Micro-fluidic chip for sequencing single virus genome and sequencing method

Abstract

The invention relates to a micro-fluidic chip for sequencing a single virus genome and a sequencing method. The micro-fluidic chip comprises a first functional area, a delay pipeline, a fusion pipeline, a second functional area and a fusion electrode, the first functional area is used for generating first liquid drops of virus-in-oil and lysate; two ends of the delay pipeline are respectively connected with the first functional area and the fusion pipeline; the second functional area is used for generating second liquid drops of the oil bag amplification reaction liquid, and the second functional area is connected with the fusion pipeline to enable the second liquid drops to enter the fusion pipeline; a fusion point is arranged on the downstream of the joint of the fusion pipeline and the second functional area, and the fusion electrode can act on the fusion point to fuse the first liquid drop and the second liquid drop. According to the micro-fluidic chip disclosed by the invention, single-virus whole genome amplification can be completely completed on a single chip, so that the operation steps are simplified, the complexity of sample treatment is reduced, pollution and sample loss are effectively avoided, and the efficiency, the stability and the reliability are improved.

Description

Technical Field

[0001] The present invention relates to the field of microfluidics technology, and in particular to a microfluidics chip and a sequencing method for single virus genome sequencing. Background Art

[0002] With the rapid development of molecular biology and sequencing technology, single-virus genomics (SVG) has gradually become an important tool for studying uncultured viral communities. SVG research covers multiple frontier fields, including environmental virome analysis, human virome research, and exploration of viral ecological functions. Current single-virus genome sequencing technologies (such as microfluidic gel bead-based sequencing platforms) can obtain genetic information at the level of single virus particles and reveal the diversity of uncultured viruses. However, these technologies have obvious limitations in terms of operational complexity, sample loss, and contamination risks, which restricts their application in large-scale high-throughput viral genome analysis.

[0003] At present, single virus sequencing technology can be divided into two categories: one is flow cytometry based on flow cell sorting technology combined with multiple displacement amplification technology. This method has low throughput and limited detection ability for very small virus particles, especially when dealing with uncultured viruses. In addition, the labeling process of virus particles in flow cytometry is complicated, which may interfere with the natural structure or function of the virus and increase the uncertainty of the experiment. The other is a single virus genome sequencing platform based on microfluidic technology. The microfluidic technology platform has the advantages of high throughput, high efficiency and high accuracy. However, its operation process is relatively complicated, involving multiple sample processing and reinjection steps, which can easily introduce contamination or cause sample loss. At the same time, droplets are prone to fusion during multiple operations, affecting the integrity of the data and the analysis results. In high-throughput analysis, this multi-step process is particularly time-consuming and lacks stability.

[0004] Therefore, traditional microfluidic chips still need further improvement to better meet the detection requirements of single virus genome sequencing. Summary of the invention

[0005] Based on this, it is necessary to provide a microfluidic chip that can improve the efficiency, stability and reliability of single virus genome sequencing.

[0006] A microfluidic chip for single virus genome sequencing comprises a first functional area, a delay pipe, a fusion pipe, a second functional area and a fusion electrode; the first functional area is used to generate a first droplet of oil-encapsulated virus and lysis solution; the two ends of the delay pipe are respectively connected to the first functional area and the fusion pipe, so that the first droplet enters the fusion pipe after a preset time; the second functional area is used to generate a second droplet of oil-encapsulated amplification reaction solution, and the second functional area is connected to the fusion pipe so that the second droplet can enter the fusion pipe; a fusion point is provided downstream of the connection between the fusion pipe and the second functional area, the fusion electrode can act on the fusion point to fuse the first droplet and the second droplet, and a droplet collection port is provided at the end of the fusion pipe.

[0007] When the microfluidic chip of the present invention is used for single virus genome sequencing, a single virus suspension, a lysate and an oil phase can be introduced into the microfluidic chip, a first droplet of oil-enclosed virus and lysate is generated through the first functional area, and the first droplet is made to reach the fusion channel through the delay channel; an amplification reaction solution and an oil phase are introduced into the microfluidic chip, a second droplet of oil-enclosed amplification reaction solution is generated through the second functional area, and the second droplet is made to enter the fusion channel; in the fusion channel, two different droplets merge, and due to the inconsistency of the droplet volumes, the second droplet will catch up with the first droplet, and finally form a close pairing, and the paired droplets continue to move forward in the fusion channel and reach the fusion point; a high-frequency voltage is applied on both sides of the fusion point through a fusion electrode, and the electric field is used to induce the fusion of the first droplet and the second droplet, and through the action of the electric field, the two droplets that are closely adjacent to each other fuse, and then the amplification reaction of the virus genome can be started under specific temperature conditions; finally, the reaction products are collected from the droplet collection port for purification, library construction, sequencing and other operations.

