WO2020228257A1 - Oil phase composition for preparing digital pcr microdroplets and application thereof - Google Patents

Abstract

The present invention provides an oil phase composition for preparing digital PCR microdroplets, consisting of the following components: 1) a macromolecular nonionic surfactant having a low hydrophilic lipophilic balance value and having polydimethylsiloxane as a backbone (2-5%w/w); 2) a polyethyleneglycol micromolecular nonionic surfactant having a high hydrophilic lipophilic balance value (0.05-0.2%w/w); 3) an anti-evaporation agent (1-5%w/w); and 4) the balance a base oil. The digital PCR microdroplets prepared using the oil phase composition provided by the present invention have the advantages of a uniform size, good thermostability, no evaporation, no fusion, and no inhibition of PCR amplification.

Description

Oil phase composition for preparing digital PCR microdrops and application thereof Technical field

The invention belongs to the technical field of molecular biology. Specifically, the present invention relates to an oil phase composition for preparing digital PCR droplets and its application.

Background technique

Droplet digital PCR (droplet digital polymerase chain reaction, ddPCR) technology is a method for absolute quantification of nucleic acid molecules. Compared with quantitative PCR (quantitative PCR, qPCR), it has unparalleled advantages in precision, accuracy and sensitivity. At the same time, ddPCR eliminates the dependence on the quantitative standard curve and improves the tolerance to amplification inhibitors. Therefore, ddPCR is called the third generation PCR technology.

The working principle of ddPCR is to process the reaction solution into droplets before PCR amplification, that is, to disperse the reaction solution containing nucleic acid template into tens of thousands of nano-grade droplets, each droplet does not contain or contains one to several to be tested Nucleic acid target molecules, and each droplet serves as an independent PCR reaction chamber. After PCR amplification, the droplet containing the nucleic acid target molecule to be detected produces a fluorescent signal, and the droplet containing the nucleic acid target molecule to be detected does not produce a fluorescent signal. Finally, according to the Poisson distribution principle and the proportion of positive droplets, the initial concentration or copy number of the nucleic acid target molecule to be detected is calculated.

Microfluidic technology is a widely used method to realize the dropletization of reaction liquids, but it has limitations, such as: microfluidic chips are expensive to manufacture, complicated to operate, and low droplet generation throughput. Therefore, we use the interface vertical vibration method to generate droplets (see CN104324769B). This method requires that the density of the oil phase is less than that of water, so that the generated reaction liquid droplets settle quickly, thereby reducing collisions between the droplets; at the same time, the droplets should have good thermal stability and uniformity to ensure that the subsequent PCR reaction proceeds and Absolute quantitative detection of nucleic acid target molecules to be tested.

In recent years, oil-phase formulations combining mineral oil and surfactants have been widely used in ddPCR. Surfactants play an important role in the formation and stability of droplets. They reduce the tension of the oil-water interface and form an interface film by adsorption on the interface to ensure the stability of the droplets. However, we found in our work that although the currently disclosed formula can generate uniformly sized reaction liquid droplets, its thermal stability cannot meet the requirements of the ddPCR reaction (up to 95°C), so a new type of oil phase formula is needed.

Summary of the invention

In view of the above-mentioned problems, the present invention provides an oil phase composition. The droplets produced by using this oil phase composition have uniform size, good thermal stability, no evaporation, no fusion, no inhibition of PCR amplification, and can ensure subsequent PCR reactions. The progress.

The present invention provides an oil phase composition for preparing digital PCR microdroplets, which is characterized in that it contains the following components: 1) a macromolecule with a low hydrophilic-lipophilic balance value and a polydimethylsiloxane backbone Non-ionic surfactant; 2) Polyethylene glycol small molecule non-ionic surfactant with high hydrophilic and lipophilic balance; 3) Anti-evaporation agent; 4) The balance is mineral oil.

Preferably, the macromolecular nonionic surfactant with low hydrophilic-lipophilic balance value and polydimethylsiloxane as the skeleton has cetyl polyethylene glycol/polypropylene glycol-10/1 polydimethylsiloxane Siloxane or similar structure.

