EB (Epstein-Barr) virus quantification method based on flow analysis of polydisperse gel microspheres

Abstract

The invention discloses an EB (Epstein-Barr) virus quantitative method based on polydisperse gel microsphere flow analysis, which comprises the following steps: mixing a sample reagent with an agarose solution to generate polydisperse agarose liquid drops; the agarose liquid drops are placed in an isothermal environment for incubation, nucleic acid amplification is carried out, and polydisperse agarose gel microspheres are obtained; after nucleic acid amplification is completed, the agarose gel microspheres are cooled and solidified, and the agarose gel microspheres are obtained; carrying out demulsification and dyeing treatment on the agarose gel microspheres; performing flow cytometry analysis on the dyed agarose gel microspheres to obtain a forward scattering light signal and a lateral scattering light signal; according to the forward scattering light signal and the lateral scattering light signal, respectively obtaining the volume size and granularity difference of the polydispersed agarose gel microspheres, and combining with a multi-volume nucleic acid calculation formula to obtain an EB virus quantitative result of the sample reagent. The EB virus quantitative method provided by the invention has the advantages of being universal in laboratories, simple and convenient to operate, friendly in resources and the like.

Description

Technical Field

[0001] The invention relates to the technical field of nucleic acid detection, and in particular to an EB virus quantitative method based on polydisperse gel microsphere flow analysis. Background Art

[0002] At present, Epstein-Barr virus infection has become an important issue that cannot be ignored in the field of public health. Epstein-Barr virus (EBV) is one of the most common viruses in humans, belongs to the family of herpesvirus, and widely infects people around the world. The virus is mainly transmitted through saliva, infects B lymphocytes, and causes latent infection in the host. Although most EBV-infected people have no obvious symptoms, EBV can be reactivated in the case of impaired immune system or immunosuppression, thereby causing a variety of diseases related to immune dysfunction, especially in organ transplant patients. EBV is not only closely related to infectious mononucleosis (IM), but is also common in patients after chemotherapy or stem cell transplantation, especially in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). These patients need to monitor EBV DNA levels regularly, especially those who receive bone marrow transplantation or high-intensity chemotherapy. When signs of EBV infection are obvious, antiviral treatment or adjustment of immunosuppressive treatment strategies should be carried out in a timely manner. In addition, EBV infection is closely related to a variety of malignant tumors (such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, etc.) and post-immunosuppressive lymphoproliferative disorders (PTLD).

[0003] Post-transplant lymphoproliferative disease (PTLD) is a common complication in transplant patients, especially in patients with kidney transplantation, liver transplantation and bone marrow transplantation. The occurrence of PTLD is closely related to the latent infection of EBV. Especially under immunosuppressive therapy, the host immune surveillance function is impaired, resulting in the activation of EBV and the abnormal proliferation of lymphocytes, which may eventually evolve into malignant tumors. The clinical manifestations of PTLD are usually atypical, and common symptoms include nonspecific fever, lymphadenopathy and organ failure, so early diagnosis is crucial. The biopsy pathological classification organized by the WHO is still the gold standard for the diagnosis of PTLD, but the optimal staging scheme has not yet been determined. The commonly used early diagnostic measure is to detect the DNA load of EB virus in peripheral blood and take intervention measures to reduce the viral load based on the test results. By monitoring the DNA load of EB virus, the risk of PTLD can be effectively predicted, especially when the viral DNA load rises sharply after transplantation, timely intervention can help significantly improve the prognosis of patients.

[0004] Clinically, EBV DNA load detection has become an important tool for assessing EBV infection and the risk of PTLD. By quantitatively analyzing the EBV DNA load in the patient's peripheral blood, doctors can monitor the level of viral replication in real time and intervene in time when the viral load increases, thereby avoiding or slowing the occurrence of PTLD. Traditional EBV DNA detection methods mainly include real-time quantitative PCR (qPCR) and digital PCR (ddPCR). These two methods have their own advantages and disadvantages, and there are certain limitations in clinical applications. Although real-time fluorescence quantitative (qPCR) can provide higher sensitivity and accuracy, it relies on the establishment of a standard curve and can only achieve relative quantification. In order to measure the number of nucleic acid copies, a standard curve must be generated by a standard sample, and the results are calculated based on the curve. However, this process not only increases the complexity of the experiment, but may also introduce errors due to differences in experimental conditions or reagents, affecting the accuracy of the test results. In addition, qPCR may have problems with decreased sensitivity and accuracy in low copy number samples, especially complex samples, which limits its wide application. In contrast, digital PCR (ddPCR), as an absolute quantitative technology, does not require a standard curve, can directly perform absolute quantification of nucleic acids, and has strong anti-interference ability. However, ddPCR detection relies on uniform droplet generation and high-cost, complex equipment, such as dedicated droplet generators and droplet readers, which limits its application in low-cost, low-threshold scenarios. More importantly, ddPCR has limited applicability in large-scale high-throughput detection, as it takes a long time to generate a large number of droplets, further limiting its widespread application. LAMP (loop-mediated isothermal amplification) technology, as a simple and efficient detection method, has been widely used in the detection of various pathogens in many studies. In early studies, Iwata et al. combined traditional LAMP technology with agarose gel electrophoresis to successfully detect EBV infection, demonstrating its superior detection capabilities in clinical applications.

[0005] Therefore, how to overcome the shortcomings of existing nucleic acid detection methods in terms of cost, ease of operation and laboratory portability is a problem that technicians in this field need to solve. Summary of the invention

[0006] The embodiment of the present invention provides an EB virus quantitative method based on polydisperse gel microsphere flow analysis, aiming to design an EB virus quantitative method that is universal in laboratories, easy to operate and resource-friendly.

[0007] The embodiment of the present invention provides a method for quantifying EB virus based on polydisperse gel microsphere flow analysis, comprising:

[0008] Obtaining a sample reagent to be subjected to EB virus quantification, and mixing the sample reagent with an agarose solution to generate polydisperse agarose droplets;

[0009] placing the agarose droplets in an isothermal environment for incubation to perform nucleic acid amplification to obtain polydisperse agarose gel microspheres;

[0010] After the nucleic acid amplification is completed, the agarose gel microspheres are cooled and solidified to obtain agarose gel microspheres;

[0011] Demulsifying and dyeing the agarose gel microspheres;

[0012] The dyed agarose gel microspheres were analyzed by flow cytometry to obtain forward scattered light signals and side scattered light signals;

[0013] The volume size and particle size difference of the polydispersed agarose gel microspheres are respectively obtained according to the forward scattered light signal and the side scattered light signal, and the EB virus quantitative result of the sample reagent is calculated in combination with the multi-volume nucleic acid calculation formula.

[0014] In order to simplify the steps of generating uniform droplets, the embodiment of the present invention proposes a strategy of using polydisperse volumes, and the flocculent precipitation generated by isothermal amplification is used to differentiate the side scattered light signal, so that the forward scattered light signal and the side scattered light signal output by the flow cytometer can be cleverly used to respectively reflect the volume size and particle size difference of the polydisperse agarose gel microspheres, thereby realizing digital nucleic acid detection. The embodiment of the present invention combines the loop-mediated isothermal amplification technology and the high-throughput detection capability of the flow cytometer, without the need for expensive special equipment, and only using vortexes can generate batch droplets, so nucleic acid quantification can be performed efficiently and easily under common laboratory conditions. Compared with traditional qPCR technology and ddPCR technology, the embodiment of the present invention not only reduces equipment requirements, but also simplifies experimental operations and improves operability. Therefore, the embodiment of the present invention provides a new direction for the innovation and application of nucleic acid detection technology, and develops a digital nucleic acid detection method for detecting Epstein-Barr virus DNA load, especially in high-throughput screening and large-scale clinical testing, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0016] Figure 1 A schematic diagram of a flow chart of a method for quantifying EB virus based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0017] Figure 2A system schematic diagram of an EB virus quantification method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0018] Figure 3 A droplet processing flow chart of the EB virus quantification method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0019] Figure 4 A first experimental comparison diagram of the EB virus quantitative method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0020] Figure 5 A second experimental comparison diagram of the EB virus quantitative method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0021] Figure 6 A first verification schematic diagram of the EB virus quantification method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0022] Figure 7 A second verification schematic diagram of the EB virus quantification method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0023] Figure 8 A third verification schematic diagram of the EB virus quantification method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0024] Fig. 9 A fourth verification schematic diagram of the EB virus quantification method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0025] Fig.10 A first flow analysis scatter plot of the EB virus quantitative method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0026] Fig.11 A second flow analysis scatter plot of the EB virus quantitative method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention;

[0027] Fig.12 This is a performance comparison chart of the EB virus quantification method based on polydisperse gel microsphere flow analysis provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] It should be understood that when used in this specification and the appended claims, the terms "include" and "comprises" indicate the presence of described features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or combinations thereof.

