Nucleic acid purification device and application thereof in extraction of total DNA (deoxyribonucleic acid) of microorganisms in crude oil and / or thickened oil

Abstract

The invention relates to the technical field of microorganism nucleic acid extraction, and discloses a nucleic acid purification device for extracting microorganism total DNA in crude oil and / or thick oil and application of the nucleic acid purification device. The nucleic acid purification device consists of four parts, namely a rough filtration column, a purification column, a first collection pipe and a second collection pipe, the rough filtration column, the purification column, the first collection pipe and the second collection pipe can be mutually and stably accommodated or buckled to form a whole; wherein the second collection pipe, the purification column and the rough filtration column are sequentially arranged from outside to inside and are mutually embedded, and the purification column and the first collection pipe are connected in series at the upper part and the lower part in the second collection pipe. Due to the continuous and semi-automatic design, the device has high applicability to low-biomass samples such as crude oil and / or thickened oil samples, and the large-volume rapid extraction and double-filtration system has the functions of convenience, rapidness, good enrichment and purification effects and the like and also achieves the purpose of reducing the cost.

Description

Technical Field

[0001] The invention relates to the technical field of microbial nucleic acid extraction, and in particular to a nucleic acid purification device and application thereof in extracting total DNA of microorganisms in crude oil and / or heavy oil. Background Art

[0002] In the field of molecular biology, the extraction of microbial DNA from environmental samples is an important way to study the characteristics of microbial communities. How to efficiently separate and purify is a key content in DNA extraction technology. Using nucleic acid purification columns to separate and purify microbial DNA has always been one of the common methods in DNA extraction technology, and it is also the most commonly used method in commercial kits on the market.

[0003] The common nucleic acid purification column on the market is a cylindrical tubular structure with a cap at one end: a hollow cylindrical structure with a cap of about 4 cm in length (outer diameter of about 1 cm), and a nucleic acid adsorption membrane at the bottom. The nucleic acid purification column body is nested in another hollow cylinder (collection tube) without a cap and about 3 cm in length. The diameter of the collection tube is slightly larger than the nucleic acid purification column body. The two parts are generally referred to as a nucleic acid purification column. The general process of extracting DNA using a common nucleic acid purification column method is to first lyse cells with chemical reagents or bioenzyme preparations to obtain a crude DNA solution, and then transfer this solution to a nucleic acid purification column for purification to remove impurities such as cell debris and prions.

[0004] However, for some extreme environmental samples such as crude oil, the content of microorganisms is low, and the viscosity makes the limitations of ordinary purification columns prominent. First, in terms of convenience, taking crude oil samples as an example, to obtain the same large mass of DNA, it is necessary to increase the number of repeated extractions by dozens of times, making the experimental operation more cumbersome and amplifying human errors; second, in terms of timeliness, it is necessary to repeat the filtration and separation steps of ordinary nucleic acid purification devices many times, and the purification steps that originally only take 5-10 minutes for samples with large biomass will be extended to 1 hour due to repeated operations; third, in terms of nucleic acid quality and purity, the viscosity and low density of crude oil make it easy for crude oil oil phase impurities to adhere and difficult to remove, which has a greater impact on the purification function of general nucleic acid purification columns; fourth, in terms of economy, the characteristics of "small amount and multiple times" make traditional disposable nucleic acid purification columns extremely expensive when applied to large volume samples, and are prone to nucleic acid environmental pollution, while for low biomass samples, more consumption and investment are required. In summary, ordinary nucleic acid purification devices are less suitable for extreme environmental samples including crude oil.

[0005] Therefore, there is an urgent need in the art to develop a nucleic acid purification device for extreme environment samples such as crude oil to simplify the operation process of such nucleic acid separation and purification and improve its efficiency. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of ordinary nucleic acid purification devices and further enhance the crude oil DNA extraction effect. The present invention aims to design a nucleic acid purification device for extracting total microbial DNA from crude oil and / or heavy oil.

[0007] The purpose of the present invention is also to design such a nucleic acid purification device for crude oil and / or heavy oil samples to adapt to the two patented methods for extracting total deoxyribonucleic acid from crude oil, namely, the invention patent number ZL 201710116846.5 (CN106754895B) published by this unit, "Method and kit for extracting total deoxyribonucleic acid from crude oil" and "Preparation and application of kit for extracting genomic deoxyribonucleic acid from heavy oil" (PCT / CN2019 / 104518), so as to further improve the extraction efficiency of total microbial DNA in crude oil and / or heavy oil samples.

[0008] In order to achieve the above object, the first aspect of the present invention provides a nucleic acid purification device, which is composed of four parts, namely a coarse filtration column, a purification column, a first collection tube and a second collection tube;

[0009] The coarse filtration column, the purification column, the first collection tube and the second collection tube can stably accommodate or buckle with each other to form a whole; wherein, the second collection tube, the purification column and the coarse filtration column are arranged in sequence from the outside to the inside and interlocked with each other, and the purification column and the first collection tube are connected in series to the upper and lower parts of the second collection tube.

[0010] Preferably, the centrifugal rotor of an existing commercial centrifuge (adapted to 50mL and 100mL centrifuge tubes) is used as a template to design relevant dimensions. The nucleic acid purification device includes a coarse filtration column, a purification column, a first collection tube and a second collection tube. The top of the nucleic acid purification device is a sample loading port, which is equipped with a nut cover type sealing cover. The inner diameter of the sample loading port is 32mm. Below the sealing plug is the coarse filtration column. After being filtered by the coarse filtration column, the sample enters the purification column, and then enters the first collection tube after secondary filtration by the purification column to obtain a DNA solution.

[0011] Preferably, the structures of the first collection tube and the second collection tube are respectively adapted to the structures of the purification column: the inner diameter of the second collection tube is the same as the upper outer diameter of the purification column so that the purification column can be tightly attached to the inner wall of the second collection tube and the contact surface is made of a frosted surface to prevent the purification column from tilting or freely rotating; the inner diameter of the first collection tube is the same as the lower outer diameter of the purification column so that the nucleic acid purification column can be tightly attached to the inner wall of the first collection tube and the contact surface is made of a frosted surface to prevent the purification column from tilting or freely rotating; the structures of the filter membranes for the two filtrations are respectively adapted to the structures of the coarse filtration column and the purification column so that the filter membranes are stably nested in the bottom of the coarse filtration column and the purification column, respectively.

[0012] Preferably, the nucleic acid purification device of the present invention comprises four main components that are independent of each other but can be tightly fastened to each other, the first part is a coarse filtration column; the second part is a purification column, the coarse filtration column is connected and wrapped by the purification column; the third part is a first collection tube, the first collection tube is closely connected to the bottom end of the purification column; the fourth part is a second collection tube; the second collection tube as a whole wraps the first, second and third parts, and the matching nut cover type sealing cover.