Preferably, the macromolecular nonionic surfactant with low hydrophilic lipophilic balance value and polydimethylsiloxane as the skeleton is selected from ABIL ^TM EM 90, ABIL ^TM EM 180, or ABIL ^TM WE09 One or more.

Preferably, the content of the low hydrophilic-lipophilic balance value of the macromolecular nonionic surfactant with polydimethylsiloxane as the skeleton is 2-5% w/w. Preferably, the polyethylene glycol small molecule nonionic surfactant with a high hydrophilic-lipophilic balance value is selected from one or more of Tween series, Nonnard series, and Triton series.

Preferably, the content of the high hydrophilic-lipophilic balance value of polyethylene glycol small molecule nonionic surfactant is 0.05-0.2% w/w.

Preferably, the anti-evaporation agent is selected from polyisobutenol with a molecular weight of 1300.

Preferably, the anti-evaporation agent is selected from one or more of polyisobutylene H300 and polyisobutylene PB1300.

Preferably, the content of the anti-evaporation agent is 1-5% w/w.

Preferably, the base oil is selected from one or more of mineral oil, silicone oil, dodecane, tetradecane, hexadecane, and octadecane.

The present invention also provides a method for preparing digital PCR droplets, characterized in that the method includes the following steps:

1) Configure the aqueous solution, including template DNA, forward primer, reverse primer, double-stranded DNA fluorescent dye or TaqMan fluorescent probe, DNA polymerase and PCR reaction buffer; mix the above liquids uniformly in a certain ratio, The obtained solution is used as an aqueous solution for later use;

2) Configure the oil phase liquid, mix the components of the oil phase composition uniformly according to the formula amount, and use the obtained solution as the oil phase liquid for later use;

3) Add the water phase solution to the droplet generating device, add the oil phase liquid to the collecting device, and use the droplet generating device to prepare and obtain digital PCR droplets.

The invention also provides the use of the oil phase composition in preparing digital PCR microdroplets.

The beneficial effects of the present invention are: 1) The present invention provides an oil phase composition for preparing digital PCR droplets, and the droplets prepared by the present invention have uniform size, good thermal stability, no evaporation and no fusion. , Does not inhibit the advantages of PCR amplification; 2) The present invention is also suitable for the formation of uniform and stable droplets between the microfluidic device and the water phase reaction liquid; 3) The present invention can be combined with the water phase reaction liquid with ordinary water-in-oil droplets Preparation methods, such as stirring, shaking, etc. to form a thermally stable emulsion system.

Description of the drawings

Figure 1 is a fluorescence picture of the reaction liquid droplets prepared from the oil phase composition in Example 1 after PCR amplification.

Figure 2 is a fluorescence picture of the reaction liquid droplets prepared from the oil phase composition in Example 2 after PCR amplification.

Figure 3 is a fluorescence picture of the reaction liquid droplets prepared from the oil phase composition in Example 3 after PCR amplification.

Figure 4 is a bright field picture of PCR reaction liquid droplets prepared from the oil phase composition in Example 4.

Figure 5 is a bright field picture of PCR reaction liquid droplets prepared from the oil phase composition in Example 5.

Figure 6 is a fluorescence picture of the reaction liquid droplets prepared from the oil phase composition in Example 5 after PCR reaction.

Figure 7 is a fluorescence picture of the reaction liquid droplets prepared from the oil phase composition in Example 15 after PCR reaction.

8 is a bright field picture of PCR reaction liquid droplets prepared by the oil phase composition in Example 7.

Figure 9 is a fluorescence picture of the reaction liquid droplets prepared from the oil phase composition in Example 24 after PCR reaction.

Figure 10 is a fluorescence picture of the reaction liquid droplets prepared from the oil phase composition in Example 25 after PCR reaction.

11 is a fluorescence picture of the reaction liquid droplets prepared from the oil phase composition in Example 26 after PCR reaction.

