Oil phase composition for microdroplet type digital PCR (Polymerase Chain Reaction) ten million microdroplet generation and application of oil phase composition

Abstract

The invention relates to the technical field of biology, and discloses an oil phase composition for microdroplet type digital PCR (Polymerase Chain Reaction) ten million microdroplet generation and an application of the oil phase composition. The oil phase composition comprises base oil and a surfactant, the base oil comprises fluorocarbon oil generation oil and fluorocarbon oil detection oil, and the surfactant comprises a hydrocarbon polymer and a water phase stabilizer. In the preparation and detection process of the droplet type digital PCR million droplets, the uniformity and stability of the generated million water-in-oil droplets with the diameter of less than 25 microns after PCR amplification can be ensured through the matching of the oil phase compositions.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biotechnology, and in particular to an oil-phase composition for generating tens of millions of droplets in droplet digital PCR and its application. Background Art

[0002] Digital PCR (dPCR) is the third-generation PCR technology following real-time fluorescence PCR technology. It is a brand-new method for nucleic acid detection and quantification, featuring ultra-high sensitivity detection, and can perform accurate absolute quantification without relying on control samples and standard curves. The sample is dispersed into discrete and independent reaction units (droplets / micro-wells). Some units do not contain nucleic acid templates, while others contain one or more template copies. Each unit undergoes independent PCR amplification to the end point, and then a droplet reader is used to read to determine the fraction of positive partitions. It is estimated by modeling the concentration as a Poisson distribution. For a given mean value, the Poisson distribution has a constant variance. Finally, based on the fluorescence amplitude of each unit being specified as positive or negative by a threshold, the number of positive droplets and negative droplets is used to calculate the concentrations of target DNA and reference DNA sequences (Poisson-based 95% confidence interval). The sample partitioning that occurs in dPCR detection allows for reliable measurement of small-fold differences in target DNA sequences between samples, thereby improving the measurement accuracy.

[0003] According to the different forms of reaction units, dPCR can be mainly divided into two categories: chip-based and droplet-based. The former is based on an integrated fluidic pathway chip. By physically dividing the chip into tens of thousands of micro-wells, the reaction sample is distributed into independent micro-wells for reaction, also known as the "physical partitioning method". Then, the entire chip is photographed by a high-resolution fluorescence imaging system to obtain the fluorescence images of the samples in all micro-wells to complete the acquisition of result data. The latter is to micro-dropletize the PCR reaction system, use a droplet generator to generate tens of thousands of independent water-in-oil droplets, complete the partitioning of the original system, and then use flow cytometry to analyze the signals of each droplet. Both types of dPCR directly calculate the number of positive droplets according to the Poisson distribution principle to obtain the initial copy number concentration of the target molecule. However, currently, the number of micro-wells and water-in-oil droplets in these two types of dPCR on the market is basically below 50,000, usually around 20,000. The detection sensitivity calculated according to the Poisson distribution is about 0.2 - 0.5%, which limits the application of dPCR. Moreover, the limited number of independent reaction units (droplets / micro-wells) results in a small dynamic detection range. Some samples with high concentrations need to be diluted twice before loading, which is prone to missed detection of samples with low mutant abundances.

[0004] For droplet digital PCR, commonly used oil-phase reagents on the market currently include fluorocarbon oil, mineral oil, and mixtures with non-ionic biocompatible surfactants, which are used to prepare monodisperse water-in-oil droplets with less than 50,000 droplets. There is no reported formulation of droplet generation oil and surfactant for droplet generation reactions at higher orders of magnitude. Summary of the Invention

[0005] To solve the problems in the prior art, the present invention provides an oil-phase composition for generating tens of millions of droplets in droplet digital PCR. By combining the generation oil and the detection oil, the uniformity and stability of the water-in-oil droplets with a diameter of less than 25 microns generated after PCR amplification are ensured.

