 2 to 50 microns.

[0008] Based on the above technical solution, the diameter of the optical detection area is 2 to 10 mm.

[0009] In a second aspect, the present application provides a fluorescence detection system, the fluorescence detection system comprising:

[0010] The above-mentioned microfluidic chip is used to hold samples;

[0011] A laser light source, which is disposed below the optical detection area and is used to provide excitation light for the sample;

[0012] A first filter, which is disposed between the laser light source and the optical detection area and has a transmission wavelength equal to the excitation wavelength of the sample; and

[0013] The second filter is arranged directly above the optical detection area, and its transmission wavelength is the emission wavelength of the sample.

[0014] On the basis of the above technical solution, the fluorescence detection system further includes a fluorescence signal processing unit, which is used to capture the fluorescence image above the second filter and analyze the concentration of the target detection object in the sample based on the fluorescence image.

[0015] In a third aspect, the present application provides applications of the above-mentioned microfluidic chip and the above-mentioned fluorescence detection system in the field of fluorescence detection.

[0016] In a fourth aspect, the present application provides a detection method based on the above-mentioned fluorescence detection system, comprising the following steps:

[0017] Inject standard solutions of different concentrations into the optical detection area of ​​the microfluidic chip, place the microfluidic chip in a fluorescence detection system, turn on the laser light source, take a fluorescence image of the standard solution, and make a standard curve based on the relationship between the fluorescence intensity and the concentration of the standard solution;

[0018] The sample to be tested is injected into the optical detection area of ​​the microfluidic chip, and the microfluidic chip is placed in a fluorescence detection system. The laser light source is turned on to capture the fluorescence image of the sample to be tested, and the concentration of the sample to be tested is calculated based on the fluorescence intensity.

[0019] On the basis of the above technical solution, the sample to be tested is modified with a fluorescent probe, the fluorescent probe is rhodamine 6G, the transmission wavelength of the first filter is 532nm, and the transmission wavelength of the second filter is 600nm.

[0020] Based on the above technical solution, the sample to be tested is a nucleic acid extract.

[0021] Compared with the prior art, this application has the following advantages and beneficial effects:

[0022] 1. The microfluidic chip provided in the present application can enhance the intensity of the excitation light through the microcavity effect and focusing effect of the microspheres, thereby improving the fluorescence signal of the sample and improving the sensitivity and detection limit of the fluorescence detection technology.

[0023] 2. The fluorescence detection system provided in this application only uses a laser light source, two filters, and a simple microfluidic chip. It has a simple structure, is easy to carry, and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1Schematic diagram of the fluorescence detection system and detection method provided in this application; wherein, Figure 1 a is a schematic diagram of the process of nucleic acid detection; Figure 1 b is a schematic diagram of the fluorescence detection system provided in Example 1; Figure 1 c shows the mobile app interface and photos taken with the phone.

[0026] Figure 2 The schematic diagram of the fluorescence detection system provided in this application; wherein, Figure 2 a is a schematic diagram of the detection light path of the fluorescence detection system. Figure 2 b is a schematic diagram of the structure of the microfluidic chip. Figure 2 c is a schematic diagram of the cavity effect; Figure 2 d is a schematic diagram of the focusing effect.

[0027] Figure 3 It is a schematic diagram of the fluorescence experiment results; Figure 3 a shows the fluorescence image obtained from the Rhodamine 6G solution experiment; Figure 3 b shows the fluorescence image obtained from the SARS-CoV-2 recombinant plasmid experiment; Figure 3 c shows Figure 3 a. Figure 3 b The result after digital processing; Figure 3 d shows the comparison results of noise amplification and signal amplification.