Figure 12 is a fluorescence picture of the reaction liquid droplets prepared from the oil phase composition in Example 27 after PCR reaction.

Fig. 13 shows the evaporation of droplets in the central area of the droplet collection device after PCR amplification in Examples 4-27.

14 is a fluorescence picture of the reaction liquid droplets prepared from the oil phase composition in Example 28 after PCR reaction.

Detailed ways

Hereinafter, specific embodiments of the present invention will be described in more detail with reference to the accompanying drawings. Although specific embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

The disclosed subject matter can be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. In fact, for those skilled in the art to which the subject matter of the present disclosure pertains, many modifications and other embodiments of the subject matter of the present disclosure that have the benefit of the teachings given in the description included herein will come to mind. Therefore, it should be understood that the subject matter of the present disclosure is not limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the subject matter disclosed.

Although specific terms are used in this article, they are only used in a general and descriptive sense, not for limiting purposes. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the subject described in this disclosure belongs.

Use standard nomenclature to describe compounds. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

In this article, the digital PCR droplet refers to an independent reaction unit after the PCR reaction solution is dispersed. Its volume can be miniaturized to several picoliters, and its number can be as high as millions.

In this article, the hydrophilic-lipophilic balance (HLB) value refers to the ratio between the hydrophilic group and the lipophilic group in the surfactant molecule; the higher the HLB value, the stronger the hydrophilicity, the lower the HLB value, the higher the affinity The oiliness is stronger.

In this article, the macromolecular nonionic surfactant with low HLB value and polydimethylsiloxane as the skeleton refers to the cetyl polyethylene glycol/polypropylene glycol-10/1 polydimethylsiloxane An alkane or similar surfactant that does not generate ions in an aqueous solution has an HLB value of about 4-6, preferably about 5.

In this article, ABIL ^TM EM 90, ABIL ^TM EM 180, or ABIL ^TM WE 09 refers to the cetyl polyethylene glycol/polypropylene glycol-10/1 polyethylene glycol produced by Germany/Evonik Goschmidt The product name of siloxane or similar structure is a typical low HLB macromolecular nonionic surfactant with polydimethylsiloxane as the skeleton.

In this article, polyethylene glycol small molecule nonionic surfactants with high HLB value refer to Tween series, Nonnard series, and Triton series nonionic surfactants with a molecular weight of less than 1500, and their HLB value About 13-16.

In this article, polyisobutylene H300 refers to polyisobutylene with a molecular weight of 1300 in Japan; polyisobutylene PB1300 refers to polyisobutylene with a molecular weight of 1300 in Korea.

In this article, a variety of methods for preparing digital PCR droplets known in the art can be used, including but not limited to the interface vertical vibration method. In the interface vertical vibration method, the PCR reaction liquid is used as the first liquid, and the oil phase composition is used as the second liquid. The first liquid is located in the dropletization device, and the second liquid is located in the collection device. Digital PCR droplets.

In this article, PCR amplification can be performed by conventional reaction procedures, including but not limited to: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 20 seconds, 55°C annealing extension for 40 seconds, a total of 40 cycles.

Specifically, the present invention relates to the following embodiments:

1. An oil phase composition for the preparation of digital PCR microdroplets, characterized in that the oil phase composition comprises the following components: 1) a low HLB large polydimethylsiloxane backbone Molecular nonionic surfactant; 2) Polyethylene glycol small molecule nonionic surfactant with high HLB value; 3) Anti-evaporation agent; 4) The balance is mineral oil.

2. The oil phase composition as described above, characterized in that the low HLB value macromolecular nonionic surfactant with polydimethylsiloxane as the skeleton has cetyl polyethylene glycol/ Polypropylene glycol-10/1 polydimethylsiloxane or similar structure.

3. The oil phase composition according to the above, characterized in that the macromolecular nonionic surfactant with low HLB value and polydimethylsiloxane as the skeleton is selected from ABIL ^TM EM 90, ABIL ^TM One or more of EM 180 or ABIL ^TM WE 09.

