CN114941023A - Droplet digital PCR oil phase composition, droplet digital PCR composition and its application and kit - Google Patents

Abstract

The invention provides a microdroplet type digital PCR oil phase composition, a microdroplet type digital PCR composition and an application and a kit thereof, and relates to the field of biotechnology. The microdroplet digital PCR oil phase composition provided by the present invention includes fluorinated oil and PFPE-PEG-PFPE in a specific proportion, and the microdroplets formed by the oil phase composition and the water phase are uniform in size and have good stability, and are suitable for PCR The effect of amplification is small, and it can be used in droplet digital PCR as an oil phase. The droplet digital PCR composition provided by the present invention includes the above-mentioned droplet digital PCR oil phase composition and water phase composition, wherein the water phase composition contains a specific concentration of hot-start Taq enzyme, MgCl ₂ , KCl, Tris- HCl, BSA, dNTP, PEG2000, betaine, DMSO, glycerol, protamine DNA, and Proclin‑300, the composition forms more stable droplets.

Description

Droplet digital PCR oil phase composition, droplet digital PCR composition and its application and kit

technical field

The invention relates to the field of biotechnology, in particular to a droplet type digital PCR oil phase composition, a droplet type digital PCR composition and its application and kit.

Background technique

Digital PCR is dPCR. After a certain dilution of the nucleic acid template, it is randomly allocated to a large number of reaction units for amplification reaction. The Poisson distribution formula calculates the original concentration or content of the sample by PCR technology. Since the introduction of digital PCR technology, related technologies and industrialization have developed very rapidly. To date, there are three main categories of digital PCR technologies: micro-reaction chambers/well plates, large-scale integrated microfluidic chips, and droplet digital PCR systems. Droplet digital PCR has high fluorescence intensity, easy detection, and high sensitivity. The rapid and sensitive detection of infectious nucleic acid viruses is of great significance to the monitoring and control of epidemics.

Droplet digital PCR is a water-in-oil droplet, and the key is to maintain the stability of the water-in-oil droplet during PCR amplification. The metastable state in the presence of surfactants is a system that does not conform to thermodynamic equilibrium. Therefore, in the application of droplet digitization, the droplets tend to coalesce or rupture. In view of the above two phenomena, the usual solution is to add surfactants in the oil, which stabilize the oil-water interface of the droplets through different polar components at both ends, balance the driving force of the homogenization of the system, and stabilize the dispersion in the The metastable state ensures that the droplets are not easily coagulated. And in order to ensure the stability of the amplification and storage stability of the droplets, it is often necessary to add a stabilizer to the water phase to act together with the surfactant in the oil phase to maintain the stability of the droplets.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The first objective of the present invention is to provide a droplet digital PCR oil phase composition.

The second object of the present invention is to provide a droplet digital PCR composition, the droplets prepared with the composition are more stable.

The third object of the present invention is to provide the above-mentioned droplet digital PCR oil phase composition or the application of the droplet digital PCR composition in droplet digital PCR.

The fourth object of the present invention is to provide a droplet type digital PCR kit.

In a first aspect, the present invention provides a droplet digital PCR oil phase composition, which, in parts by mass, comprises: 92-98 parts of fluorinated oil and 2-8 parts of PFPE-PEG-PFPE.

As a further technical solution, in parts by mass, including: 93-97 parts of fluorinated oil and 3-7 parts of PFPE-PEG-PFPE;

Preferably, in parts by mass, it includes: 95 parts of fluorinated oil and 5 parts of PFPE-PEG-PFPE.

In a second aspect, the present invention provides a droplet digital PCR composition, including the droplet digital PCR oil phase composition and water phase composition;

The solutes of the aqueous composition include hot-start Taq enzyme, MgCl ₂ , KCl, Tris-HCl, BSA, dNTP, PEG2000, betaine, DMSO, glycerol, protamine DNA and Proclin-300;

In the aqueous phase composition, the concentration of hot-start Taq enzyme is 0.08-0.12 U/μL, the concentration of MgCl ₂ is 3-4 mM, the concentration of KCl is 100-150 mM, the concentration of Tris-HCl is 16-24 mM, and the concentration of BSA The concentration of 2-3mg/mL, the concentration of dNTP is 0.3-0.5mM, the volume fraction of PEG2000 is 0.6%-0.8%, the concentration of betaine is 0.3-0.5M, the volume fraction of DMSO is 1.6%-2.4%, The volume fraction of glycerol was 0.2%-0.3%, the concentration of protamine DNA was 0.4-0.6 ng/μL, and the volume fraction of Proclin-300 was 0.03%-0.05%.