[0030] It should also be understood that the terms used in this specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. As used in the specification of the present invention and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" are intended to include plural forms.

[0031] It should be further understood that the term "and / or" used in the present description and the appended claims refers to any and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0032] See below Figure 1 The embodiment of the present invention provides an EB virus quantitative method based on polydisperse gel microsphere flow analysis, which specifically includes: steps S101 to S106.

[0033] Step S101, obtaining a sample reagent to be used for EB virus quantification, and mixing the sample reagent with an agarose solution to generate polydisperse agarose droplets;

[0034] Step S102, placing the agarose droplets in an isothermal environment for incubation to perform nucleic acid amplification to obtain polydisperse agarose gel microspheres;

[0035] Step S103, after the nucleic acid amplification is completed, the agarose gel microspheres are cooled and solidified to obtain agarose gel microspheres;

[0036] Step S104, performing demulsification and dyeing treatment on the agarose gel microspheres;

[0037] Step S105, performing flow cytometry analysis on the stained agarose gel microspheres to obtain forward scattered light signals and side scattered light signals;

[0038] Step S106, respectively obtaining the volume size and particle size difference of the polydispersed agarose gel microspheres according to the forward scattered light signal and the side scattered light signal, and calculating the EB virus quantitative result of the sample reagent in combination with the multi-volume nucleic acid calculation formula.

[0039] In this embodiment, polydisperse droplets are first used to replace uniform droplets as reaction units, and the precipitate produced after LAMP amplification causes changes in the particle size of agarose gel microspheres, replacing the traditional fluorescent signal as an indication of the positive and negative nature of the droplets, and then the agarose gel microspheres are cooled and solidified, and demulsification and staining are performed after cooling and solidification. Subsequently, based on flow cytometry, the size and particle size of the cells are measured respectively by outputting forward scattered light signals and side scattered light signals, thereby defining the characteristics of the cell population. Among them, forward scattered light (FSC) is used to measure cell size, and a strong FSC signal indicates that the cells are larger; side scattered light (SSC) is used to measure particle size and internal structure complexity, and a strong SSC signal indicates that the particle size is larger or the internal structure is more complex.

[0040] This embodiment cleverly uses the FSC signal and SSC signal of the flow cytometer to reflect the volume size and particle size differences of the polydisperse agarose gel microspheres. Through the multi-volume nucleic acid calculation formula, the absolute quantitative detection of digital nucleic acid is further realized. This embodiment combines polydisperse agarose droplets, isothermal amplification and flow cytometry analysis to provide a new solution for low-cost, high-sensitivity and laboratory-general nucleic acid detection.

[0041] Here, conventional nucleic acid detection uses real-time fluorescence quantitative PCR technology and ddPCR technology. In the PCR reaction system, it needs to undergo three basic reaction steps of denaturation, annealing and extension, and the total reaction time is about 2 hours. This embodiment uses a loop-mediated isothermal amplification reaction (Loop-mediated Isothermal Amplification, LAMP), which is characterized by designing 4 specific primers for 6 regions of the target gene, and amplifying under the action of chain displacement DNA polymerase (Bst DNA polymerase) at a constant temperature of 60-65 ° C, and hundreds of millions of times of nucleic acid amplification can be achieved in less than 1 hour. LAMP has the characteristics of simple operation, strong specificity, and easy product detection. LAMP amplification (loop-mediated isothermal amplification) reduces the requirements for hardware equipment. During the isothermal amplification DNA synthesis process, the pyrophosphate ions in the deoxyribonucleic acid triphosphate substrate (dNTP) react with the magnesium ions in the reaction solution to produce a large amount of magnesium pyrophosphate precipitation. This precipitation leads to differences in particle size on the droplet surface, so that the SSC output by the flow cytometer can be used as an indicator of the positive and negative state of the droplet. There is no need for dedicated droplet generation instruments and droplet reading instruments, which reduces the hardware requirements for droplet reading equipment and simplifies experimental operations.

[0042] This embodiment combines polydisperse droplet LAMP isothermal amplification and flow cytometry to reduce the hardware requirements of experimental equipment. By using a common vortex oscillator in the laboratory to generate droplets, polydisperse agarose droplets encapsulating 0 or more nucleic acid molecules can be quickly batch-produced, without the need for a special droplet generator to generate uniform, preset droplets, greatly reducing the cost of droplet generation and the difficulty of droplet reading. On the other hand, this embodiment uses the FSC and SSC parameters output by a common flow cytometer in the laboratory for the calculation of droplet volume and the judgment of yin and yang, respectively, which reduces the hardware requirements for droplet reading and simplifies experimental operations.

[0043] This embodiment develops the flow cytometer output parameters FSC and SSC for the calculation of agarose microbead volume and the judgment of positive and negative, respectively, and then substitutes them into the multi-volume Poisson distribution nucleic acid concentration calculation model to obtain the target nucleic acid concentration of the sample.

[0044] In addition, since the Beckman CytoFLEX series flow cytometer has a very high throughput and can analyze more than 10,000 events per second, this embodiment is more suitable for large-scale sample analysis. This embodiment is also suitable for single-cell transcription analysis and rare gene mutation analysis, etc., providing new possibilities for research in related fields and realizing the research of low-cost digital nucleic acid detection methods suitable for high-throughput analysis.

[0045] In addition, microfluidic chips with cross-focusing channels have become mainstream equipment for generating microdroplets (including agarose droplets). However, due to the high viscosity of agarose droplets, the generation process is significantly different from aqueous droplets, and the operation of making uniform agarose droplets is complicated and slow. From the results of uniform agarose droplets generated by mineral oil and droplet generators in actual experiments, due to the slow speed of agarose solution injection into the chip, uniform and stable agarose droplets of the required size can be prepared without squeezing or fusion by adjusting the oil flow rate, and it takes at least 1 hour to generate 25 μL agarose droplets, and agarose will slowly gel at room temperature, so it is necessary to use a graphene heating film to keep the syringe warm, and the pipeline and droplet generator also need to be heated on the heating table, and covered with a towel for insulation. This results in a low flux, and the device for generating agarose droplets on a microfluidic chip is complex, expensive and time-consuming. In order to simplify the step of uniform droplet generation, the present embodiment proposes a strategy using polydisperse volume. Previous studies have shown that the use of polydisperse droplets for digital nucleic acid detection is feasible and effective. Therefore, this embodiment selects polydisperse agarose droplets as an alternative to improve the high throughput of the experiment, reduce equipment and material costs, and simplify the manufacturing process. Considering the convenience of operation, this embodiment uses vortex-generated polydisperse droplets as amplification reaction units in subsequent experiments.

[0046] Commercial digital droplet PCR instruments require dedicated equipment for droplet generation and fluorescence reading units, which leads to high costs and complex instruments, thus limiting the application of digital nucleic acid detection technology and reducing the portability and versatility of its experiments. Figure 2 This embodiment breaks through these limitations through the following innovations. The flow chart and the corresponding process droplet micrograph are as follows: Figure 2 As shown, Figure 2 In the experiment, (a) shows that the sample reagent is mixed in agarose solution and fluorinated oil and then vortexed to generate polydisperse droplets. After isothermal incubation at 65°C, the droplets containing the target will amplify the target nucleic acid and generate precipitation. After low-temperature incubation at 4°C, the sol-gel switching property of agarose is used to form stable agarose gel microspheres after amplification, which are demulsified and stained, providing a solution for downstream flow analysis and digital nucleic acid quantification. (b) shows the microscopic images of the droplets in the experimental process, including microscopic images after vortexing to generate polydisperse agarose droplets; bright field images of polydisperse agarose droplets after amplification, where the red arrow indicates the positive droplets that produce precipitation and the green arrow indicates the negative droplets that do not produce precipitation; droplet images after low-temperature incubation; bright field images of agarose gel microspheres after demulsification; and finally, the bright field and fluorescence field microscopic images of agarose gel microspheres resuspended in 0.01% (v / v) Tween 20 solution after demulsification and washing.

[0047] In a specific embodiment, the step of obtaining a sample reagent to be subjected to EB virus quantification and mixing the sample reagent with an agarose solution to generate polydisperse agarose droplets comprises:

[0048] The sample reagent is mixed with the agarose solution, and under the action of fluorinated oil and a surfactant, a batch of polydispersed agarose droplets are generated by a vortex oscillation method.