4. The oil phase composition as described above, characterized in that the content of the macromolecular nonionic surfactant with low HLB value and polydimethylsiloxane as the skeleton is 2-5% w/ w.

5. The oil phase composition as described above, characterized in that the high HLB value polyethylene glycol small molecule nonionic surfactant is selected from Tween series, Nonnard series, and Triton series One or more of.

6. The oil phase composition according to the above, characterized in that the content of the high HLB value polyethylene glycol small molecule nonionic surfactant is 0.05-0.2% w/w.

7. The oil phase composition as described above, characterized in that the anti-evaporation agent is selected from polyisobutylene with a molecular weight of 1300.

8. The oil phase composition as described above, characterized in that the anti-evaporation agent is selected from one or more of polyisobutylene H300 and polyisobutylene PB1300.

9. The oil phase composition as described above, characterized in that the content of the anti-evaporation agent is 1-5% w/w.

10. The oil phase composition as described above, wherein the base oil is selected from one or more of mineral oil, silicone oil, dodecane, tetradecane, hexadecane, and octadecane .

11. A method for preparing digital PCR droplets, characterized in that the method comprises the following steps:

1) Configure the aqueous solution, including template DNA, forward primer, reverse primer, double-stranded DNA fluorescent dye or TaqMan fluorescent probe, DNA polymerase and PCR reaction buffer; mix the above liquids evenly in a certain ratio, The obtained solution is used as an aqueous solution for later use;

2) Configure the oil phase liquid, mix the components of the oil phase composition described above uniformly according to the formula amount, and use the obtained solution as the oil phase liquid for later use;

3) Add the water phase solution to the droplet generating device, add the oil phase liquid to the collecting device, and use the droplet generating device to prepare and obtain digital PCR droplets.

12. The use of the oil phase composition described above in the preparation of digital PCR droplets.

The following specific examples are used to elaborate and illustrate the implementation of the present invention, but the following content should not be construed as any limitation on the present invention.

Example

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

In the following specific embodiments, the sources of some materials and reagents are as follows:

Mineral oil, Tween 80, Span 80, Brij L4, Triton X-100, and polyisobutylene H300 were purchased from Sigma;

ABIL EM 90, ABIL EM 180, ABIL WE 09 were purchased from Germany/Evonik Schmidt;

The positive and negative primers and fluorescent probes were synthesized by Shanghai Shenggong Biotechnology Co., Ltd., and the nucleotide sequence is shown in Table 1.

TransScript Supermix was purchased from Beijing Quanshijin Company;

The template DNA is extracted from Saccharomyces cerevisiae Hansen ATCC 204508/S288C;

The remaining reagent materials, unless otherwise specified, are all conventionally commercially available.

Preferably, the final concentrations of the primers whose nucleotide sequences are shown in Table 1 are 750 nmol/L.

Preferably, the final concentration of the fluorescent probe whose nucleotide sequence is shown in Table 1 is 250 nmol/L.

Preferably, the labeling group at the 5'end of the fluorescent probe is FAM (6-carboxy-fluorescein), and the labeling group at the 3'end of the fluorescent probe is BHQ1.

The PCR reaction solution was used as the first liquid, and the oil phase composition in Examples 1-28 was used as the second liquid. The first liquid (20μL) was located in the dropletization device, and the second liquid (1000μL) was located in the collection device. Digital PCR droplets were prepared by vibration method. Transfer the collection device containing the droplets to the BIO-GENER PCR machine (Hangzhou Baiheng) for PCR amplification. The reaction procedure is: 95°C pre-denaturation for 5 minutes, 95°C denaturation for 20 seconds, 55°C annealing and extension for 40 seconds, a total of 40 cycles .

