WO2021042323A1

WO2021042323A1 - Method for extracting heavy oil genomic deoxyribonucleic acid, and kit and application thereof

Info

Publication number: WO2021042323A1
Application number: PCT/CN2019/104518
Authority: WO
Inventors: 万云洋; 穆红梅; 熊驷骏; 李文宏
Original assignee: 中国石油大学(北京)
Priority date: 2019-09-05
Filing date: 2019-09-05
Publication date: 2021-03-11
Also published as: CN113924363B; CN113924363A

Abstract

Provided are a method for extracting heavy oil genomic deoxyribonucleic acid, and a kit and an application thereof. The kit comprises: a snail enzyme, a prion enzyme K, a DNA extraction solution containing Triton X-100, a decontamination solution, a prion precipitation solution and an adsorption column. The described method is mainly for high-density and high-viscosity oils, and combines the application of a snail enzyme, an extraction solution containing Triton X-100 and an SDC solution. The described kit can be applied to the extraction of heavy oil genomic DNA.

Description

Method, kit and application for extracting heavy oil genome deoxyribonucleic acid

Technical field

The invention belongs to the technical field of microbial geology, and relates to a method, kit and application for extracting heavy oil genome deoxyribonucleic acid (DNA).

Background technique

Commonly used internationally °API≥32

°API is 20-30(

Is 0.8654-0.9340), °API is 10-20(

0.9340-1) and °API≤10(

), respectively indicate light crude oil, medium crude oil, heavy crude oil and extra-heavy crude oil. According to China’s oil and natural gas industry standard "Reservoir Classification" (SY/T 6169-1995), ^{crude oil with a viscosity> 50-10000 mPa·s and a relative density greater than 0.9200 g·cm -3} (20°C) is an ordinary heavy oil; viscosity> 10000-50000, the crude oil with relative density greater than 0.9500g·cm ^-3 (20°C) is extra-heavy oil; the crude oil with viscosity greater than 50000 and relative density greater than 0.9800g·cm ^-3 (20°C) is super-heavy oil. High viscosity and high density are the most important characteristics of heavy oil, as well as the main indicators that distinguish it from ordinary thin oil. Due to the high content of non-hydrocarbons, asphaltenes and other components in heavy oil, which affects the collection of biomass during the pretreatment process and the interference of subsequent DNA extraction, which in turn increases the difficulty of DNA extraction, more targeted pretreatment methods and cells are selected. The method of cracking is very important. At present, there have been reports on methods for extracting DNA from reservoir samples, but no reports on heavy oil have been seen.

Summary of the invention

In order to overcome the above shortcomings of the prior art and further enhance the effect of heavy oil DNA extraction, the purpose of the present invention is to provide a kit for extracting heavy oil genome deoxyribonucleic acid and a method for extracting heavy oil genome deoxyribonucleic acid using the kit; The purpose of the present invention is also to provide the application of the kit in the extraction of heavy oil genome deoxyribonucleic acid. The present invention improves cell lysis and DNA extraction liquid components, increases the effectiveness of heavy oil DNA extraction; simplifies and shortens some extraction steps and time such as shaking and thermal reaction, and enhances DNA extraction efficiency; at the same time, it improves the environmental protection of the pretreatment process and uses green Reagents replace conventional organic reagents such as petroleum ether and isooctane.

The purpose of the present invention is achieved through the following technical solutions:

In one aspect, the present invention provides a method for extracting heavy oil genome deoxyribonucleic acid, which comprises the following steps:

Step 1: Add snailase, prionase K, DNA extract containing Triton X-100 (Triton X-100), and decontamination solution to the heavy oil to lyse cells;

Step 2: After standing still, centrifugation removes the sediment to obtain the upper layer liquid, and the prion precipitation liquid is added to the upper layer liquid to remove the organic macromolecular components, and the upper layer liquid is collected by centrifugation;

Step 3: Transfer the upper layer collected by centrifugation to the adsorption column for adsorption, and then rinse with the rinsing solution and elution with the DNA elution solution to collect the heavy oil genome deoxyribonucleic acid.