[0006] An embodiment of the present invention provides an oil-phase composition for generating tens of millions of droplets in droplet digital PCR, including a base oil and a surfactant:

[0007] The base oil includes a fluorocarbon oil generation oil and a fluorocarbon oil detection oil, and the surfactant includes a hydrocarbon polymer and an aqueous phase stabilizer.

[0008] Optionally, the fluorocarbon oil is one of HFE7500 fluorinated oil or FC-40.

[0009] Optionally, the hydrocarbon polymer in the surfactant is perfluoropolyether-polyethylene glycol.

[0010] Optionally, the mass of the perfluoropolyether-polyethylene glycol accounts for 1.5 - 5.5% of the total mass of the generation oil.

[0011] Optionally, the aqueous phase stabilizer in the surfactant is polyethylene glycol, betaine, bovine serum albumin, or trehalose.

[0012] Optionally, the reaction concentration of the polyethylene glycol is 0.2 - 2%, the reaction concentration of the betaine is 0.1 - 2M, the reaction concentration of the bovine serum albumin is 0.1 - 1 μg / μL, and the reaction concentration of the trehalose is 0.1 - 8.5%.

[0013] Furthermore, the present invention proposes the application of the above-mentioned generation oil and detection oil formulations and the combination with the aqueous phase stabilizer in the preparation of droplet digital PCR droplets, or in the detection of droplet digital PCR droplets.

[0014] Advantages of the Present Invention:

[0015] The present invention discloses an oil-phase reagent formulation for generating tens of millions of microdroplets in droplet digital PCR, including: a generation oil / detection oil formulation composed of a fluorocarbon oil and a hydrocarbon polymer, and an aqueous-phase stabilizer formulation. Using the oil-water phase formulation of the droplet dPCR disclosed in the present invention is beneficial to maintaining the stability of tens of millions of water-in-oil droplets during PCR amplification. Compared with the traditional oil phase on the market for generating 50,000 or fewer droplet numbers, it can effectively avoid the problems of droplet fusion and fragmentation, and significantly improve the detection sensitivity of dPCR. At the same time, the detection oil of the present invention can well cooperate with the generation oil phase to maintain the uniformity of tens of millions of droplets, and can be widely applied to dPCR detection based on Taqman probes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the microscopic examination result after droplet generation in Example 1 of the present invention;

[0017] Figure 2 It is a schematic diagram of the droplet fluorescence detection result in Example 2 of the present invention;

[0018] Figure 3 It is the dPCR flow scatter result in Example 2 of the present invention, including a fluorescence intensity scatter plot and a droplet number statistical table calculated by software;

[0019] Figure 4 It is a schematic diagram of the microscopic examination result after droplet generation in Comparative Example 1 of the present invention;

[0020] Figure 5 It is a schematic diagram of the droplet fluorescence real-time detection result in Comparative Example 2 of the present invention;

[0021] Figure 6 It is the dPCR flow scatter result in Comparative Example 2 of the present invention, including a fluorescence intensity scatter plot and a droplet number statistical table calculated by software. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0022] The present invention will be further described in detail below with reference to the drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than limiting the present invention. Additionally, it should be noted that for the sake of description, only parts related to the present invention are shown in the drawings, rather than all structures.

[0023] This embodiment provides an oil-phase composition for generating tens of millions of microdroplets in droplet digital PCR, including a generation oil, a detection oil, and an aqueous-phase stabilizer, for generating uniform-sized water-in-oil microdroplets with a diameter of 15 - 25 μm in tens of millions.