Table 1 The nucleic acid sequences of the primers and fluorescent probes in the PCR reaction solution of Examples 1-28

Table 2 The composition of the PCR reaction solution of Examples 1-28

name	Volume/final concentration

Template DNA	1μL
Forward primer (F)	1.5μL (750nmol/L)
Reverse primer (R)	1.5μL (750nmol/L)
TransScript Supermix	10μL
Probe	0.5μL (250nmol/L)
ddH 2 O	5.5μL
total capacity	20μL

Example 1

Using the oil phase composition reported in the literature 4% EM 90, 1% Span 80 (Wu N, et al. A PMMA microfluidic droplet platform for in vitro protein expression using crude E. coli S30 extract. Lab on a Chip, 2009, 9:3391-3398) Prepare digital PCR droplets. After PCR amplification, the result is shown in Figure 1: The PCR droplets are partially fused, and because Span 80 has background fluorescence, the negative droplets cannot be counted.

Example 2

Using the oil phase composition reported in the literature, 5% w/w span 80, 0.5% w/w Brij L4 (Pandit K R, et al. Assets of Surfactants for efficient droplet PCR in mineral oil using the pendant drop technology.Colloids and Surfaces B: Biointerfaces, 2015, 126:489-495) Prepare digital PCR droplets, and after PCR amplification, the result is shown in Figure 2: PCR droplets are fused in a large area.

Example 3

Utilizing the oil phase composition reported in the literature 4.5%v/v Span 80, 0.4%v/v Tween 80, 0.05%v/v Triton X-100 (Williams R, et al. Amplification of complex gene libraries by emulsion PCR .Nature methods, 2006, 3(7): 545-550) Prepare digital PCR droplets, and after PCR amplification, the result is shown in Figure 3: PCR droplets are fused in a large area.

Table 3 Oil phase composition formulas of Examples 4-23 (w/w)

To	ABIL EM 90	Tween 80	mineral oil
Example 4	1%	0	99%
Example 5	2%	0	98%
Example 6	5%	0	95%
Example 7	10%	0	90%
Example 8	15%	0	85%
Example 9	1%	0.05%	98.95%

Example 10	2%	0.05%	97.95%
Example 11	5%	0.05%	94.95%
Example 12	10%	0.05%	89.95%
Example 13	15%	0.05%	84.95%
Example 14	1%	0.1%	98.9%
Example 15	2%	0.1%	97.9%
Example 16	5%	0.1%	94.9%
Example 17	10%	0.1%	89.9%
Example 18	15%	0.1%	84.9%
Example 19	1%	0.2%	98.8%
Example 20	2%	0.2%	97.8%
Example 21	5%	0.2%	94.8%
Example 22	10%	0.2%	89.8%
Example 23	15%	0.2%	84.8%

Large-scale screening and comparison of the oil phase compositions of Examples 4-23 found that the droplets prepared with 1% w/w EM 90 collided and merged during the sedimentation process (Figure 4), adding 0.05-0.2% w /w Tween 80 did not improve the droplet fusion phenomenon; 2-5% w/w EM 90 can prepare droplets of uniform size (Figure 5), but after PCR amplification, the droplets fuse in a large area (Figure 6). Adding 0.05-0.2% w/w Tween 80 to make the droplets remain intact after PCR amplification (Figure 7); 10-15% w/w EM 90 makes the oil phase composition too viscous and causes the droplet preparation process Many small droplets were generated (Figure 8), and the addition of 0.05-0.2% w/w Tween 80 did not improve the generation of small droplets.

It was found that WE 09 and EM 180 both have cetyl polyethylene glycol/polypropylene glycol-10/1 polydimethylsiloxane or similar structures. The oil phase composition of Example 24 contained 5% w/w EM 180 and 0.1% w/w Tween 80 (Figure 9); the oil phase composition of Example 25 contained 5% w/w WE 09 and 0.1% w/ w Tween 80 (Figure 10), the droplets remain intact after PCR amplification.

It was found that both the Nonnard series surfactants and Triton series surfactants belong to high HLB value polyethylene glycol small molecule nonionic surfactants. The oil phase composition of Example 26 contained 5% w/w EM 90 and 0.1% w/w Nonnard P-40 (Figure 11); the oil phase composition of Example 27 contained 5% w/w EM 90 and 0.1% w/w 0.1% w/w Triton X-100 (Figure 12), the droplets remained intact after PCR amplification.