[0049] When generating polydisperse droplets, the sample reagent is first mixed with the agarose solution, and then a batch of polydisperse agarose droplets are generated by vortex oscillation under the action of fluorinated oil and surfactant. Figure 3 Part (a) shows the actual droplets after vortex oscillation and standing. The generated polydisperse droplets are the micro-reaction space for subsequent nucleic acid amplification.

[0050] In a specific embodiment, the agarose droplets are placed in an isothermal environment for incubation to perform nucleic acid amplification to obtain polydisperse agarose gel microspheres, comprising:

[0051] The agarose droplet was placed in an isothermal environment at 60-70°C and incubated for 40-50 minutes for nucleic acid amplification.

[0052] When performing isothermal amplification, the generated agarose droplets are placed in an isothermal environment at 65°C and incubated for 45 minutes for nucleic acid amplification. In the positive droplets containing the target nucleic acid, the nucleic acid amplification reaction occurs, and the generated magnesium pyrophosphate forms a flocculent precipitate visible under bright field (such as Figure 2 The positive gel microspheres have a larger particle size and a more complex internal structure, resulting in a larger SSC signal in flow cytometry analysis, while the negative droplets (such as Figure 2 There was no nucleic acid amplification in the sample (indicated by the green arrow in the figure), no precipitation was produced, and the SSC signal of flow cytometric analysis was smaller than that of the positive gel microspheres.

[0053] In a specific embodiment, after the nucleic acid amplification is completed, the agarose gel microspheres are cooled and solidified to obtain agarose gel microspheres, comprising:

[0054] The agarose gel microspheres obtained by nucleic acid amplification are cooled to 2-6° C. to obtain the agarose gel microspheres.

[0055] During cooling and solidification, after amplification is completed, the sample is quickly cooled to 4°C. The sol-gel properties of agarose are used to solidify the droplets to form stable agarose gel microspheres, which fix the reaction products and provide physical stability for subsequent operations.

[0056] In a specific embodiment, the demulsification and dyeing treatment of the agarose gel microspheres includes:

[0057] The agarose gel microspheres are washed to remove the oil phase, and the agarose gel microspheres after the oil phase is removed are stained with dye for nucleic acid.

[0058] During demulsification and staining, the oil phase is removed, the agarose gel microspheres are recovered and washed, and then SYBR Green I dye is used to stain nucleic acids. Figure 3 (b), (c) and (d) in the figure, where (b) shows the liquid after the demulsifier is added for the first time and mixed by pipetting; (c) shows the stratified liquid phase after the bottom oil phase is removed and 0.1mL of 0.01% (v / v) Tween 20 solution is added and centrifuged; (d) shows the agarose gel microsphere suspension after the oil phase is removed. It should be noted that the agarose gel microspheres in the figure are samples taken after centrifugation enrichment.

[0059] In a specific embodiment, the stained agarose gel microspheres are subjected to flow cytometry analysis to obtain forward scattered light signals and side scattered light signals, including:

[0060] The stained agarose gel microspheres were analyzed by flow cytometry.

[0061] The stained agarose gel microspheres were analyzed by flow cytometry, and the FSC and SSC signals of the flow cytometer were used to reflect the volume and particle size differences of the polydispersed agarose gel microspheres. The SSC signal was used to classify the gel microspheres into negative and positive, and combined with the multi-volume nucleic acid calculation formula, the EB virus quantification could be achieved.

[0062] Furthermore, before the step of using a flow cytometer to perform flow cytometric analysis on the stained agarose gel microspheres, the method includes:

[0063] The stained agarose gel microspheres are blown and then introduced into the flow cytometer.

[0064] Here, the agarose gel microsphere suspension needs to be blown before entering the flow cytometer. Positive agarose gel microspheres show fluorescence under the FITC channel of the fluorescence microscope. At the same time, in flow cytometry analysis, the FITC (fluorescein, a commonly used fluorescent marker) signal can be used as a positive and negative indicator to facilitate subsequent quantitative analysis.

[0065] In general, this embodiment integrates nucleic acid amplification, flow analysis signal conversion and digital detection into a single system based on the physical properties of agarose droplets, and finally applies the digital nucleic acid detection method provided in this embodiment to the detection of EB virus DNA load. Preferably, in order to avoid cross contamination and specific amplification, droplet generation, amplification, demulsification washing and flow analysis are all performed independently in different laboratory rooms. Figure 2 The microscopic images shown further demonstrate the various steps of the method, including droplet generation, positive and negative determination after amplification, gelation, and fluorescent signal verification after staining.

[0066] In one embodiment, the step of obtaining the volume size and particle size difference of polydispersed agarose gel microspheres according to the forward scattered light signal and the side scattered light signal respectively includes:

[0067] When the forward scattered light signal is larger, it is determined that the volume of the agarose gel microsphere is larger;

[0068] When the forward scattered light signal is smaller, it is determined that the volume of the agarose gel microsphere is smaller.

[0069] In another embodiment, the method of obtaining the volume and positive / negative properties of polydispersed agarose gel microspheres according to the forward scattered light signal and the side scattered light signal, respectively, further comprises:

[0070] When the side scattered light signal reaches a preset threshold, the agarose gel microspheres are determined to be positive;

[0071] When the side scattered light signal is lower than a preset threshold, the agarose gel microspheres are determined to be negative.

[0072] In the early stage of the experiment, according to the principle of LAMP reaction, this example respectively studied the effect of agarose on isothermal amplification, the thermal stability of droplets and the effect of the mechanical strength of gel microbeads on the experiment.

[0073] First, it is necessary to verify whether the agarose solution will have a significant effect on isothermal amplification. To this end, this embodiment designed a set of control experiments to evaluate the effect of adding or not adding agarose solution on the LAMP amplification reaction. The Staphylococcus aureus isothermal nucleic acid amplification kit was used in the experiment. Specifically, the experiment was divided into two groups: one group added agarose solution to the amplification reaction, and the other group did not add agarose solution, and the part to which agarose solution should be added was replaced with enzyme-free water. The experimental results showed that the proportion of fluorescent negative droplets in the two groups of experiments was not much different, such as Figure 4The bright field image and corresponding fluorescence image of part (a) in the figure are shown in Figure 1. Part (a) shows the effect of agarose solution on LAMP amplification reaction. The bright field and fluorescence field microscopic images of the amplified droplet without adding agarose solution and the bright field and FITC field fluorescence microscopic images of the amplified droplet with adding 2% agarose solution are shown in Figure 1. Figure 4 The bar graph in part (b) shows the digital droplet LAMP nucleic acid quantification data after droplet amplification of the two groups in (a), and the data are mean ± standard deviation, n = 3. This shows that the addition of agarose solution to the amplification reaction has little effect on isothermal amplification and can be almost ignored.

[0074] It is worth noting that due to the different transmittance of the droplets in the system with ordinary enzyme-free water and the system with 2% agarose solution, there are some differences in imaging. However, these differences did not have a significant effect on the results of quantitative analysis. To further verify this, the quantitative results of the two groups of experiments were analyzed in detail. The results showed that there was no significant difference in the absolute quantitative results between the experimental group with added agarose solution and the control group without added agarose solution. The radius of the droplets after amplification in the control group with the same components and added enzyme-free water was 18.40±0.37μm (n=100), and 2213 droplets were counted in each group, and the absolute quantitative result was 6.300±0.284×10 4 copies / μL (n=3); the radius of the droplet after amplification in the experimental group with agarose solution was 20.08±0.18μm (n=100), 3498 droplets were counted in each group, and the absolute quantitative result was 6.591±0.125×10 4 copies / μL (n=3). Specifically, the concentrations of the three parallel experiments after droplet amplification without the addition of agarose solution were 6.143×10 4 , 6.632×10 4 and 6.124×10 4 copies / μL, with an average value of 6.300±0.284×10 4 copies / μL (n=3); after droplet amplification with agarose solution, the concentrations of the three parallel experiments were 6.574×10 4 , 6.724×10 4 and 6.474×10 4 copies / μL, with an average value of 6.591±0.125×10 4copies / μL (n=3). An independent sample t-test was performed on the two groups of data, and the t value was 1.6016 and the P value was 0.1845 (P>0.05), indicating that the difference between the two groups of data was not statistically significant. It can be seen that the addition of agarose solution will not significantly affect the quantitative results of the LAMP amplification reaction. This further shows that it is feasible to add agarose solution to the amplification system, and its effect on the experimental results can be ignored.