[0028] Figure 4 is the experimental result of the recombinant plasmid solution; among them, Figure 4 a shows the fluorescence images of recombinant plasmid solutions with different concentrations; Figure 4 b shows the standard curve of the recombinant plasmid solution. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0030] like Figure 2 a. Figure 2As shown in Figure b, the microfluidic chip provided by the present application is provided with a disc-shaped optical detection area, and a layer of densely stacked microspheres is provided at the bottom of the optical detection area to enhance the fluorescence signal of the sample. Specifically, the microfluidic chip is a PDMS-glass bonded chip, the size of the PDMS layer is 20×20×5mm, the glass bottom plate is 25×25×1mm, and a cylindrical chamber with a diameter of 8mm is provided in the center of the microfluidic chip, i.e., the optical detection area; the microspheres are polystyrene microspheres with a diameter of 8μm, and the arrangement is densely stacked, with a single layer covering the entire bottom of the optical detection area. The microfluidic chip can utilize the enhancement effect of the microspheres on the fluorescence signal of the surrounding liquid to improve the detection sensitivity and detection limit level of nucleic acid samples. Compared with traditional microfluidic chips without microspheres, the detection limit using this microfluidic chip can reach 250copies / mL. The method of spreading the microspheres all over the bottom of the optical detection area is as follows: 10 μL of microsphere dispersion is dropped onto the glass bottom plate of the cylindrical chamber, and then gently tilted left and right to evenly spread the microsphere dispersion on the bottom of the cylindrical chamber, and then left to stand at room temperature for about 1-2 hours, and the microspheres are spread all over the bottom of the cylindrical chamber by the evaporation self-assembly effect of the microspheres. The diameter of polystyrene microspheres is 8 μm, and the cost is 1,200 yuan / 5 mL, which is low cost and can be efficiently and mass-produced.

[0031] Figure 2 c. Figure 2 d shows the principle of microspheres enhancing the fluorescence signal of samples, namely the microcavity effect ( Figure 2 c) and focusing effect ( Figure 2 d). The microcavity effect means that each microsphere is an optical microcavity in a whispering gallery mode. After the excitation light enters such an optical microcavity, part of the incident angle of light will be continuously reflected on the surface of the microsphere. When the length of the arc through which the incident light is reflected is an integer multiple of the wavelength, the light intensity at the reflection position will be enhanced, thereby enhancing the fluorescence signal on the surface of the microsphere. The focusing effect means that the microsphere will focus the incident light at the focal point like a lens. The focal point is located in the liquid, and the excitation light signal here is enhanced, thereby enhancing the fluorescence signal at this location.

[0032] like Figure 1As shown in FIG. 1 , the fluorescence detection system provided by the present application includes a laser light source, a first filter, a microfluidic chip, and a second filter. The optical detection area of ​​the microfluidic chip is used to hold samples, and a laser light source for providing sample excitation light is provided below the optical detection area; the first filter is provided between the laser light source and the optical detection area, and its transmission wavelength is the excitation wavelength of the sample; the second filter is provided directly above the optical detection area, and its transmission wavelength is the emission wavelength of the sample. The excitation light of the fluorescence detection system changes the optical path after being reflected by a high-efficiency reflector, and then passes through the first filter to filter the incident light with a large wavelength difference from the excitation light. The filtered incident light enters the sample, and the fluorescent molecules in the sample are excited to emit fluorescent signals. The fluorescent signals pass through the second filter to filter out the fluorescent signals. The fluorescence detection system also includes a fluorescent signal processing unit, which collects fluorescent signals by shooting the fluorescent images above the second filter, and analyzes the concentration of the target detection object in the sample according to the fluorescent images. Further, the fluorescent signal processing unit is a smart phone, which shoots fluorescent images through the mobile phone camera, and uses an APP to analyze the captured fluorescent images. The APP can perform fluorescent area recognition and RGB analysis on fluorescent images taken by mobile phones, and calculate the sample concentration based on the RGB analysis results. The fluorescence detection system can be used for fluorescence detection of viral nucleic acid samples. Based on the effect of laser excitation of samples to produce fluorescent signals, combined with smart phones, on-site analysis of sample fluorescence signals can be achieved. Compared with ordinary fluorescence detection equipment, the fluorescence detection system provided in this application is more portable, accurate and sensitive. The use of microspheres to amplify the fluorescence signal can reduce the detection limit of new coronavirus nucleic acid to 250 copies / mL.

[0033] The detection method based on the above-mentioned fluorescence detection system provided in the present application includes the following steps: injecting standard solutions of different concentrations into the optical detection area of ​​the microfluidic chip, placing the microfluidic chip in the fluorescence detection system, turning on the laser light source, taking a fluorescence image of the standard solution, and making a standard curve based on the relationship between the fluorescence intensity and the concentration of the standard solution; injecting the sample to be tested into the optical detection area of ​​the microfluidic chip, placing the microfluidic chip in the fluorescence detection system, turning on the laser light source, taking a fluorescence image of the sample to be tested, and calculating the concentration of the sample to be tested based on the fluorescence intensity.

[0034] The present application verifies the superiority of the above fluorescence detection system by detecting fluorescent samples. The fluorescence detection system can detect extremely low concentrations of rhodamine 6G solution (10 -6 g / L), and the fluorescence signal collected was nearly twice that collected by the fluorescence detection system without microspheres. Compared with the traditional microfluidic fluorescence detection method, the detection method of the present application has higher sensitivity and detection limit level, and its further promotion and application is of great significance to the portability and intelligence of nucleic acid detection.