In the method of the present invention, Triton X-100 is used, which can effectively dissolve cell membranes and improve the quality of DNA amplified by polymerase chain reaction after DNA extraction; because the heavy oil system is complex and the extraction is the total genome Therefore, the use of the snail enzyme of the present invention is more conducive to the breaking of fungal cells.

In the above method, preferably, the step 1 further includes a step of pre-treating the heavy oil. The pre-processing is to add a pretreatment reagent to the heavy oil for treatment to obtain the pre-treated heavy oil.

In the method of the present invention, γ-valerolactone is used as a pretreatment reagent. Different from isooctane, γ-valerolactone is a green and non-toxic chemical that can reduce oil viscosity and is easy to collect microorganisms.

In the above method, preferably, the method specifically includes the following steps:

Step 1: Add 10-20μL of snail enzyme to every 300-500mg of heavy oil, shake at 30-37℃, 90-120rpm for 20-30min, and then add 10-20μL of prionase K and 1-2mL in sequence The DNA extract containing Triton X-100 is shaken at 30-37°C and 90-120 rpm for 20-30 minutes, and finally 100-200 μL of decontamination solution is added, and placed at 50-65°C for 30-60 minutes;

Or, directly add 10-20μL of snailase, 10-20μL of prionase K, 1-2mL containing Triton X-100, 100-200μL of decontamination solution to the heavy oil, and place it at 50-65℃ for 30- 60min;

Step 2: Centrifuge at room temperature and 8000rpm (7870.72×g) for 2-3min to remove the precipitate to obtain the supernatant, transfer the supernatant to a new centrifuge tube, add the same volume of prion precipitation to the supernatant, and Centrifuge for 2-3min at 8000-10000rpm, transfer the upper layer to a new centrifuge tube, repeat the operation of adding the prion precipitation solution once, and collect the upper layer by centrifugation;

Step 3: Transfer the upper layer collected by centrifugation to the adsorption column for adsorption, put the adsorption column into the collection tube, centrifuge at 10000-12000rpm (12298-17709.12×g) for 1-2min to remove the waste liquid, and then transfer it to the adsorption column Add pre-cooled rinsing solution to rinse, centrifuge at 10000-12000 rpm for 1 min to remove waste liquid, repeat the rinsing step once; place the adsorption column at room temperature for 10-20 minutes to dry the residual rinsing in the adsorption membrane in the adsorption column Finally, drop the DNA eluate into the middle of the adsorption membrane in the adsorption column, let it stand at room temperature for 5-10 minutes, centrifuge at 10000-12000 rpm for 2-3 minutes, and collect the centrifugal supernatant to be thick oil Genomic DNA.

In the above method, preferably, the step one further includes the step of pretreating the heavy oil, the pretreatment is to add 150-250 μL of pretreatment reagent to every 300-500 mg of heavy oil, and vortex to mix. After homogenization, the lower layer liquid is removed to obtain the pretreated heavy oil.

In the above method, preferably, the mass volume concentration of the snail enzyme is 10-20 mg/mL (a snail enzyme aqueous solution).

In the above method, preferably, the pretreatment reagent is γ-valerolactone.

In the above method, preferably, the mass volume concentration of prion K is 5-20 mg/mL (aqueous solution of prion K).

In the above method, preferably, each liter of DNA extraction solution containing Triton X-100 includes 50-200mmol of Trishydroxymethylaminomethane hydrochloride, 50-200mmol of Triton X-100, 50 -200mmol of Na ₃ PO ₄ and 0.5-2mol of NaCl.

In the above method, preferably, the decontamination solution is a sodium deoxycholate (SDC) solution with a mass volume concentration of 50-100 mg/mL (aqueous solution of sodium deoxycholate).

In the method of the present invention, a solution of sodium deoxycholate (SDC) is used as the decontamination solution. The sodium deoxycholate of the present invention does not have a polar head group, and its polar groups are distributed in various parts of the molecular chain. Compared with conventional anionic detergents, the sodium deoxycholate of the present invention has better solubility and is easier to prepare, and it can be used with other extraction reagents of the present invention to make the extraction effect of heavy oil total DNA better.

