Reaction system based on microdroplet type digital PCR and application thereof
By using an oil phase of base oil and silicone surfactants, and an aqueous phase of PCR reaction reagents and nonionic surfactants in microdroplet digital PCR, stable and uniform droplets are generated, solving the droplet fusion/fission problem, improving detection sensitivity and reducing costs.
Patent Information
- Application Number
- CN202510995318.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-18
- Publication Date
- 2025-11-07
AI Technical Summary
Existing droplet digital PCR technology suffers from droplet fusion/fission during PCR thermal cycling, resulting in poor quantitative accuracy and reagent waste. Fluorinated oil is costly, and silicone oil droplets are uneven in size, affecting detection results.
The reaction system employs an oil phase containing base oil and silicone surfactants, and an aqueous phase containing PCR reaction reagents and nonionic surfactants to generate uniform and stable droplets, which is suitable for high-temperature PCR amplification and reduces costs.
It improves the detection sensitivity and amplification result reliability of digital PCR, reduces droplet volume variation, lowers reagent costs, and is applicable to most commercially available PCR reaction solutions.
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Figure CN120905366A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of microfluidic technology, in particular to a reaction system based on droplet digital PCR and application thereof, more particularly to a reaction system based on droplet digital PCR, a digital PCR amplification method and a digital PCR amplification kit. BACKGROUND
[0002] Droplet digital PCR (ddPCR) is a highly sensitive and accurate method for absolute quantification of nucleic acid molecules. In ddPCR, the sample is divided into many discrete sub-reaction units using microfluidic methods, so that each unit contains a single or no target molecules, and then PCR amplification and detection are performed. These sub-reaction units are usually small droplets of water phase wrapped in oil phase. The miniaturization of droplet microfluidic reaction volume brings the advantages of high cost efficiency, high throughput analysis, high detection sensitivity, high sample utilization rate and high reaction efficiency. With the development of microfluidic technology, using microfluidic droplet method can easily reduce the solution volume of biochemical analysis to picoliter or even femtoliter, which has the ability of adjustable size and controllable number.
[0003] However, there are still some challenges in the practice of ddPCR technology. One of the main problems is that during PCR thermal cycling, unconstrained droplets can closely aggregate together, leading to easy fusion / fission of droplets. This is due to the movement of droplets caused by thermal expansion and compression during PCR process in continuous thermal cycling. Fusion / fission of droplets leads to polydispersity of droplets, which not only affects the quantitative accuracy of ddPCR, but also causes waste of reagents and rare samples. Secondly, fluorine oil is high in cost, and droplets generated by low-cost silicon oil have the problem of uneven size.
[0004] Therefore, there is still a need for a low-cost and stable oil phase formula that can resist the negative effects of temperature on droplets during PCR heating and cycling, stabilize the droplet morphology, and ensure the retention rate of droplets after heating and the amplification effect of PCR. SUMMARY
[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, one object of the present application is to provide a low-cost and high-temperature-resistant droplet digital PCR reaction system.
[0006] Specifically, the present application provides the following technical solutions:
[0007] In a first aspect, the present application provides a reaction system based on droplet digital PCR. According to embodiments of the present application, the reaction system comprises: an oil phase comprising base oil and a silicon surfactant; and an aqueous phase comprising PCR reaction reagents and a non-ionic surfactant. In some examples of the present application, the above-mentioned reaction system is suitable for high-temperature PCR amplification at 95°C, has no inhibition on amplification, generates droplets with uniform size, is stable in high-temperature form, and has good biocompatibility. In addition, the reaction system is suitable for most commercial PCR reaction solutions, has low cost, and is highly available.
[0008] In a second aspect, the present application provides a method for digital PCR amplification. According to embodiments of the present application, the method comprises: generating droplets based on the reaction system of the first aspect of the present application; and performing PCR amplification on the droplets. In some examples of the present application, the method for digital PCR amplification avoids the problem that the reliability of the digital PCR amplification result is not high due to too few or too many droplets after amplification, and improves the detection sensitivity.