[0035] The fluorescence detection system and detection method involved in the present application are further described in detail below with reference to the accompanying drawings and specific embodiments.

[0036] Example 1

[0037] Figure 1 a is a schematic diagram of the nucleic acid detection process. A throat swab is used to obtain a patient's body fluid sample, and the RNA on the throat swab is extracted. The extract is then mixed with the Cas12 solution. After the mixture is allowed to stand for 30 minutes, 200 μL is injected into the optical detection area of ​​the microfluidic chip and placed in the fluorescence detection system for detection. Figure 1 b is a schematic diagram of the fluorescence detection system. The internal optical path includes a laser light source, a reflector, and two filters, and the microfluidic chip is placed between the two filters. Figure 1 cThe left side is the mobile app interface, and the right side is the photo taken by the mobile phone.

[0038] Figure 2 a is a schematic diagram of the detection light path, in which there is a layer of self-assembled microspheres (each microsphere has a diameter of 8 μm) at the bottom of the optical detection area of ​​the microfluidic chip containing the sample. Figure 2 b is a schematic diagram of the structure of the microfluidic chip, where the microspheres cover a circular area with a diameter of about 6-8 mm. Figure 2 As shown in c, each microsphere is an optical microcavity in a whispering gallery mode. After the excitation light passes through such an optical microcavity, the fluorescence signal on the surface of the microsphere can be enhanced. Figure 2 d is a schematic diagram of the focusing effect, showing the light-gathering effect of the microspheres. Part of the incident light is gathered by the microspheres to form a stronger excitation light intensity at the focus, thereby enhancing the fluorescence signal at the focus. The two effects work together to enhance the fluorescence signal of the sample.

[0039] 1. Fabrication of microfluidic chips by standard UV lithography:

[0040] First, draw the template pattern according to the designed chip structure; then, carve the template pattern on the mask, and use ultraviolet lithography technology to evenly apply SU8-2050 photoresist on the silicon wafer. After ultraviolet exposure, the silicon wafer is rinsed with developer to obtain the PDMS template. After pouring the unsolidified PDMS on the template and baking it in an oven at 75°C for one hour, remove the solidified PDMS to obtain the PDMS channel layer; then use a plasma cleaner to bond the PDMS channel layer to a glass slide coated with a layer of PDMS to obtain a microfluidic chip.

[0041] 2. Make microsphere layer by self-assembly method:

[0042] The microspheres used in this embodiment were purchased from Zhongke Leiming (Beijing) Technology Co., Ltd., 8μm polystyrene microspheres (PS), 1200 yuan / 5mL. The purchased PS microsphere solution was diluted 10 times with deionized water to obtain a PS microsphere stock solution, and then a 10μL pipette was used to drop 5μL of the PS microsphere stock solution on the bottom of the optical detection area of ​​the prepared microfluidic chip. Slightly tilt the microfluidic chip to flatten the droplets and evenly disperse the microspheres in the droplets. After that, place the microfluidic chip on a horizontal table and let it stand. After the water evaporates, the microspheres will self-assemble and arrange in the optical detection area to form a layer of densely packed microspheres.

[0043] 3. Prepare Rhodamine 6G solution:

[0044] The solid powder of Rhodamine 6G used in this embodiment was purchased from McLean (R817245 Rhodamine 6G, high purity, 95%). When preparing the mother solution of Rhodamine 6G, weigh 0.1g of Rhodamine 6G powder and dissolve it in 100mL of deionized water to obtain 1g / L of Rhodamine 6G mother solution, which is transferred into a polyethylene bottle and can be stored for 6 months at 0-4°C in the dark. When the Rhodamine 6G standard solution needs to be used for the experiment, take out the Rhodamine 6G mother solution, dilute the Rhodamine 6G mother solution with deionized water, and obtain the Rhodamine 6G standard solution of the specified concentration. The diluted Rhodamine 6G standard solution needs to be used on the same day.

[0045] 4. Obtaining the COVID-19 recombinant plasmid solution:

[0046] The new coronavirus recombinant plasmid solution used in this example was purchased from GenScript Biotech Co., Ltd., and the sequence is shown in Seq_1.