In the above method, preferably, the prion precipitation liquid is a mixed liquid of phenol, chloroform and isoamyl alcohol, and the mixing volume ratio of phenol, chloroform and isoamyl alcohol is (24-26):(23-25):( 0.5-1.5).

In the above method, preferably, the rinsing liquid is chromatographically pure ethanol, and the volume concentration of the ethanol is 70%.

In the above method, preferably, the DNA eluate is sterile ultrapure water.

In the above method, preferably, the adsorption membrane used in the adsorption column is a silica gel membrane.

On the other hand, the present invention also provides a kit for extracting heavy oil genome deoxyribonucleic acid. The kit includes: snailase, prionase K, DNA extraction solution containing Triton X-100, decontamination solution, prion Precipitation liquid and adsorption column.

In the above kit, preferably, the kit further includes a pretreatment reagent, and the pretreatment reagent is γ-valerolactone.

In the above kit, preferably, the kit further includes a rinse solution and a DNA elution solution.

In the above kit, preferably, the kit further includes glass beads, and the glass beads are monodisperse glass beads with a particle size of 1.5-2.0 mm.

In another aspect, the present invention also provides the application of the above kit in the extraction of heavy oil genome deoxyribonucleic acid.

The beneficial effects of the present invention:

The method for extracting heavy oil genomic DNA provided by the present invention has more advantages for the extraction of heavy oil. First of all, the present invention can directly extract heavy oil as it is, or select γ-valerolactone as the pretreatment reagent according to the oil condition. The pretreatment reagent is a green reagent, which is more environmentally friendly than using isooctane, petroleum ether and other reagents for pretreatment. Secondly, the present invention is specific to heavy oil samples. The combined application of snail enzyme, Triton X-100-containing extract and SDC solution has better cell lysis and decontamination of microorganisms in high-density and viscous oils. effect. Finally, the present invention simplifies the extraction steps, and also shortens the extraction time. The entire extraction process can be completed within one and a half hours at the fastest, and the extraction concentration is also high, the amount of dimer produced is minimal, and the subsequent qPCR detection, high Throughput sequencing and metagenomic analysis. The kit simplifies the operation method, and can be conveniently used for the extraction of heavy oil genomic DNA to achieve the purpose of efficiently extracting the total deoxyribonucleic acid in the heavy oil. Compared with the applicant's previous genomic DNA extraction method for ordinary crude oil (ZL 20170116846.5), the present invention has a shorter time (a savings of more than 100 minutes compared to the same period last year), a better effect, and is more environmentally friendly and economical.

Description of the drawings

Figure 1 is an electrophoresis chart of PCR product amplification of genomic DNA extracted in Example 1 of the present invention (wherein: 1 to 4 are parallel samples of the same oil sample; M is a molecular weight marker Marker);

Figure 2 is the electrophoresis pattern of PCR product amplification of genomic DNA extracted in Examples 2 and 3 of the present invention (wherein: 5 to 6 are parallel samples of the same oil sample in Example 2; 7-8 are the same oil sample in Example 3 Parallel samples; M is the molecular weight marker Marker).

Fig. 3 is an electrophoresis pattern of PCR product amplification of genomic DNA extracted in Example 4 of the present invention (a, 9 to 10 in Fig. 3 are parallel samples of the same oil sample in Example 4) and the genome extracted in Comparative Example 2 of the present invention The electrophoresis patterns of DNA PCR product amplification (b, 11 to 12 in Fig. 3 are the parallel samples of the same oil sample in Comparative Example 2; M is the comparison chart of the molecular weight marker Marker).

detailed description

In order to have a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention are now described in detail below, but they should not be understood as limiting the scope of implementation of the present invention.

A nucleic acid prion detector was used to determine the concentration and purity of the DNA extract (A260/A280), and the concentration of the DNA extract was determined in combination with the amount of Qubit.