[0024] Among them, the generation oil phase and the detection oil phase reagents are HFE7500 or FC-40 fluorinated oil (3M TM) Dissolve 1.5 - 5.5% (w / w) perfluoropolyether - polyethylene glycol (PEG - PFPE 2 ), PEG - PFPE 2 It is a non - ionic fluorosurfactant composed of a non - polar, lipophilic hydrocarbon chain part and a polar, hydrophilic group, making it both hydrophilic and lipophilic. Components such as nucleic acids and buffers in the dPCR reaction system are insoluble in fluorinated oil, which can well separate the oil and water phases in dPCR; in addition, fluorinated oil can dissolve oxygen, which has a certain effect on controlling the generation of bubbles in the reaction. PEG - PFPE 2 The PFPE fluorocarbon in PEG - PFPE provides good stability for the water phase in the fluorocarbon oil - in - water droplets; while the head PEG part can prevent the adsorption of biological materials and reduce the reagent loss in actual operation; the triblock copolymer can further improve the surface coverage rate of the surfactant to obtain the best oil - in - water droplet stability and biocompatibility, ensuring the smooth independent reaction of 10 million droplets. The detection oil is used to separate the droplets and push them through the detection area. Using the same fluorinated oil as the generation oil can avoid the risk of droplet fusion caused by oil - phase incompatibility.

[0025] Furthermore, the aqueous phase stabilizer consists of 0.2 - 2% polyethylene glycol (molecular weight between 800 - 8000), 0.1 - 2M betaine, 0.1 - 1 μg / μL bovine serum albumin (BSA), and 0.1 - 8.5% trehalose. The addition of the stabilizer can prevent the fusion between droplets at the oil - water interface, further improve the stability of droplets in PCR amplification, and does not adsorb with the sample nucleic acids and other biological macromolecules in the droplets. Among them, as a hydrophilic polymer, PEG may reduce the internal flow of droplets through its thickening effect, reduce the shear force caused by the change of temperature gradient, thereby reducing the droplet breakage rate and fusion rate; betaine reduces the activity of aqueous phase molecules by forming a high - viscosity environment and buffers the aqueous phase separation caused by the temperature change in PCR; as a protein, BSA can adsorb at the oil - water interface, reduce the oil - water interface tension, and form a protective layer; trehalose can protect the oil - water structure from being damaged under high - temperature conditions, jointly ensuring that the droplets still maintain a uniform morphology after PCR amplification (the highest temperature is 98°C), greatly reducing breakage and fusion.

[0026] Example 1: Observation of the morphology of ten - million - droplet generation and amplification using the generation oil - phase formula and aqueous - phase stabilizer of the present invention

[0027] (1) Prepare the generation oil: Mix 9.6 mL of HFE7500 fluorinated oil and 0.4 mL of PEG - PFPE 2 (4%, w / w, Dolomite) thoroughly to obtain 10 mL of droplet generation oil;

[0028] (2) Prepare reaction reagents: an aqueous phase stabilizer, plasmids with known concentrations containing the EGFR-T790M mutation and wild-type plasmids, forward and reverse amplification primers (F / R), wild-type and mutant TaqMan probes (WP / MP), reaction buffer TaqMan Genotyping Master Mix (Thermo Fisher). The specific sequences are shown in Table 1, and prepare the reaction system as shown in Table 2;

[0029] Table 1 Primer and probe sequences for digital PCR detection of EGFR-T790M mutation ("+" represents a locked nucleic acid modified base)

[0030] T790M-F 5’-CCTCACCTCCACCGTGCA-3’ T790M-R 5’-AGGCAGCCGAAGGGCA-3’ T790M-WP 5’-VIC-T+CATC+A+C+GCA+GCTC-MGB-3’ T790M-MP 5’-FAM-T+CATC+A+T+GCA+GC+TC-MGB-3’

[0031] Table 2 Aqueous phase reaction system for digital PCR

[0032]

[0033] (3) Inject 10 mL of the generating oil into the fuel tank of the fully automatic sample processing system. Add the aqueous phase reaction system into a 0.2 mL PCR eight-strip tube and start droplet generation;

[0034] (4) Perform PCR amplification on the generated water-in-oil droplets (milky white). The reaction program is: pre-denaturation at 95 °C for 10 min; denaturation at 95 °C for 15 s, annealing and extension at 62 °C for 1 min, for a total of 45 cycles; extension at 98 °C for 5 min, incubation at 12 °C;

[0035] (5) After the amplification is completed, aspirate 1 μL of the droplets and observe the droplet morphology under a microscope.

