CN111607635B - Blood genome DNA extraction method based on 3D printing special-shaped functional body and application kit thereof - Google Patents
Blood genome DNA extraction method based on 3D printing special-shaped functional body and application kit thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The invention discloses a blood genome DNA extraction method based on a 3D printing special functional body and an application kit thereof, belonging to the field of nucleic acid extraction and separation. The invention uses the lysate to carry out the lysis treatment on the sample, and uses the 3D printing special functional body to carry out the combination, separation and purification of nucleic acid. According to the method, the 3D printing functional body is quickly transferred in the lysate, the cleaning solution and the eluent by holding the device by hand or machine, time consuming and complicated steps such as pipetting and centrifuging are not needed, the manual labor intensity is low, the cost is low, the flux is high, the obtained nucleic acid is high in quality, the requirements on equipment and operation space are small, and the method has an automatic and high-flux downstream application prospect. Furthermore, the invention also provides an application kit of the nucleic acid extraction process. The invention solves the problems of complex preparation, time consumption, low flux and the like of target nucleic acid aiming at the existing PCR in-vitro amplification technology, and has wide application prospect of rapid detection of blood pathogens.
Description
Technical Field
The invention belongs to the field of nucleic acid extraction and separation, and particularly relates to a blood genome DNA extraction method based on a 3D printing special functional body and an application kit thereof.
Background
Diseases transmitted through blood often cause significant infectious diseases, which present global public health safety issues. Prevention of such infectious diseases is mainly based on three principles: the most effective prevention means is to find the infectious agent and cut off the transmission as soon as possible by detecting the pathogenic agent, and seek a convenient, rapid, high-flux and high-sensitivity pathogenic agent detection method, which is a key problem for improving the detection efficiency and accuracy. The pathogen detection methods commonly used at present are as follows: etiology detection, immunology detection and molecular biology detection.
The etiology detection is commonly performed by a thick and thin blood film staining microscopic examination method, and the method has the advantages of simple operation, low cost, capability of identifying pathogenic species and the like, is widely used for etiology diagnosis of diseases, and is one of the most commonly used methods at present. However, the blood membrane staining microscopic examination method is time-consuming and labor-consuming, has high requirements on microscopic examination personnel and low sensitivity, and is easy to cause the continuous transmission of diseases caused by missed examination of pathogens. As another common method for pathogen detection, the immunochromatography technology has the advantages of simple operation, portability and low equipment requirement. However, the method has higher economic cost, obvious detection result difference due to the environment, humidity and sample state, high false negative or false positive rate and low detection sensitivity.
PCR (polymerase chain reaction) technology is used as a common technology for molecular biological detection, is amplified based on nucleic acid as a target, has the advantages of high sensitivity, good specificity and rapid detection compared with etiology detection and immunological diagnosis, and is widely applied to in-vitro PCR diagnosis of disease pathogens at present. However, extraction of genomic DNA from blood for in vitro PCR diagnosis of disease pathogens has become a bottleneck limiting the application of molecular detection techniques in clinic.
Liquid phase extraction means such as nucleic acid extraction, phenol-chloroform extraction and trizol method from complex biological samples are gradually eliminated due to heavy pollution and large sample demand. The solid phase separation, especially the magnetic separation, can avoid complicated centrifugal operation and a plurality of manual operation errors in the traditional nucleic acid separation process by means of the property that the magnetic nano particles are combined with the nucleic acid and can rapidly move in an externally applied magnetic field, and is considered to have the prospect of downstream high-flux and automatic application. However, the aggregation and sedimentation effects inherent to the nanoparticles are difficult to avoid, and severely restrict the efficiency and stability of nucleic acid extraction. The magnetic nanoparticles have the problems of difficult desorption of nucleic acid and low nucleic acid extraction efficiency, and although the problem is reported to be avoided by directly using a complex of the magnetic nanoparticles and the nucleic acid as a PCR template, the magnetic particles have strong inhibition effect on the PCR amplification process and the elution process is unavoidable. For non-magnetic solid phase separation technology, such as traditional column extraction, the solid phase carrier combined with nucleic acid is separated from the lysate system, and complicated operations such as multi-step centrifugation or repeated pipetting are still needed, so that labor intensity and flux are low.