As a further technical solution, in the aqueous phase composition, the concentration of hot-start Taq enzyme is 0.1 U/μL, the concentration of MgCl ₂ is 3.5 mM, the concentration of KCl is 125 mM, the concentration of Tris-HCl is 20 mM, and the concentration of BSA is 20 mM. 2.5mg/mL, dNTP concentration of 0.4mM, PEG2000 volume fraction of 0.7%, betaine concentration of 0.4M, DMSO volume fraction of 2%, glycerol volume fraction of 0.25%, fish sperm DNA concentration It is 0.5ng/μL, and the volume fraction of Proclin-300 is 0.04%.

As a further technical solution, the solute of the aqueous composition further includes a primer pair, a probe and a fluorescein sodium salt.

As a further technical solution, the concentration of the primer pair is 600-1000nM, preferably 800nM;

Preferably, the concentration of the probe is 160-240nM, preferably 200nM;

Preferably, the concentration of the fluorescein sodium salt is 80-120 nM, preferably 100 nM.

In a third aspect, the present invention provides the droplet digital PCR oil phase composition or the application of the droplet digital PCR composition in droplet digital PCR.

In a fourth aspect, the present invention provides a droplet digital PCR kit, comprising the droplet digital PCR oil phase composition or the droplet digital PCR composition.

Compared with the prior art, the present invention has the following beneficial effects:

The microdroplet digital PCR oil phase composition provided by the present invention includes fluorinated oil and PFPE-PEG-PFPE in a specific proportion, and the microdroplets formed by the oil phase composition and the water phase are uniform in size and have good stability, and are suitable for PCR The effect of amplification is small, and it can be used in droplet digital PCR as an oil phase.

The droplet digital PCR composition provided by the present invention includes the above-mentioned droplet digital PCR oil phase composition and water phase composition, wherein the water phase composition contains a specific concentration of hot-start Taq enzyme, MgCl ₂ , KCl, Tris- HCl, BSA, dNTP, PEG2000, betaine, DMSO, glycerol, protamine DNA and Proclin-300, the water-phase composition and the oil-phase composition have a cooperative effect, and the water-in-oil droplets formed by mixing the two are more stable, And the PCR amplification has high sensitivity, strong specificity and high amplification efficiency, and is suitable for the amplification of various nucleic acids.

Description of drawings

In order to illustrate the specific embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without creative efforts.

Fig. 1 is the droplet generated by silicone oil;

Figure 2 is a droplet generated by mineral oil;

Fig. 3 is the droplet that fluorinated oil generates;

Fig. 4 is the droplet generated by the oil phase composition provided by Comparative Example 1;

Fig. 5 is the droplet generated by the oil phase composition provided by Comparative Example 2;

Figure 6 is the droplet generated by the oil phase composition provided by Comparative Example 3;

Figure 7 is the droplet generated by the oil phase composition provided by Comparative Example 4;

Figure 8 is the droplet generated by the oil phase composition provided by Comparative Example 5;

Figure 9 is the droplet generated by the oil phase composition provided in Example 1;

Figure 10 is the droplet generated by the oil phase composition provided in Example 2;

Figure 11 is the droplet generated by the oil phase composition provided in Example 3;

Figure 12 is a fluorescence picture after PCR amplification of microdroplets prepared by using the oil phase and water phase prepared in Example 5;

Figure 13 is a fluorescence picture after PCR amplification of microdroplets prepared by using the oil phase and water phase prepared in Example 6;

Figure 14 is a fluorescence picture after PCR amplification of microdroplets prepared by using the oil phase and water phase prepared in Example 4;

Fig. 15 is the fluorescence picture that utilizes the oil phase and the water phase prepared by Example 4 to generate microdroplets after PCR amplification and place them for 7 days;

16 is a fluorescence picture after PCR amplification of microdroplets prepared by using the oil phase and water phase prepared in Comparative Example 6;

Figure 17 is a fluorescence picture after PCR amplification of microdroplets prepared by using the oil phase and water phase prepared in Comparative Example 7;

FIG. 18 is a fluorescent picture after PCR amplification of microdroplets prepared by using the oil phase and the water phase prepared in Comparative Example 8. FIG.