[0013] Preferably, the coarse filter column of the first part is a cylindrical structure, with the upper end being the total sample loading port of the device (without a cover), and the lower end being the filter membrane as the bottom. When the coarse filter column is placed in the purification column of the second part, it can fit tightly against the inner wall of the purification column. The purification column can also fit tightly against the inner wall of the second collection tube and fit into the first collection tube to firmly limit it between the second collection tube and the purification column. These four parts can be tightly fastened and combined into a whole at the same time. The lower part of the coarse filter column is a filter membrane, which intercepts insoluble impurities and does not specifically bind to DNA. The diameter of the filter membrane is 32 mm. The bottom of the purification column is equipped with an adsorption membrane, which can specifically adsorb DNA and has a diameter of 6 mm.

[0014] Preferably, the coarse filtration column, the purification column, the first collection tube and the second collection tube are all of similar cylindrical structure, and the outer diameters and inner diameters of the three are in sequence. From inside to outside (or the inner diameter is from small to large), they are respectively the first collection tube, the coarse filtration column, the purification column and the second collection tube. Through the interaction of the frosted surface where the inner and outer tube walls meet, the four parts can be firmly joined together and can be freely disassembled and removed.

[0015] Preferably, the bottom of the coarse filtration column of the first part is a coarse filtration membrane for removing impurities that are insoluble in crude oil, and the purification column of the second part is an adsorption membrane for purifying nucleic acids.

[0016] Preferably, the large capacity and dual-filtration structure of the nucleic acid purification device enable the extraction of microbial nucleic acid from common environmental samples, which usually requires repeated operations by a general nucleic acid purification system, to be completed in a single operation.

[0017] Preferably, the nucleic acid purification device provided by the present invention can greatly improve the enrichment effect of microbial nucleic acid for the extraction of microbial deoxyribonucleic acid from extreme environment samples such as crude oil, thereby improving the extraction efficiency.

[0018] Preferably, the addition of a small first collection tube inside the nucleic acid purification device provided by the present invention can avoid the problem of excessive relative loss of a small volume of target DNA solution due to pipetting and storage in a large volume of the second collection tube.

[0019] The second aspect of the present invention provides the use of the nucleic acid purification device for extracting total DNA of microorganisms in crude oil and / or viscous oil according to the first aspect in nucleic acid extraction, wherein the nucleic acid extraction in the application comprises the following extraction steps:

[0020] (1) taking a sample with a volume of 20-30 mL; the sample is a crude oil sample and / or a heavy oil sample;

[0021] If the sample is a low-biomass sample I, the sample I is centrifuged to discard the supernatant and retain the precipitate; the sample I is an extreme environment sample (crude oil (light oil (LO), medium oil (MO), ordinary heavy oil (OHO), extra heavy oil (EHO), super heavy oil (SHO), formation water, etc.);

[0022] If the sample is sample II with a higher degree of solidification, the sample II is ground or fragmented before processing; the sample II is soil (including highly saline and alkaline soil) or rock minerals;

[0023] (2) adding 20-30 mL of extraction buffer, 500-750 μL of washing enzyme I, 500-750 μL of washing enzyme II and 5-7.5 mL of decontamination solution in sequence, maintaining a constant temperature at 65° C. for 30-45 min, and then maintaining a constant temperature at 90° C. for 30-45 min, and transferring the supernatant to a new centrifuge tube after a first centrifugation for 3-5 min; the relative centrifugal force of the first centrifugation is 12298×g-17709.12×g;

[0024] Add the same volume of protein precipitate as the upper layer, and centrifuge for a second time for 5-10 minutes; the relative centrifugal force of the second centrifugation is 12298×g-17709.12×g;

[0025] (3) combining the coarse filtration column, the purification column and the second collection tube in the nucleic acid purification device to obtain a combined structure;

[0026] The supernatant collected by the second centrifugation is completely transferred to the coarse filter column in the combined structure, and the waste liquid in the second collection tube is discarded and returned to the second collection tube for a third centrifugation for 5-10 minutes; wherein the volume of the supernatant is 20-30 mL; and the relative centrifugal force of the third centrifugation is 12298×g-17709.12×g;

[0027] (4) discarding the coarse filtration column and adding 1 mL of pre-cooled rinsing solution to the purification column for rinsing, performing a fourth centrifugation for 5-10 min, discarding the waste liquid in the second collection tube, and repeating the rinsing step once; the relative centrifugal force of the fourth centrifugation is 12298×g-17709.12×g;

[0028] The purification column is placed at room temperature for 10-20 minutes to dry the residual rinsing liquid in the adsorption membrane;

[0029] (5) Finally, a first collection tube is placed at the bottom of the purification column, and 50-200 μL of DNA elution solution is added dropwise to the middle part of the adsorption membrane of the purification column, and the mixture is allowed to stand at room temperature for 5-10 minutes, and then centrifuged for a fifth time for 3-5 minutes to obtain the total DNA of microorganisms in the crude oil and / or heavy oil; the relative centrifugal force of the fifth centrifugation is 12298×g-17709.12×g.

[0030] Preferably, the nucleic acid extraction in the application comprises the following extraction steps:

[0031] (1) taking a sample with a volume of 20-30 mL; the sample is a crude oil sample;

[0032] If the sample is a low-biomass sample I, the sample I is centrifuged to discard the supernatant and retain the precipitate; the sample I is an extreme environment liquid sample;

[0033] If the sample is a solid sample II, the sample II is ground or fragmented before processing; the sample II is soil, rock or mineral in an extreme environment;

[0034] (2) adding 20-30 mL of extraction buffer, 500-750 μL of washing enzyme I, 500-750 μL of washing enzyme II and 5-7.5 mL of decontamination solution in sequence, maintaining a constant temperature at 65° C. for 30-45 min, and then maintaining a constant temperature at 90° C. for 30-45 min, and transferring the supernatant to a new centrifuge tube after a first centrifugation for 3-5 min; the relative centrifugal force of the first centrifugation is 12298×g-17709.12×g;

[0035] Add the same volume of protein precipitate as the upper layer, and centrifuge for a second time for 5-10 minutes; the relative centrifugal force of the second centrifugation is 12298×g-17709.12×g;

[0036] (3) combining the coarse filtration column, the purification column and the second collection tube in the nucleic acid purification device to obtain a combined structure;

[0037] The supernatant collected by the second centrifugation is completely transferred to the coarse filter column in the combined structure, and the waste liquid in the second collection tube is discarded and returned to the second collection tube for a third centrifugation for 5-10 minutes; wherein the volume of the supernatant is 20-30 mL; and the relative centrifugal force of the third centrifugation is 12298×g-17709.12×g;

[0038] (4) discarding the coarse filtration column and adding 1 mL of pre-cooled rinsing solution to the purification column for rinsing, performing a fourth centrifugation for 5-10 min, discarding the waste liquid in the second collection tube, and repeating the rinsing step once; the relative centrifugal force of the fourth centrifugation is 12298×g-17709.12×g;

[0039] The purification column is placed at room temperature for 10-20 minutes to dry the residual rinsing liquid in the adsorption membrane;

[0040] (5) Finally, a first collection tube is placed at the bottom of the purification column, and 50-200 μL of DNA elution solution is added dropwise to the middle part of the adsorption membrane of the purification column, and the mixture is allowed to stand at room temperature for 5-10 minutes, and then centrifuged for a fifth time for 3-5 minutes to obtain the total DNA of microorganisms in the crude oil and / or heavy oil; the relative centrifugal force of the fifth centrifugation is 12298×g-17709.12×g.