Based on the above, development of a simple, high-throughput and rapid nucleic acid extraction and detection method is an important point of research. The control step of high flux in the whole process, namely nucleic acid extraction, is limited, so that the primary problem to be solved is to ensure the extraction quality and improve the flux at the same time, and the purposes of high flux, rapidness and simplicity are achieved.
Disclosure of Invention
Aiming at the problems of complex process, time consumption, low flux and the like in the existing extraction of genome DNA from blood, the invention provides a blood genome DNA extraction method based on a 3D printing special-shaped functional body.
In order to solve the technical problems, the invention adopts the following technical scheme: a blood genome DNA extraction method based on 3D printing special-shaped functional bodies comprises the following steps:
(1) Carrying out cracking treatment on a blood sample to be detected by using a cracking liquid;
(2) Stretching the nucleic acid binding area of the 3D printing special functional body into the solution obtained in the step (1) by hand or machine holding to bind nucleic acid;
(3) 3D printing the special functional body in the step (2) is completed through handheld or machine-held movement, and the nucleic acid binding area extends into the cleaning liquid to clean the nucleic acid;
(4) Taking out the 3D printing special-shaped functional body which is finished in the step (3) through hand holding or machine holding, and drying;
(5) And (3) placing the nucleic acid binding region of the 3D printing special functional body which is subjected to the step (4) into an eluent by hand or machine holding, and eluting to obtain the eluent, namely blood genome DNA.
Specifically, in the above technical solution, the blood sample to be tested in the step (1) includes whole blood separated from the human or animal body, red blood cells removed from the supernatant, and dried blood spots.
Specifically, in the above technical solution, the 3D printing special functional body is a micro-component with an umbrella-shaped structure, which is obtained by using photosensitive resin or thermoplastic plastic as a raw material and by a 3D printing technology, and comprises a nucleic acid binding region and a handle region.
Specifically, in the above-mentioned technical solution, the 3D printing special-shaped functional body contains 1 or at least two nucleic acid binding regions, which are at least two nucleic acid binding regions, and the handle regions are connected together side by means of a connecting region; the nucleic acid binding region is a cone with the following dimensions: 2-5mm (diameter of cone bottom) ×5-20mm (height); the handle area is a cylinder or a cuboid, and one end of the handle area is connected with the center of the cone bottom of the nucleic acid binding area to form an umbrella-shaped structure; the surface of the nucleic acid binding area is flat or has an uneven microstructure, and the uneven microstructure comprises a thread lining structure, a groove structure, a porous structure and a protruding structure; the uneven microstructure can be of any shape and distributed on any part of the outer surface of the nucleic acid binding area; the porous structure can be nano-sized and micro-sized, and the shape of the porous structure can be sphere, square body or irregular shape; the nucleic acid binding region is loaded or unloaded with a particulate material comprising inorganic salt particles and a metal particulate material, the inorganic salt particles comprising silica, titania, manganese dioxide; the nucleic acid binding region is modified with or without functional groups including hydroxyl, carboxyl, amino. The handle area is in the shape of a cylinder or a cuboid, the diameter of the cylinder is 2-5mm, the length of the cuboid is 2-5mm, and the width of the cuboid is 1-5mm; the connecting area is cuboid, the size is 6.5-10cm (length) ×2-10mm (width) ×1-5mm (height), and the overall height h of the 3D printing special-shaped functional body is 2.1cm-10cm.
Furthermore, the special functional body is accurately prepared on a printer by utilizing a 3D printing technology, and the nucleic acid binding area, the handle area and the connecting area can be integrally prepared or prepared separately; when the parts are prepared separately, the assembly of the nucleic acid binding region and the handle region can be performed by adhesion.