[0075] In the digital nucleic acid detection method using the polydisperse droplet strategy, the thermal stability of the droplets is crucial because it affects whether the agarose droplets will break, fuse or evaporate during the heating process, which directly affects the accuracy of the quantitative calculation. In order to ensure the stability of the droplet morphology during the experiment, this example conducts a detailed analysis of the droplets from droplet generation, isothermal amplification, low-temperature incubation to demulsification and washing operations, such as Figure 4Part (c) in the figure shows the stability of agarose droplets in the entire FlowJo-LAMP digital analysis process. It shows the bright field droplet microscopy image after generating monodisperse droplets and the bright field microscopy image of gel microspheres after amplification, demulsification and washing. The charts show the particle size distribution of droplets before amplification and gel microspheres after demulsification and washing after amplification, respectively. The dotted line is the corresponding Gaussian distribution fitting result. The "Before" group shows the bright field droplet microscopy image after generating uniform droplets, while the "After" group shows the bright field microscopy image of agarose gel microspheres after amplification, demulsification and washing. The right side shows the distribution of droplet size before and after the operation, and the dotted line is the Gaussian distribution fitting curve. It can be seen from the microscopic image that the agarose droplets after the operation have a good morphology, and no obvious droplet fusion or gel microsphere breakage is observed. In order to quantify this observation, the droplet size distribution before and after the operation was analyzed, and the radius size of 300 droplets and gel microspheres was counted. The average radius of the droplets before the amplification operation was 32.88±0.90μm, and the coefficient of variation (CV value) of the particle size was 2.76%; while the average radius of the gel microspheres after the operation was 34.28±1.35μm, and the coefficient of variation (CV value) of the particle size was 3.69%. The volume of the agarose gel microspheres increased slightly after the operation. It is speculated that agarose, as a natural polymer, has strong hydrophilicity, so it is easy to absorb water and swell in aqueous solution. Subsequent resuspension in 0.01% (v / v) Tween 20 solution, the surfactant can reduce the surface tension of the droplets, so that the surface of the gel microspheres is in contact with water, increasing its water absorption. As water penetrates into the gel microspheres, the molecular network of the agarose gel expands, causing the size of the gel microspheres to increase. Although the particle size of the agarose gel microspheres increased after the operation, the droplet morphology remained good and no obvious rupture or fusion was observed. It can be inferred that this expansion is controllable and limited. The CV value also increased slightly, but the change was small and did not significantly affect the experimental results, indicating that the experimental operation had little effect on the droplet morphology and the stability of the droplets was well maintained. In addition, the agarose gel microspheres in the experiment have good mechanical strength and can withstand operations such as washing, blowing, pipetting and centrifugation. It should be added that the solubility of the magnesium pyrophosphate (Mg2P2O7) precipitate produced during the amplification process is extremely low in water, and it can be considered that it is almost insoluble in water. Therefore, although the precipitate will form in the reaction system, its solubility in the aqueous phase when resuspended has almost no effect on the experimental process. Since the solubility of magnesium pyrophosphate is almost zero, the effect on the stability of the precipitate can be ignored when resuspended in a solution containing 0.01% (v / v) Tween 20, which can ensure that the precipitate will not dissolve back into the liquid phase during the experimental operation. This result proves that the experimental operation process including droplet generation, isothermal amplification, low-temperature incubation and emulsification and washing steps can effectively maintain the stability and morphological consistency of the droplets.Through this series of operations, the fusion of droplets is less likely to occur, and the reliability and repeatability of the experimental results are guaranteed.

[0076] Since the LAMP reaction can produce obvious precipitated floccules under bright field, the precipitate generated after amplification is used to cause changes in the particle size of agarose gel, replacing the traditional fluorescent signal as an indication of the positive and negative nature of the droplets. In flow cytometry analysis, side scattered light (SSC) is used to measure the particle size and the complexity of the internal structure. A strong SSC signal indicates a larger particle size or a more complex internal structure. This embodiment uses this characteristic and speculates that after the positive droplets containing the target are amplified, the generation of precipitates causes the positive agarose gel microspheres to have an increased particle size and a more complex internal structure, thereby serving as an indication of the positive and negative droplets.

[0077] To verify this hypothesis, this example designed an experiment to distinguish the positive and negative nature of the droplets by the presence or absence of precipitated flocs. First, this example conducted a preliminary experiment using a Staphylococcus aureus isothermal nucleic acid amplification kit. The amplification reagent containing the target was vortexed to generate polydisperse agarose droplets, which were then amplified, cooled and solidified, washed, and stained. Next, bright field imaging of agarose gel microspheres resuspended in 0.01% (v / v) Tween20 solution was performed, as shown in Figure 2. Figure 5 (a) and (b) in the figure show the bright field microscopic image and FITC channel fluorescence field microscopic image of agarose gel microspheres resuspended in 0.01% (v / v) Tween 20 solution after amplification, washing, centrifugation and staining, respectively.

[0078] In bright field imaging, the negative gel microspheres after amplification staining appear transparent and without precipitation, such as Figure 5 (a)(i) shows a local magnified bright field microscopic image of the negative gel microspheres after amplification staining, while the positive gel microspheres show an image of scattered precipitates, such as Figure 5 (a)(ii) shows a local magnified bright field microscopic image of the positive gel microspheres after amplification staining. Compared with the negative gel microspheres, the positive gel microspheres have larger particle size and irregular magnesium pyrophosphate precipitation formed inside, showing different permeability from the negative gel microspheres. Further analysis of the fluorescence field image shows that the negative gel microspheres after amplification staining have no fluorescent signal under the FITC channel, as shown in Figure 2. Figure 5 (b)(i) shows a local magnified FITC channel fluorescence field microscopic image of the negative gel microspheres after amplification staining, while the positive gel microspheres show obvious fluorescence signals, such as Figure 5(b)(ii) shows a local magnified FITC channel fluorescence field microscopic image of the positive gel microspheres after amplification staining. The positive gel microspheres have both precipitation and fluorescence signals, while the negative gel microspheres have neither precipitation nor fluorescence.

[0079] The preliminary experiment qualitatively proved that the positive gel microspheres can generate precipitation, resulting in an increase in particle size, and show fluorescence in the FITC channel after staining with SYBR Green I. Combined with the characteristics of SSC signal used to measure particle size and internal structure in flow cytometry analysis, it is speculated that the generation of precipitation and particle size differences can indicate the positive and negative nature of the gel microspheres. Based on this, this embodiment further designed a quantitative experiment to verify this conjecture.

[0080] This example explores whether the SSC signal can distinguish the formation of precipitation and the difference in particle size, so that it can replace the FITC parameter to distinguish the positive and negative of agarose gel microspheres, thereby further realizing the absolute quantification of nucleic acids in label-free polydisperse agarose droplets, further simplifying the experiment, and developing a label-free, easy-to-operate, low-cost, highly versatile digital nucleic acid detection technology suitable for high-throughput screening. This example uses a flow cytometer to perform data analysis on the SSC signal and FITC signal of the polydisperse agarose gel microspheres stained after amplification. This experiment uses the FITC fluorescence signal as the gold standard to determine the true positive and negative nature of the droplets, and explores the correlation between the SSC signal and the fluorescence intensity of the gold standard FITC signal. The signal scatter plot of the SSC signal and the FITC fluorescence intensity and the marginal histogram of the corresponding parameters (n=11601), the four quadrant divisions are shown as follows Figure 5 (c) is a scatter plot of the SSC parameter and fluorescence intensity of the agarose gel microspheres stained with SYBR Green I and the corresponding marginal histogram. The four quadrants Q1, Q2, Q3, and Q4 are used to distinguish false positives, true positives, false negatives, and true negatives, respectively. Two obvious clusters are found with good dispersion. Q1, Q2, Q3, and Q4 represent false positives, true positives, false negatives, and true negatives, accounting for 2.41%, 64.0%, 1.56%, and 32.1%, respectively. It is concluded that the accuracy of the SSC parameter as a droplet positive and negative determination is 96.1%, indicating that it has a high accuracy.

[0081] The true negatives in the Q4 quadrant (such as Figure 5 (d)(i), which shows the dot density plot of SSC parameters and fluorescence intensity of true negative agarose gel microspheres) and true positive in the Q2 quadrant (such as Figure 5(d)(ii), which shows the SSC parameters and fluorescence intensity of true positive agarose gel microspheres) The correlation between the SSC signal of the droplet and the FITC fluorescence signal, and a dot density diagram was drawn. The results show that the negative droplet gel microspheres have no precipitate generated, the particle size is small, the SSC signal value is small, there is no double-stranded DNA replication, and the fluorescence intensity is also small. At this time, the fluorescence signal value is the background fluorescence value. The Pearson's r of the obtained SSC signal and FITC signal is 0.369, which is a medium to weak positive correlation. It is judged to be a true negative gel microsphere, indicating that there is a certain linear relationship between the two, but it is not very strong. The SSC signal and FITC signal may be affected by other factors. For example, the FITC signal may be interfered by background noise. Especially in the case of weak fluorescence, low concentration or low number of target molecules will lead to high background noise, thereby affecting the correlation between the two.