[0008] The microfluidic chip of the present invention enables the amplification of a single virus whole genome to be completed entirely on a single chip, greatly simplifies the operating steps, reduces the complexity of sample processing, and effectively avoids pollution and sample loss, greatly improves the efficiency, stability and reliability of the results of the experiment, and provides a more concise and efficient solution for single virus sequencing technology. Moreover, the present invention introduces a delay pipeline in the chip design, and by setting a delay area, the droplets can stay in the pipeline for a longer time, thereby ensuring sufficient lysis time, even under the condition of high-speed flow of droplets, the lysis efficiency can be guaranteed, the problem of incomplete lysis is avoided, and the high efficiency of virus lysis is ensured, which lays the foundation for subsequent genome amplification.

[0009] In one embodiment, the length of the delay pipe is greater than 160 mm, and the preset time is greater than 15 s.

[0010] In one embodiment, the delay pipeline includes a plurality of straight pipelines and arc-shaped pipelines, the plurality of straight pipelines are arranged in parallel at intervals, and the arc-shaped pipeline connects adjacent straight pipelines in sequence.

[0011] In one of the embodiments, it further includes a first spacer oil input pipeline, which is connected to the fusion pipeline, and the connection point is located upstream of the connection point between the second functional area and the fusion pipeline.

[0012] In one embodiment, the first functional area includes a first oil pipeline, a virus suspension input pipeline and a lysis reagent input pipeline. After the virus suspension input pipeline merges with the lysis reagent input pipeline, it intersects with the first oil pipeline in a cross shape to form a first droplet output pipeline, and the first droplet output pipeline is connected to the delay pipeline.

[0013] In one embodiment, the second functional area includes a second oil pipeline, an amplification reaction liquid input pipeline and a second spacer oil input pipeline. The second oil pipeline and the amplification reaction liquid input pipeline intersect in a cross shape to form a second droplet output pipeline. The second spacer oil input pipeline is connected to the second droplet output pipeline, and the second droplet output pipeline is connected to the fusion pipeline.

[0014] In one embodiment, the number of the fusion points is more than two, and the diameter of the fusion pipe located in the fusion point area is more than 50 μm larger than that in the non-fusion point area.

[0015] The present invention also provides a single virus genome sequencing method for non-disease diagnosis and treatment purposes, using the above-mentioned microfluidic chip, the sequencing method comprises the following steps:

[0016] Inputting a single virus suspension, a lysate and an oil phase into the microfluidic chip, generating the first droplet of the oil-encapsulated virus and the lysate through the first functional area, and allowing the first droplet to reach the fusion channel through the delay channel;

[0017] Inputting the amplification reaction solution and the oil phase into the microfluidic chip, generating the second droplet of the oil-encapsulated amplification reaction solution through the second functional area, and allowing the second droplet to enter the fusion channel;

[0018] Applying a high-frequency voltage to the fusion point through the fusion electrode, inducing the fusion of the first droplet and the second droplet by using the electric field, and then initiating an amplification reaction of the viral genome;

[0019] The products after the amplification reaction are collected from the droplet collection port for purification, library construction and sequencing.

[0020] In one embodiment, the lysis solution comprises 0.6-1.2 M NaOH and 0.05 wt % -0.2 wt % surfactant.

[0021] In one embodiment, the amplification reaction solution comprises 90-110 mM Tris-Acetate, 9-11 mM MgCl2, 9-11 mM (NH4)2SO4, 3-5 mM DTT, 0.4-0.6 U / μl Phi29, 15-25 mM Random Primer and 0.8-1.2 mM dNTP.

[0022] The sequencing method of the present invention greatly simplifies the operation process, reduces the risk of sample loss and cross-contamination, significantly improves the processing efficiency and the accuracy of the analysis results, and provides a more concise and efficient solution for single virus sequencing technology by completing virus encapsulation, lysis and whole genome amplification on a single microfluidic chip at the same time. Moreover, it can well ensure the efficient lysis of the virus on the chip, simplify the traditional two-step fusion process, reduce droplet loss, improve experimental efficiency, and can conveniently and accurately control the droplet generation frequency to ensure a stable pairing effect. Through multi-point fusion and fluid dynamics design, it solves the fusion problem under high-speed droplet flow. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A schematic diagram of the structure of a microfluidic chip according to an embodiment;

[0024] Figure 2 for Figure 1 Schematic diagram of the working principle of the microfluidic chip shown;

[0025] Figure 3 for Figure 1 A photomicrograph of part A of the microfluidic chip shown;

