A multifunctional microfluidic system for rapid nucleic acid detection and its application

Abstract

This invention provides a multifunctional microfluidic system for rapid nucleic acid detection and its application. The system includes: an array microcavity chip for high-throughput rapid screening of nucleic acid samples, and a microfluidic droplet chip or microfluidic microcavity chip for rapid quantification of nucleic acid gene copy numbers. The array microcavity chip, microfluidic droplet chip, or microfluidic microcavity chip share a rapid thermal cycling platform, enabling a single test to be completed within 15 minutes. This invention can achieve both high-throughput rapid screening of samples and rapid and accurate quantification of viral load in confirmed cases, with advantages such as short detection time and high detection accuracy. In COVID-19 nucleic acid testing, it can perform high-throughput rapid screening of nucleic acid samples from suspected positive patients and can also perform rapid, sensitive, and accurate absolute quantitative analysis of viral load in infected cases, effectively assessing the viral load in patients and possessing profound clinical testing significance.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid detection, and more specifically to a multifunctional microfluidic system for rapid nucleic acid detection and its applications. Background Technology

[0002] Currently, the novel coronavirus infection caused by the SARS-CoV-2 virus has swept the globe. Its high transmissibility and mortality rate constantly threaten human lives and have had a significant impact on global economic and social stability. However, due to the current lack of effective vaccines or treatments, early diagnosis and timely isolation are crucial to curbing the spread of COVID-19. Therefore, rapid on-site testing in key epidemic prevention sites is essential for identifying infected individuals (asymptomatic or symptomatic) and tracing potential close contacts. Furthermore, viral load analysis helps in risk assessment of COVID-19 patients, providing a solid research foundation for personalized treatment, disease detection, and drug screening. Unfortunately, there are currently no multifunctional, rapid diagnostic tools and methods. While antigen testing based on immunoassay is a relatively popular technology, it is simple, fast, and inexpensive, but the delay in antibody production hinders early diagnosis. Therefore, the gold standard for COVID-19 testing remains PCR (Polymerase Chain Reaction) nucleic acid testing, which has excellent accuracy.

[0003] Currently, most PCR methods are still deployed in centralized laboratories, resulting in a long turnaround time (at least 3 hours) and potentially 1-2 days by the time the test results are received. While some novel nucleic acid detection methods based on isothermal amplification can effectively shorten the detection time, they still cannot replace PCR methods due to their complex reaction systems, low amplification specificity, and low sensitivity. Furthermore, many isothermal amplification-based nucleic acid detection methods cannot simultaneously detect multiple nucleic acid targets within a single reaction system.

[0004] To address the aforementioned shortcomings, microfluidic chip-based PCR methods have gradually emerged. Combining microfluidic technology, these methods effectively reduce the heat capacity of the reaction system, allowing for rapid heat exchange with external thermal cycling devices, thus shortening the traditional detection time from hours to minutes. However, most rapid PCR technologies rely on endpoint detection for qualitative analysis or utilize semi-quantitative real-time PCR principles. These methods cannot provide reliable nucleic acid concentration results reflecting genomic copy number differences, i.e., absolute quantitative analysis results related to viral load. Furthermore, even some commercially available on-site nucleic acid diagnostic devices can only test a single sample during a single operation, and are costly. Therefore, in the face of pandemics like COVID-19, a multifunctional, rapid, efficient, accurate, and sensitive microfluidic nucleic acid detection system urgently needs to be developed. Besides rapid, high-throughput screening of suspected cases, it should also accurately quantify the viral load in positive patients, enabling effective and regular monitoring of the disease progression and guiding personalized treatment plans and drug screening evaluation. Summary of the Invention

[0005] The purpose of this invention is to provide a multifunctional microfluidic system for rapid nucleic acid detection and its application, thereby solving the problems of low nucleic acid detection efficiency and limited information content in the detection results caused by the long reaction time and low detection accuracy of nucleic acid amplification reaction in the prior art.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] According to a first aspect of the present invention, a multifunctional nucleic acid rapid detection microfluidic system is provided, the multifunctional nucleic acid rapid detection microfluidic system comprising: an array microcavity chip for high-throughput rapid screening of nucleic acid samples to be tested, and a microfluidic droplet chip or microfluidic microcavity chip for rapid quantification of nucleic acid gene copy number, wherein the array microcavity chip, the microfluidic droplet chip or the microfluidic microcavity chip share a rapid thermal cycling platform, and the multifunctional nucleic acid rapid detection microfluidic system can complete a single detection within 15 minutes.