[0082] The positive droplets contain the target, and the DNA double strands are replicated and magnesium pyrophosphate precipitates are generated during the amplification process. The gel microspheres have larger particle sizes, more complex internal structures, and larger SSC values. At the same time, the fluorescence signal after SYBR Green I binds to double-stranded DNA is also strong, which is judged as a true positive gel microsphere. The Pearson's r of the obtained SSC signal and the FITC signal is 0.529, and there is a medium-intensity positive correlation between the SSC signal and the fluorescence signal. This relationship shows that with the change of the internal structure of the droplet and the synchronous DNA amplification, the SSC signal and the FITC signal have a medium-intensity consistency, and the change trends of the two are synchronized. This relationship may be affected by the heterogeneity of particle size. In the experiment, there are differences in the particle sizes of the positive and negative droplet gel microspheres, and there are also differences between the positive gel microspheres. Even if there is precipitation generation that leads to an increase in particle size, the heterogeneity of agarose gel microspheres may cause gel microspheres of different particle sizes to respond differently to the SSC signal, thereby causing the dispersion of the SSC signal. Through this experiment, the feasibility and accuracy of the SSC parameter as an indicator for distinguishing the positive and negative nature of droplets were verified.

[0083] In the above experiment, this example preliminarily confirmed that the physical properties of agarose gel microspheres, especially the magnesium pyrophosphate precipitate generated during the amplification process, mainly determined the particle size and internal structure complexity of the gel microspheres, and preliminarily verified the feasibility and accuracy of the SSC parameter as an indicator for distinguishing the yin and yang of droplets. In order to confirm that the binding of dyes to double-stranded DNA may affect the physical properties of gel microspheres, especially in terms of particle size and internal structure complexity, specifically, this example speculates that after the SYBR Green I dye binds to DNA, it may change the scattering properties of agarose gel microspheres, thereby affecting the SSC signal intensity in flow cytometry analysis.

[0084] To further verify this hypothesis, this example designed unlabeled agarose gel microspheres without SYBR Green I staining, and performed flow cytometry analysis of SSC signals and FITC fluorescence signals after amplification and demulsification, aiming to evaluate the actual effect of dye binding to DNA double strands on particle complexity. The FITC-SSC correlation dot plot and marginal histogram of the flow cytometer (such as Figure 5 (e), which shows a scatter plot of the SSC parameter and fluorescence intensity of agarose gel microspheres not stained with SYBR Green I after amplification and the corresponding marginal histogram) (n=35274) shows that the particle signals of the agarose gel microspheres not stained with SYBR Green I are all within the background fluorescence intensity range (i.e., the unstained droplets have no fluorescence). Whether it is a negative droplet or a positive droplet, the fluorescence intensity of all particles does not exceed the background signal level, but the SSC signal still shows good dispersion, showing two obvious cluster distributions.

[0085] This result shows that the particle size and scattering properties of agarose gel microspheres are mainly determined by the physical properties of agarose itself, rather than the changes caused by the binding of dyes to DNA double strands. In particular, the formation of magnesium pyrophosphate precipitates in the amplification reaction is the main factor that leads to the increase in particle size and more complex internal structure of positive gel microspheres, thereby enhancing the SSC signal in flow cytometry analysis.

[0086] In summary, the experiment in this example verifies that the particle complexity of agarose gel microspheres mainly comes from its physical properties, mainly the precipitate produced by the isothermal amplification reaction, rather than the binding of SYBR Green I dye to the DNA double strand. Therefore, the effect of the dye on the particle complexity can be ruled out, further supporting the dominant role of magnesium pyrophosphate precipitation on particle size and SSC signal of flow cytometry analysis, and providing a more in-depth explanation for the relationship between SSC and FITC signals in flow cytometry analysis.

[0087] The above content discusses the use of SSC signals to replace fluorescent signals to distinguish the positive and negative nature of agarose gel microspheres. In order to achieve EB virus quantification, it is necessary to further explore the relationship between FSC signals and droplet volume. This embodiment designs the following experiment, using a flow cytometer to analyze the FSC (forward scatter) signal value of agarose droplets to explore its correlation with droplet volume.

[0088] Six groups of monodisperse agarose droplets of different sizes were prepared in this embodiment. Mineral oil was used as the continuous phase in the experiment, and a droplet generator was used to generate agarose droplets. This selection is based on the following reasons: First, the droplet generator can accurately control the size and distribution of the droplets in conjunction with a pneumatic pump, thereby ensuring the uniformity of the agarose droplets. By controlling parameters such as fluid flow rate, pressure, and channel size, monodisperse droplets of different sizes can be prepared to avoid size deviations caused by manual operation. Secondly, mineral oil has low volatility and can form a stable oil-in-water droplet structure, reducing the impact of the external environment on the agarose solution. Finally, the viscosity of mineral oil is similar to that of agarose solution, and high-viscosity mineral oil can provide sufficient shear force to generate more stable droplets. This characteristic helps to reduce the tension difference at the oil-water interface and ensure stability during droplet generation.

[0089] In this example, the ImageJ tool was used to perform statistical analysis on the droplet diameters. The results showed that the diameters of the six groups of monodisperse agarose droplets all conformed to the Gaussian distribution. 200 droplets were counted in each group, and the average diameters were 43.12±0.95μm, 46.63±1.20μm, 55.34±1.25μm, 58.86±1.18μm, 65.91±1.44μm, and 72.16±1.96μm, respectively. The coefficient of variation (CV value) was less than 3%, as shown in Figure 2. Figure 6 As shown in part (a) of the figure, six groups of bright field images of monodisperse agarose droplets of different sizes and corresponding particle size distribution histograms are shown. The Qo is fixed at 1 μL / min by a droplet generator, and the size of the agarose droplets is changed by changing Qw (Qw / Qo and agarose solution concentration are (i)-(vi) 1:20 μL / min, 0.75%; 1:15 μL / min, 0.75%; 1:12 μL / min, 0.75%; 1:10 μL / min, 0.75%; 1:8 μL / min, 0.75%; 1:8 μL / min, 1%). The dotted line is the Gaussian fitting distribution result, n=200. The results show that the monodisperse droplets prepared in this example have high uniformity in size and can be used for subsequent analysis.

[0090] Agarose gel microspheres were obtained by cooling, solidification, demulsification and washing. Subsequently, the FSC-Area, FSC-Height and FSC-Width signals of the six groups of gel microspheres were analyzed by flow cytometry. The number of microspheres analyzed in each group was 39905, 8403, 17059, 6005, 18661 and 7301, respectively. After iterative processing, box plots of FSC-Area, FSC-Height and FSC-Width and droplet volume were drawn, as shown in the figure. Figure 7As shown in (a), the box range is 25 and 75 points, the upper and lower whiskers represent the standard deviation, the box represents the mean, and the middle horizontal line represents the median line.

[0091] The FSC-Area box plot shows that as the droplet diameter increases from 43 μm to 72 μm, the FSC-Area signal value gradually increases. The linear relationship between droplet volume and FSC-Area is shown in Figure 8 As shown in part (c), the Pearson'r correlation coefficient is 0.972, and the data trend is relatively smooth, but there is a little more overlap between different diameter groups.

[0092] The FSC-Height box plot shows the signal changes of FSC-Height. As the diameter increases, the FSC-Height signal shows a relatively linear upward trend, and the range of the box plot is relatively narrow, showing good stability and repeatability. Especially in the middle diameter range, there is less overlap between the groups, indicating that FSC-Height has a good ability to distinguish microspheres of different diameters. The linear relationship between droplet volume and FSC-Height is shown in the figure below. Figure 6 As shown in part (b) of the figure, the dotted line is the fitting curve, the data are the mean ± standard deviation, the Pearson correlation coefficient is 0.985, and the linear relationship is more significant, indicating that FSC-Height is a better indicator for characterizing the droplet volume.

[0093] In contrast, the box plot of FSC-Width shows that its signal distribution is slightly irregular. Although it generally increases with increasing diameter, the linearity is poor and the box plot range is large. In particular, the discreteness of the signal is more obvious in droplets with smaller and larger diameters. This indicates that the stability and linearity of FSC-Width are low. The linear relationship between droplet volume and FSC-Width (such as Figure 8 The Pearson correlation coefficient of (as shown in part (d)) is only 0.620, which further verifies that its linear relationship with the droplet volume is weak and it is difficult to accurately characterize the droplet volume.