[0041] It should be noted that in step (1), the "low biomass sample" refers to an extreme environment sample.

[0042] Preferably, the application of the nucleic acid purification device provided by the present invention in nucleic acid extraction is suitable for environmental samples with generally low biomass. Taking the method and kit for extracting total deoxyribonucleic acid from crude oil disclosed in patent CN106754895B published by this unit as an example, the extraction process is simplified to further improve the extraction efficiency of microbial DNA in crude oil samples. The specific extraction method steps and reagent ratios refer to the above patent, and step three is modified to a certain extent to adapt to the present nucleic acid purification device.

[0043] According to a particularly preferred embodiment, the specific changes to step three are as follows:

[0044] Step 3: According to the nucleic acid purification device of the present invention, the three parts of the coarse filter column, the purification column and the second collection tube are combined, and all the supernatant collected by the second centrifugation (about 20-30 mL) is transferred to the coarse filter column in the assembled nucleic acid purification device, centrifuged at room temperature (12298×g-17709.12×g) for 5 minutes, the waste liquid in the second collection tube is discarded and returned to the second collection tube, and then the coarse filter column of the first part of the device is discarded and 1 mL of pre-cooled rinsing solution is added to the purification column for rinsing, and the third centrifugation is carried out for 5 minutes under the relative centrifugal force of 12298×g-17709.12×g. n remove the waste liquid, repeat the rinsing step once; place the purification column at room temperature for 10-20 minutes to dry the residual rinsing liquid in the adsorption membrane in the adsorption column; finally, place the first collection tube at the bottom of the purification column, drop 50-200 μL of DNA elution solution to the middle part of the adsorption membrane in the purification column, let it stand at room temperature for 5-10 minutes, and centrifuge for the fifth time for 2-3 minutes under the condition of relative centrifugal force of 12298×g-17709.12×g. The filtrate obtained after centrifugation in the first collection tube is the total DNA (i.e., deoxyribonucleic acid) of microorganisms in crude oil and / or heavy oil.

[0045] Preferably, the components and proportions of the enzyme preparation and chemical preparation (flushing enzyme I, flushing enzyme II, extraction buffer, decontamination solution, protein precipitation solution, rinsing solution and DNA elution solution) refer to patent CN106754895B.

[0046] The above technical solution of the present invention has at least the following beneficial effects compared with the prior art:

[0047] The nucleic acid purification device of the present invention comprises four independent but combined parts: a coarse filter column, a purification column, a first collecting tube and a second collecting tube. Since the coarse filter column, the nucleic acid sample preparation column and the first collecting tube are all similar cylindrical structures, and the inner and outer diameters of the three are adapted in sequence, the large capacity and double filtering structure can be firmly connected together through the sealing plug and the collecting tube (the first collecting tube and the second collecting tube) holding component, so that the microbial nucleic acid extraction target that needs to be repeated multiple times by the general nucleic acid purification system for ordinary environmental samples can be completed in a single operation under the condition of greatly reducing the loss of DNA solution. When it is necessary to perform batch extraction of microbial deoxyribonucleic acid of extreme environmental samples such as crude oil, the experimental continuity and semi-automatic functions of the present invention can effectively reduce the original large number of repeated continuous experimental operations, and at the same time greatly reduce the human errors and accidental errors generated by multiple continuous repeated operations. The present invention greatly improves the enrichment and purification effect of its microbial nucleic acid, thereby greatly improving the extraction efficiency. In addition, it can also greatly reduce the use of general disposable nucleic acid purification columns, reducing resource waste and cost investment. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is a structural schematic diagram and model diagram of a coarse filter column of a nucleic acid purification device for extracting total DNA of microorganisms from crude oil and / or viscous oil in the present invention;

[0049] Figure 2 It is a structural schematic diagram and a model diagram of a purification column of a nucleic acid purification device for extracting total DNA of microorganisms from crude oil and / or viscous oil in the present invention;

[0050] Figure 3 It is a schematic diagram of the first collecting tube of the nucleic acid purification device for extracting total DNA of microorganisms in crude oil and / or heavy oil in the present invention;

[0051] Figure 4 It is a schematic diagram and a model diagram of the second collecting tube of the nucleic acid purification device for extracting total DNA of microorganisms in crude oil and / or viscous oil in the present invention;

[0052] Figure 5 It is a schematic diagram and a model diagram of the overall structure of the nucleic acid purification device for extracting total DNA of microorganisms in crude oil and / or viscous oil in the present invention;

[0053] Figure 6 is an agarose gel electrophoresis diagram comparing the performance of the present invention and a common nucleic acid purification device when treating a heavy oil sample 1 in Example 1 of the present invention;

[0054] Figure 7 is an agarose gel electrophoresis diagram comparing the performance of the present invention and a common nucleic acid purification device when treating a heavy oil sample 2 in Example 2 of the present invention;

[0055] Figure 8 is an agarose gel electrophoresis diagram comparing the performance of the present invention and a conventional nucleic acid purification device when treating a bacteria-heavy oil sample 1 mixture in Example 3 of the present invention;

[0056] Fig. 9 is an agarose gel electrophoresis diagram comparing the performance of the present invention and a conventional nucleic acid purification device when treating a bacteria-heavy oil sample 2 mixture in Example 4 of the present invention;

[0057] Fig.10 It is an agarose gel electrophoresis diagram of the heavy oil sample 1 in Examples 1 and 3 of the present invention extracted by three different kits.

[0058] Description of Reference Numerals

[0059] 100. Coarse filter column 101. Filter membrane

[0060] 102. Second step 200. Purification column

[0061] 201. Adsorption film 202. Neck shrinkage structure

[0062] 2021, Funnel body 2022, Funnel neck

[0063] 203, cylindrical part 204, first step

[0064] 300, first collecting tube 400, second collecting tube

[0065] 500, Sealing cover DETAILED DESCRIPTION

[0066] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0067] The present invention is described in detail below with reference to specific schematic diagrams. These specific implementation cases are limited examples without violating the claims of the present invention and are not limited to the applicability of this patent to environmental samples in other fields.

[0068] As mentioned above, the first aspect of the present invention provides a nucleic acid purification device, which is composed of four parts, namely a coarse filtration column 100, a purification column 200, a first collection tube 300 and a second collection tube 400;

[0069] The coarse filtration column 100, the purification column 200, the first collection tube 300 and the second collection tube 400 can stably accommodate or buckle with each other to form a whole; wherein, the second collection tube 400, the purification column 200 and the coarse filtration column 100 are arranged in sequence from the outside to the inside and interlocked with each other, and the purification column 200 and the first collection tube 300 are arranged side by side in the upper and lower parts of the second collection tube 400.