[0047] 5. Acquisition of sample fluorescence signal:

[0048] A laser light source with a specific wavelength is used to excite the fluorescence signal of the sample. The laser light source was purchased from Ji'an Chaonai Technology Co., Ltd. The laser light source is cylindrical, 13*47mm in size, and has a power of 400mW. Since the excitation wavelengths of different fluorescent markers are different, the emission wavelengths of the laser light sources used for different samples are also different. The excitation light emitted by the laser light source changes its propagation direction after being reflected by the reflector, and the stray light is filtered through the first filter to obtain an excitation light with a relatively single wavelength. The transmission wavelength of the first filter is the excitation wavelength of the sample. The transmitted excitation light then enters the sample and excites the sample to emit fluorescence. The light signal directly above the sample is more complex, so a second filter needs to be set, and the transmission wavelength of the second filter is the emission wavelength of the sample. The fluorescence signal of the sample can be obtained above the second filter.

[0049] 6. Fluorescence signal processing:

[0050] The fluorescence signal passing through the second filter is received by the smartphone camera on the top of the fluorescence detection system housing. Use a smartphone to take a photo of the sample signal area inside the fluorescence detection system, and then use the self-developed smartphone APP to analyze and process the obtained photos. First open the mobile phone APP, and after the database is loaded, that is, the words "LoadingOpenCV Libraries..." disappear at the bottom, you can proceed to the next step. Then click the "Select Photo" button to enter the album, select the sample photo taken, and it will automatically return to the APP interface after selecting the photo. Then click the "Pixel Read" button, the reading APP will automatically identify the fluorescent area where the sample is located in the original image and intercept a 50*50mm fluorescent image, and display it in the APP interface, and then analyze its RGB information, bring it into the standard curve, and convert the sample nucleic acid concentration.

[0051] APP image analysis principle: APP will read the pixel values ​​of the three HSV channels of each pixel point in order from left to right (x-axis) and from top to bottom (y-axis). Since the rest of the image is black or close to black except for the sample area, when the H value of a pixel point at a certain position is between 30 and 100, the position will be used as the base point (x0, y0), and 20 pixels will be used as the step size to judge the pixel values ​​in the square area from x0 to x0+100 and y0 to y0+100. If the H values ​​of the 6*6=36 pixels are all between 30 and 100, the point will be used as the upper left base point to intercept a 50*50 image, and the average BGR value of this small image will be calculated. Then the value will be substituted into the standard curve to calculate the nucleic acid concentration and judge whether it is negative or positive.

[0052] 7. Rhodamine 6G solution experiment:

[0053] In this embodiment, four experimental groups were set up, namely, a deionized water without ball group, a deionized water with ball group, and a rhodamine 6G (10 -6 g / L) without ball group, rhodamine 6G (10 -6 g / L) with a ball group, the liquid volume is 200μL, and a laser light source (400mW) with a wavelength of 532nm is used as the excitation light source for fluorescence experiments. The camera parameters of the smartphone are ISO: 400 and shutter time: 1 / 17s. The first filter transmits a wavelength of 532nm, and the second filter transmits a wavelength of 600nm. The fluorescence images obtained in the experiment are as follows Figure 3As shown in a, the fluorescence signal of the deionized water without microspheres is the weakest, and the fluorescence signal of the rhodamine 6G with microspheres is the strongest. At the same time, it can be seen that the fluorescence signal of the deionized water with microspheres group is slightly larger than that of the deionized water without microspheres group. The reason may be that the incident light is scattered when passing through the microsphere layer at the bottom of the liquid. Compared with the liquid without microspheres, when the incident light passes through the liquid with microspheres, more light with a wavelength of 600nm is scattered, which increases the background noise. The four groups of experimental results were digitized to obtain the RGB information of the image. Figure 3 c. Figure 3 As shown in Figure d, by comparing the experimental results of the two groups of deionized water, it can be found that the fluorescence signal intensity of the group with microspheres is 1.1 times that of the group without microspheres, indicating that the use of microspheres increased the background noise by 1.1 times. The fluorescence intensity of the Rhodamine 6G group with microspheres is 1.8 times that of the group without microspheres. Considering that deionized water also has a fluorescent signal, and the use of microspheres will enhance the fluorescence signal of deionized water, the actual fluorescence intensity of the two groups of Rhodamine 6G should be the RGB difference between it and the corresponding deionized water group. After this conversion, the fluorescence intensity of the Rhodamine 6G group with microspheres is 2.2 times that of the group without microspheres, that is, the use of microspheres increases the concentration of 10 -6 The fluorescence signal of the 100 g / L Rhodamine 6G solution was amplified by 2.2 times.