Detailed ways

The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. If no specific conditions are specified, follow the general conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

In a first aspect, the present invention provides a droplet digital PCR oil phase composition, which, in parts by mass, comprises: 92-98 parts of fluorinated oil and 2-8 parts of PFPE-PEG-PFPE.

In the present invention, the mass fraction of fluorinated oil can be, for example, but not limited to 92 parts, 93 parts, 94 parts, 95 parts, 96 parts, 97 parts or 98 parts; the mass fraction of PFPE-PEG-PFPE can be, for example, For, but not limited to, 2, 3, 4, 5, 6, 7, or 8 servings.

The inventors have found that the droplets formed by the oil phase composition comprising a specific ratio of fluorinated oil and PFPE-PEG-PFPE and the water phase are uniform in size, have good stability, and have little impact on PCR amplification, and can be used as oil. Correspondingly used in droplet digital PCR.

In some preferred embodiments, in parts by mass, including: 93-97 parts of fluorinated oil and 3-7 parts of PFPE-PEG-PFPE;

Preferably, in parts by mass, it includes: 95 parts of fluorinated oil and 5 parts of PFPE-PEG-PFPE.

By further optimizing and adjusting the distribution ratio of each component in the oil phase composition of the microdroplet digital PCR, the size of the microdroplets prepared by mixing the oil phase combination with the water phase is more uniform and the stability is better.

In a second aspect, the present invention provides a droplet digital PCR composition, including the droplet digital PCR oil phase composition and water phase composition;

The solutes of the aqueous composition include hot-start Taq enzyme, _MgCl2 , KCl, Tris-HCl, BSA, dNTPs, PEG2000, betaine, DMSO, glycerol, protamine DNA and Proclin-300.

In the aqueous phase composition, the concentration of the hot-start Taq enzyme can be, for example, but not limited to, 0.08U/μL, 0.09U/μL, 0.10U/μL, 0.11U/μL or 0.12U/μL; the concentration of MgCl ₂ For example, it can be, but not limited to, 3mM, 3.2mM, 3.4mM, 3.6mM, 3.8mM or 4mM; the concentration of KCl can be, for example, but not limited to, 100mM, 110mM, 120mM, 130mM, 140mM or 150mM; the concentration of Tris-HCl For example, it can be, but not limited to, 16mM, 18mM, 20mM, 22mM or 24mM; the concentration of BSA can be, for example, but not limited to, 2mg/mL, 2.2mg/mL, 2.4mg/mL, 2.6mg/mL, 2.8mg/mL or 3mg/mL. The concentration of dNTP can be, for example, but not limited to, 0.3 mM, 0.34 mM, 0.38 mM, 0.42 mM, 0.46 mM or 0.5 mM; the volume fraction of PEG2000 can be, for example, but not limited to, 0.6%, 0.64%, 0.68%, 0.72% , 0.76% or 0.8%; the concentration of betaine can be, for example, but not limited to, 0.3M, 0.34M, 0.38M, 0.42M, 0.46M or 0.5M; the concentration volume fraction of DMSO can be, but not limited to, 1.6% , 1.8%, 2.0%, 2.2% or 2.4%; the volume fraction of glycerol can be, for example, but not limited to, 0.2%, 0.22%, 0.24%, 0.26%, 0.28%, or 0.3%; the concentration of fish sperm DNA, for example, can be is, but not limited to, 0.4ng/μL, 0.44ng/μL, 0.48ng/μL, 0.52ng/μL, 0.56ng/μL or 0.6ng/μL; the volume fraction of Proclin-300 can be, for example, but not limited to 0.03% , 0.035%, 0.04%, 0.045% or 0.05%.

The droplet digital PCR composition provided by the present invention includes the above-mentioned droplet digital PCR oil phase composition and water phase composition, wherein the water phase composition contains a specific concentration of hot-start Taq enzyme, MgCl ₂ , KCl, Tris- HCl, BSA, dNTP, PEG2000, betaine, DMSO, glycerol, protamine DNA and Proclin-300, the droplet digital PCR composition forms water-in-oil droplets that are more stable and have high sensitivity for PCR amplification , strong specificity, high amplification efficiency, suitable for the amplification of various nucleic acids.