[0026] Figure 4 for Figure 1 Part B of the microfluidic chip is shown as a real micrograph;

[0027] Figure 5 for Figure 1 A micrograph of part C of the microfluidic chip shown;

[0028] Figure 6 This is a graph showing the results of testing the virus lysis efficiency of different alkali concentrations and treatment times in EP tubes;

[0029] Figure 7 This is a graph showing the effect of Tris-acetate on Phi29 enzyme activity in EP tube testing;

[0030] Figure 8This is a graph showing the results of exploring the optimal activity conditions of the Phi29 enzyme. DETAILED DESCRIPTION

[0031] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively below, and preferred embodiments of the present invention are provided. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0033] In the study of single virus whole genome amplification, microfluidic chip technology is a common platform for precisely controlling droplet operations. Despite its flexibility and small-scale operation advantages, traditional microfluidic schemes still face many challenges in the process of virus lysis and genome amplification preparation, which seriously limits the efficiency and simplicity of experiments.

[0034] (1) The multi-step fusion process is complex and time-consuming:

[0035] Initial fusion of virus droplets and lysis solution: The first step of the experiment is to encapsulate the virus and lysis solution (such as alkaline lysis solution or enzyme lysis solution) into droplets to achieve virus lysis and release the viral genome. The choice of lysis solution is crucial because it directly determines the difficulty and complexity of subsequent processing.

[0036] Interference of lysis buffer on amplification reaction: Whether using alkaline lysis buffer or enzymatic lysis buffer, the residues after lysis will interfere with the amplification reaction. Alkaline lysis buffer will destroy the enzyme activity in the amplification reaction buffer, while the residual enzyme in the enzymatic lysis buffer will interfere with the function of polymerase (such as Phi29). Therefore, the droplets after lysis must be additionally treated to avoid these interferences.

[0037] The second step is fusion: the lysis solution is fused with the neutralizer or inactivator, that is, the lysed droplets need to be fused with the neutralizer or inactivator droplets to neutralize the alkaline lysis solution or inactivate the residual enzyme. This step is crucial, but it increases the complexity and time cost of the experiment.

[0038] The third step is fusion: the treated droplets fuse with the amplification buffer, that is, after neutralization or inactivation, the droplets fuse with the amplification buffer to enter the whole genome amplification stage. It is very difficult to ensure the completeness of this step of fusion, especially when the first two steps have caused the droplet volume to change or material to remain.

[0039] (2) Operational complexity and risks

[0040] High precision in droplet control: Each fusion step requires highly precise control of droplet size, flow rate, and fusion timing. Any slight deviation will result in insufficient droplet fusion, affecting the experimental results.

[0041] Risk of droplet loss: During multiple fusion processes, if the operation is improper or the conditions are unstable, some of the droplet materials may not be able to fully participate in the fusion, resulting in the loss of genomic samples and affecting the efficiency of the amplification reaction.

[0042] The experiment is time-consuming: Each step of the droplet fusion operation requires a lot of time to be processed precisely, which significantly prolongs the entire experimental process and reduces laboratory efficiency.

[0043] (3) Complexity of multi-chip operation

[0044] At least two microfluidic chips are usually required to complete these multi-step droplet fusion operations. One is used for lysis and preliminary processing, and the other is used to fuse the virus droplets with the amplification reaction solution. The transfer of droplets between chips is very complicated and must rely on a precise fluid control system, which not only increases the cost of the experiment, but also places higher demands on the technical level of the operator. Each droplet transfer between chips requires careful calibration and monitoring, further increasing the complexity of the experiment and the possibility of human error.

[0045] (4) Inherent problems with enzymatic lysis and alkaline lysis solutions

[0046] Enzyme lysis solution: After the virus is lysed, the residual enzyme in the enzyme lysis solution will interfere with the amplification reaction, so it must be inactivated before amplification. If the inactivation is not complete, the residual enzyme will seriously interfere with the amplification efficiency of the polymerase, increasing the complexity of the experiment and the risk of error.

[0047] Alkaline lysis solution: Alkaline lysis solution can quickly and effectively lyse viruses, but its highly alkaline environment has a destructive effect on the enzymes in the amplification reaction solution. After lysis, the alkaline liquid must be eliminated by a neutralizer, which further increases the experimental steps and prolongs the time.

[0048] In summary, in this multi-step fusion operation process, there are many complex factors involved, not only the challenge of droplet operation accuracy, but also the precise control of droplet size, flow rate, substance concentration, etc. during each fusion process. Due to the complexity and high operational difficulty of these technical links, so far, only a very small number of laboratories have been able to try to apply this technology, and the actual application scope is extremely limited. However, single-virus whole genome amplification is an indispensable step in single-virus sequencing. The high complexity of this technology has led to the rapid development of single-cell sequencing technology in recent years, but the development of single-virus sequencing has stagnated. Therefore, there is an urgent need for a simpler, more reliable solution that can complete all operations on one chip to solve these problems.