In particular, the techniques described aboveIn the scheme, the lysate in the step (1) refers to a buffer solution capable of releasing nucleic acid in a sample into the solution, and the method comprises the following steps: CTAB lysate and NaHCO 3 Lysates, chelex lysates, proteinase K lysates or SDS lysates.
Specifically, in the technical solution described above, the CTAB lysate includes: 1-3% CTAB, 0.5-5M NaCl, 0.01-0.05M EDTA, 0.05-0.5M Tris-HCl, 0.05-0.5% mercaptoethanol, said NaHCO 3 The lysate comprises: 0.05-1.00M NaHCO 3 0.5-10% SDS, the Chelex lysate includes: 0.5-20% Chelex-100 and 0.2-5M DTT, the proteinase K lysate comprising: 20-500 mmol of Tris-HCl, 10-50 mmol of EDTA, 100-1000 mmol of NaCl, 0.1-10% of SDS and 5-30 mu g/mL of proteinase K; the SDS lysate comprises the following components: 1-20% SDS,0-30 mug/mL proteinase K.
Specifically, in the technical scheme described above, RNA digestive enzymes are added or not added to the lysate. The treatment time of the cracking process is 1min-24h, and the treatment temperature is 0-100 ℃.
In the technical scheme, the nucleic acid binding in the step (2) is specifically that a nucleic acid binding region of the 3D printed special functional body is stretched into the lysate obtained in the step (1), an auxiliary binding solvent is added or not added, and the lysate is stirred by shaking the 3D printed special functional body, wherein the auxiliary binding solvent can be one or a mixed solution of isopropanol and absolute ethanol, and the volume of the auxiliary binding solvent is 0.6-0.8 times of the volume of the lysate; the 3D printing special functional body is used for combining nucleic acid, and the combining time is 5s-5min.
In the above technical solution, in step (3), the washing of the nucleic acid is performed, specifically, the handle region end of the 3D printed special functional body combined with the target nucleic acid is manually or mechanically moved into a container preset with a washing liquid, so that the nucleic acid binding region extends into the washing liquid to perform the washing of the nucleic acid, and the 3D printed special functional body is stirred in the washing liquid. The washing in the step (3) is generally carried out 1 to 5 times; the cleaning time is 2s-1min each time; preferably, the cleaning solution comprises 70-80% alcohol.
In the technical scheme, the drying process in the step (4) can be performed at room temperature or under heating, and the drying process time is 1min-2h;
in the above technical solution, the eluting in the step (5) is 5s-5min, the eluent may be water with proper concentration or high concentration convenient for storage, PBS, TE buffer, downstream PCR reaction solution, etc., preferably, the invention adopts TE buffer, and the specific components of the TE buffer are: 10mM Tris-HCl,1mM EDTA (pH=8.0).
The invention also provides an application kit of the blood genome DNA extraction method based on the 3D printing special functional body. Specifically, the kit comprises the following components:
(1) 3D printing of the contoured function;
(2) Lysate solution
(3) Proteinase K;
(4) RNA digestive enzyme
(5) Cleaning liquid;
(6) Eluting the eluent;
(7) A fixing frame;
(8) And an operating instruction.
The 3D printing special-shaped functional body is a micro-component which is formed by a nucleic acid binding area and a handle area and is provided with an umbrella-shaped structure and is obtained by a 3D printing technology by taking photosensitive resin or thermoplastic plastic as a raw material, and a plurality of micro-components with the umbrella-shaped structure are connected side by side through a connecting area; the lysate comprises CTAB lysate and NaHCO 3 One of lysate, chelex lysate, proteinase K lysate or SDS lysate; the cleaning liquid comprises 70-80% alcohol; the eluent comprises one of water, PBS, TE buffer solution and downstream PCR reaction solution; the fixing frame is a structure capable of fixing and arranging a plurality of 3D printing special functional bodies.