All the heavy oil samples involved in the examples are collected from Liaohe Oilfield, L2YHC23, density is 0.994g/cm ³ , viscosity is 5860mPa·s, content of gum + asphaltene is 46.8%, according to Chinese standard SY/T 6169-1995 It belongs to ordinary heavy oil and belongs to heavy crude oil according to the international common °API classification method.

Unless otherwise specified, the raw materials and reagents used in the examples are all commercially available.

Example 1

This embodiment provides a kit for extracting heavy oil genome deoxyribonucleic acid. The kit includes: pretreatment reagent, glass beads, snail enzyme, prionase K, DNA extract containing Triton X-100, and decontamination The components and materials of liquid, prion precipitation, rinse, DNA eluate, adsorption column, collection tube, centrifuge tube, etc. The content and material requirements of each component are as follows:

Pretreatment reagent: γ-valerolactone;

Glass beads: 20 monodisperse glass beads with a particle size of 1.5mm;

Snail enzyme: mass volume concentration 20mg/mL;

Prionase K: mass volume concentration 20mg/mL;

Extraction solution (pH8.0): Each liter of extraction solution contains 100mmol of tris-hydroxymethylaminomethane hydrochloride (Tris-HCl), 100mmol of Triton X-100 (Triton-X), 100mmol of Na ₃ PO ₄ and 1.5 mol NaCl;

Decontamination solution: 100mg/mL SDC solution with mass volume concentration;

Prion precipitation solution: a mixed solvent of phenol/chloroform/isoamyl alcohol with a volume ratio of 25:24:1;

Rinsing solution: chromatographically pure grade ethanol with a volume concentration of 70%;

DNA eluate: sterile ultrapure water;

Adsorption column: contains silica gel adsorption membrane;

Collection tube and centrifuge tube: EP tube.

Using the above kit can conveniently apply the following extraction method in this embodiment to achieve the purpose of efficiently extracting total deoxyribonucleic acid in heavy oil.

This implementation also provides a method for extracting heavy oil genome deoxyribonucleic acid, which includes the following steps:

Step 1: Take 500mg of heavy oil, add 200-300μL of γ-valerolactone, vortex and mix, remove the lower layer to obtain pretreated heavy oil;

Step two, take the pretreated heavy oil into a centrifuge tube, add 10-20μL of snailase, shake at 37℃, 120rpm for 30min, and then add 10-20μL of prion K and 1-2mL of snailase in turn Pass the DNA extraction solution of X-100, shake at 37°C and 120rpm for 30min, finally add 100-200μL of decontamination solution, place it at 65°C for 1h, and centrifuge at room temperature and 6000-12000rpm for 3min to remove the precipitate to obtain the supernatant , Transfer the upper layer to a new centrifuge tube, add the same volume of prion precipitation to the upper layer, centrifuge at 6000-12000 rpm for 3 minutes, transfer the upper layer to a new centrifuge tube, repeat adding the prion precipitation solution Operate once and collect the supernatant by centrifugation;

Step 3: Transfer 500-1000μL of the upper layer liquid collected by centrifugation to the adsorption column for adsorption, put the adsorption column into the collection tube, centrifuge at 6000-12000 rpm for 1 min to remove the waste liquid, repeat this step until the upper layer has mostly passed Adsorption column filtration (note: the prion precipitation layer cannot be absorbed when sucking the upper layer liquid, so keep the upper layer liquid with a thickness of 3-5mm above the prion precipitation layer), and then add 500-1000μL low-temperature pre-cooled rinsing solution to the adsorption column for rinsing. Centrifuge at 6000-12000rpm for 1 min to remove the waste liquid, repeat the rinsing step; place the adsorption column at room temperature for 10 minutes to dry the residual rinsing liquid in the adsorption membrane in the adsorption column; and finally to the middle part of the adsorption membrane in the adsorption column Add 110 μL of DNA eluate dropwise in the air, let it stand for 8 min at room temperature, and centrifuge at 10000-12000 rpm for 2 min. The supernatant after centrifugation is collected, which is the heavy oil genome deoxyribonucleic acid (DNA).