[0070] Preferably, the coarse filtration column 100 comprises a coarse filtration column 100 body and a filter membrane 101. Preferably, the purification column 200 comprises a purification column 200 body and an adsorption membrane.

[0071] Preferably, the inner diameter of the upper portion of the purification column 200 is the same as the outer diameter of the coarse filtration column 100 , and the purification column 200 can carry the coarse filtration column 100 inside.

[0072] Preferably, the first collection tube 300 is a one-piece snap-on small centrifuge tube with a cover.

[0073] Preferably, the inner diameter of the first collecting tube 300 matches and engages with the outer diameter of the lower portion of the purification column 200 , and the first collecting tube 300 is built into the entire nucleic acid purification device.

[0074] Preferably, the second collection tube 400 is a large centrifuge tube with a detachable nut-type cover.

[0075] Preferably, the inner diameter of the second collecting tube 400 matches the outer diameter of the upper portion of the purification column 200 , and the second collecting tube 400 carries and accommodates the combination of the first collecting tube 300 , the purification column 200 and the coarse filtration column 100 .

[0076] According to a particularly preferred specific implementation manner, the coarse filter column 100 is a cylindrical structure, and the inner diameter of the coarse filter column 100 is 32 mm, the height is 35 mm, and the wall thickness is 1 mm.

[0077] Preferably, the bottom of the purification column 200 is a neck structure 202 , and the open end of the first collection tube 300 is embedded in the neck structure 202 of the purification column 200 .

[0078] According to a particularly preferred embodiment, the necking structure 202 includes a funnel body 2021 and a funnel neck 2022 that are integrally formed.

[0079] According to a particularly preferred embodiment, the angle between the funnel body 2021 and the horizontal plane is 30°, and the width of the funnel neck 2022 is 6 mm.

[0080] According to a particularly preferred embodiment, the purification column 200 further includes a cylindrical portion 203 disposed above the neck structure 202 and in contact with the coarse filtration column 100 .

[0081] Preferably, the inner diameter of the cylindrical portion 203 of the purification column 200 is 34 mm, the height is 65 mm, and the wall thickness is 1 mm.

[0082] Preferably, there is a height gap between the purification column 200 and the bottom of the second collecting tube 400 to form a receiving space for the first collecting tube 300 .

[0083] Preferably, the purification column 200 fits tightly against the inner wall of the second collecting tube 400 and is embedded in the first collecting tube 300 to firmly limit it between the second collecting tube 400 and the purification column 200, so that the four parts of the coarse filtration column 100, the purification column 200, the first collecting tube 300 and the second collecting tube 400 can be tightly fastened and combined into a whole at the same time.

[0084] Preferably, the nucleic acid purification device further comprises a sealing cover 500 covering the inlet end of the coarse filtration column 100 .

[0085] The present invention has no special requirements on the covering method of the sealing cover 500, and it only needs to make the purification device sealed. For example, the sealing cover 500 can be connected to the outer wall of the second collection tube 400 through a threaded cover to seal the purification device.

[0086] Preferably, the inlet end of the purification column 200 has a first step 204 , and the first step 204 matches with the open end of the second collecting tube 400 so that the inlet end of the purification column 200 abuts against the open end of the second collecting tube 400 .

[0087] Preferably, the inlet end of the coarse filtration column 100 has a second step 102 , and the second step 102 matches the open end of the purification column 200 so that the inlet end of the coarse filtration column 100 abuts against the first step 204 .

[0088] In the present invention, the diameter of the filter membrane 101 matches the inner diameter of the coarse filter column 100 , and the diameter of the filter membrane 101 can be selected according to the inner diameter of the coarse filter column 100 . Exemplarily, the diameter of the filter membrane 101 is 32 mm.

[0089] According to a particularly preferred embodiment, the filter membrane 101 has an average pore size of 22 μm and a thickness of 1 mm.

[0090] In the present invention, the diameter of the adsorption membrane 201 matches the inner diameter of the necking structure 202 of the purification column 200. The diameter of the adsorption membrane 201 can be selected according to the inner diameter of the necking structure 202 of the purification column 200. For example, the diameter of the adsorption membrane 201 is 6 mm.

[0091] Preferably, the adsorption membrane 201 has an average pore size of 0.7 μm and a thickness of 2.5 mm.

[0092] Preferably, the length of the necking structure 202 is 12 mm.

[0093] Preferably, the first collecting tube 300 has an inner diameter of 8 mm, a height of 42 mm, and a wall thickness of 1 mm.

[0094] Preferably, the second collecting tube 400 has an outer diameter of 38 mm, a height of 110 mm, and a wall thickness of 1 mm.

[0095] like Figure 1 As shown, Figure 1 A and Figure 1 b represents a schematic diagram and a top view of the structure of the coarse filter column 100 according to the present invention, Figure 1 C and Figure 1d represents a model diagram of the coarse filter column 100 involved in the present invention. The coarse filter column 100 is composed of a hollow cylinder and a filter membrane. The side wall of the hollow cylinder is not hollowed out, the top is open, and the bottom is a circular hollow grid. The grid is equipped with a double-layer circular filter membrane with a pore size of 22μm. The diameter of the filter membrane needs to be close to the inner diameter of the column to ensure that the reaction liquid of the device of the present invention is smoothly filtered through the filter membrane during use.

[0096] like Figure 2 As shown, the left part is a schematic diagram and a top view of the purification column 200 of the present invention, and the right part is a model diagram of the purification column 200 of the present invention, which is also composed of a main column and a filter membrane. The main column is also a hollow cylindrical structure, with no hollowing of the side wall and an open top, but the difference from the coarse filter column 100 is that the circular grid at its bottom is reduced in size and a double-layer grid is designed, the bottom grid is a fixed grid, carrying a 0.7μm adsorption membrane, and the second grid is a movable grid with a slightly smaller inner diameter to facilitate snapping and fixing the filter membrane.

[0097] like Figure 3 The figure shows a schematic diagram of the first collection tube 300 involved in the present invention. The first collection tube 300 needs to be prepared by the user and can generally be replaced by a common 1.5mL or 2.0mL centrifuge tube.

[0098] like Figure 4 As shown, the left part is a schematic diagram of the second collection tube 400 of the present invention, and the right part is a model diagram of the second collection tube 400 of the present invention. The second collection tube 400 comprises two parts, namely a cylindrical open column and a detachable nut-type top cover, and the top cover needs to be tightened and have good sealing performance after the second collection tube 400 accommodates the purification column 200 and the coarse filtration column 100.

[0099] like Figure 5 As shown, the left part is a schematic diagram of the overall structure of the nucleic acid purification device provided by the present invention, and the right part is a model diagram of the nucleic acid purification device provided by the present invention. The nucleic acid purification device includes a hollow coarse filter column 100, a purification column 200, a first collection tube 300, and a second collection tube 400. Among them, the coarse filter column 100 and the purification column 200 also respectively include their own bodies and corresponding filter membranes. The coarse filter column 100 is equipped with a filter membrane with a pore size of 42mm, and the purification column 200 is equipped with a 0.7μm adsorption membrane. The two corresponding filter membrane diameters of the nucleic acid purification device during normal use must be tightly fitted to the inner diameter of the column body of the nucleic acid purification device.