[0009] In a third aspect, the present application provides a kit for digital PCR amplification. According to embodiments of the present application, the kit comprises: an oil phase comprising base oil and a silicon surfactant; and an aqueous phase comprising PCR reaction reagents and a non-ionic surfactant. In some examples of the present application, the kit is used in the process of digital PCR amplification, has low cost, and has higher detection sensitivity.
[0010] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS
[0011] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.
[0012] Figure 1 is a microscope photo of droplets generated based on the reaction system of the present application (before PCR amplification) provided by embodiments 4, 9, 10, and 12 of the present application;
[0013] Figure 2 is a microscope photo of droplets generated based on the reaction system of the present application (before PCR amplification) provided by embodiments 4, 5, 7, 9, and 10 of the present application;
[0014] Figure 3 is a microscope photo of droplets generated based on the reaction system of the present application (before PCR amplification) provided by embodiment 19 of the present application;
[0015] Figure 4is a microscope photo of the droplet generated based on the reaction system of the present application (after PCR amplification) provided in Example 19 of the present application;
[0016] Figure 5 is a FAM scatter plot of the target nucleic acid molecule after PCR amplification of the droplet generated based on the reaction system of the present application provided in Example 19 of the present application. DETAILED DESCRIPTION
[0017] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference signs represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by reference to the accompanying drawings are exemplary and are only for the purpose of explaining the present application, and cannot be understood as limiting the present application.
[0018] At present, there are still some deficiencies in the microdroplet digital PCR technology, such as non-uniform droplet generation, easy deformation at high temperature, etc., thereby resulting in low reliability of PCR results. In order to obtain stable and uniform droplets at high temperature, in one aspect of the present application, a reaction system is provided. The system comprises: an oil phase, the oil phase comprising a base oil and a silicon surfactant; and an aqueous phase, the aqueous phase comprising PCR reaction reagents and a non-ionic surfactant. The oil phase and the aqueous phase are prepared into uniform and stable PCR reaction droplets based on a microfluidic device, which can increase the reliability of PCR amplification results and improve the detection sensitivity of PCR amplification products.
[0019] In some examples of the present application, the base oil comprises at least one of saturated alkane silicone oil and mineral oil;
[0020] In some examples of the present application, the silicon surfactant is selected from at least one of cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethyl siloxane alcohol, cyclopentasiloxane, trimethylsilyloxy silicate and polyether modified heptamethyl trisiloxane. In a preferred example of the present application, the above-mentioned silicon surfactant is selected from cyclopentasiloxane and trimethylsilyloxy silicate.
[0021] wherein the mass ratio of the above-mentioned base oil to the silicon surfactant is (2-8): 1, which can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1, preferably 4:1.
[0022] In some examples of the present application, the PCR reaction reagents include amplification primers, probes, dNTPs, amplification templates and amplification buffers.
[0023] In some examples of the present application, the above-mentioned non-ionic surfactant is selected from Tween 80, Tween 60, Poloxamer TM 188、 at least one of FS-30. The addition of non-ionic surfactant in the aqueous phase can increase the viscosity of the aqueous phase droplet and increase the thermal stability of the microfluidic chip in the amplification process. Importantly, the surfactant added in the aqueous phase of the reaction system of the present application does not affect the biological activity of the target nucleic acid molecule in the process of digital PCR. In a preferred example of the present application, the non-ionic surfactant is selected from FS-30 and Poloxamer TM 188.
[0024] In some examples of the present application, the mass ratio of the above-mentioned PCR reaction reagent and the above-mentioned non-ionic surfactant is (3-9): 1, which can be 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 or 9:1, preferably 4:1.
[0025] The digital PCR reaction droplet prepared based on the above-mentioned aqueous phase composition and oil phase composition is uniform in size, stable in high temperature cycle (95-98℃), good in biocompatibility, weak in fluidity, can be perfectly preserved in the chip after heating, and has no side effects on the amplification of the target nucleic acid molecule (DNA or RNA) of digital PCR, so that high-temperature amplification can be directly carried out in the generated chip, solving the problem that high temperature causes droplet fusion or breakage, high-temperature thermal motion causes droplet to escape, evaporate or deform, so that the amount of droplet after amplification is too small or changes too much, resulting in low reliability of digital PCR amplification result, and improving the detection sensitivity.