The 3D printing special functional body has any one of the structures as described above, preferably a structure containing 8 nucleic acid binding areas for matching with 8 rows of tubes as shown in fig. 1 (b), and can be broken off for use when single use is needed; the fixing frame is a structure capable of fixing and arranging a plurality of 3D printing special-shaped functional bodies, so that the plurality of 3D printing special-shaped functional bodies can move along with the fixing frame at the same time and are matched with a porous plate or a plurality of centrifuge tubes.
The beneficial effects are that: the method for obtaining the blood DNA can efficiently, simply, conveniently and rapidly extract and separate the target DNA with high flux, and has the advantages obviously advanced in the prior art:
1) According to the blood DNA extraction method provided by the invention, the 3D printing special-shaped functional body is adopted to separate and purify nucleic acid, and the 3D printing special-shaped functional body is quickly transferred by mechanical force to finish the separation, so that the bottleneck restriction that the traditional non-magnetic nucleic acid solid-phase separation relies on multi-step centrifugation and pipetting operation is avoided, and the labor force is remarkably saved; meanwhile, when the 3D printing special-shaped functional body can be matched with an 8-row tube, a 96-hole plate or a 384-hole plate for use, high-flux operation is realized, which is an important breakthrough of low flux in the current extraction technology;
2) The blood DNA extraction method provided by the invention adopts the 3D printing special functional body to separate and purify the nucleic acid, and is a solid-phase nucleic acid separation method. In solid phase nucleic acid separation, magnetic separation is generally considered to be rapid and have high flux, and the method of the present invention can obtain a separation speed faster than magnetic separation, a simpler operation process and a more stable separation effect. Nucleic acid separation is carried out by utilizing the 3D printing special functional body, and the nucleic acid can be separated and purified from the lysate within 1-5min without complex and time-consuming operations such as pipetting, centrifuging and the like; magnetic separation typically requires about 14.5 minutes and multiple pipetting operations (Zou Y, mason MG, wang Y, wee E, turni C, blackall PJ, et al (2017) Nucleic acid purification from plants, animals and microbes in under 30 seconds.PLoS Biol 15 (11): E2003916.). Meanwhile, the problems of insufficient nucleic acid combination, impurity encapsulation and the like caused by the problems of sedimentation, aggregation and the like of magnetic bead particles are completely avoided by adopting 3D printing, so that the stability and quality of nucleic acid extraction are effectively ensured;
3) The 3D printing special-shaped functional body provided by the invention has cheap and easily available raw materials; the printing process is convenient and quick, and the economic cost can be obviously reduced;
4) The blood DNA extraction method provided by the invention does not depend on complex operations such as centrifugation, pipetting and the like, can realize the separation of the artificial low-labor-density high-flux nucleic acid, and can realize the whole process within 1m 2 The operation is completed in the operating environment, the space is saved, and the use flexibility is high; meanwhile, the blood extraction method based on the 3D printing special-shaped functional body can also realize the transfer of the 3D printing special-shaped functional body among the lysate, the cleaning liquid and the eluent through the programmed equipment, thereby realizing automation, further reducing the labor cost, and being beneficial to promoting the downstream molecular technology, especially the accurate and rapid diagnosis of pathogenic pathogens in blood, such as the clinical application of the PCR in-vitro diagnosis of plasmodium.
Drawings
Fig. 1 is a schematic structural diagram of a 3D printing special-shaped functional body according to the present invention, (a) is a single 3D printing special-shaped functional body, and (b) is an eight-link 3D printing special-shaped functional body.
FIG. 2 is an agarose gel electrophoresis chart of example 2 of the present invention, in which lanes 1-4 are samples 1-4 of filter paper blood of 3D7 strain of plasmodium falciparum, lanes 5-8 are samples 5-8 of filter paper blood of a patient infected with plasmodium vivax, and lane M is DL2000 Marker.