In some preferred embodiments, in the aqueous composition, the concentration of hot-start Taq enzyme is 0.1 U/μL, the concentration of MgCl ₂ is 3.5 mM, the concentration of KCl is 75 mM, the concentration of Tris-HCl is 20 mM, The concentration of BSA was 2.5mg/mL, the concentration of dNTP was 0.4mM, the volume fraction of PEG2000 was 0.7%, the concentration of betaine was 0.4M, the volume fraction of DMSO was 2%, the volume fraction of glycerol was 0.5%, and the The concentration of DNA was 0.5 ng/μL, and the volume fraction of Proclin-300 was 0.04%.

By further optimizing and adjusting the concentration of the water-phase composition components in the microdroplet digital PCR composition, the stability of the microdroplets prepared by the composition is better, and the specificity, sensitivity and amplification of nucleic acid amplification are improved. efficiency.

In some preferred embodiments, the solute of the aqueous composition further includes a primer pair, a probe and fluorescein sodium salt, so as to realize the detection or quantification of the target nucleic acid.

In some preferred embodiments, the concentration of the primer pair can be, for example, but not limited to, 600nM, 700nM, 800nM, 900nM or 1000nM, preferably 800nM; the concentration of the probe can be, for example, but not limited to 160nM, 180nM, 200nM, 220nM or 240nM, preferably 200nM; the concentration of the fluorescein sodium salt can be, for example, but not limited to, 80nM, 90nM, 100nM, 110nM or 120nM, preferably 100nM.

In a third aspect, the present invention provides the droplet digital PCR oil phase composition or the application of the droplet digital PCR composition in droplet digital PCR.

The droplet digital PCR oil phase composition provided by the present invention can form droplets with uniform size and good stability with the water phase, and the droplets have little influence on PCR amplification; the droplet digital PCR composition provided by the present invention , including the oil phase composition and the water phase composition of specific components, through the synergistic effect of the two, the prepared water-in-oil droplets are more stable, and have high sensitivity and specificity for PCR amplification. High efficiency. Therefore, both the oil phase composition of droplet type digital PCR and the composition of droplet type digital PCR provided by the present invention can be applied to the droplet type digital PCR.

In a fourth aspect, the present invention provides a droplet digital PCR kit, comprising the droplet digital PCR oil phase composition or the droplet digital PCR composition.

The kit is suitable for the amplification of various nucleic acids, and the PCR amplification has high sensitivity, strong specificity and good amplification efficiency.

The present invention is further described below through specific examples and comparative examples, however, it should be understood that these examples are only used for more detailed description, and should not be construed to limit the present invention in any form.

It should be noted that the target fragments of primer pairs, probes and plasmid templates used in the following examples or test examples are as follows, wherein the plasmid is pUC57.

Example 1

A droplet type digital PCR oil phase composition, calculated in parts by mass, comprises 95 parts of fluorinated oil and 5 parts of gemini surfactant.

The gemini surfactant is PFPE-PEG-PFPE.

Example 2

A droplet type digital PCR oil phase composition, in parts by mass, comprises 92 parts of fluorinated oil and 8 parts of gemini surfactant.

The gemini surfactant is PFPE-PEG-PFPE.

Example 3

A microdroplet digital PCR oil phase composition, calculated in parts by mass, comprises 98 parts of fluorinated oil and 2 parts of gemini surfactant.

The gemini surfactant is PFPE-PEG-PFPE.

Comparative Example 1

A microdroplet digital PCR oil phase composition, in parts by mass, comprises 95 parts of fluorinated oil and 5 parts of ionic surfactant.

The ionic surfactant is dodecyldimethylcarboxybetaine.

Comparative Example 2

A microdroplet digital PCR oil phase composition, in parts by mass, comprises 95 parts of fluorinated oil and 5 parts of ionic surfactant.

The ionic surfactant is dodecyl dimethyl ammonium bromide.

Comparative Example 3

A droplet type digital PCR oil phase composition, in parts by mass, comprises 95 parts of fluorinated oil and 5 parts of nonionic surfactant.

The nonionic surfactant is Abil EM-90.

Comparative Example 4

A droplet type digital PCR oil phase composition, in parts by mass, comprises 95 parts of fluorinated oil and 5 parts of nonionic surfactant.

Among them, the nonionic surfactant is Span80.

Comparative Example 5

A droplet digital PCR oil phase composition, the difference from Example 1 is that it contains 85 parts of fluorinated oil and 15 parts of gemini surfactant.