[0049] like Figure 1 As shown, a microfluidic chip 100 for single virus genome sequencing according to an embodiment of the present invention includes a first functional area 10 , a delay channel 20 , a fusion channel 30 , a second functional area 40 and a fusion electrode 50 .

[0050] The first functional area 10 is used to generate the first droplet of the oil-in-virus and lysate. The two ends of the delay pipe 20 are respectively connected to the first functional area 10 and the fusion pipe 30, and the first droplet can reach the fusion pipe 30 through the delay pipe 20, so that the first droplet enters the fusion pipe 30 after a preset time. The second functional area 40 is used to generate the second droplet of the oil-in-amplification reaction solution, and the second functional area 40 is connected to the fusion pipe 30 so that the second droplet enters the fusion pipe 30. A fusion point 31 is provided downstream of the connection between the fusion pipe 30 and the second functional area 40, and the fusion electrode 50 can act on the fusion point 31 to fuse the first droplet and the second droplet, and a droplet collection port 32 is provided at the end of the fusion pipe 30.

[0051] The working principle of the microfluidic chip 100 of the present invention when used for single virus genome sequencing is as follows: Figure 2As shown, a single virus suspension, a lysate and an oil phase are introduced into the microfluidic chip 100, and an oil-encapsulated virus (virus) and a lysate (Lysis The first droplet of the amplification reaction solution and the oil phase (oil) are introduced into the microfluidic chip 100, and a second droplet of the oil-encapsulated amplification reaction solution is generated through the second functional area 40, and the second droplet is allowed to enter the fusion channel 30; in the fusion channel 30, the two different droplets merge, and due to the inconsistency of the droplet volumes, the second droplet will catch up with the first droplet, and finally form a tight pairing, and the paired droplets continue to move forward in the fusion channel 30 and reach the fusion point 31; a high-frequency voltage is applied to both sides of the fusion point 31 through the fusion electrode 50, and the electric field is used to induce the fusion of the first droplet and the second droplet. Through the action of the electric field, the two droplets that are closely together fuse, and then the amplification reaction of the viral genome is started at a specific temperature; finally, the reaction products are collected from the droplet collection port 32 for purification, library construction, sequencing and other operations.

[0052] The microfluidic chip 100 of the present invention enables the amplification of a single virus whole genome to be completed entirely on a single chip, greatly simplifies the operating steps, reduces the complexity of sample processing, and effectively avoids pollution and sample loss, greatly improves the efficiency, stability and reliability of the experiment, and provides a more concise and efficient solution for single virus sequencing technology. Moreover, in the early stage of single virus genome amplification, the virus and the lysate need to be wrapped in the same droplet, and the lysate needs enough time to fully contact the virus to complete the lysis. However, since the flow rate of the droplet is usually faster on the microfluidic chip, the virus lysis time may be insufficient, affecting the lysis efficiency and subsequent genome amplification. The present invention introduces a delay pipeline 20 in the chip design, and by setting a delay area, the droplet can stay in the pipeline for a longer time, thereby ensuring the sufficiency of the lysis time, even under the condition of high-speed flow of the droplet, the lysis efficiency can be guaranteed, and the problem of incomplete lysis is avoided, ensuring the high efficiency of virus lysis, and laying the foundation for subsequent genome amplification.

[0053] In a specific example, the length of the delay pipe 20 is greater than 160 mm, such as 160 mm, 170 mm, 180 mm, etc., but not limited to this, and the preset time is greater than 15 s, such as 15 s, 16 s, 17 s, 18 s, etc., but not limited to this, so as to ensure sufficient lysis time.

[0054] In a specific example, if Figure 1 As shown, the delay pipe 20 includes a plurality of straight pipes and arc pipes. The plurality of straight pipes are arranged in parallel at intervals, and the arc pipes connect adjacent straight pipes in sequence to form an S-shaped bent pipe, thereby increasing the length and reducing the occupied space.