[0008] Preferably, the thermal cycling platform can be a thermal cycling temperature control platform made of one or more materials selected from silicon, glass, quartz glass, etc.

[0009] Preferably, in order to achieve rapid heat exchange between the reaction system and the thermal cycling platform, the substrates of the array microcavity chip and the microfluidic droplet chip are both made of one or more materials selected from silicon, glass, quartz glass, plastic and other materials, and the substrate thickness of each reaction chip is less than 1 mm.

[0010] The array microcavity chip includes at least four independent millimeter-scale reaction chambers, each capable of holding 1–10 μL of reaction system. The diameter of each reaction chamber is 2–5 mm, and the height of each reaction chamber is 2–5 mm.

[0011] The array microcavity chip uses a thin glass substrate to achieve rapid heat exchange, thereby completing a rapid nucleic acid amplification reaction; multiple millimeter-scale through-holes are constructed and then bonded to the thin glass substrate.

[0012] The microfluidic droplet chip includes: a reagent loading shell, a droplet generation structure layer, a glass cover, and a substrate layer; wherein, the droplet generation structure layer and the glass cover are arranged side by side on the substrate layer, the reagent loading shell includes: a liquid phase loading chamber and an oil phase loading chamber for loading liquid and oil phases respectively; the droplet generation structure layer includes: a liquid phase inlet and an oil phase inlet connected to the liquid phase loading chamber and the oil phase loading chamber respectively below, and a cross-shaped nozzle for generating droplets; the glass cover is fixed to the substrate layer with double-sided tape or UV-curable adhesive and forms a microdroplet storage cavity between the two, wherein the microdroplet storage cavity can accommodate 10,000 to 50,000 micrometer-sized sample reaction units.

[0013] In the microfluidic droplet chip, the thickness of the droplet generation structure layer is 50–200 μm, the inner diameter of the liquid phase inlet and the oil phase inlet is 30–150 μm, and the diameter of the generated droplets is 40–150 μm; the height of the microdroplet storage cavity is 100–200 μm, and the area of ​​the microdroplet storage cavity is 2–5 cm². 2 .

[0014] The microfluidic droplet chip has the function of dividing the nucleic acid amplification reaction system into independent reaction units at the micrometer scale. Preferably, the storage cavity in the microfluidic droplet chip can accommodate 10,000 to 50,000 micrometer-scale sample reaction units. More preferably, the sample reaction units in the storage cavity are in the form of water-in-oil droplets, and the diameter of the droplets is 40 to 150 μm.

[0015] The advantages of the microfluidic droplet chip include the following aspects: First, it uses a thin glass substrate, thereby achieving rapid heat exchange; second, it adopts a glass cavity structure, which prevents droplet fusion caused by oil phase absorption due to the porous structure of traditional PDMS materials, making the droplets more stable; third, the glass material can withstand the high temperature of PCR thermal cycling denaturation, and its hardness does not fluctuate with temperature; fourth, the reagent loading shell makes it easier to connect to a standard Luer connector gas pump for droplet generation.

[0016] The storage cavity of the microfluidic droplet chip is prepared using double-sided adhesive of a fixed thickness or fixed with UV-curable adhesive, specifically a sandwich structure. Double-sided adhesive is applied to three edges of the glass cover plate, which is then bonded to the thin glass substrate. While open to the atmosphere, when water-in-oil droplets are generated, the edges open to the atmosphere will not flow out due to the surface tension of the oil phase, ensuring stability during droplet storage. The preferred height of this storage cavity is 100–200 μm. The upper surface of the storage cavity is made of one or more materials selected from silicon, glass, quartz glass, etc., and the total surface area of ​​the storage cavity is 2–5 cm². Furthermore, the sample inside the storage cavity does not directly contact the rapid thermal cycling platform; the rapid thermal cycling platform exchanges heat with the sample inside the storage cavity through the dielectric layer.

[0017] The contact surface of the rapid thermal cycling platform is made of silicon-based material, which has good thermal conductivity and is compatible with semiconductor processes. This allows temperature sensing and heating electrodes to be fabricated on it for temperature control. The storage chamber or chip can be placed on the heating surface. The dielectric layer can be made of thermally conductive silicone or liquid thermally conductive silicone oil to ensure that the system in the reaction chamber can make better contact with the silicon-based heating plate through the thin glass substrate. Since both the silicon-based heating plate and the thin glass substrate are rigid, a dielectric layer is needed to effectively ensure thermal contact between the two.