In addition, it was found that in Examples 4-27, there was droplet evaporation in the central area of the droplet collection device after PCR amplification (Figure 13). The oil phase composition of Example 28 contains 5% w/w EM 90, 0.1% w/w Tween 80, and 5% w/w polyisobutylene H300 (Figure 14). Adding an anti-evaporation agent to the oil phase composition eliminates the evaporation of droplets in the central area of the droplet collection device.

The foregoing merely illustrates the principle of the present invention. It should be understood that the scope of the present invention is not intended to be limited to the exemplary aspects described herein, but should include all currently known and future developed equivalents. In addition, it should be pointed out that several improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should also be regarded as the scope of the present invention.

Claims (12)

1. An oil phase composition for preparing digital PCR microdroplets, characterized in that the oil phase composition comprises the following components: 1) Low hydrophilic-lipophilic balance value with polydimethylsiloxane as the backbone 2) Polyethylene glycol small molecule non-ionic surfactant with high hydrophilic-lipophilic balance value; 3) Anti-evaporation agent; 4) The balance is base oil.
2. The oil phase composition of claim 1, wherein the low hydrophilic-lipophilic balance value of the macromolecular nonionic surfactant with polydimethylsiloxane as the skeleton has cetyl poly Ethylene glycol/polypropylene glycol-10/1 polydimethylsiloxane or similar structure.
3. The oil phase composition of any one of claims 1-2, wherein the low hydrophilic-lipophilic balance value of the macromolecular non-ionic surface active with polydimethylsiloxane as the skeleton The agent is selected from one or more of ABIL ^TM EM 90, ABIL ^TM EM 180, or ABIL ^TM WE 09.
4. The oil phase composition according to any one of claims 1 to 3, wherein the low hydrophilic-lipophilic balance value of the macromolecular non-ionic surface active with polydimethylsiloxane as the skeleton The content of the agent is 2-5% w/w.
5. The oil phase composition according to any one of claims 1 to 4, wherein the polyethylene glycol small molecule nonionic surfactant with high hydrophilic-lipophilic balance value is selected from the Tween series, One or more of the Nonnard series and the Triton series.
6. The oil phase composition according to any one of claims 1 to 5, wherein the content of the polyethylene glycol small molecule nonionic surfactant with high hydrophilic-lipophilic balance value is 0.05-0.2 %W/w.
7. The oil phase composition according to any one of claims 1-6, wherein the anti-evaporation agent is selected from polyisobutylene with a molecular weight of 1300.
8. The oil phase composition according to any one of claims 1-7, wherein the anti-evaporation agent is selected from one or more of polyisobutylene H300 and polyisobutylene PB1300.
9. The oil phase composition according to any one of claims 1-8, wherein the content of the anti-evaporation agent is 1-5% w/w.
10. The oil phase composition according to any one of claims 1-9, wherein the base oil is selected from mineral oil, silicone oil, dodecane, tetradecane, hexadecane, and octadecane One or more of.
11. A method for preparing digital PCR droplets, characterized in that the method comprises the following steps:

    1) Configure the aqueous solution, including template DNA, forward primer, reverse primer, double-stranded DNA fluorescent dye or TaqMan fluorescent probe, DNA polymerase and PCR reaction buffer; mix the above liquids evenly in a certain ratio, The obtained solution is used as an aqueous solution for later use;

    2) The oil phase liquid is prepared, the components of the oil phase composition according to any one of claims 1-10 are uniformly mixed according to the formula amount, and the obtained solution is used as the oil phase liquid for later use;

    3) Add the water phase solution to the droplet generating device, add the oil phase liquid to the collecting device, and use the droplet generating device to prepare and obtain digital PCR droplets.
12. The use of the oil phase composition according to any one of claims 1-10 in the preparation of digital PCR droplets.

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