[0055] In a specific example, the microfluidic chip 100 also includes a first spacer oil input pipeline 60, which is connected to the fusion pipeline 30, and the connection is located upstream of the connection between the second functional area 40 and the fusion pipeline 30. High-speed droplet generation is the key to achieving high-throughput single virus sequencing. However, in the case of high-speed flow of droplets, traditional technology is difficult to control the proportion and pairing of droplets, resulting in poor fusion effect. This is because in high-speed flowing liquid, droplets are difficult to pair in precise proportions, thereby affecting subsequent fusion and amplification steps. In order to solve this problem, the present invention innovatively introduces a spacer oil channel. By controlling the flow rate of the spacer oil, we can freely adjust the frequency of droplet generation so that the droplets to be fused can be stably paired in the pipeline. This innovative design ensures that each pair of droplets is in an ideal proportion and state when entering the fusion channel, effectively improving the accuracy of droplet pairing and fusion effect. This technology not only solves the instability problem in traditional droplet pairing, but also significantly improves the overall efficiency of the experiment.

[0056] In a specific example, the first functional area 10 includes a first oil pipeline 11, a virus suspension input pipeline 12 and a lysis reagent input pipeline 13. After the virus suspension input pipeline 11 and the lysis reagent input pipeline 12 merge, they intersect with the first oil pipeline 13 in a cross shape to form a first droplet output pipeline 14. Figure 3 for Figure 1 The microphotograph of part A of the microfluidic chip 100 is shown.

[0057] In a specific example, as shown in the figure, the second functional area 40 includes a second oil pipeline 41, an amplification reaction liquid input pipeline 42 and a second spacer oil input pipeline 43, the second oil pipeline 41 and the amplification reaction liquid input pipeline 42 intersect in a cross shape to form a second droplet output pipeline, and the second spacer oil input pipeline 43 is connected to the second droplet output pipeline, such as Figure 4 for Figure 1 The microphotograph of part B of the microfluidic chip 100 is shown. Thus, before the second droplets enter the fusion channel 30, a spacer oil channel is also introduced to adjust the spacing and frequency between the second droplets to ensure that these droplets form a stable 1:1 pairing with the first droplets.

[0058] In a specific example, the number of fusion points 31 is more than two, and the diameter of the fusion pipe 30 located in the fusion point 31 area is increased by more than 50 μm, such as 50 μm, 60 μm, etc., compared with the non-fusion point area. Figure 5 for Figure 1The actual microphotograph of part C of the microfluidic chip 100 is shown. Under high-speed flow conditions, the traditional electrofusion technology relies on fixed fusion points, which easily causes the droplets to flow away directly without having time to fuse. This problem is more prominent, especially when the droplet flow rate is fast, which not only affects the efficiency of fusion, but also greatly increases the failure rate of the experiment. The present invention adopts two innovative designs to improve the fusion efficiency: 1) Multiple fusion point design: multiple electrofusion points are set in the chip. If a pair of droplets fails to fuse successfully at the first fusion point, they can continue to fuse at subsequent fusion points. This design greatly improves the success rate of fusion and ensures that each pair of droplets can find opportunities to complete fusion in multiple fusion points. 2) Flow rate control at the fusion point: We have made an innovative structural design of the pipeline at the fusion point. By widening the pipeline diameter at the fusion point and using the Bernoulli effect in fluid dynamics, the flow rate of the droplets drops sharply when passing through the widened area. This design provides sufficient time for electrofusion and ensures that the droplets can complete full fusion at the fusion point. In addition, as the droplet flow rate decreases, the droplets behind can catch up with the droplets in front, achieving secondary pairing of unpaired droplets, thereby further improving the success rate of fusion. In this way, the fusion problem of droplets under high-speed flow is effectively solved, ensuring the efficiency and reliability of the fusion process.

[0059] A single virus genome sequencing method according to an embodiment of the present invention uses the above-mentioned microfluidic chip 100, and the sequencing method includes the following steps S1 to S4:

[0060] S1, a single virus suspension, a lysate and an oil phase are input into the microfluidic chip 100, a first droplet of oil-encapsulated virus and lysate is generated through the first functional area 10, and the first droplet is made to pass through the delay pipe 20 to reach the fusion pipe 30;

[0061] S2, inputting the amplification reaction solution and the oil phase into the microfluidic chip 100, generating a second droplet of the oil-encapsulated amplification reaction solution through the second functional area 40, and allowing the second droplet to enter the fusion channel 30;

[0062] S3, applying a high-frequency voltage through the fusion electrode 50 at the fusion point 31 of the fusion pipe 30, inducing the fusion of the first droplet and the second droplet by using the electric field, and then starting the amplification reaction of the viral genome;

[0063] S4. Collect the products after the amplification reaction from the droplet collection port for purification, library construction and sequencing.

[0064] It can be understood that the above-mentioned high-frequency voltage refers to an electrical signal with a frequency higher than 1K Hz. The virus sample is prepared into a single virus suspension, and the virus suspension is diluted to ensure that the concentration of single virus particles in the liquid reaches the target concentration. Usually, according to the Poisson distribution principle, the probability of containing a single virus particle per unit volume is high to avoid the situation where multiple virus particles exist in the same droplet at the same time. The flow of liquid in the microfluidic chip is carried out by a microfluidic drive device, which can be hydraulically driven or pneumatically driven.