[0018] Preferably, the thickness of the dielectric layer is 50 to 300 μm, and it is made of any one of the following materials or a composite of multiple materials: polydimethylsiloxane (PDMS), epoxy resin, glass, quartz glass, polycarbonate (PC), polymethyl methacrylate (PMMA).

[0019] Preferably, the microfluidic droplet chip is also equipped with a reagent loading shell, which is fabricated using 3D printing, engraving, injection molding, or other processing techniques. The material is photosensitive resin or any plastic material suitable for microfluidic fabrication. The reaction liquid storage chamber and oil phase storage chamber of the loading shell are surrounded by standard Luer connectors.

[0020] According to the present invention, the microfluidic droplet chip can be replaced with a microfluidic microcavity chip. This microfluidic microcavity chip is another type of digital PCR chip, with tens of thousands of chambers on the order of hundreds or tens of micrometers, with a diameter of about 100 μm. These tens of thousands of very small microcavities are filled through microfluidic channels to achieve partitioning of the reaction system; it can be said that the microfluidic microcavity is another form of method for dispersing the reaction system.

[0021] The microcavity array on a microfluidic microcavity chip is more like a well plate array, consisting of millimeter-scale microcavities, with each cavity containing multiple different reaction systems, which are added using a pipette. In contrast, a microfluidic microcavity chip divides a reaction system into thousands of small reaction units, which is another common digital PCR chip structure. After pipetting, the reaction system is drawn into thousands of micrometer-scale chambers by a pump or negative pressure self-aspiration. The micrometer-scale independent reaction units achieve the segmentation function in the form of microcavities, with a diameter of 20–200 μm and a height of 20–200 μm.

[0022] The reaction chip is selected from, but is not limited to, any one or more materials selected from dimethylsiloxane (PDMS), polycarbonate (PC), and methyl methacrylate (PMMA). Furthermore, the reaction chip is composed of at least two materials bonded together by plasma bonding, ultrasonic welding, chemical bonding, or other methods.

[0023] According to a second aspect of the present invention, a multifunctional nucleic acid rapid detection microfluidic system as described above is provided for the rapid quantitative detection of nucleic acids, rapid detection of nucleic acid mutations or methylation, and rapid and accurate quantification of nucleic acid copy numbers. This system, as a universal platform in conjunction with reaction chips and rapid nucleic acid reaction systems, can be widely used in the field of nucleic acid detection, including but not limited to virus detection, sequence detection of target molecules such as nucleic acids (RNA and DNA), and detection of nucleic acid mutations or methylation.

[0024] According to a preferred embodiment of the present invention, the method includes: (1) providing a multifunctional nucleic acid rapid detection microfluidic system as described above; (2) loading the nucleic acid detection reaction system into an array microcavity chip using a pipette, and using a rapid thermal cycling platform to achieve rapid qualitative screening of the presence of nucleic acid-specific targets, determining whether the nucleic acid sample carries the relevant targets, thereby rapidly screening suspected cases and improving detection efficiency; (3) for nucleic acid samples confirmed to contain targets, dispersing the nucleic acid detection reaction system into tens of thousands of micron-level sample reaction units using a microfluidic droplet chip or a microfluidic microcavity chip; (4) performing nucleic acid amplification on the dispersed sample reaction units using a rapid thermal cycling platform, and then determining the nucleic acid copy number in the initial nucleic acid sample by counting the number of reaction units with positive fluorescent signals in the chamber, thereby achieving rapid and accurate absolute quantification of nucleic acid targets.

[0025] In one specific embodiment, the nucleic acid amplification reaction sequentially includes reverse transcription, pre-denaturation and denaturation, annealing and extension. Given that the nucleic acid amplification reaction system uses a DNA rapid polymerase with hot-start characteristics and a rapid thermal cycling platform, preferably, the thermal cycling parameters can be set as follows: reverse transcription temperature 42–50°C, time 30 s–5 min, cycle number 1; pre-denaturation 95°C, time 30 s–1 min, cycle number 1; two-step PCR program set as denaturation temperature 95°C, time 2–5 s, annealing and extension temperature 55–65°C, time 4–12 s, cycle number 40–45; or, three-step PCR program set as denaturation temperature 95°C, time 2–5 s, annealing temperature 55–65°C, time 4–10 s, extension temperature 72°C, time 2–5 s, cycle number 40–45.