[0036] The results are shown in Figure 1 , Figure 1 The diameter of the droplets in

[0037] Example 2: Use the detection oil phase formulation and aqueous phase stabilizer of the present invention for droplet counting and droplet signal detection

[0038] (1) Prepare the detection oil: Mix 19.2 mL of HFE7500 fluorinated oil and 0.8 mL of PEG-PFPE 2 (4%, w / w, Dolomite) thoroughly to obtain 20 mL of droplet detection oil, and inject it into the fuel tank of the biochip analyzer. Place the PCR eight-strip tube containing droplets after PCR amplification in Example 1 into the biochip analyzer and start droplet signal detection.

[0039] (2) Collect droplet signals: PCR reactions are carried out inside each droplet, and the biochip analyzer can detect the fluorescence signal intensity when each droplet passes through.

[0040] When the droplet contains wild-type template, the degradation of WP probe causes the separation of VIC fluorescence emission group and fluorescence quenching group, and the droplet shows VIC signal, which is captured by PMT2 channel (the received fluorescence wavelength is 543nm ± 15nm, indicating that the droplet contains EGFR wild-type plasmid DNA);

[0041] When the droplet contains mutant template, the degradation of MP probe causes the separation of FAM fluorescence emission group and fluorescence quenching group, and the droplet shows FAM signal, which is captured by PMT1 channel (the received fluorescence wavelength is 510nm ± 10nm, indicating that the droplet contains T790M mutant plasmid DNA);

[0042] When the droplet does not contain plasmid template, the probe is intact and its fluorescence signal is absorbed by the quenching group, and the droplet shows the basic fluorescence signals of PMT1 and PMT2;

[0043] (3) Generate droplet number count: Through the Flowjo flow cytometry analysis software owned by the instrument, the droplets in the PCR octuplet tube are automatically counted.

[0044] Figure 2 The results show that the peak shape of each droplet T is complete when passing through the detection area, showing a single peak shape, further verifying the integrity of the droplet after PCR amplification. Among them, PMT1 represents the FAM fluorescence signal, PM2 represents the VIC fluorescence signal, the coincidence of PMT1 and PMT2 signals indicates NA droplets, and the stronger PMT2 signal than the PMT1 signal peak indicates WT droplets (MT droplets have a stronger PMT1 signal than the PMT2 signal peak, not shown in the figure). Figure 3 On the left is the fluorescence intensity scatter plot. Among them, the droplets aggregated near the PMT2 axis contain VIC fluorescence signals, that is, the copy number of wild-type EGFR DNA, circled with an oval and marked "WT"; the droplets aggregated near the PMT1 axis contain FAM fluorescence signals, that is, the copy number of T790M mutant DNA, circled with an oval and marked "MT". Figure 3 On the right are the results of the total droplet number calculated by the software and the detected nucleic acid copy number. Figure 3The quantitative results of the sample DNA showed that the number of generated droplets was approximately 7.8 million, meeting the droplet generation level of tens of millions. The calculated mutation percentage of the sample was 9.4%, within the theoretical value range of 10% ± 5%, and the detection result was accurate (the number of droplets of wild-type DNA was 4596, and the number of droplets of DNA containing the T790M mutation was 432). This indicates that the detection oil in this example has good performance when used in combination with the generation oil, and can complete the generation of tens of millions of water-in-oil droplets, the integrity of droplet amplification, and Taqman probe-based dPCR detection.

[0045] Comparative Example 1: Commercial dPCR generation oil was used for droplet generation, morphology observation, and dPCR detection.