Perform PCR amplification on the heavy oil genomic DNA extracted in this example to verify its purity, and the specific steps are as follows:

The forward primer (Primer1) is 515F: 5'-GTGYCAGCMGCCGCGGTAA-3',

The reverse primer (Primer2) is 806R: 5'-GGACTACVSGGGTATCTAAT-3'.

Among them: Y, M, V, S are degenerate bases.

Y represents that the base can be selected from C or T;

M represents that the base can be selected from A or C;

V represents that the base can be selected from A, C or G;

S represents that the base can be selected from C or G;

Therefore, the base sequence of the forward primer can be selected from one of the following sequences:

5'-GTGCCAGCAGCCGCGGTAA-3' (as shown in SEQ ID NO:1);

5'-GTGCCAGCCGCCGCGGTAA-3' (as shown in SEQ ID NO: 2);

5'-GTGTCAGCAGCCGCGGTAA-3' (as shown in SEQ ID NO: 3);

5'-GTGTCAGCCGCCGCGGTAA-3' (as shown in SEQ ID NO: 4).

The base sequence of the reverse primer can be selected from one of the following sequences:

5'-GGACTACACGGGTATCTAAT-3' (as shown in SEQ ID NO: 5);

5'-GGACTACCCGGGTATCTAAT-3' (as shown in SEQ ID NO: 6);

5'-GGACTACGCGGGTATCTAAT-3' (as shown in SEQ ID NO: 7);

5'-GGACTACAGGGGTATCTAAT-3' (as shown in SEQ ID NO: 8);

5'-GGACTACCGGGGTATCTAAT-3' (as shown in SEQ ID NO: 9);

5'-GGACTACGGGGGTATCTAAT-3' (as shown in SEQ ID NO: 10).

The amplified products were electrophoresed on a 1% agarose gel with a voltage of 100V for 45 minutes and photographed with an ultraviolet gel imaging system. L2YHC23-1 and L2YHC23-2 are a set of parallel samples, and L2YHC23p is the average value and standard deviation of the oil sample (Table 1). The DNA extracted in Example 1 is relatively pure, the ratio of A260/A280 is about 1.6, and the concentration is also relatively high (Table 1). No. 1 and No. 2 electrophoresis (Figure 1) showed that the DNA amplification product had a single and clear band. It can be seen that the DNA extracted by the present invention has a better effect.

Table 1 Concentration and purity of genomic DNA extracted from heavy oil in Example 1

Comparative example 1

Take 500mg of heavy oil, the pretreatment method is the same as in Example 1, add 200-300μL of γ-valerolactone, vortex and mix, remove the lower layer to obtain the pretreatment liquid, and the rest of the extraction steps follow the QIAGEN kit method get on. L2YHC23-3 and L2YHC23-4 are a set of parallel samples, and L2YHC23p is the average and standard deviation of the oil sample (Table 2). The purity and concentration of the DNA extracted by the QIAGEN kit method (Table 2) are lower than those of the method of the present invention (Example 1), and the difference seems to be small. However, subsequent sequencing shows that only the method of the present invention can be used for subsequent experiments.

Table 2 Concentration and purity of genomic DNA extracted from heavy oil in Comparative Example 1

Example 2

The only difference between this example and Example 1 is the lack of step one, that is, no pretreatment reagent (γ-valerolactone) is used, and heavy oil is directly used for extraction, and the rest of the operations are completely the same. L2YHC23-5 and L2YHC23-6 are a set of parallel samples, and L2YHC23p is the average value and standard deviation of the oil sample (Table 3). The extraction results and verification results show that the concentration of DNA extracted by the present invention is relatively high (Table 3), and the electrophoresis bands No. 5 and No. 6 (Figure 2) are single and clear, indicating that the heavy oil DNA extraction effect is better.