[0026] According to a third aspect of the present invention, an application of the aforementioned multifunctional nucleic acid rapid detection microfluidic system in the rapid nucleic acid detection of SARS-CoV-2 is also provided. On the one hand, it can rapidly and with high throughput screen suspicious samples; on the other hand, it can perform rapid absolute quantitative analysis of nucleic acid targets in positive samples after isolation, enabling assessment of viral load and monitoring of patient disease progression.

[0027] As described in the background section, in order to speed up the nucleic acid detection process, existing technologies usually avoid nucleic acid amplification methods based on PCR principles and instead use technologies based on isothermal amplification principles. However, their specificity and sensitivity are not as good as those based on PCR principles. Secondly, most rapid PCR or integrated nucleic acid detection systems have not demonstrated their practical application significance and functionality, such as high-throughput nucleic acid screening capabilities, which are crucial in the COVID-19 detection process. Most existing products can only test a single sample at a time and require about one hour to complete the test.

[0028] To address the problems existing in the prior art, the key inventive point of this invention lies in simultaneously providing an array microcavity chip and a microfluidic droplet chip or microfluidic microcavity chip in a single detection system. Different chips can be used to meet different detection needs, enabling rapid, high-throughput screening on the one hand, and rapid quantitative analysis of positive samples on the other. This invention achieves this through the combination of digital PCR and rapid PCR technologies, which can be applied to the rapid monitoring of viral nucleic acid load in positive patients, providing personalized treatment plans and being used for drug screening, which is of great significance. Furthermore, this invention employs a rapid thermal cycling platform, which can complete 40 thermal cycles within a few minutes, comparable in time to isothermal amplification methods or antigen-antibody detection methods, but with better specificity and sensitivity than PCR technology.

[0029] Both array microcavity chips and microfluidic droplet chips utilize thin substrate materials to achieve rapid heat conduction. Furthermore, the design is simple and easy to operate for sample addition. For microfluidic droplet chips, the glass chamber provides advantages such as resistance to evaporation and fusion of droplets during rapid thermal cycling in PCR.

[0030] Furthermore, this invention overcomes the public's prejudice that traditional PCR technology is time-consuming and therefore requires isothermal amplification. However, nucleic acid amplification detection based on PCR technology is still the gold standard for COVID-19 detection. This invention uses a rapid heating and cooling platform combined with microfluidic technology to prepare various chips to meet a range of current detection needs, enabling rapid screening of multiple samples and rapid absolute quantification of viral nucleic acid load to assess the patient's disease progression.

[0031] According to the technical solution of the present invention, 40 thermal cycles of PCR can be achieved within 15 minutes, and qualitative screening of 9 nucleic acid samples can be completed. Finally, the present invention also combines digital PCR technology with rapid PCR by introducing microfluidic droplet chips, so as to achieve absolute quantification of nucleic acid detection. This has higher quantitative accuracy than fluorescence quantitative PCR or endpoint-based qualitative detection PCR. In this way, viral nucleic acid load can be assessed quickly and accurately on a rapid heating and cooling platform, and the viral load in the patient's body can be reflected accordingly, which has profound clinical detection significance.

[0032] In summary, the multifunctional rapid nucleic acid detection microfluidic system and its application provided by this invention can achieve high-throughput rapid screening of samples to be tested, as well as rapid and accurate quantification of viral load in confirmed cases. It also boasts advantages such as short detection time and high detection accuracy. In particular, when using this multifunctional rapid nucleic acid detection microfluidic system for COVID-19 nucleic acid testing, it can perform high-throughput rapid screening of nucleic acid samples from suspected positive patients and complete rapid, highly sensitive, and highly accurate absolute quantitative analysis of viral load in infected cases, effectively assessing the viral load in patients and thus promoting the implementation of personalized COVID-19 treatment plans, which has profound clinical testing significance. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of a multifunctional nucleic acid rapid detection microfluidic system provided according to Embodiment 1 of the present invention;

[0034] Figure 2 Is it like this? Figure 1 A schematic diagram of the individual structure of the array microcavity chip in the multifunctional nucleic acid rapid detection microfluidic system shown.

[0035] Figure 3 Is it like this? Figure 1A schematic diagram of the individual structure of the microfluidic droplet chip in the multifunctional nucleic acid rapid detection microfluidic system shown.