[0054] 8. COVID-19 recombinant plasmid solution experiment:

[0055] This example experiments on the COVID-19 recombinant plasmid solution, and also sets up four groups of experiments, namely, blank group without balls (DEPC treated water, Thermo Fisher AM9920), blank group with balls (DEPC treated water + microspheres), solution group without balls (recombinant plasmid solution, 500 copies / mL), solution group with balls (recombinant plasmid solution + microspheres, 500 copies / mL). The experimental results are shown in Figure 3 As shown in b, similar to the experimental results of rhodamine 6G solution, the use of microspheres not only increases the fluorescence signal of the recombinant plasmid solution, but also increases the fluorescence signal of DEPC-treated water, i.e., background noise. Figure 3 c. Figure 3 As shown in Figure d, after digital processing of the experimental results, it can be seen that the background noise of the blank ball group increased by 1.17 times. After subtracting the noise signal, the fluorescence signal of the solution ball group increased by 1.73 times. The experiment shows that the microspheres amplified the fluorescence signal of the recombinant plasmid solution with a concentration of 500 copies / mL by 1.73 times. The fluorescence intensity of recombinant plasmid solutions of different concentrations was measured using a microfluidic chip. Six groups of solutions were used, with concentrations of 250 copies / mL, 500 copies / mL, 5000 copies / mL, 5×10 4 copies / mL, 5×10 5 copies / mL, 5×106 copies / mL. Figure 4 a shows the fluorescence images of recombinant plasmid solutions with different concentrations, and the fluorescence intensity changes with the concentration. Figure 4 As shown in Figure b, the horizontal axis is the logarithm of the concentration and the vertical axis is the fluorescence intensity. It can be seen that the fluorescence intensity of the solution is linearly correlated with the logarithm of the concentration, indicating that the RGB analysis method can obtain the concentration of the sample.

[0056] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A fluorescence detection system, characterized in that: The fluorescence detection system comprises: A microfluidic chip is used to hold samples. The microfluidic chip is provided with an optical detection area. The optical detection area is in a round cake shape, and the bottom of the optical detection area is a glass bottom plate. A layer of densely packed microspheres is provided on the glass bottom plate. A laser light source, which is disposed below the optical detection area and is used to provide excitation light for the sample; A first filter, which is disposed between the laser light source and the optical detection area and has a transmission wavelength equal to the excitation wavelength of the sample; and A second filter, which is disposed directly above the optical detection area and has a transmission wavelength equal to the emission wavelength of the sample; The fluorescence detection system further includes a fluorescence signal processing unit, which is used to capture a fluorescence image above the second filter and analyze the concentration of the target detection object in the sample according to the fluorescence image; The method of analyzing the concentration of the target detection object in the sample according to the fluorescent image specifically includes the following steps: performing fluorescent area recognition and RGB analysis on the fluorescent image, and calculating the sample concentration according to the RGB analysis result; The sample is a liquid sample, which can generate a fluorescent signal under the action of excitation light. When the sample is injected into the optical detection area, the microsphere is located in the sample.

2. The fluorescence detection system according to claim 1, characterized in that: The microspheres are polystyrene microspheres.

3. The fluorescence detection system according to claim 1, characterized in that: The diameter of the microspheres is 2 to 50 microns.

4. The fluorescence detection system according to claim 1, characterized in that: The diameter of the optical detection area is 2 to 10 mm.

5. Application of the fluorescence detection system according to any one of claims 1 to 4 in the field of fluorescence detection.

6. A detection method using the fluorescence detection system according to claim 1, characterized in that: The steps include: Inject standard solutions of different concentrations into the optical detection area of ​​the microfluidic chip, place the microfluidic chip in a fluorescence detection system, turn on the laser light source, take a fluorescence image of the standard solution, and make a standard curve based on the relationship between the fluorescence intensity and the concentration of the standard solution; The sample to be tested is injected into the optical detection area of ​​the microfluidic chip, and the microfluidic chip is placed in a fluorescence detection system. The laser light source is turned on to capture the fluorescence image of the sample to be tested, and the concentration of the sample to be tested is calculated based on the fluorescence intensity.

7. The detection method according to claim 6, characterized in that: The method of calculating the concentration of the sample to be tested according to the fluorescence intensity includes: identifying the fluorescent area where the sample is located in the fluorescent image and intercepting a fluorescent image of a specific size, analyzing its RGB information, and bringing it into the standard curve to convert the sample nucleic acid concentration.

8. The detection method according to claim 6, characterized in that: The sample to be tested is a nucleic acid extract.

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