[0094] Comprehensive analysis of the signal characteristics of FSC-Area, FSC-Height and FSC-Width and their linear relationship with droplet volume showed that FSC-Height showed the best linearity, stability and discrimination ability (Pearson'r=0.985). Therefore, it is most appropriate to use FSC-Height signal as the main parameter when characterizing droplet volume.

[0095] In one embodiment, the quantitative result of EB virus of the sample reagent obtained by combining the multi-volume nucleic acid calculation formula includes:

[0096] The EB virus quantitative result of the sample reagent is calculated according to the following formula:

[0097]

[0098] Wherein, Fk,c represents the multi-volume nucleic acid calculation formula, k represents the proportionality coefficient between the polydispersed agarose gel microspheres and the forward scattered light signal, C represents the original nucleic acid concentration, Φ represents the forward scattered light signal, j represents the positive agarose gel microspheres, and N represents the number of agarose gel microspheres.

[0099] Digital PCR (dPCR) is a method that can achieve absolute quantification of nucleic acids. It can be mainly divided into microwell-based dPCR and droplet-based dPCR according to the different ways of forming independent units. The generated droplets can be divided into two cases. One is an empty droplet that does not contain nucleic acids. After amplification, the fluorescence signal is still weak, which is defined as a negative droplet. The other is a droplet containing one or more nucleic acids. After amplification, the fluorescence signal is enhanced and is defined as a positive droplet. The probability distribution of the number of nucleic acid molecules contained in each droplet follows a Poisson distribution, so the original concentration of the nucleic acid sample can be calculated using a uniform volume model.

[0100] (1) Uniform volume droplet detection model (Model I)

[0101] Assuming that the initial number of DNA molecules contained in the droplet is X, then the probability distribution function P of X = t (t = 0, 1, 2, ...) conforms to the Poisson probability model:

[0102]

[0103] Where λ represents the average number of nucleic acid molecules contained in each droplet. Assuming that the original concentration of the nucleic acid sample is C and the droplet volume is V0, then

[0104] By counting the number of droplets and droplet information, we can get the total number of droplets N (total) and the number of negative droplets N (neg), then the probability of not encapsulating nucleic acid molecules is:

[0105]

[0106] Thus, the average number of nucleic acid molecules encapsulated by each droplet can be obtained as:

[0107]

[0108] Combining the above formula, we can get the uniform droplet nucleic acid concentration calculation model:

[0109]

[0110] The premise for the above uniform droplet nucleic acid concentration calculation model formula to be valid is that the droplet volume is a preset size. In order to meet this condition, ddPCR usually uses droplet microfluidics technology to produce uniform droplets, which increases the cost of detection. It is still difficult to reproduce ddPCR in different laboratories.

[0111] Some researchers have proposed a method of generating droplets using vortex oscillation, and established a polydisperse (multi-volume) droplet model to calculate the original concentration of nucleic acids. This can greatly simplify the droplet generation steps, reduce the investment cost of ddPCR, and is more suitable for promoting ddPCR technology.

[0112] (2) Polydisperse (multi-volume) droplet detection model (Model II)

[0113] Group the multi-volume droplets by volume, then the volume is V i The number of DNA molecules encapsulated by the uniform droplet is λ = CV i , similarly V i The probability distribution function P of the number of DNA molecules contained in a droplet of volume conforms to the Poisson probability model:

[0114]

[0115] For an empty droplet, the probability is:

[0116]

[0117] Thus, the probability of positive droplets can be obtained as:

[0118]

[0119] Assuming the total number of positive droplets is N and the total number of negative droplets is M, then

[0120]

[0121] Among them, j represents positive and i represents negative. The left side of the formula is the total volume of droplets, and the right side is the total number of positive droplets / positive droplet probability, which is also the total volume of droplets, that is, the equation holds. The most suitable C found by the above formula is the corresponding original concentration of nucleic acid.

[0122] In terms of droplet signal reading, a direct method is to analyze the fluorescence image of stationary droplets in the microchamber, but it requires a lot of labor costs and is not suitable for instant nucleic acid detection. Another method is to collect fluorescence signals through photomultiplier tubes to achieve droplet signal detection. Although this method has high throughput and high degree of automation, it requires the use of lasers and photomultiplier tubes for signal excitation and detection, which makes the composition of the instrument complicated and costly. The need for cost-effective, reliable sample partitioning and signal reading remains unmet.

[0123] Some studies have used the precipitation produced by loop-mediated isothermal amplification (LAMP) to replace the fluorescent signal of PCR as an amplification signal. LAMP is similar to PCR. It can amplify nucleic acids at a constant temperature. Moreover, because there are more primer combinations than PCR (2 to 3 groups), the amplification rate is faster and more suitable for real-time nucleic acid detection. During its amplification, a precipitate visible under bright field, magnesium pyrophosphate, is produced, which can be used to replace fluorescent probes as an amplification signal. However, it may be because the image analysis based on multi-volume droplets is more complicated, a large number of images need to be taken, time-consuming and complex algorithm training and calculation are required, the cost is high and the operation is complicated, and the experimental portability is poor. In order to develop low-cost digital nucleic acid detection technology, this embodiment proposes a new method for digital nucleic acid detection based on polydisperse agarose droplets and flow cytometry, and verifies that it can be used for quantitative analysis of nucleic acids and viral DNA load detection. This method combines polydisperse droplet LAMP isothermal amplification and flow cytometry to reduce the hardware requirements of experimental equipment. This embodiment uses a common vortex oscillator in the laboratory to generate droplets, which can quickly batch-produce polydisperse agarose droplets that encapsulate zero or more nucleic acid molecules, without the need for a special droplet generator to generate uniform, preset droplets, greatly reducing the cost of droplet generation and the difficulty of droplet reading. On the other hand, this embodiment uses the FSC and SSC parameters output by a common flow cytometer in the laboratory for calculation of droplet volume and determination of positive and negative properties, respectively, reducing the hardware requirements for droplet reading and simplifying experimental operations.

[0124] (3) Polydisperse (multi-volume) droplet detection model based on flow cytometry (Model III)

[0125] According to the formulas for the total number of positive droplets N and the total number of negative droplets M, this embodiment uses the FSC signal and SSC signal output by a flow cytometer commonly used in laboratories for calculation of droplet volume and judgment of positive and negative properties, respectively. Φ is the volume-related value of the gel microspheres measured by the flow cytometer for the FSC signal. There is also a related proportionality coefficient k between the volume V of the polydisperse gel microspheres and the FSC signal Φ. The determination of k needs to be calibrated with a known gold standard concentration. Therefore, the volume V of each polydisperse agarose gel microsphere can be indirectly determined by k and Φ. ∑Φ is the sum of the measured value Φ of all gel microspheres multiplied by the proportionality coefficient k. Then the left side of the equation for the formulas for the total number of positive droplets N and the total number of negative droplets M is as follows:

[0126]

[0127] The right side of the above formula is as follows:

[0128]

[0129] After moving the terms, use Newton's iteration method to solve the function of k and C, as shown below:

[0130]

[0131] This example uses the above-constructed detection method to verify the performance of digital nucleic acid detection based on polydisperse agarose droplets and flow cytometric analysis. It has been verified above that the FSC and SSC signals of the flow cytometer can be cleverly used to digitally reflect the volume size and particle size differences of polydisperse agarose gel microspheres, respectively. By substituting these signals into the polydisperse droplet nucleic acid calculation formula, six groups of samples containing different concentrations of target nucleic acids were quantitatively analyzed using the Staphylococcus aureus isothermal nucleic acid amplification kit.

[0132] As the target concentration increases, the proportion of positive droplets (located in the upper right quadrant, i.e., Q2 area) gradually increases, the distribution gradually becomes more dense, and the positive density also increases. In addition, the FITC fluorescence intensity signal and SSC signal also show a trend of gradual enhancement. In contrast, the proportion of negative droplets (located in the lower left quadrant, i.e., Q3 area) gradually decreases, and its scatter point density is significantly reduced. The changes in the upper axis marginal histogram (FITC fluorescence intensity signal) show that as the target concentration increases, the area of ​​the FITC signal intensity peak (left peak) of the negative droplets gradually decreases, and the proportion of negative droplets continues to decrease; on the contrary, the area of ​​the FITC signal intensity peak (right peak) of the positive droplets expands, and the peak intensity increases, indicating that the proportion of positive droplets increases with the increase of target concentration. The changes in the right axis marginal histogram (SSC signal) show that as the target concentration increases, the area of ​​the SSC signal intensity peak (lower peak) of the negative droplets decreases significantly, further proving that the number of negative droplets decreases; at the same time, the area of ​​the SSC signal intensity peak (upper peak) of the positive droplets gradually increases, and the distribution range of the signal is also wider, indicating that the distribution of the positive droplets in terms of side scattering intensity becomes more significant.