[0036] Figure 4 The temperature curves of the rapid thermal cycling reaction parameters provided according to Embodiment 2 of the present invention are shown.

[0037] Figure 5 This is the result of rapid multi-target (RdRp, N, and RNase P genes) detection of COVID-19 standard samples using the array microcavity chip provided by the present invention in Example 2.

[0038] Figure 6 This is a diagram showing the identification results of the array microcavity chip provided by the present invention for distinguishing between positive and negative thresholds in Example 2.

[0039] Figure 7 This is a diagram showing the repeatability and stability of the RdRp gene, a target of COVID-19, detected by the array microcavity chip provided by the present invention in Example 2.

[0040] Figure 8 This is a diagram showing the repeatability and stability of the COVID-19 target N gene detected by the array microcavity chip provided by the present invention in Example 2.

[0041] Figure 9 This is a diagram showing the repeatability and stability of the quality control gene RNase P detected by the array microcavity chip provided by the present invention in Example 2.

[0042] Figure 10 This is a diagram showing the repeatability and stability of the RdRp gene, a target of COVID-19, detected by the microfluidic droplet chip provided by the present invention in Example 2.

[0043] Figure 11 This is a diagram showing the repeatability and stability of the microfluidic droplet chip provided by the present invention for detecting the N gene, a target of the novel coronavirus, in Example 2.

[0044] Figure 12 This is a diagram showing the repeatability and stability of the quality control gene RNase P detected by the microfluidic droplet chip provided by the present invention in Example 2.

[0045] Figure 13 This is the detection result of a clinical sample using the multifunctional nucleic acid rapid detection microfluidic system provided by the present invention, as shown in Example 3. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0047] Example 1: A Multifunctional Microfluidic System for Rapid COVID-19 Nucleic Acid Detection

[0048] According to this preferred embodiment, a multifunctional microfluidic system for rapid COVID-19 nucleic acid detection is provided, such as... Figure 1 As shown, the system mainly includes an array microcavity chip 100 and a microfluidic droplet chip 200. By using reaction chips with different structures, it is possible not only to achieve rapid and high-throughput nucleic acid sample screening, but also to simultaneously achieve rapid, sensitive, and accurate absolute quantification of the copy number of nucleic acid-specific target genes.

[0049] like Figure 2 The image shows an array microcavity chip 100 according to this preferred embodiment. The array microcavity chip mainly includes a microcavity array structure layer 1 and a thin glass substrate layer 2. The microcavity array structure layer 1 includes nine independent reaction system loading chambers, each with a diameter of 3 mm (preferably 2-5 mm). The overall chamber height is consistent with the chip thickness, being 3 mm (preferably 2-5 mm). However, it should be understood that this is merely an example and not a limitation; the number of reaction system loading chambers in the array microcavity chip 100 may be more or fewer.

[0050] like Figure 3 As shown, a microfluidic droplet chip 200 is provided according to this preferred embodiment, which is mainly assembled from a reagent loading shell 3, a microfluidic droplet generation structure layer 4, a base layer 5, an upper glass cover plate 6, and double-sided adhesive tape 7.

[0051] In this embodiment, the reagent loading shell 3 is made of transparent photosensitive resin material by 3D printing, including: liquid phase loading chamber 11, oil phase loading chamber 12, and the interface with the gas valve or external gas circuit is a standard Luer connector structure. The loading chambers 11 and 12 are preferably inverted cone shape, and the volume of liquid (liquid phase or oil phase) that can be stored is 40μL, but it can also be preferably 20 to 200μL.

[0052] The microfluidic droplet generation structure layer 4 has the function of dividing the nucleic acid amplification reaction system into thousands of independent reaction units. It mainly includes a liquid phase inlet 8, an oil phase inlet 9, and a cross-shaped nozzle 10. When the reaction system is injected through the liquid phase inlet 8, it is subjected to oil phase shearing from the oil phase inlet 9 to generate microdroplets with a size on the order of micrometers and uniform size. The diameter of the microdroplets is 90 μm, but it can also be preferably 40 to 150 μm.

[0053] The upper glass cover plate 6 is fixed to the base layer 5 with double-sided adhesive tape 7, thereby forming a microdroplet storage cavity between the upper glass cover plate 6 and the base layer 5. The microdroplet storage cavity can hold 10,000 to 50,000 micron-sized sample reaction units.