[0026] In another aspect of the present application, based on the above-mentioned reaction system, a digital PCR amplification method is developed. The method comprises: based on a microfluidic device, droplet generation processing is carried out on the reaction system of any of the examples; and the generated droplet is subjected to PCR amplification.
[0027] In some examples of the present application, in the above-mentioned reaction system, the mass ratio of the above-mentioned oil phase and the above-mentioned aqueous phase is (3-8): 1, which can be 3:1, 4:1, 5:1, 6:1, 7:1 or 8:1, preferably 4:1. The droplet generated based on the above-mentioned ratio does not fuse or break after the PCR cycle heating program of 95℃.
[0028] In some examples of the present application, the diameter of the droplet generated based on the above-mentioned ratio is not more than 100μm; which can be 50μm, 60μm, 70μm, 80μm, 90μm or 100μm, preferably 50-90μm. In some examples of the present application, droplets of different sizes can be generated by adjusting the pressure.
[0029] In the above-mentioned PCR amplification, the amplification program is selected from: pre-denaturation: 95℃, 5min; amplification: 95℃ 15s→60℃ 30s, 45 cycles.
[0030] After the amplification product is obtained by performing the above amplification, further comprising nucleic acid detection on the PCR amplification product.
[0031] Based on the above method, the sample utilization rate is high and the detection sensitivity is high, and the application range is wide.
[0032] In another aspect of the present application, a digital PCR amplification kit is provided. The kit comprises: an oil phase comprising a base oil and a silicon surfactant; and an aqueous phase comprising a PCR reaction reagent and a non-ionic surfactant.
[0033] In some examples of the present application, the base oil described above comprises at least one of saturated alkane silicone oil and mineral oil.
[0034] In some examples of the present application, the silicon surfactant described above is selected from at least one of cetyl polyethylene glycol / polypropylene glycol-10 / 1 dimethyl siloxanol, cyclopentasiloxane, trimethylsilyloxy silicate and polyether modified heptamethyl trisiloxane.
[0035] In some examples of the present application, the PCR reaction reagent described above comprises a target nucleic acid molecule, an amplification primer, a probe, a dNTP, an amplification template and an amplification buffer.
[0036] In some examples of the present application, the non-ionic surfactant described above is selected from Tween 80, Tween 60, Poloxamer TM 188、 FS-30.
[0037] The kit described above is low in price, and the aqueous phase and oil phase system is stable, suitable for storage and transportation. In the digital PCR amplification using the kit, the detection sensitivity can be significantly improved.
[0038] It should be noted that the features and technical effects described in this paper for different aspects can be mutually borrowed, and will not be repeated here.
[0039] The present application is illustrated by way of examples below, but this should not be understood as limiting the scope of the subject matter of the present application to the examples below. Any technology implemented based on the above description of the present application falls within the scope of the present application. The compounds or reagents used in the following examples can be obtained commercially or prepared by conventional methods known to those skilled in the art; the experimental instruments used can be obtained commercially.
[0040] In the following detailed description, the sources of some materials and reagents are as follows:
[0041] Saturated alkane silicone oil, mineral oil, Tween 80, Tween 60, purchased from Sigma Company;
[0042] Ceteth-10 phosphate was purchased from Shanghai Kain Chemical Co., Ltd.
[0043] Trimethylsiloxysilicate was purchased from Hangzhou Siyou New Material Technology Co., Ltd.
[0044] Ceteth-10 phosphate was purchased from Chemical Book Co.
[0045] Polyether-modified heptamethyltrisiloxane was purchased from Yixing Biological Co.
[0046] FS-30 was purchased from Chemours Co.
[0047] Poloxamer TM Taq DNA polymerase, aTaq Mix were purchased from Thermo Fisher Scientific.