Example 4

A droplet digital PCR composition, comprising the oil phase composition of Example 1 and a water phase, the water phase composition is shown in Table 1.

Table 1

Example 5

A droplet type digital PCR composition includes the oil phase composition of Example 1 and an aqueous phase, and the aqueous phase composition is shown in Table 2.

Table 2

Example 6

A droplet type digital PCR composition includes the oil phase composition of Example 1 and an aqueous phase, and the aqueous phase composition is shown in Table 3.

table 3

component Final concentration MgCl₂ 3mM KCl 150mM Tris-HCl 16mM BSA 3mg/mL dNTPs 0.3mM glycerin 0.3% betaine 0.3M PEG2000 0.8% DMSO 1.6% Fish sperm DNA 0.6ng/μL Proclin-300 0.05% Hot start Taq enzyme 2U/reaction Primer F 1000nM Primer R 1000nM Probe P 160nM Plasmid template 5μL/reaction Fluorescein sodium salt 120nM Nuclease-free water Make up to 25 μL

Comparative Example 6

A droplet type digital PCR composition includes the oil phase composition of Example 1 and an aqueous phase, and the aqueous phase composition is shown in Table 3.

Table 4

component Final concentration MgCl₂ 1mM KCl 200mM Tris-HCl 10mM BSA 6mg/mL dNTPs 0.4mM glycerin 0.8% betaine 0.8M PEG2000 0.3% DMSO 3% Fish sperm DNA 0.8ng/μL Proclin-300 0.04% Hot start Taq enzyme 2.5U/reaction Primer F 800nM Primer R 800nM Probe P 200nM Plasmid template 5μL/reaction Fluorescein sodium salt 100nM Nuclease-free water Make up to 25 μL

Comparative Example 7

A droplet digital PCR composition, the difference from Example 4 is that betaine is not included in the aqueous phase.

Comparative Example 8

A droplet digital PCR composition, the difference from Example 4 is that PEG2000 is not included in the aqueous phase.

Test Example 1

The inventors used silicone oil, mineral oil and fluorinated oil as the oil phase, respectively, and the water phase (in the water phase, the hot-start Taq enzyme concentration was 0.1 U/μL, the MgCl concentration was ₃ mM, the KCl concentration was 75 mM, and the Tris-HCl concentration was 0.1 U/μL). The concentration of 20 mM, the concentration of BSA is 0.5 mg/mL, the concentration of dNTP is 0.2 mM, and the volume fraction of DMSO is 1%) to prepare droplets. The preparation method of the droplets is as follows:

First, 20 μL of the oil phase was added to the chip, centrifuged at 1200 rpm for 1 min, and then 10 μL of the aqueous phase was added to the chip, and centrifuged at 1200 rpm for 1 min.

The results are shown in Figures 1, 2 and 3. It can be seen from the figure that the use of fluorinated oil is more stable than the use of silicone oil or mineral oil droplets, with less rupture and fusion, and the size of the droplets prepared by using fluorinated oil as the oil phase and the water phase is more uniform, so fluorine is initially selected. The carburetor serves as the oil phase for droplet digital PCR.

Test Example 2

After the oil was identified, the surfactants suitable for use in droplet digital PCR were screened from ionic surfactants, nonionic surfactants and gemini surfactants. Wherein, the preparation method of the oil phase is as follows: the surfactant is dissolved in the fluorinated oil and shaken, so that it is fully dissolved and mixed.

The oil phase compositions provided in Examples 1-3 and Comparative Examples 1-5 were used as the oil phase, and the microdroplets were prepared by mixing with the water phase (the water phase was the same as in Test Example 1) according to the method of Test Example 1. The results are shown in Figure 4 - As shown in Figure 11.

As can be seen from the figure, the use of the gemini surfactant PFPE-PEG-PFPE is more stable than the use of ionic or nonionic surfactant droplets, with less breakup and fusion. Concentration optimization found that 95 parts of fluorinated oil and 5 parts of gemini surfactant can generate stable and uniform droplets.

Test Example 3

Droplet digital PCR was carried out with the compositions provided in Examples 4-6 and Comparative Examples 6-8, and the steps were as follows:

PCR master mix: including hot-start Taq enzyme, MgCl ₂ , KCl, Tris-HCl, BSA, dNTP, PEG2000, betaine, DMSO, glycerol, protamine DNA, Proclin-300.

Aqueous phase preparation: Add plasmid template, primer pair and probe to PCR master mix.