[0054] Under the shearing action of the oil phase, the microdroplets generated in the microfluidic droplet generation structure layer 4 are then pushed by the oil phase into the microdroplet storage cavity formed between the upper glass cover plate 6 and the base layer 5. Specifically, the cavity is first filled with the oil phase, which guides the droplets into the glass cavity on the one hand, and seals the edge of the glass cavity by surface tension on the other. Since the microdroplets are surrounded by the oil phase, and near the edge of the storage cavity, the oil phase will be confined between the upper glass cover plate 6 and the base layer 5 due to the presence of surface tension, so that the droplets remain stationary in the microdroplet storage cavity.

[0055] Each edge of the upper glass cover 6 is adhered with double-sided adhesive of a fixed thickness to ensure that the height of the microdroplet storage cavity is 100 μm, which can also preferably be 100–200 μm. It should be understood that each droplet chip has only one microdroplet storage cavity. Preferably, the microdroplet storage cavity of a single microfluidic droplet chip contains 20,000–25,000 microdroplets, which can also preferably be 10,000–50,000. It should also be understood that the main function of the microfluidic droplet chip is to store the reaction system in a dispersed form within a fixed cavity; besides the water-in-oil microdroplet form shown in the embodiment, it can also be in the form of a microcavity.

[0056] like Figure 2 and Figure 3 As shown, the structural layer material of the array microcavity chip 100 and the microfluidic droplet chip 200 is polydimethylsiloxane (PDMS), and the structural layer and the glass substrate are bonded by plasma bonding.

[0057] However, it should be understood that these reaction chips can be made of any one or a combination of materials selected from polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC), epoxy resin, glass, and quartz glass. Preferably, the reaction chip is made of at least two materials bonded together by a method such as plasma bonding, ultrasonic welding, or chemical bonding.

[0058] Example 2: A method for rapid detection of COVID-19 nucleic acid using multiple functions

[0059] To verify the repeatability and stability of the multifunctional nucleic acid rapid detection microfluidic system provided in Example 1, this example uses the system to perform repeatability tests on the serially diluted COVID-19 RNA standard provided by the National Institute of Metrology.

[0060] Since the standard contains only dual targets of RdRp and N, to better simulate the testing of real case samples, the target RNase P extracted from human saliva nucleic acid was uniformly mixed with the initial concentration of the standard as a quality control target in the entire nucleic acid detection amplification process, followed by serial dilution. According to the initial template concentration values ​​provided in the National Institute of Metrology's standard instructions, the diluted RdRp and N target concentrations were 10, 100, 1000, 5000, 10000 copies / μL and 20, 200, 2000, 10000, 20000 copies / μL, respectively. Since the concentration of the RNase P target was not quantified, the dilution gradient was used as the x-axis of the test results, with a dilution gradient parameter of 10. -4 10 -3 10 -2 2×10 -2 10 -1 Each test should be repeated at least three times.

[0061] As described in Example 1, the system includes both an array microcavity chip 100 and a microfluidic droplet chip 200. On the same rapid thermal cycling platform, this example uses the array microcavity chip 100 for rapid qualitative analysis of nucleic acids and the microfluidic droplet chip 200 for rapid, sensitive, and accurate absolute quantitative analysis of nucleic acids, thereby effectively quantifying the nucleic acid copy number in the sample and assessing the viral load in the patient.

[0062] Specifically, we employ optimized thermal cycling parameters for rapid detection of multifunctional COVID-19 nucleic acid. The specific thermal cycling temperature and time parameters are as follows: Figure 4 As shown, the reverse transcription temperature was 50℃, the time was 2 min, and the cycle number was 1; the pre-denaturation temperature was 95℃, the time was 30 s, and the cycle number was 1; the three-step PCR method included: denaturation temperature 95℃, time 3 s, annealing temperature 60℃, time 8 s, extension temperature 72℃, time 2 s, and the cycle number was 40.

[0063] like Figure 5 As shown, by selecting an appropriate concentration of standard samples for testing, it was found that the fluorescence intensity in the chamber containing the target gene was significantly stronger than that in the negative control. To effectively distinguish between positive and negative results for samples loaded into the chamber, at least 10 repetitive tests were performed on the negative control, and a threshold for distinguishing between positive and negative fluorescence intensity was subsequently obtained. Figure 6 As shown. The specific threshold calculation method follows the 3-sigma principle, which is to add three times the standard deviation to the mean of the fluorescence intensity values ​​of multiple replicates of the negative control as the threshold for distinguishing between positive and negative fluorescence intensity.