[0065] It can be understood that the sequencing method also includes the following steps: inputting spacer oil through the first spacer oil input pipeline 60 and the second spacer oil input pipeline 43, and adjusting the spacing and frequency of the two droplets by controlling the flow rate of the spacer oil, so that the droplets to be fused can be stably paired 1:1 in the pipeline.

[0066] In a specific example, the lysate contains 0.6-1.2 M NaOH and 0.05 wt%-0.2 wt% surfactant, such as 0.8 M NaOH and 0.1 wt% surfactant, and the surfactant may be Tween 20 or Triton X-100 but is not limited thereto.

[0067] Thus, by optimizing the lysis solution, the virus can be fully lysed in a relatively short time. Even under the condition of high-speed flow of droplets, the lysis efficiency can be guaranteed, and the problem of incomplete lysis caused by insufficient time can be avoided. By combining the delay pipe 20 with the optimization of the lysis solution, the high efficiency of virus lysis is further ensured, laying a better foundation for subsequent genome amplification. The introduced surfactant has the following multiple functions: (1) reducing interfacial tension. The surfactant is arranged in a directional manner on the liquid interface, with the polar part in contact with the water phase and the non-polar part in contact with the oil phase, thereby reducing the interfacial tension of the water-oil interface. The effect of reducing interfacial tension: it is easier to form droplets, and the droplet generation process is smoother; it reduces the energy required for droplet generation, making the droplet size more uniform. (2) stabilizing the droplet interface. The surfactant molecules form a protective layer on the water-oil interface of the droplets, preventing the water phase droplets from contacting and merging with each other. Effect: preventing direct contact between droplets, avoiding fusion or rupture; improving the independence of droplets, ensuring that droplets are not disturbed when flowing in the pipeline. (3) inhibiting droplet deformation. During the droplet generation and flow process, the droplets may be deformed or broken due to external shear forces. Surfactants can inhibit droplet deformation in the following ways: dynamic restoration of interfacial tension. When the droplet deformation causes the interface area to increase, the surfactant quickly migrates to the newly formed interface to restore the surface tension; anti-shear force. The surfactant protective layer can absorb part of the shear force and reduce damage to the droplets. (4) Prevent droplet surface contamination. When droplets flow in the pipe, they may contact the pipe wall or be contaminated by external particles. The surfactant protective layer can effectively reduce these effects: pipe wall lubrication. The surfactant reduces the adhesion of the droplets to the pipe wall and avoids droplet breakage or residue; pollution protection. The stable interface formed by the surfactant prevents external pollutants from entering the droplets.

[0068] In one embodiment, the amplification reaction solution contains 90-110mM Tris-Acetate. Specifically, the amplification reaction solution contains 90-110mM Tris-Acetate, 9-11mM MgCl2, 9-11mM (NH4)2SO4, 3-5mM DTT, 0.4-0.6U / μl Phi29, 15-25mM Random Primer and 0.8-1.2mM dNTP. Preferably, the amplification reaction solution contains 100mM Tris-Acetate, 10mM MgCl2, 10mM (NH4)2SO4, 4mM DTT, 0.5U / μl Phi29, 20mMRandom Primer and 1mM dNTP. Preferably, Tris-Acetate and amplification buffer are mixed in a volume ratio of 1:2. It can be understood that the specific type of amplification reaction solution can be selected and adjusted according to the specific amplification method, for example, it can be MDA reaction solution, PCR reaction solution, LAMP reaction solution, RCA reaction solution, SDA reaction solution, NASBA reaction solution, etc.

[0069] The present invention has studied and obtained the above-mentioned amplification reaction liquid system, and by introducing Tris-Acetate, it can immediately neutralize the alkaline environment when it is fused with untreated alkaline lysate. Tris-Acetate can not only quickly neutralize the alkaline components in the lysate, but also ensure that the fused droplets are in the optimal pH conditions required for amplification. This design directly avoids the separate neutralization or inactivation steps that must be performed in traditional methods, simplifies the two-step fusion into a single-step fusion, and the virus droplets can be directly fused with the amplification reaction liquid after lysis, without the need for intermediate neutralization or inactivation steps, greatly simplifying the operation process. This single-step fusion method not only saves experimental time, but also significantly reduces droplet loss, and improves the stability and accuracy of the experimental results.

[0070] In summary, the technical solution of the present invention has the following significant advantages:

[0071] Simplified operation process: Traditional sequencing platforms based on microfluidic gel beads require multiple steps, and each step has the risk of sample loss or contamination. This solution significantly simplifies the operation process by completing encapsulation, lysis and amplification on a single chip, improving the stability and operation efficiency of the system.