[0100] The nucleic acid purification device provided by the present invention can be used to extract nucleic acid from organisms, can be used to efficiently purify nucleic acid, and is suitable for large volume or low biomass samples. The material of the nucleic acid purification device (except the filter membrane) is preferably medical grade polypropylene material, and the thickness is preferably 1-2 mm.

[0101] As mentioned above, the second aspect of the present invention provides the use of the nucleic acid purification device for extracting total DNA of microorganisms in crude oil and / or heavy oil according to the first aspect in nucleic acid extraction, characterized in that the nucleic acid extraction in the application comprises the following extraction steps:

[0102] (1) taking a sample with a volume of 20-30 mL; the sample is a crude oil sample and / or a heavy oil sample;

[0103] If the sample is a low-biomass sample I, the sample I is centrifuged to discard the supernatant and retain the precipitate; the sample I is an extreme environment sample (crude oil (light oil (LO), medium oil (MO), ordinary heavy oil (OHO), extra heavy oil (EHO), super heavy oil (SHO), formation water, etc.);

[0104] If the sample is sample II with a higher degree of solidification, the sample II is ground or fragmented before processing; the sample II is soil (including highly saline and alkaline soil) or rock minerals;

[0105] (2) adding 20-30 mL of extraction buffer, 500-750 μL of washing enzyme I, 500-750 μL of washing enzyme II and 5-7.5 mL of decontamination solution in sequence, maintaining a constant temperature at 65° C. for 30-45 min, and then maintaining a constant temperature at 90° C. for 30-45 min, and transferring the supernatant to a new centrifuge tube after a first centrifugation for 3-5 min; the relative centrifugal force of the first centrifugation is 12298×g-17709.12×g;

[0106] Add the same volume of protein precipitate as the upper layer, and centrifuge for a second time for 5-10 minutes; the relative centrifugal force of the second centrifugation is 12298×g-17709.12×g;

[0107] (3) combining the coarse filtration column 100, the purification column 200 and the second collection tube 400 in the nucleic acid purification device to obtain a combined structure;

[0108] The supernatant collected by the second centrifugation is completely transferred to the coarse filter column 100 in the combined structure, and the third centrifugation is performed for 5-10 minutes, and the waste liquid in the second collection tube 400 is discarded and returned to the second collection tube 400; wherein the volume of the supernatant is 20-30 mL; and the relative centrifugal force of the third centrifugation is 12298×g-17709.12×g;

[0109] (4) discarding the coarse filtration column 100 and adding 1 mL of pre-cooled rinsing solution to the purification column 200 for rinsing, performing a fourth centrifugation for 5-10 min, discarding the waste liquid in the second collection tube 400, and repeating the rinsing step once; the relative centrifugal force of the fourth centrifugation is 12298×g-17709.12×g;

[0110] The purification column is placed at room temperature for 10-20 minutes to dry the residual rinsing liquid in the adsorption membrane 201;

[0111] (5) Finally, a first collecting tube 300 is placed at the bottom of the purification column, and 50-200 μL of DNA elution solution is dripped into the middle part of the adsorption membrane of the purification column 200, and the mixture is allowed to stand at room temperature for 5-10 minutes. The mixture is centrifuged for a fifth time for 3-5 minutes to obtain the total DNA of microorganisms in the crude oil and / or heavy oil; the relative centrifugal force of the fifth centrifugation is 12298×g-17709.12×g.

[0112] Preferably, the nucleic acid extraction in the application comprises the following extraction steps:

[0113] (1) taking a sample with a volume of 20-30 mL; the sample is a crude oil sample;

[0114] If the sample is a low-biomass sample I, the sample I is centrifuged to discard the supernatant and retain the precipitate; the sample I is an extreme environment liquid sample;

[0115] If the sample is sample II with a higher degree of solidification, the sample II is ground or fragmented before processing; the sample II is soil, rock or mineral in an extreme environment;

[0116] (2) adding 20-30 mL of extraction buffer, 500-750 μL of washing enzyme I, 500-750 μL of washing enzyme II and 5-7.5 mL of decontamination solution in sequence, maintaining a constant temperature at 65° C. for 30-45 min, and then maintaining a constant temperature at 90° C. for 30-45 min, and transferring the supernatant to a new centrifuge tube after a first centrifugation for 3-5 min; the relative centrifugal force of the first centrifugation is 12298×g-17709.12×g;

[0117] Add the same volume of protein precipitate as the upper layer, and centrifuge for a second time for 5-10 minutes; the relative centrifugal force of the second centrifugation is 12298×g-17709.12×g;

[0118] (3) combining the coarse filtration column 100, the purification column 200 and the second collection tube 400 in the nucleic acid purification device to obtain a combined structure;

[0119] The supernatant collected by the second centrifugation is completely transferred to the coarse filter column 100 in the combined structure, and the third centrifugation is performed for 5-10 minutes, and the waste liquid in the second collection tube 400 is discarded and returned to the second collection tube 400; wherein the volume of the supernatant is 20-30 mL; and the relative centrifugal force of the third centrifugation is 12298×g-17709.12×g;

[0120] (4) discarding the coarse filtration column 100 and adding 1 mL of pre-cooled rinsing solution to the purification column 200 for rinsing, performing a fourth centrifugation for 5-10 min, discarding the waste liquid in the second collection tube 400, and repeating the rinsing step once; the relative centrifugal force of the fourth centrifugation is 12298×g-17709.12×g;

[0121] The purification column 200 is placed at room temperature for 10-20 minutes to dry the residual rinsing liquid in the adsorption membrane;

[0122] (5) Finally, a first collecting tube 300 is placed at the bottom of the purification column, and 50-200 μL of DNA elution solution is dripped into the middle part of the adsorption membrane of the purification column 200, and the mixture is allowed to stand at room temperature for 5-10 minutes, and then centrifuged for 3-5 minutes to obtain the total DNA of microorganisms in the crude oil and / or heavy oil; the relative centrifugal force of the fifth centrifugation is 12298×g-17709.12×g.

[0123] Preferably, in step (1), the extreme environment samples include reservoir environment samples such as crude oil and formation water.

[0124] Preferably, the crude oil includes light oil (LO), medium oil (MO), ordinary heavy oil (OHO), extra heavy oil (EHO), and super heavy oil (SHO).

[0125] Preferably, in step (1), the sample I is an extreme environment sample such as crude oil (light oil (LO), medium oil (MO), ordinary heavy oil (OHO), extra heavy oil (EHO), super heavy oil (SHO), formation water, etc.

[0126] Preferably, in step (1), the sample II is extreme environment soil (including highly saline and alkaline soil) or rock minerals.