Table 3 Concentration and purity of genomic DNA extracted from heavy oil in this implementation 2

Example 3

Compared with Example 1, this example omitted step one and directly used heavy oil for extraction. The specific process is as follows: take 500 mg of heavy oil in a centrifuge tube, add 10-20 μL of snail enzyme, and then add 10-20 μL of Prion K and 1-2 mL of DNA extract containing Triton X-100, finally add 100-200 μL of decontamination solution, place it at 65°C for 1 h, and centrifuge at room temperature and 8000 rpm for 3 min to remove the precipitate to obtain the upper layer. Transfer the upper layer to a new centrifuge tube, add the same volume of prion precipitation to the upper layer, centrifuge at 8000 rpm for 3 minutes, transfer the upper layer to a new centrifuge tube, and repeat the operation of adding prion precipitation once. The supernatant was collected by centrifugation.

L2YHC23-7 and L2YHC23-8 are a set of parallel samples, and L2YHC23p is the average value and standard deviation of the oil sample (Table 4). The extraction results and verification results show that the concentration of DNA extracted by the present invention is higher (Table 4), and the electrophoresis bands No. 7 and No. 8 (Figure 2) are the clearest and brightest, indicating that the heavy oil DNA extraction effect is better.

Table 4 Concentration and purity of genomic DNA extracted from heavy oil in Example 3

Example 4

Compared with Example 3, this example uses the same heavy oil (different storage conditions after collection), and the dosage is exactly the same as the experimental operation. L2YHC23-9 and L2YHC23-10 are a set of parallel samples, and L2YHC23p is the average value and standard deviation of the oil sample (Table 5). The extraction results and verification results show that the concentration and purity of the DNA extracted by the present invention are higher (Table 5), and the electrophoresis bands No. 9 and No. 10 (a in Fig. 3) are clearer and brighter, indicating that the extraction effect of heavy oil DNA is better. Great.

Table 5 Concentration and purity of genomic DNA extracted from heavy oil in Example 4

Comparative example 2

Compared with Example 4, this comparative example 2 uses the same heavy oil, and the extraction method refers to the method of invention patent ZL201710116846.5. L2YHC23-11 and L2YHC23-12 are a set of parallel samples. L2YHC23p is the average and standard deviation of the oil sample (Table 6). The extraction and verification results show that the concentration and purity of DNA extracted by this method are higher than those of the method of the present invention (implementation). Example 4) is low (Table 6), and the electrophoretic bands No. 11 and No. 12 (b in Fig. 3) are not as bright as the method of the invention (a in Fig. 3), indicating that the extraction effect of the invention is better. In addition, the application of the present invention takes less time than our previous invention to extract DNA, and can be completed within 90 minutes at the fastest, and can save at least 100 minutes compared with the previous invention. At the same time, the present invention does not use isooctane and other organic reagents in the pretreatment process, which is better than the previous invention. More environmentally friendly.

Table 6 Concentration and purity of genomic DNA extracted from heavy oil in this comparative example 2

The genomic DNA extracted by the method of the present invention can meet the analysis requirements of subsequent qPCR detection, high-throughput sequencing and metagenomics.

Claims

A method for extracting heavy oil genome deoxyribonucleic acid, which comprises the following steps:

Step 1: Add snailase, prionase K, DNA extract containing Triton X-100, and decontamination solution to the heavy oil to lyse the cells;

Step 2: After standing still, the sediment is removed by centrifugation to obtain the upper liquid, and the prion sediment is added to the upper liquid to remove the organic macromolecular components, and the upper liquid is collected by centrifugation;

Step 3: Transfer the upper layer collected by centrifugation to the adsorption column for adsorption, and then rinse with the rinsing solution and elution with the DNA elution solution to collect the heavy oil genome deoxyribonucleic acid.
The method according to claim 1, wherein, before the first step, it further comprises a step of pretreating the heavy oil, and the pretreatment is to add a pretreatment reagent to the heavy oil for treatment to obtain the pretreated heavy oil .
The method according to claim 1, wherein the method specifically comprises the following steps:

Step 1: Add 10-20μL of snail enzyme to every 300-500mg of heavy oil, shake at 30-37℃, 90-120rpm for 20-30min, and then add 10-20μL of prionase K and 1-2mL in sequence The DNA extract containing Triton X-100 is shaken at 30-37°C and 90-120 rpm for 20-30 minutes, and finally 100-200 μL of decontamination solution is added, and placed at 50-65°C for 30-60 minutes;

Or, directly add 10-20μL of snailase, 10-20μL of prionase K, 1-2mL of DNA extract containing Triton X-100, and 100-200μL of decontamination solution to every 300-500mg of heavy oil. , Placed at 50-65℃ for 30-60min;

Step 2: Centrifuge for 2-3min at room temperature and 8000-10000rpm to remove the precipitate to obtain the supernatant. Transfer the supernatant to a new centrifuge tube. Add the same volume of prion precipitation to the supernatant at 8000-10000rpm. Centrifuge for 2-3min under conditions, transfer the upper layer to a new centrifuge tube, repeat the operation of adding prion precipitation solution, and collect the upper layer by centrifugation;

Step 3: Transfer the upper layer liquid collected by centrifugation to the adsorption column for adsorption, put the adsorption column into the collection tube, centrifuge at 10000-12000rpm for 1-2min to remove the waste liquid, and then add the pre-cooled rinsing solution to the adsorption column Carry out rinsing, centrifuge at 10000-12000rpm for 1-2min to remove the waste liquid, repeat the rinsing step; place the adsorption column at room temperature for 10-20min to dry the residual rinsing liquid in the adsorption membrane in the adsorption column; finally transfer to the adsorption column The DNA eluate suspended in the middle part of the adsorption membrane in the medium, left standing at room temperature for 5-10min, centrifuged at 10000-12000rpm for 2-3min, and the supernatant collected after centrifugation is the heavy oil genome deoxyribonucleic acid.
The method according to claim 3, wherein, before the step one, it further comprises a step of pretreating the heavy oil, and the pretreatment is to add 150-250 μL of pretreatment reagent to every 300-500 mg of heavy oil, After vortexing and mixing, the lower layer liquid is removed to obtain the pretreated heavy oil.
The method according to any one of claims 1 to 4, wherein the mass volume concentration of the snail enzyme is 10-20 mg/mL.
The method according to claim 2 or 4, wherein the pretreatment reagent is γ-valerolactone.
The method according to any one of claims 1 to 4, wherein the mass volume concentration of prion K is 5-20 mg/mL.
The method according to any one of claims 1 to 4, wherein each liter of DNA extraction solution containing Triton X-100 comprises 50-200mmol of tris hydrochloride, 50-200mmol of Triton X-100, 50-200 mmol of Na 3 PO 4 and 0.5-2 mol of NaCl.
The method according to any one of claims 1 to 4, wherein the decontamination liquid is a sodium deoxycholate solution with a mass volume concentration of 50-100 mg/mL.
The method according to any one of claims 1 to 4, wherein the prion precipitation liquid is a mixed liquid of phenol, chloroform and isoamyl alcohol, and the mixing volume ratio of phenol, chloroform and isoamyl alcohol is (24-26) : (23-25): (0.5-1.5).
The method according to any one of claims 1 to 4, wherein the rinsing liquid is chromatographically pure ethanol, and the volume concentration of the ethanol is 70%;

Preferably, the DNA eluate is sterile ultrapure water;

Preferably, the adsorption membrane used in the adsorption column is a silica gel membrane.
A kit for extracting heavy oil genome deoxyribonucleic acid, wherein the kit includes: snailase, prionase K, DNA extraction solution containing Triton X-100, decontamination solution, prion precipitation solution and adsorption column.
The kit according to claim 12, wherein the kit further comprises a pretreatment reagent, and the pretreatment reagent is γ-valerolactone;

Preferably, the kit further includes a rinse solution and a DNA elution solution.
The kit according to claim 12, wherein the kit further comprises glass beads, and the glass beads are monodisperse glass beads with a particle size of 1.5-2.0 mm.
The use of the kit according to any one of claims 12-14 in the extraction of heavy oil genome deoxyribonucleic acid.