[0048] The template DNA, forward and reverse primers, and fluorescent probes were synthesized by Shanghai Sangon Biological Technology Co., Ltd., and the nucleotide sequences are shown in Table 1.
[0049] The remaining reagent materials, unless otherwise specified, were all commercially available.
[0050] The oil phase composition in Examples 1-28 was used as solution A, and the aqueous phase surfactant reaction solution in PCR Examples 1-28 was used as solution B. Solution A (40-80 μL) was placed in the droplet device, and solution B (10-20 μL) was placed in the collection device. The digital PCR instrument of the company was used to make microdroplets and perform PCR amplification. The reaction program was as follows: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 60℃ annealing and extension for 30 s, a total of 45 cycles.
[0051] Table 1 Nucleic acid sequences of primers and fluorescent probes in PCR reaction solutions of Examples 1-28
[0052]
[0053] Table 2 PCR amplification program
[0054]
[0055] Table 3 Composition of PCR reaction solutions of Examples 1-28
[0056] Component Volume (μL) aTaq Mix 10 Forward Primer 0.5 Reverse Primer 0.5 Probe 0.5 Template 3 Internal Control 0.4 Surfactant 2 ddH20 3.1 Total 20
[0057] Example 1
[0058] An oil phase composition was prepared comprising 90% w / w silicone oil, 10% w / w cetostearyl alcohol; and a water phase composition was prepared according to Table 2, with the surfactant being Tween 80. The two compositions were used to generate droplets using a droplet generator, and it was observed under a microscope that the droplets were of varying sizes.
[0059] Example 2
[0060] An oil phase composition was prepared comprising 90% w / w silicone oil, 10% w / w cetostearyl alcohol; and a water phase composition was prepared according to Table 2, with the surfactant being Tween 80. The two compositions were used to generate droplets using a droplet generator, and it was observed under a microscope that the droplets were of varying sizes.
[0061] Example 3
[0062] An oil phase composition was prepared comprising 90% w / w silicone oil, 10% w / w cetostearyl alcohol; and a water phase composition was prepared according to Table 2, with the surfactant being Poloxamer TM 188. The two compositions were used to generate droplets using a droplet generator, and it was observed under a microscope that the droplets were of varying sizes.
[0063] Example 4
[0064] An oil phase composition was prepared comprising 90% w / w silicone oil, 10% w / w cetostearyl alcohol; and a water phase composition was prepared according to Table 2, with the surfactant being FS-30. The two compositions were used to generate droplets using a droplet generator, and it was observed under a microscope that the droplets were of varying sizes.
[0065] Table 4 Oil phase composition formulations
[0066]
[0067] In Examples 5-12, the oil phase composition was prepared according to Table 4, and the water phase composition was prepared according to Table 2, with the surfactant consisting of 1 μL Tween 80 and 1 μL Poloxamer TM 188.
[0068] In Examples 13-20, the oil phase composition was prepared according to Table 4, and the water phase composition was prepared according to Table 2, with the surfactant consisting of 1 μL Tween-60 and 1 μL FS-30.
[0069] In Examples 21-28, the oil phase composition was prepared according to Table 4, and the water phase composition was prepared according to Table 2, with the surfactant consisting of 1 μL and 1 μL FS-30.
[0070] The two compositions were used to generate droplets by the droplet generation device. It was observed under a microscope that the droplet sizes of Examples 5, 6, 7, 9, 13, 14, 15, 17, 21, 22, 23, and 25 were not uniform, as shown in Figure 1 .
[0071] After all the examples were subjected to PCR heating and amplification according to Table 3, it was found under a microscope that Examples 8, 10, 12, 16, 18, 20, 24, 26, and 28 were broken, as shown in Figure 2 .
[0072] The two compositions were used to generate droplets by the droplet generation device. It was observed under a microscope that the droplet sizes of Examples 11, 19, and 27 were uniform, as shown in Figure 3 .