[0127] Preferably, in step (2), the extraction buffer is tris-HCl, Triton X-100 (Triton-X), sodium phosphate (Na 3 PO 4 ) and sodium chloride (NaCl).

[0128] According to a preferred embodiment, the concentration of the tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) is 100 mmol / L and the pH is 8.0.

[0129] Preferably, the concentration of Triton-X is 100 mmmol / L, and the sodium phosphate (Na 3 PO 4 ) is 100mmmol / L, and the concentration of the mixed solution of sodium chloride (NaCl) is 1.5mol / L.

[0130] Preferably, in step (2), the mass volume concentration of the flushing enzyme I is 20 mg / L, the mass volume concentration of the flushing enzyme II is 20 mg / L, and the mass volume concentration of the decontamination liquid is 100 mg / L.

[0131] Preferably, in step (2), the decontamination solution is sodium deoxycholate (SDC) solution.

[0132] Preferably, in step (2), the prion precipitation solution is a mixed solvent of phenol / chloroform / isoamyl alcohol in a volume ratio of 25:24:1.

[0133] Preferably, in step (4), the volume concentration of the rinsing solution is 70% ethanol.

[0134] Preferably, in step (5), the DNA elution solution is sterile ultrapure water.

[0135] Figure 6-Figure 9 , Fig.10 In the figure, “M” indicates a nucleic acid marker; Fig.10 The agarose gel electrophoresis diagrams of the heavy oil sample 1 in Examples 1 and 3 of the present invention extracted by three different kits are shown in FIG. Fig.10 In the data, SG1, SG2 and SG3 represent three parallel extractions of heavy oil sample 1 using the Ezup column bacterial genomic DNA extraction kit (Sangong), MK1, MK2, MK3, MK4, MK5 and MK6 represent six parallel extractions of heavy oil sample 1 using the bacterial genomic DNA extraction kit (McLean), Patent 1, Patent 2 and Patent 3 represent three parallel extractions of heavy oil sample 1 using the "Preparation and Application of Kit for Extracting Heavy Oil Genomic Deoxyribonucleic Acid and Its Application" (PCT / CN2019 / 104518), and "BC" represents 50 ng of Bacillus cereus DNA.

[0136] The present invention will be described in detail below through examples.

[0137] In the following examples, unless otherwise specified, the instruments, reagents, materials, etc. involved are conventional instruments, reagents, materials, etc. and can be obtained through regular commercial channels.

[0138] Heavy oil sample 1: L1YHC23 (Liaohe oilfield heavy oil sample, viscosity 5860mPa·s and density 0.996g / cm 3 (20℃)).

[0139] Heavy oil sample 2: Ji 103 (Xinjiang oilfield heavy oil sample, viscosity 14141mPa·s and density 0.953g / cm 3 (47.5℃)).

[0140] The nucleic acid purification device described in the present invention is independently developed and is proposed to be named BCO.

[0141] The model of the common nucleic acid purification device is (BS-SC-06, 2mL).

[0142] Filter membrane: pore size is 22μm.

[0143] Adsorption membrane: pore size is 0.7 μm.

[0144] Flushing enzyme I: Protease K at a mass volume concentration of 20 mg / mL.

[0145] Flushing enzyme II: snail enzyme with a mass volume concentration of 20 mg / mL.

[0146] Extraction buffer: Tris-HCl at a concentration of 100 mmol / L, pH 8.0, Triton-X at a concentration of 100 mmol / L, sodium phosphate at a concentration of 100 mmmol / L 3 PO 4 ) and a mixture of sodium chloride (NaCl) with a concentration of 1.5 mol / L.

[0147] Decontamination solution: sodium deoxycholate (SDC) solution with a mass volume concentration of 100 mg / mL.

[0148] Protein precipitation solution: a mixed solvent of phenol / chloroform / isoamyl alcohol in a volume ratio of 25:24:1.

[0149] Rinsing solution: chromatographic grade ethanol with a volume concentration of 70%.

[0150] DNA elution buffer: sterile ultrapure water.

[0151] In the following examples, the concentration and purity of the DNA extract were determined using an ultraviolet spectrophotometer (Hangzhou Haipei Instrument Co., Ltd., Aurora-900), where purity was measured by OD 260 / OD 280 To indicate; polymerase chain reaction and agarose gel electrophoresis were used to further verify the test results.

[0152] The following examples are all improved on the basis of the invention patent (CN 106754895B) published by our unit.

[0153] Example 1

[0154] Nucleic acid extraction of heavy oil sample 1:

[0155] Step 1: Sampling. The experiment was divided into two groups: the nucleic acid purification device of the present invention (hereinafter referred to as the "large extraction group") and the common nucleic acid purification device (hereinafter referred to as the "conventional group"). The large extraction group took 20g of the heavy oil sample 1, and the conventional group took 0.5g of the heavy oil sample 1;

[0156] Step 2: Add the extraction reagent system. The following reagents were added to the large extraction group and the conventional group respectively. The reagent addition amounts of the large extraction group and the conventional group for nucleic acid extraction of heavy oil sample 1 are shown in Table 1.

[0157] Table 1

[0158] Reagent name Large group Conventional group Extraction buffer 20mL 500μL Flushing enzyme Ⅰ 500μL 20μL Flushing enzyme II 500μL 20μL Decontamination liquid 5mL 200μL

[0159] Step 3: Purify nucleic acid. Keep the mixture from the previous step at 65°C for 30 min, then at 90°C for 30 min, centrifuge (room temperature, 17709.12×g) for 3 min and transfer the supernatant (the crude oil phase is on the upper layer) to a new centrifuge tube;

[0160] Add the same volume of prion precipitate as the upper layer, centrifuge for the second time (room temperature, 17709.12×g) for 5 min and transfer the supernatant to the coarse filtration column 100 (combined coarse filtration column 100, purification column 200 and second collection tube 400).

[0161] Step 4: rinsing and elution. All supernatants are subjected to a third centrifugation (room temperature, 17709.12×g) for 5 min, and the waste liquid in the second collection tube 400 is discarded and returned to the second collection tube 400;

[0162] The coarse filtration column 100 is discarded and 1 mL of pre-cooled rinsing solution is added to the purification column 200 for rinsing. The waste liquid in the second collection tube 400 is removed by a fourth centrifugation (room temperature, 17709.12×g) for 5 min, and the rinsing step is repeated once;

[0163] The purification column 200 is placed at room temperature for 10 minutes to dry the residual rinsing liquid in the adsorption membrane in the purification column 200; the first collection tube 300 is placed in the device, and 100 μL of DNA elution solution is added dropwise to the middle part of the adsorption membrane in the purification column 200, and the mixture is allowed to stand at room temperature for 10 minutes. The mixture is centrifuged (room temperature, 17709.12×g) for 3 minutes. The solution obtained in the first collection tube 300 is the total DNA (deoxyribonucleic acid) of microorganisms in the crude oil and / or heavy oil.