[0064]

[0065] Where T is the fluorescence intensity value of the threshold. The mean fluorescence intensity of the negative control was obtained from multiple replicates. This represents the standard deviation of the results from multiple repetitions.

[0066] Furthermore, once the relevant thresholds are determined, the positive and negative results of multiple samples can be rapidly screened. To evaluate the detection sensitivity and stability of the microcavity array chip, the aforementioned gradient-dilution standards were used for testing. Figures 7-9 The results show the fluorescence results of each target at different standard gradient dilutions on the left and the specific fluorescence intensity values ​​on the right. As can be seen from the figure, the detection sensitivity of the triple target is at least 10 copies / reaction, which has good detection sensitivity and can effectively distinguish between positive and negative templates.

[0067] In this embodiment, a microfluidic droplet chip is used, employing the aforementioned preferred thermal cycling reaction parameters for rapid, sensitive, and accurate absolute quantitative analysis of nucleic acids. This effectively quantifies the nucleic acid copy number in a sample, thereby assessing the viral load in the patient. It should be noted that the same thermal cycling reaction parameters are used on both types of chips in this embodiment, enabling qualitative or quantitative amplification reactions within 15 minutes. To evaluate the accuracy and sensitivity of rapid quantification, the aforementioned gradient dilution standards were also used for testing. Figures 10-12 To illustrate the detection results, the left side of the image shows fluorescence images corresponding to different concentrations at various dilution gradients. It can be seen that as the concentration decreases and the dilution gradient increases, the number of positive droplets and their proportion appearing in the field of view gradually decrease. The right side shows the corresponding concentration fitting results. As can be seen from the figure, the detection results of the triple target have good consistency with the standard concentration. 2 The fact that the microfluidic droplet chip has a detection efficiency of >0.99 and can detect a minimum of 5 copies / reaction concentration of standard demonstrates that the microfluidic droplet chip exhibits good repeatability and stability in the rapid nucleic acid detection system for COVID-19, and that the detection sensitivity can reach at least 5 copies / reaction.

[0068] Specifically, the total number of droplets n was counted using the FAM, VIC, and CY5 channels of a fluorescence microscope. t and the number of droplets with fluorescent signals n p The copy number N of the initial nucleic acid template can be obtained through Poisson distribution statistics, and the concentration c of the target in the sample can be calculated based on the droplet volume (where: V d V represents the volume of a single droplet. s V represents the volume of nucleic acid template added. r (This represents the volume of the entire reaction system).

[0069]

[0070] Example 3 uses this multifunctional nucleic acid rapid detection microfluidic system and method to detect clinical samples.

[0071] In this embodiment, the multifunctional nucleic acid rapid detection microfluidic system provided in Example 1 and the multifunctional nucleic acid rapid detection microfluidic method provided in Example 2 were used to test clinical samples. The microcavity array chip enables high-throughput, efficient, and rapid determination of the positive or negative status of samples. Subsequently, in conjunction with a microfluidic droplet chip, rapid, sensitive, and accurate absolute quantification of viral load within the sample is achieved.

[0072] like Figure 13 The image shows the test results of 13 clinical samples. Samples 1-10 are nucleic acid samples from 10 confirmed COVID-19 patients, and samples 11-13 are nucleic acid samples extracted from throat swabs of 3 healthy individuals. Figure 13 It was found that the fluorescence intensity values ​​of nucleic acid samples 11–13 from healthy individuals did not exceed the threshold in both the FAM and VIC channels. Furthermore, after screening for positive cases in clinical samples, this platform can be used to perform a rapid, sensitive, and accurate absolute quantification.

[0073] Table 1 shows the copy number of target nucleic acids in each sample calculated according to the Poisson distribution equation. Absolute quantification of the target nucleic acid copy number can be obtained by statistically analyzing the positive and negative droplets within the microfluidic droplet chip. The quantification results show good consistency with traditional RT-qPCR. It is worth noting that RT-qPCR can only determine target concentration through the Ct value, and its quantification error for target nucleic acid copy number is relatively large, only achieving relative quantification. However, based on the digital readout of the microfluidic droplet chip, a single-copy discrimination can be achieved to obtain an accurate digital copy number, more accurately characterizing the target concentration in the nucleic acid samples of positive patients. This can reflect viral load, which is beneficial for guiding personalized treatment plans, monitoring disease progression, and drug screening research.