[0073] In Examples 11 and 19, the generated droplets were subjected to PCR heating, and the specific heating program is shown in Table 3. After PCR amplification, it was found under a microscope that some droplets were broken.
[0074] In Example 27, the generated droplets were subjected to PCR heating, and the specific heating program is shown in Table 3. After PCR amplification, it was found under a microscope that the droplet sizes were uniform, and no breaking occurred, as shown in Figure 4 .
[0075] In Example 27, the droplets after the PCR program were placed under a biofilm scanner to scan, and the results of the template amplification in Table 1 were obtained, as shown in Figure 5 . The template amplification was successful, and the positive and negative droplets could be clearly distinguished. This partition has good biocompatibility.
[0076] In summary, based on the reaction system of the present application, a large number of stable and uniform water-in-oil droplets can be generated. After the PCR amplification of the droplets, no fusion, breaking, or deformation occurs. After the amplification of the target nucleic acid molecules based on the reaction system, the FAM scatter plot presents obvious positive and negative differentiation, indicating that the reaction system of the present application has excellent biocompatibility.
[0077] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.
[0078] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely divergences of the principles and application of the present application and that numerous modifications, changes, substitutions, and alterations can be made thereto without departing from the spirit and scope of the present application, which is defined by the following claims and their equivalents.
Claims
1. A reaction system based on droplet digital PCR, characterized by, comprising: an oil phase comprising a base oil and a silicone surfactant; a water phase comprising PCR reagents and a non-ionic surfactant.
2. The reaction system of claim 1, wherein, The base oil comprises at least one of a saturated alkane silicone oil and a mineral oil.
3. The reaction system of claim 1, wherein, The silicone surfactant is selected from at least one of cetyl peg / ppg-10 / 1 dimethicone, cyclopentasiloxane, trimethylsiloxy silicate and polyether modified heptamethyl trisiloxane; Preferably, the silicone surfactant is selected from a mixture of cetyl peg / ppg-10 / 1 dimethicone and trimethylsiloxy silicate.
4. The reaction system according to any one of claims 1 to 3, characterized in that, In the oil phase, the total mass ratio of the base oil to the silicone surfactant is (2-8):1, preferably 4:
1.
5. The reaction system of claim 1, wherein, The PCR reagents comprise amplification primers, probes, dNTPs, amplification templates and amplification buffer.
6. The reaction system of claim 1, wherein, The non-ionic surfactant is selected from the group consisting of Tween 80, Tween 60, Poloxamer TM 188、 at least one of FS-30.
7. The reaction system according to claim 1 or 5 or 6, characterized in that, The mass ratio of the PCR reagents to the non-ionic surfactant is (3-9):1, preferably 4:
1.
8. A method of digital PCR amplification, characterized by, comprising: based on a microfluidic device, performing droplet generation on the reaction system of any one of claims 1-7; performing PCR amplification on the droplets.
9. The method of claim 8, wherein, The mass ratio of the oil phase to the water phase is (3-8):1, preferably 4:1; Optionally, the droplet diameter is no more than 100 pm; preferably 50-90 pm; Optionally, the PCR amplification reaction conditions are: pre-denaturation: 95°C, 5 min; amplification: 95°C 15s→60°C 30s, 45 cycles; Optionally, the method further comprises: performing nucleic acid detection on the PCR amplification product.
10. A digital PCR amplification kit characterized in that, comprising: an oil phase comprising a base oil and a silicone surfactant; and a water phase comprising PCR reagents and a non-ionic surfactant; Optionally, the base oil comprises at least one of a saturated alkane silicone oil and a mineral oil. Optionally, the silicone surfactant is selected from at least one of cetyl peg / ppg-10 / 1 dimethicone, cyclopentasiloxane, trimethylsiloxy silicate and polyether modified heptamethyl trisiloxane. Optionally, the PCR reagents comprise target nucleic acid molecules, amplification primers, probes, dNTPs, amplification templates and amplification buffer. Optionally, the non-ionic surfactant is selected from Tween 80, Tween 60, Poloxamer TM 188、 at least one of FS-30.