[0164] Test Example 1

[0165] The concentration and purity of the extracted product in Example 1 were detected by ultraviolet spectrophotometer, and the quality of the extracted product was further verified by polymerase chain reaction and agarose gel electrophoresis. The concentration and purity of the genomic DNA extracted from the heavy oil sample 1 in Example 1 are shown in Table 2.

[0166] The primers used in the polymerase chain reaction are as follows:

[0167] The forward primer is 341F: 5′-ACTCCTACGGGAGGCAGCAG-3′,

[0168] The reverse primer was 518R: 5′-ATTACCGCGGCTGCTGG-3′.

[0169] Figure 6 1 is an agarose gel electrophoresis diagram comparing the performance of the present invention and the conventional nucleic acid purification device when treating the heavy oil sample 1 in Example 1 of the present invention. The extraction results and verification results show that the DNA concentration extracted by the heavy oil extraction group (the present invention) is higher (Table 2), and the No. 1 electrophoresis band corresponding to the heavy oil sample 1 extraction group is clearer and brighter than the No. 2 electrophoresis band corresponding to the heavy oil sample 1 conventional group (such as Figure 6 As shown), under the same method, the present invention can obtain more total genomic DNA with relatively higher purity through one extraction, and it can be simpler and faster and consume less manpower and material resources when the purpose is to obtain a large number of sample genomes in batches. This further shows that the present invention is more applicable to heavy oil samples than ordinary nucleic acid purification devices.

[0170] Table 2

[0171] Group DNA concentration (ng / μL) <![CDATA[A 260 / A 280 ]]> <![CDATA[A 260 / A 230 ]]> Electrophoresis bands Heavy oil sample 1 large group 149.109 1.25 0.52 Figure 6 Strip 1 Heavy oil sample 1 conventional group 40.673 1.02 0.44 Figure 6 Strip 2

[0172] Example 2

[0173] Nucleic acid extraction of heavy oil sample 2.

[0174] The nucleic acid is extracted according to the method of Example 1, except that the heavy oil sample 2 is different. Specifically, the heavy oil sample 2 is:

[0175] Ji 103 (density 0.953g / cm 3 , viscosity 14169mPa·s, Xinjiang oilfield heavy oil sample)

[0176] The remaining materials, reagents, steps and detection are exactly the same as those in Example 1.

[0177] Test Example 2

[0178] The concentration and purity of the genomic DNA extracted from the heavy oil sample 2 in this Example 2 are shown in Table 3.

[0179] Figure 7 This is an agarose gel electrophoresis diagram comparing the performance of the present invention and the conventional nucleic acid purification device when treating the heavy oil sample 2 in Example 2 of the present invention. The extraction results and verification results show that the DNA concentration extracted by the large extraction group of the heavy oil sample 2 (the present invention) is higher (Table 3), and the No. 3 electrophoresis band corresponding to the large extraction group is clearer and brighter than the No. 4 electrophoresis band corresponding to the conventional group of the heavy oil sample 2 ( Figure 7 ), under the same method, the total genomic DNA obtained by the present invention is more and the purity is relatively higher in one extraction, and it is more simple, quick and less labor- and material-intensive when the purpose is to obtain a large number of sample genomes in batches. This further shows that the present invention is more applicable to heavy oil samples from different oil fields than ordinary nucleic acid purification devices.

[0180] Table 3

[0181] Group DNA concentration (ng / μL) <![CDATA[A 260 / A 280 ]]> <![CDATA[A 260 / A 230 ]]> Electrophoresis bands Heavy oil sample 2 large group 112.949 1.09 0.57 Figure 7 Strip 3 Heavy oil sample 2 conventional group 56.488 1.05 0.47 Figure 7 Strip 4

[0182] Example 3

[0183] Nucleic acid extraction from the mixture of heavy oil sample 1 and bacterial solution.

[0184] The nucleic acid was extracted according to the method of Example 1, except that bacterial liquid was added to the sample of this example, specifically:

[0185] Bacterial liquid: Cupidesulfovibrio liaohensis (OD 600 =0.58)

[0186] Preparation of bacteria-heavy oil sample 1 mixture: about 150 mL of Liaohe Zhongshida Desulfurized Vibrio bacterial liquid was thoroughly mixed, centrifuged and discarded the supernatant, and a small amount of sterile culture medium was added to make a cell resuspension. After being thoroughly mixed again, it was loaded into weighed 20g, 0.5g, 0.5g, and 0.5g L1YHC23 heavy oil sample tubes according to the volume ratio of 20:0.5:0.5:0.5, thereby making bacteria-heavy oil sample 1 large extraction group and bacteria-heavy oil sample 1 conventional group samples.

[0187] The heavy oil sample 1 is the same as that in Example 1, and the other materials, reagents, steps and detection are completely consistent with those in Example 1.

[0188] Test Example 3

[0189] The concentration and purity of the genomic DNA of the mixture of bacteria and heavy oil sample 1 extracted in this Example 3 are shown in Table 4.

[0190] Figure 8This is an agarose gel electrophoresis diagram comparing the performance of the present invention and a conventional nucleic acid purification device when treating a mixture of bacteria-heavy oil sample 1 in Example 3 of the present invention. The extraction results and verification results show that the concentration of DNA extracted by the present invention is higher (Table 4), and the No. 5 electrophoresis band corresponding to the bacteria-heavy oil sample 1 large extraction group is clearer and brighter than the No. 6, No. 7 and No. 8 electrophoresis bands corresponding to the bacteria-heavy oil sample 1 conventional group (such as Figure 8 ), under the same method, the total genomic DNA obtained by the present invention is more and the purity is relatively higher in one extraction, and it is more simple, quick and less labor- and material-intensive when the purpose is to obtain a large number of sample genomes in batches, which further indicates that the present invention is more applicable to heavy oil samples than ordinary nucleic acid purification devices.

[0191] Table 4

[0192]

[0193] Example 4

[0194] Nucleic acid extraction from the mixture of heavy oil sample 2 and bacterial solution.

[0195] The heavy oil sample 2 is the same as that in Example 2; the preparation method of the bacteria-heavy oil sample 2 mixture is the same as that in Example 3.

[0196] The remaining materials, reagents, steps and detection are exactly the same as those in Example 3.

[0197] Test Example 4

[0198] The concentration and purity of the genomic DNA of the mixture of bacteria and heavy oil sample 2 extracted in Example 4 are shown in Table 5.

[0199] Fig. 9 : This is an agarose gel electrophoresis diagram comparing the performance of the present invention and a conventional nucleic acid purification device when treating a mixture of bacteria-heavy oil sample 2 in Example 4 of the present invention. The extraction results and verification results show that the concentration of DNA extracted by the present invention is higher (Table 5), and the No. 5 electrophoresis band corresponding to the bacteria-heavy oil sample 2 large extraction group is clearer and brighter than the No. 9, No. 10 and No. 11 electrophoresis bands corresponding to the bacteria-heavy oil sample 2 conventional group (e.g. Fig. 9 ), under the same method, the total genomic DNA obtained by the present invention is more and the purity is relatively higher in one extraction, and it is more simple, quick and less labor- and material-intensive when the purpose is to obtain a large number of sample genomes in batches, which further indicates that the present invention is more applicable to heavy oil samples than ordinary nucleic acid purification devices.