[0074] Table 1. Results of rapid quantitative nucleic acid testing of samples from confirmed patients and healthy individuals.

[0075]

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A multifunctional nucleic acid rapid detection microfluidic system, characterized in that, The multifunctional nucleic acid rapid detection microfluidic system comprises: an array microcavity chip for high-throughput rapid screening of nucleic acid samples to be detected, and a microfluidic droplet chip or a microfluidic microcavity chip for rapid quantitative detection of nucleic acid gene copy number, the array microcavity chip, the microfluidic droplet chip or the microfluidic microcavity chip share a rapid thermal cycling platform, and the multifunctional nucleic acid rapid detection microfluidic system can complete single detection within 15 minutes; The rapid thermal cycling platform is made of one or more selected from silicon and quartz glass, the substrate of the array microcavity chip, the microfluidic droplet chip or the microfluidic microcavity chip is made of any one or more selected from silicon, quartz glass and plastic, and the thickness of the substrate of each chip is less than 1 mm, so that rapid heat exchange of the rapid thermal cycling platform and the detection system in the array microcavity chip, the microfluidic droplet chip or the microfluidic microcavity chip is realized; The rapid thermal cycling platform and the detection system in the array microcavity chip, the microfluidic droplet chip or the microfluidic microcavity chip realize rapid heat exchange through a medium layer with a thickness of 50-300 μm, and the material of the medium layer is selected from any one or more of polydimethylsiloxane, epoxy resin, quartz glass, polycarbonate and polymethyl methacrylate; The array microcavity chip comprises at least four mutually independent millimeter-scale reaction chambers, each reaction chamber can accommodate 1-10 μL of a reaction system, the diameter of each reaction chamber is 2-5 mm, and the height of each reaction chamber is 2-5 mm; The microfluidic droplet chip comprises: a reagent loading shell, a droplet generation structure layer, a glass cover plate and a substrate layer; wherein the droplet generation structure layer and the glass cover plate are arranged side by side on the substrate layer, the reagent loading shell comprises: a liquid phase loading chamber and an oil phase loading chamber for loading liquid phase and oil phase respectively; the droplet generation structure layer comprises: a liquid phase sample inlet and an oil phase sample inlet connected with the liquid phase loading chamber and the oil phase loading chamber respectively below, and a cross-shaped structure nozzle for generating droplets; the glass cover plate is fixed on the substrate layer by double-sided adhesive or ultraviolet curing adhesive and forms a microdroplet storage cavity therebetween, and the microdroplet storage cavity can accommodate 10,000-50,000 micron-scale sample reaction units; The microfluidic microcavity chip comprises a plurality of micron-scale independent reaction chambers, the microfluidic microcavity chip realizes the segmentation function in the form of a microcavity, the diameter of each independent reaction chamber is 20-200 μm, and the height is 20-200 μm.

2. The multifunctional nucleic acid rapid detection microfluidic system according to claim 1, characterized in that, In the microfluidic droplet chip, the thickness of the droplet generation structure layer is 50-200 μm, the inner diameter size of the liquid phase sample inlet and the oil phase sample inlet is 30-150 μm, and the diameter of the generated droplet is 40-150 μm; the height of the microdroplet storage cavity is 100-200 μm, and the area of the microdroplet storage cavity is 2-5 cm 2 .

3. Use of the multifunctional nucleic acid rapid detection microfluidic system according to any one of claims 1-2 in the preparation of a nucleic acid rapid detection system for COVID-19.

4. Use according to claim 3, characterized in that, The detection comprises the following steps: The nucleic acid detection reaction system is loaded into the array microcavity chip by a pipette gun, rapid qualitative screening of the presence of nucleic acid specific targets is realized by the rapid thermal cycling platform, whether the nucleic acid sample carries the related target is judged, suspected cases are rapidly ruled out, and the detection efficiency is improved. For confirming the nucleic acid sample containing the target, the nucleic acid detection reaction system is dispersed into tens of thousands of micron-sized sample reaction units by a microfluidic droplet chip or a microfluidic microcavity chip; The dispersed sample reaction units are subjected to nucleic acid amplification by a rapid thermal cycling platform, and the number of reaction units with positive fluorescent signals in the statistical chamber is counted, so that the nucleic acid copy number in the initial nucleic acid sample is determined, and rapid and accurate absolute quantification of the nucleic acid target is realized.

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