FIG. 3 is an agarose gel electrophoresis chart of example 3 of the present invention, in which lanes 1-8 are samples 1-8 of blood of tussah pupae infected with baculovirus, and lane M is DL2000 Marker.
In the figure, 1, nucleic acid binding region; 2. a handle region; 3. and a connection region.
Detailed Description
The invention is further described below in connection with the following examples. It should be noted that the following description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The materials, actual and experimental equipment related to the embodiment of the invention accord with the commercial products in the biotechnology field unless specified.
The primers related to the embodiment of the invention are synthesized by entrusted bioengineering companies, and the specific primer information is as follows:
SEQ ID NO:1：
5’-TCAAAGATTAAGCCATGCAAGTGA-3’
SEQ ID NO:2：
5’-CCTGTTGTTGCCTTAAACTTC-3’
SEQ ID NO:3：
5’-TTTTTATAAGGATAACTACGGAAAAGCTGT-3’
SEQ ID NO:4：
5’-TACCCGTCATAGCCATGTTAGGCCAATACC-3’
example 1
Photosensitive resin (PAA) is used as a raw material, and a light curing reaction is carried out under 400-800nm illumination by adopting a DLP digital image projection 3D printing technology, so that a 3D printing special-shaped functional body with a regular structure is prepared: the 3D printing special-shaped functional body is similar to an umbrella-shaped structure, the single 3D printing special-shaped functional body is composed of a nucleic acid binding area 1 and a handle area 2, the nucleic acid binding area 1 is a cone, the handle area 2 is a cylinder, and the center position of the cone bottom of the nucleic acid binding area 1 is connected with one bottom surface of the handle area 2. The kit specifically comprises 1 monomer functional body with a single nucleic acid binding area 1, wherein the width of the nucleic acid binding area 1 is 5mm, the height of the nucleic acid binding area 1 is 10mm, the outer surface of the nucleic acid binding area 1 is provided with a thread and groove structure, and the height of the handle area 2 is 35mm. A single 3D print contoured function is shown in fig. 1 (a).
The whole blood from human and pig blood was collected from laboratory frozen stock at 20. Mu.L each, placed in two EP tubes, and suspended by adding 1 XPBS at 60. Mu.L. Adding 80 mu L of 2 Xlysate (200 mmol Tris-HCl, 50mmol EDTA, 1000mmol NaCl and 2% SDS) into an EP tube, shaking and mixing 5 mu L of proteinase K solution, standing in a 55 ℃ water bath for 30 minutes, adding 60 mu L of absolute ethyl alcohol, putting into a 3D printing special-shaped functional body, enabling a nucleic acid binding area of the 3D printing special-shaped functional body to extend into the solution, holding a handle area, gently shaking the 3D printing special-shaped functional body and mixing the solution, transferring the 3D printing special-shaped functional body into an 8-row EP tube with 100 mu L preset with a cleaning buffer (75% alcohol) after 10 seconds, enabling the nucleic acid binding area to extend into the cleaning buffer, holding the handle area, gently shaking the 3D printing special-shaped functional body up and down for 5 seconds, and repeatedly transferring 2-3 times in the cleaning buffer; taking out the 3D printing special-shaped functional body from the cleaning Buffer, throwing away residual liquid drops on the 3D printing special-shaped functional body, drying in a blast incubator at 37 ℃ for 2 minutes, placing in 40 mu L elution Buffer (TE) to enable a nucleic acid binding area to extend into the elution Buffer, holding a handle area, and gently shaking for 30s to perform elution. The resulting eluate was subjected to UV spectrophotometry to determine DNA concentrations of 45.060 ng/. Mu.L and 53.273 ng/. Mu.L, respectively, A260/280 of 1.806 and 1.798, and A260/230 of 1.882 and 1.756, respectively.
This example demonstrates that the method of the present invention can rapidly extract genomic DNA from an animal blood sample with high purity, less contamination and good quality.