[0200] Table 5

[0201]

[0202] The evaluation of the examples is shown in Table 6.

[0203] It can be seen from the above results that from Examples 1-4, combined with the crude oil nucleic acid extraction method previously published by this unit (patent CN106754895B), the extraction effect of the present invention is significantly improved compared with that of ordinary nucleic acid purification devices, which is a relatively large improvement for crude oil and even heavy oil samples. The main reason is that the general purification device or general extraction method is not compatible with the sample type. The characteristics of this sample incompatibility are mainly manifested in: "small amount and multiple times", low biomass, complex sample chemical composition and complex physical properties.

[0204] Table 6

[0205]

[0206] In terms of convenience, the first stage filtration of the present invention (coarse filter column 100 with double filtration design) eliminates the time of manual operation to transfer the lower layer (or middle layer) clear liquid (the upper layer is the oil phase after centrifugation), greatly reducing human errors and single operation time. When extracting low-biomass samples with large mass, ordinary nucleic acid purification devices need to increase the number of experimental repetitions by dozens of times to improve the success rate or total mass. Therefore, the present invention has obvious convenience advantages over ordinary nucleic acid purification devices.

[0207] In terms of timeliness, the present invention has the advantages of continuity and semi-automatic operation. The time saved by a single extraction is about 10 minutes, which can be magnified when the ordinary nucleic acid purification device is repeatedly operated multiple times, so that it can also have a more prominent advantage in timeliness.

[0208] In terms of nucleic acid quality and purity, the test results prove that the quality and purity of the product of the present invention are superior to those of the nucleic acid purification device. The shortcoming of the present invention is that the difference in mass multiples of the original sample consumed by the two nucleic acid purification devices cannot be reflected in the same multiples of the extracted product quality. This may be due to the low biomass and complex components of the sample. However, from the practical results, the advantages of the present invention in the quality and purity of extracted nucleic acid are absolutely superior in overcoming the low success rate of difficult-to-extract samples.

[0209] In terms of economy, when processing samples of the same mass, if the sample mass is less than 4g, the cost of an ordinary nucleic acid purification device of about 2.5 yuan / g is more economical, but when the sample mass is more than 4g, the cost required by the present invention is lower than that of an ordinary nucleic acid purification device, and as the sample mass increases, the economic advantage of the present invention becomes more significant.

[0210] In summary, the present invention has a better application effect on heavy oil samples.

[0211] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A nucleic acid purification device, characterized in that: The nucleic acid purification device is composed of four parts, namely a coarse filtration column (100), a purification column (200), a first collection tube (300) and a second collection tube (400); The coarse filtration column (100), the purification column (200), the first collection tube (300) and the second collection tube (400) can be stably accommodated or buckled with each other to form a whole; wherein the second collection tube (400), the purification column (200) and the coarse filtration column (100) are arranged in sequence from the outside to the inside and interlocked with each other, and the purification column (200) and the first collection tube (300) are connected in series to the upper and lower parts of the second collection tube (400).

2. The nucleic acid purification device according to claim 1, characterized in that: The coarse filter column (100) comprises a coarse filter column (100) body and a filter membrane (101); and / or, The purification column (200) comprises a purification column (200) body and an adsorption membrane (201); and / or, The inner diameter of the upper portion of the purification column (200) is the same as the outer diameter of the coarse filtration column (100), and the purification column (200) can carry the coarse filtration column (100) inside.

3. The nucleic acid purification device according to claim 1, characterized in that: The first collection tube (300) is a one-piece snap-on small centrifuge tube with a cover; and / or, The inner diameter of the first collecting tube (300) matches and engages with the outer diameter of the lower portion of the purification column (200), and the first collecting tube (300) is built into the entire nucleic acid purification device.

4. The nucleic acid purification device according to claim 1, characterized in that: The second collection tube (400) is a large centrifuge tube with a detachable nut-type cover; and / or, The inner diameter of the second collection tube (400) matches the outer diameter of the upper portion of the purification column (200), and the second collection tube (400) carries and accommodates the combination of the first collection tube (300), the purification column (200) and the coarse filtration column (100).

5. Use of the nucleic acid purification device according to any one of claims 1 to 4 in extracting total DNA of microorganisms in crude oil, characterized in that: The nucleic acid extraction in the application comprises the following extraction steps: (1) taking a sample with a volume of 20-30 mL; the sample is a crude oil sample and / or a heavy oil sample; If the sample is a low-biomass sample I, the sample I is centrifuged to discard the supernatant and retain the precipitate; the sample I is an extreme environment sample (crude oil (light oil (LO), medium oil (MO), ordinary heavy oil (OHO), extra heavy oil (EHO), super heavy oil (SHO), formation water, etc.); If the sample is sample II with a higher degree of solidification, the sample II is ground or fragmented before processing; the sample II is soil (including highly saline and alkaline soil) or rock minerals; (2) adding 20-30 mL of extraction buffer, 500-750 μL of washing enzyme I, 500-750 μL of washing enzyme II and 5-7.5 mL of decontamination solution in sequence, maintaining a constant temperature at 65° C. for 30-45 min, and then maintaining a constant temperature at 90° C. for 30-45 min, and transferring the supernatant to a new centrifuge tube after a first centrifugation for 3-5 min; the relative centrifugal force of the first centrifugation is 12298×g-17709.12×g; Add the same volume of protein precipitate as the upper layer, and centrifuge for a second time for 5-10 minutes; the relative centrifugal force of the second centrifugation is 12298×g-17709.12×g; (3) combining the coarse filtration column, the purification column and the second collection tube in the nucleic acid purification device to obtain a combined structure; The supernatant collected by the second centrifugation is completely transferred to the coarse filter column in the combined structure, and the waste liquid in the second collection tube is discarded and returned to the second collection tube for a third centrifugation for 5-10 minutes; wherein the volume of the supernatant is 20-30 mL; and the relative centrifugal force of the third centrifugation is 12298×g-17709.12×g; (4) discarding the coarse filtration column and adding 1 mL of pre-cooled rinsing solution to the purification column for rinsing, performing a fourth centrifugation for 5-10 min, discarding the waste liquid in the second collection tube, and repeating the rinsing step once; the relative centrifugal force of the fourth centrifugation is 12298×g-17709.12×g; The purification column is placed at room temperature for 10-20 minutes to dry the residual rinsing liquid in the adsorption membrane (201); (5) Finally, a first collection tube is placed at the bottom of the purification column, and 50-200 μL of DNA elution solution is added dropwise to the middle part of the adsorption membrane of the purification column, and the mixture is allowed to stand at room temperature for 5-10 minutes, and then centrifuged for a fifth time for 3-5 minutes to obtain the total DNA of microorganisms in the crude oil and / or heavy oil; the relative centrifugal force of the fifth centrifugation is 12298×g-17709.12×g.

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