First, 20 μL of the oil phase was added to the chip, centrifuged at 1200 rpm for 1 min, and then 10 μL of the aqueous phase was added to the chip, and centrifuged at 1200 rpm for 1 min. The sample hole is sealed with a breathable film.

The microdroplet-generating chip is placed in a PCR machine for amplification.

Use a chip reader to read and photograph the chip after the amplification reaction. The results are shown in Figures 12-18.

It can be seen from the figure that, compared with the composition provided in Example 4, the size of the microdroplets prepared by the compositions provided in Comparative Examples 7 and 8 is not uniform, and the droplets are ruptured or fused in a large area, indicating that the sugar beet Alkali and PEG2000 cooperate with other water phase components of the present invention, which helps to prepare droplets of uniform and stable size; compared with Comparative Example 6, the optimized PCR premix of the present invention is more suitable for the oil phase, The droplet uniformity is good, the droplet is stable, the amplification is accurate, and the amplification efficiency is high.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: The technical solutions described in the foregoing embodiments can still be modified, or some or all of the technical features thereof can be equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention. scope.

SEQUENCE LISTING

<110> Hangzhou Bioer Technology Co., Ltd.

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Claims (10)

1. A droplet type digital PCR oil phase composition, characterized in that, in parts by mass, comprising: 92-98 parts of fluorinated oil and 2-8 parts of PFPE-PEG-PFPE. 2. droplet type digital PCR oil phase composition according to claim 1, is characterized in that, by mass fraction, comprises: 93-97 parts of fluorinated oil and 3-7 parts of PFPE-PEG-PFPE; Preferably, in parts by mass, it includes: 95 parts of fluorinated oil and 5 parts of PFPE-PEG-PFPE. 3. A droplet type digital PCR composition, characterized in that, comprising the droplet type digital PCR oil phase composition and the water phase composition according to claim 1 or 2; The solutes of the aqueous composition include hot-start Taq enzyme, MgCl ₂ , KCl, Tris-HCl, BSA, dNTP, PEG2000, betaine, DMSO, glycerol, protamine DNA and Proclin-300; In the aqueous phase composition, the concentration of hot-start Taq enzyme is 0.08-0.12 U/μL, the concentration of MgCl ₂ is 3-4 mM, the concentration of KCl is 100-150 mM, the concentration of Tris-HCl is 16-24 mM, and the concentration of BSA The concentration of 2-3mg/mL, the concentration of dNTP is 0.3-0.5mM, the volume fraction of PEG2000 is 0.6%-0.8%, the concentration of betaine is 0.3-0.5M, the volume fraction of DMSO is 1.6%-2.4%, The volume fraction of glycerol was 0.2-0.3%, the concentration of proclin DNA was 0.4-0.6 ng/μL, and the volume fraction of Proclin-300 was 0.03%-0.05%. 4. The droplet digital PCR composition according to claim 3, wherein in the aqueous phase composition, the concentration of hot-start Taq enzyme is 0.1 U/μL, the concentration of MgCl is 3.5 mM, the concentration of _KCl 125 mM, 20 mM Tris-HCl, 2.5 mg/mL BSA, 0.4 mM dNTP, 0.7% PEG2000, 0.4 M betaine, and 0.4 M DMSO 2%, the volume fraction of glycerol was 0.25%, the concentration of protamine DNA was 0.5 ng/μL, and the volume fraction of Proclin-300 was 0.04%. 5 . The droplet digital PCR composition according to claim 3 , wherein the solute of the aqueous composition further comprises primer pairs, probes and fluorescein sodium salt. 6 . 6. The droplet digital PCR composition according to claim 5, wherein the concentration of the primer pair is 600-1000 nM, preferably 800 nM. 7. The droplet digital PCR composition according to claim 5, wherein the concentration of the probe is 160-240 nM, preferably 200 nM. 8 . The droplet digital PCR composition according to claim 5 , wherein the concentration of the fluorescein sodium salt is 80-120 nM, preferably 100 nM. 9 . 9 . The application of the droplet digital PCR oil phase composition of claim 1 or 2 or the droplet digital PCR composition of any one of claims 3 to 8 in droplet digital PCR. 10 . 10. A droplet digital PCR kit, characterized in that it comprises the droplet digital PCR oil phase composition of claim 1 or 2 or the droplet digital PCR described in any one of claims 3-8 PCR composition.

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