[0133] Specifically, the positive droplets with stronger FITC signals also have higher SSC signals. This indicates that there is consistency between the FITC fluorescence intensity in the droplets and the particle size characteristics. This positive correlation is clearly reflected in the scatter plot. As the target concentration increases, the scatter points of the positive droplets (located in the upper right quadrant, i.e., the Q2 area) gradually concentrate in the high FITC and high SSC areas. In the marginal histogram, as the target concentration increases, the peak (low FITC, low SSC) area of ​​the negative droplets gradually decreases, while the peak (high FITC, high SSC) area of ​​the positive droplets gradually expands. In the upper axis histogram, the peak of the positive droplets gradually moves to the right (FITC signal increases); in the right histogram, the peak of the positive droplets gradually moves upward (SSC signal increases). This result intuitively demonstrates the positive correlation between the FITC fluorescence signal and the SSC signal through the coordinated change of the signal, laying the foundation for the digital classification of the particle size characteristics of polydisperse agarose gel microspheres by flow cytometry.

[0134] This embodiment further analyzes 20054, 20372, 9365, 30000, 14531 and 20282 polydisperse agarose gel microspheres contained in six groups of samples. In FlowJo, this embodiment processes the original FCS file, which contains the original signal data of all agarose gel microspheres, which may contain some impurities or signal noise. This embodiment needs to ensure that each gel microsphere has independent FITC-H, SSC-H and FSC-H signal values, and selects the parameters to be exported. Ultimately, the quantitative results of ddlAMP are used as the gold standard for quantitative performance detection, and the FITC-H signal is used as an auxiliary verification standard for the flow analysis method.

[0135] In order to accurately distinguish the yin and yang of agarose gel microspheres, the present embodiment performs data transformation on FITC-H and SSC-H signals, and a histogram is drawn using a logarithm (log10) with a base of 10. After data transformation, the distribution of the signal is more concentrated, and the horizontal axis is the value range of FITC-H and SSC-H signals, and the vertical axis is the frequency in each interval. For FITC-H signals, bincount is set to 100, and the original horizontal axis range is [0.01, 4000000]. After log10 transformation, the data range becomes [-2, 6.602], covering all distribution intervals, and the grid size is 0.08602. The mean of the two peaks is obtained by Gaussian mixture model (GMM), and the valley value, i.e., the lowest point between the two peaks, is found by calculating the intersection between the two Gaussian distributions. The median value of the interval is selected as the threshold, and all data less than the threshold are marked as negative (assigned as 0), and data greater than or equal to the threshold are marked as positive (assigned as 1). Fig. 9 The positive and negative thresholds of FITC-H signals in (a)-(f) are 4.8386, 4.4085, 4.5805, 4.5805, 4.4945, and 4.6665, respectively. For the SSC-H signal, the bin count is set to 100, and the original horizontal axis range is [1000,63095700]. After log10 transformation, the data range becomes [3,7.8002], covering all distribution intervals, and the grid size is 0.048002. The positive and negative thresholds of SSC-H signals in Figure 7 (a)-(f) are 4.9916, 5.0396, 5.0876, 5.1396, 5.1876, and 4.8196, respectively. Fig. 9(a)-(f) show the scatter plots of the FICT fluorescence signal and SSC signal of six groups of polydisperse agarose gel microspheres with nucleic acid concentrations from low to high: 8.24×101copies / μL, 1.65×102copies / μL, 8.24×102copies / μL, 1.65×103copies / μL, 8.24×103copies / μL, and 1.65×104copies / μL. The upper axis is the marginal histogram of the FITC signal, and the right axis is the marginal histogram of the SSC signal. The four quadrants Q1 (upper left), Q2 (upper right), Q3 (lower right), and Q4 (lower left) are respectively the false positive, true positive, false negative, and true negative divisions.

[0136] For the FSC-H signal, the FSC-H signal of each gel microsphere characterizes its corresponding volume size. Fig.10 The scatter plot of FITC-H and FSC-H signals is shown. Fig.11 A scatter plot of SSC-H and FSC-H signals is shown. Fig.10 and Fig.11 It can be found that the probability of the larger gel microspheres (i.e., larger FSC-H signals) encapsulating the target nucleic acid molecules is greater and the number is greater. Stronger positive gel microspheres may produce more magnesium pyrophosphate precipitation, making the inside of the gel microspheres more scattered and irregular, thereby increasing the SSC-H signal. This embodiment uses the FSC-H value of each gel microsphere to correspond to its relative volume, and substitutes it into the polydisperse volume nucleic acid calculation model to calculate the relative size value of EB virus quantification.

[0137] In clinical verification, EBV usually remains dormant in the body for a long time, especially in human B cells. For people with normal immune function, EBV usually does not cause symptoms. But when the immune system is suppressed, EBV can reactivate and cause disease, especially in patients after chemotherapy or stem cell transplantation. Patients with blood system diseases, organ transplantation, tumors and other immune-related diseases usually have impaired or suppressed immune system function due to the disease itself or treatment (such as chemotherapy, immunosuppressive therapy, etc.), and their immunity is significantly reduced. The weakening of immunity reduces the body's defense against viruses, especially for latent viruses such as EBV, which are easy to reactivate in the case of low immune function, leading to infection. In order to effectively monitor and prevent EBV-related complications, the load of EBV DNA in these patients must be regularly monitored. Through this test, signs of EBV infection can be detected in time, which facilitates intervention measures and improves the patient's prognosis.

[0138] In order to evaluate the performance of the Gel-LAMP method of the present embodiment in clinical samples, 39 EBV-DNA positive plasma samples were obtained for detecting the EB virus DNA nucleic acid load in whole blood samples. These 39 positive patients can be roughly divided into three stages from the age point of view, 0-20 years old accounted for about 10.3% (4 / 39), 21-60 years old accounted for about 61.5% (24 / 39), 61-80 years old accounted for about 28.2% (11 / 39). In terms of ward distribution, blood wards accounted for 43.6% (17 / 39), the highest proportion, transplant wards accounted for 28.2% (11 / 39), respiratory medicine, pediatrics, infectious diseases department accounted for 5.1% (2 / 39), neurosurgery accounted for 2.6% (1 / 39), and critical care medicine accounted for 7.7% (3 / 39). In the distribution of disease types, blood system diseases were the most prominent, accounting for 64.1% (25 / 39), of which acute myeloid leukemia accounted for 30.8% (12 / 39), acute lymphocytic leukemia accounted for 12.8% (5 / 39), chronic myelomonocytic leukemia accounted for 7.7% (3 / 39), and other blood system diseases accounted for 12.8% (5 / 39); infectious diseases accounted for 17.9% (7 / 39); and other diseases accounted for 17.9% (7 / 39).