[0046] 10 mL of droplet generation oil (#1863005, Bio-Rad) was injected into the fully automatic sample processing system, and the remaining reaction reagents and reaction conditions were the same as those in Example 1 for droplet generation and PCR amplification.

[0047] The results were as Figure 4 shown. After PCR, some of the droplets fused to form large droplets, and the droplet uniformity was poor, indicating that this generation oil cannot be directly used as the oil phase for tens of millions of droplet generation.

[0048] Comparative Example 2: Commercial dPCR generation oil and detection oil were used for droplet counting and droplet signal detection

[0049] 20 mL of droplet detection oil (#1863004, Bio-Rad) was injected into the biochip analyzer, and the uniformly sized droplets generated in Example 1 were placed in the instrument for detection. The remaining steps were the same as those in Example 2.

[0050] The signal results of individual droplets were as Figure 5 shown. The peak shape was not single, and a double peak appeared, indicating that the volume of the droplets passing through the detection area was too large, resulting in continuous fluorescence signals, indicating that the droplets had fused. Figure 6 The quantitative results showed that there was no FAM or VIC fluorescence aggregation at the corresponding positions of the droplets. Comparing with the results of Example 2, it was judged that the possible reason was that the detection oil phase did not match the generation oil phase, resulting in the inability to receive the fluorescence group signal. This result indicates that this detection oil cannot be directly used as the detection oil phase for tens of millions of droplets.

[0051] Therefore, adopting the generation oil and detection oil formulations of the microdroplet dPCR disclosed in this example is beneficial to maintaining the stability of tens of millions of water-in-oil droplets during PCR amplification, effectively avoiding problems such as droplet fusion and fragmentation, and significantly improving the detection sensitivity of dPCR; in addition, at the same time, the detection oil of the present invention can cooperate well with the generation oil phase, maintain the uniformity of tens of millions of droplets, and can be widely applied to Taqman probe-based dPCR detection.

[0052] Note that the above is only a preferred embodiment of the present invention and the technical principles applied. Those skilled in the art will understand that the present invention is not limited to the specific embodiments described herein, and various obvious changes, re-adjustments, and substitutions can be made by those skilled in the art without departing from the protection scope of the present invention. Therefore, although the present invention has been described in more detail through the above embodiments, the present invention is not limited to the above embodiments. Without departing from the concept of the present invention, more other equivalent embodiments can be included, and the scope of the present invention is determined by the scope of the appended claims.

Claims

1. An oil phase composition for generating millions of droplets in droplet digital PCR, characterized in that: Includes base oil and surfactant; The base oil includes fluorocarbon oil generation oil and fluorocarbon oil detection oil, and the surfactant includes hydrocarbon polymer and water phase stabilizer.

2. The oil phase composition according to claim 1, characterized in that The fluorocarbon oil in the base oil is one of HFE7500 fluorinated oil and FC-40.

3. The oil phase composition according to claim 1, characterized in that The hydrocarbon polymer in the surfactant is perfluoropolyether-polyethylene glycol.

4. The oil phase composition according to claim 3, characterized in that The mass of the perfluoropolyether-polyethylene glycol accounts for 1.5-5.5% of the total mass of the generated oil.

5. The oil phase composition according to claim 1, characterized in that The water phase stabilizer in the surfactant is one of polyethylene glycol, betaine, bovine serum albumin, trehalose or a combination thereof.

6. The oil phase composition according to claim 5, characterized in that The reaction concentration of the polyethylene glycol is 0.2-2%.

7. The oil phase composition according to claim 5, characterized in that The reaction concentration of the betaine is 0.1-2M.

8. The oil phase composition according to claim 5, characterized in that The reaction concentration of the bovine serum albumin is 0.1-1 μg / μL.

9. The oil phase composition according to claim 5, characterized in that The reaction concentration of the trehalose is 0.1-8.5%.

10. Use of the oil phase composition according to any one of claims 1 to 9 in preparing droplet digital PCR droplets, or in detecting droplet digital PCR droplets.

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