[0072] Reduce sample loss and contamination: Multiple sample handling and reinjection processes can easily introduce contamination or sample loss. This solution integrates all key steps on one platform, greatly reducing the risk of sample loss and cross-contamination and improving the accuracy of analytical results.

[0073] Improved processing efficiency and result accuracy: By optimizing droplet generation and fusion technology, the system's processing efficiency has been significantly improved, and up to 2.5 million virus particles can be processed per hour. In addition, with the help of surfactants (such as Tween 20, TritonX-100) and spacer oil input pipelines, the stable generation and efficient amplification of droplets are ensured, and the final genomic DNA obtained has high accuracy and integrity.

[0074] Enhanced stability and repeatability: This solution introduces a spacer oil input pipeline and surfactant to further improve the stability and amplification efficiency of the system. This makes this technical solution more stable and repeatable in large-scale high-throughput viral genome analysis, and is suitable for long-term continuous experimental operations.

[0075] The following is a specific experiment to test the effects of different conditions on sequencing.

[0076] Experiment 1: EP tube testing the virus lysis efficiency at different alkali concentrations and treatment times

[0077] If the alkali concentration for virus lysis is too high, a large amount of weak acid will be needed for neutralization, which will cause the acetic acid droplet volume to be too large. At the same time, the delay pipeline of the microfluidic chip is too long, which will cause the microfluidic pipeline pressure to be too high. Therefore, we set different alkali concentrations and reaction times to treat viruses with intact capsids, then perform MDA amplification, and finally use DNA agarose gel to determine the optimal conditions for virus capsid lysis, and determine the lowest alkali concentration and shortest lysis time for complete virus lysis. The experiment set up ten groups:

[0078] Group 1: No virus, no enzyme water replacement (negative control)

[0079] Group 2: containing virus, high temperature denaturation annealing treatment (positive control)

[0080] Groups 3-6: containing virus, lysed with 0.8M NaOH, treated for 1min, 30s, 15s, and 7s respectively

[0081] Groups 7-10: containing virus, lysed with 0.4M NaOH, treated for 1min, 30s, 15s, and 7s respectively

[0082] The results are as follows Figure 6 As shown, the higher the NaOH concentration, the better the cleavage effect. At the same time, the longer the alkali treatment time, the better the cleavage effect. After comprehensive consideration, the cleavage condition of 0.8 M NaOH for 15 s is preferred.

[0083] Experiment 2: EP tube test of the effect of Tris-acetate on phi29 enzyme activity (secondary fusion changed to primary fusion)

[0084] The virus needs alkaline lysis, and the alkali has an effect on the activity of the Phi29 enzyme, so after the virus is lysed, it needs to be neutralized with acid before adding the Phi29 enzyme. Initially, the microfluidic chip we designed required two droplet electroporation fusions, that is, the first fusion added acid, and the second fusion added Phi29 enzyme for amplification reaction, but it is cumbersome to fuse two times on a chip at the same time and prone to failure. Therefore, through research, it was found that mixing Tris-acetate with Phi29 enzyme and amplification buffer can be well compatible, thereby reducing one fusion and greatly simplifying the microfluidic steps. First, we used EP tubes to test the effect of Tris-acetate on the activity of the Phi29 enzyme. The experimental groups are as follows:

[0085] Group 1: No virus, no enzyme water replacement (negative control)

[0086] Group 2: containing virus, high temperature denaturation annealing treatment (positive control)

[0087] Group 3: Containing virus, alkaline denaturation, acid neutralization, then adding Phi29 enzyme and amplification buffer

[0088] Group 4: Contains virus, alkaline denaturation, Tris-acetate, phi29 enzyme and amplification buffer

[0089] The results are as follows Figure 7 As shown, the Phi29 enzyme can maintain activity in a Tris-acetate environment, so Tris-acetate can be mixed with the Phi29 enzyme and the amplification buffer, thereby reducing one fusion and greatly simplifying the microfluidic steps.