[0139] In this example, LAMP primers targeting the EBNA-LP gene were synthesized. A special kit was used to use a centrifugal adsorption column that specifically binds to nucleic acids for positive plasma samples. After the samples were lysed, the DNA was eluted with a buffer. Four assay methods, including qPCR, ddPCR, Gel-LAMP-FITC in this example, and Gel-LAMP-SSC, were performed on these 39 clinical samples. The test results are shown in Figure 2. Fig.12The heat map shown in part (a) shows the CT value and concentration quantitative determination results of qPCR, ddPCR, Gel-LAMP-FITC and Gel-LAMP-SSC; the color gradient indicates the change of CT value and quantitative concentration change, blue indicates low CT value (high concentration), red indicates high CT value (low concentration); the slash " / " indicates that the sample data is missing. Each row represents the test results of the same sample by different detection methods, from bottom to top, qPCR-CT, providing a reference for CT value; ddPCR as the gold standard; Gel-LAMP-FITC and Gel-LAMP-SSC are two methods proposed in this example; the range of EB virus DNA detected is 6.55-320.38 copies / μL. Among them, the area marked with " / " indicates that data is lost due to operational errors during the experiment, such as the absence of sample 25, the exhaustion of pre-experimental samples for samples 1 and 2, abnormal SSC data for samples 3, 4, 5, 6, 7, 14, 20, and 28, sample mixing leading to sample addition errors for samples 36 and 37, and too little data for samples 38 and 39. The abnormal SSC signal data of the samples may be due to the instability of SSC side scatter signal detection on these samples or excessive background noise, resulting in the inability to obtain reliable data. During the LAMP amplification process, the precipitate generated in the reaction system (such as magnesium pyrophosphate precipitate) is the main source of SSC signal detection distinction, or it may be that the precipitate is not generated or does not gather during the amplification process, resulting in scattered or weak signals. Blue indicates that the EBV DNA load in the corresponding sample is relatively large, and blue indicates that the EBV DNA load in the corresponding sample is relatively small. The qPCR results show that the CT value is basically consistent with the concentration trend of other quantitative methods (ddPCR and Gel-LAMP method) when the CT value is used as a relative quantitative reference. The Gel-LAMP-FITC and Gel-LAMP-SSC methods showed good performance in terms of data consistency and accuracy. From the color distribution, Gel-LAMP-FITC and Gel-LAMP-SSC can well distinguish the target signals between high-concentration samples (dark blue) and low-concentration samples (light red). Fig.12 (b) and (c) are the comparative scatter plots and linear fitting curves of the Gel-LAMP-FITC and Gel-LAMP-SSC detection methods and the ddPCR detection results, respectively. The fitting formula of Gel-LAMP-FITC is y=1.12x, and the correlation coefficient R 2 is 0.966; the fitting formula of Gel-LAMP-SSC is y=1.11x, and the correlation coefficient R 2 The value of Gel-LAMP was 0.973, indicating that both methods had good consistency with the ddPCR test results. Gel-LAMP technology showed good detection performance in clinical samples and was significantly consistent with the gold standard ddPCR results.

[0140] Therefore, the digital nucleic acid detection method provided in this embodiment has the following innovations:

[0141] GelLAMP can efficiently distribute all reaction reagents through ordinary vortexing in a short time, eliminating complex microfluidic chips, complicated procedures and high-cost instruments, which makes it possible to implement frequent and large-scale detection in practical applications.

[0142] Furthermore, the Beckman CytoFLEX series flow cytometer used in the detection means has a throughput of more than 10,000 events per second, which can obtain signals quickly and at high throughput. This embodiment is the first to develop the FSC and SSC signals output by the flow cytometer to obtain the volume of agarose microbeads and judge the positive and negative, and substitute them into the multi-volume Poisson distribution nucleic acid concentration calculation model to obtain the target nucleic acid concentration of the sample. In addition, the absolute quantification of the DNA load of the EB virus was successfully detected through clinical sample verification, with relatively good accuracy.

[0143] Preferably, FSC-Height, FSC-Area, FSC-Width three-dimensional signals can be further included in the model calculation to optimize the digital determination method of polydisperse volume. Regarding the selection of flow cytometer, the other functions of the instrument are currently redundant for this method, so it is planned to develop a new type of equipment integrating vortex platform, isothermal control platform, centrifugal elution and front side scattering, aiming to achieve automatic detection and automatic data processing, and then promote the research and development of digital quantitative analysis instruments. However, the method based on polydisperse droplets still needs to achieve absolute quantification with the help of standard k value correction. Therefore, in order to optimize the accuracy of the experiment, all flow cytometers should be uniformly set, including laser power, gain and detection channel range, etc., and standard microspheres (such as flow cytometer calibration microspheres) are used to calibrate instrument performance.

[0144] In general, this embodiment develops a quantitative method for EB virus based on polydisperse gel microsphere flow analysis, which can efficiently detect viral DNA through minimal and simple equipment combined with isothermal amplification technology. And this embodiment successfully detected the viral DNA load in 25 EB virus (EBV) DNA-positive plasma samples, demonstrating the potential for low-cost viral DNA load detection. The digital nucleic acid detection method provided in this embodiment can not only serve as an important tool for pathogen typing, nucleic acid detection and patient prognosis assessment, but also meet the needs of primary care in a low-cost and efficient manner, and is expected to provide new solutions for resource-limited environments.

[0145] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same and similar parts between the various embodiments can be referred to each other. For the system disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part description. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of this application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of this application.

[0146] It should also be noted that, in this specification, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "comprises", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the statement "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device including the element.

Claims

1. A quantitative method for EB virus based on polydisperse gel microsphere flow analysis, characterized in that: include: Obtaining a sample reagent to be subjected to EB virus quantification, and mixing the sample reagent with an agarose solution to generate polydisperse agarose droplets; placing the agarose droplets in an isothermal environment for incubation to perform nucleic acid amplification to obtain polydisperse agarose gel microspheres; After the nucleic acid amplification is completed, the agarose gel microspheres are cooled and solidified to obtain agarose gel microspheres; Demulsifying and dyeing the agarose gel microspheres; The dyed agarose gel microspheres were analyzed by flow cytometry to obtain forward scattered light signals and side scattered light signals; The volume size and particle size difference of the polydispersed agarose gel microspheres are respectively obtained according to the forward scattered light signal and the side scattered light signal, and the EB virus quantitative result of the sample reagent is calculated in combination with the multi-volume nucleic acid calculation formula.

2. The EB virus quantitative method based on polydisperse gel microsphere flow analysis according to claim 1, characterized in that: The method of obtaining a sample reagent to be subjected to EB virus quantification and mixing the sample reagent with an agarose solution to generate polydisperse agarose droplets comprises: The sample reagent is mixed with the agarose solution, and under the action of fluorinated oil and a surfactant, a batch of polydispersed agarose droplets are generated by a vortex oscillation method.

3. The EB virus quantitative method based on polydisperse gel microsphere flow analysis according to claim 1, characterized in that: The step of placing the agarose droplets in an isothermal environment for incubation to perform nucleic acid amplification to obtain polydisperse agarose gel microspheres comprises: The agarose droplet was placed in an isothermal environment at 60-70°C and incubated for 40-50 minutes for nucleic acid amplification.

4. The EB virus quantitative method based on polydisperse gel microsphere flow analysis according to claim 1, characterized in that: After the nucleic acid amplification is completed, the agarose gel microspheres are cooled and solidified to obtain agarose gel microspheres, comprising: The agarose gel microspheres obtained by nucleic acid amplification are cooled to 2-6° C. to obtain the agarose gel microspheres.

5. The EB virus quantitative method based on polydisperse gel microsphere flow analysis according to claim 1, characterized in that: The demulsification and dyeing treatment of the agarose gel microspheres comprises: The agarose gel microspheres are washed to remove the oil phase, and the agarose gel microspheres after the oil phase is removed are stained with dye for nucleic acid.

6. The EB virus quantitative method based on polydisperse gel microsphere flow analysis according to claim 1, characterized in that: The method of performing flow cytometry analysis on the stained agarose gel microspheres to obtain forward scattered light signals and side scattered light signals comprises: The stained agarose gel microspheres were analyzed by flow cytometry.

7. The EB virus quantitative method based on polydisperse gel microsphere flow analysis according to claim 6, characterized in that: Before the step of using a flow cytometer to perform flow cytometric analysis on the stained agarose gel microspheres, the method comprises: The stained agarose gel microspheres are blown and then introduced into the flow cytometer.

8. The EB virus quantitative method based on polydisperse gel microsphere flow analysis according to claim 1, characterized in that: The step of obtaining the volume and particle size difference of the polydispersed agarose gel microspheres according to the forward scattered light signal and the side scattered light signal comprises: When the forward scattered light signal is larger, it is determined that the volume of the agarose gel microsphere is larger; When the forward scattered light signal is smaller, it is determined that the volume of the agarose gel microsphere is smaller.

9. The EB virus quantitative method based on polydisperse gel microsphere flow analysis according to claim 1, characterized in that: The method of obtaining the volume and the positive and negative properties of the polydispersed agarose gel microspheres according to the forward scattered light signal and the side scattered light signal respectively further comprises: When the side scattered light signal reaches a preset threshold, the agarose gel microspheres are determined to be positive; When the side scattered light signal is lower than a preset threshold, the agarose gel microspheres are determined to be negative.

10. The EB virus quantitative method based on polydisperse gel microsphere flow analysis according to claim 1, characterized in that: The EB virus quantitative result of the sample reagent is calculated by combining the multi-volume nucleic acid calculation formula, including: The EB virus quantitative result of the sample reagent is calculated according to the following formula: Wherein, F(k,c) represents the multi-volume nucleic acid calculation formula, k represents the proportional coefficient between the polydispersed agarose gel microspheres and the forward scattered light signal, C represents the original nucleic acid concentration, Φ represents the forward scattered light signal, j represents the positive agarose gel microspheres, and N represents the number of agarose gel microspheres.

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