[0090] Experiment 3: Exploring the optimal activity conditions of Phi29 enzyme / Comparison experiment before and after optimization of MDA reaction system

[0091] The above results show that Tris-acetate has little effect on the activity of Phi29 enzyme, but the proportion of MDA amplification buffer is large (MDA amplification buffer: Tris-acetate = 4:1), which makes the droplets in the microfluidics particularly large and difficult to merge with the virus droplets. Therefore, we further experimented to reduce the ratio of the two as much as possible under suitable pH conditions. The experiment set up five groups:

[0092] Group 1: No viral DNA template (negative control)

[0093] Group 2: Containing viral DNA template, high temperature denaturation annealing treatment (positive control)

[0094] Group 3: Containing viral DNA template, alkaline denaturation, the volume ratio of MDA to 0.1M Tris-acetic acid was 1:1

[0095] Group 4: containing viral DNA template, alkaline denaturation, MDA volume to 0.1M Tris-acetic acid volume ratio of 2:1

[0096] Group 5: containing viral DNA template, alkaline denaturation, MDA volume to 0.1M Tris-acetic acid volume ratio of 4:1

[0097] The results are as follows Figure 8 As shown, the optimal ratio of the volume of MDA amplification buffer to the volume of 0.1M Tris-acetic acid is 4:1, but in microfluidics, the MDA droplets will be particularly large, which is not conducive to fusion with virus droplets. After comprehensive consideration, 2:1 was selected.

[0098] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0099] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A microfluidic chip for single virus genome sequencing, characterized in that: It includes a first functional area, a delay pipe, a fusion pipe, a second functional area and a fusion electrode; the first functional area is used to generate a first droplet of oil-enclosed virus and lysis solution; the two ends of the delay pipe are respectively connected to the first functional area and the fusion pipe, so that the first droplet enters the fusion pipe after a preset time; the second functional area is used to generate a second droplet of oil-enclosed amplification reaction solution, and the second functional area is connected to the fusion pipe so that the second droplet can enter the fusion pipe; a fusion point is provided downstream of the connection between the fusion pipe and the second functional area, the fusion electrode can act on the fusion point to make the first droplet and the second droplet fuse, and a droplet collection port is provided at the end of the fusion pipe.

2. The microfluidic chip according to claim 1, characterized in that: The length of the delay pipe is greater than 160 mm, and the preset time is greater than 15 s.

3. The microfluidic chip according to claim 2, characterized in that: The delay pipeline includes a plurality of straight pipelines and arc pipelines. The plurality of straight pipelines are arranged in parallel at intervals, and the arc pipeline connects adjacent straight pipelines in sequence.

4. The microfluidic chip according to claim 1, characterized in that: It also includes a first spacer oil input pipeline, which is connected to the fusion pipeline, and the connection point is located upstream of the connection point between the second functional area and the fusion pipeline.

5. The microfluidic chip according to claim 1, characterized in that: The first functional area includes a first oil pipeline, a virus suspension input pipeline and a lysis reagent input pipeline. After the virus suspension input pipeline merges with the lysis reagent input pipeline, it intersects with the first oil pipeline in a cross shape to form a first droplet output pipeline, and the first droplet output pipeline is connected to the delay pipeline.

6. The microfluidic chip according to claim 1, characterized in that: The second functional area includes a second oil pipeline, an amplification reaction liquid input pipeline and a second spacer oil input pipeline. The second oil pipeline and the amplification reaction liquid input pipeline intersect in a cross shape to form a second droplet output pipeline. The second spacer oil input pipeline is connected to the second droplet output pipeline, and the second droplet output pipeline is connected to the fusion pipeline.

7. The microfluidic chip according to any one of claims 1 to 6, characterized in that: The number of the fusion points is more than two, and the diameter of the fusion pipe located in the fusion point area is more than 50 μm larger than that in the non-fusion point area.

8. A method for sequencing a single viral genome for purposes other than disease diagnosis and treatment, characterized in that: Using the microfluidic chip according to any one of claims 1 to 7, the sequencing method comprises the following steps: Inputting a single virus suspension, a lysate and an oil phase into the microfluidic chip, generating the first droplet of the oil-encapsulated virus and the lysate through the first functional area, and allowing the first droplet to reach the fusion channel through the delay channel; Inputting the amplification reaction solution and the oil phase into the microfluidic chip, generating the second droplet of the oil-encapsulated amplification reaction solution through the second functional area, and allowing the second droplet to enter the fusion channel; Applying a high-frequency voltage to the fusion point through the fusion electrode, inducing the fusion of the first droplet and the second droplet by using the electric field, and then initiating an amplification reaction of the viral genome; The products after the amplification reaction are collected from the droplet collection port for purification, library construction and sequencing.

9. The sequencing method according to claim 8, characterized in that The lysate contains 0.6-1.2M NaOH and 0.05wt%-0.2wt% surfactant.

10. The sequencing method according to claim 8, characterized in that The amplification reaction solution contains 90-110 mM Tris-Acetate, 9-11 mM MgCl2, 9-11 mM (NH4)2SO4, 3-5 mM DTT, 0.4-0.6 U / μl Phi29, 15-25 mM Random Primer and 0.8-1.2 mM dNTP.

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