Example 2
(1) Preparation of special-shaped functional products
Photosensitive resin (PAA) is used as a raw material, and a light curing reaction is carried out under 400-800nm illumination by adopting a DLP digital image projection 3D printing technology, so that a 3D printing special-shaped functional body with a regular structure is prepared: the 3D printing special-shaped functional body is of an umbrella-shaped structure, the eight-connected 3D printing special-shaped functional body is composed of a nucleic acid binding area 1, a handle area 2 and a connecting area 3, the nucleic acid binding area 1 is a cone, the handle area 2 is a cylinder, the connecting area 3 is in a long strip shape, the center position of the cone bottom of the nucleic acid binding area 1 is connected with one bottom surface of the handle area 2, the other bottom surface of the handle area 2 is connected with the connecting area 3, and 8 single 3D printing special-shaped functional bodies are connected on the connecting area 3 side by side to form the eight-connected 3D printing special-shaped functional body. Specifically comprises an eight-linked functional body with 8 nucleic acid binding areas 1, the width of the nucleic acid binding area 1 is 3.8mm, the height is 10mm, the outer surface of the nucleic acid binding area 1 is provided with a thread and groove structure, and the height of the handle area 2 is 20mm. The octal 3D printing special function is shown in fig. 1 (b).
(2) Genomic DNA extraction
Taking an artificially cultured 3D7 strain filter paper blood sample and an artificially cultured filter paper blood sample of a patient infected with plasmodium vivax, respectively taking 4 wafer dry blood spot blood samples with the diameter of 3mm from the two samples by using a puncher, and placing the blood samples into 8 sample holes of an 8-row EP tube, wherein the numbers are 1-4 # and 5-8 # respectively. Adding 80 mu L of lysate (100 mmol of Tris-HCl, 25mmol of EDTA, 500mmol of NaCl and 1% of SDS) into an EP tube, vibrating and mixing 5 mu L of proteinase K solution, standing at room temperature for 30 minutes, adding 60mL of absolute ethyl alcohol, placing the 3D printing special functional body into the solution to enable a nucleic acid binding area of the 3D printing special functional body to extend into the solution, holding the solution by hand or a mechanical handle area, slightly shaking the 3D printing special functional body to mix the solution, transferring the 3D printing special functional body into an 8-row EP tube with 100 mu L of preset cleaning buffer (75% of alcohol) after 10 seconds, enabling the nucleic acid binding area to extend into the cleaning buffer, holding the handle area by hand or mechanical handle area, slightly shaking the 3D printing special functional body up and down, and repeatedly transferring the 3D printing special functional body in the cleaning buffer for 2-3 times; taking out the 3D printing special-shaped functional body from the cleaning Buffer, throwing away residual liquid drops on the 3D printing special-shaped functional body, drying in a blast incubator at 37 ℃ for 1 minute, placing in 40 mu L elution Buffer (TE) to enable a nucleic acid binding area to extend into the elution Buffer, holding a handle area by hand or machine, and gently shaking for 30s for elution. The obtained eluates were quantified by an ultraviolet spectrophotometer at a concentration of 19.882-25.734 ng/. Mu.L, A260/280 at a concentration of 1.775-1.893 and A260/230 at a concentration of 1.792-1.945. The rest nucleic acid is frozen at-20 ℃ for standby.
(3) The extracted DNA is used for in vitro PCR diagnosis
The extracted blood genome DNA is used as a template, nest type PCR (Nested PCR) is selected for amplifying the target plasmodium genome genes.
First step PCR: each 25. Mu.L of the system contains 0.5. Mu.L of the outer primer (10 pM) shown in SEQ ID NO. 1 and SEQ ID NO. 2, 0.25. Mu.L of template DNA 2. Mu. L, rTaq 0.25, 2.5. Mu.L of 10 XBuffer, 2. Mu.L of dNTPs and 17.25. Mu.L of deionized water. PCR conditions were 95℃for 4min; nested PCR was performed using the PCR product obtained by performing 30 cycles at 95℃for 30 seconds, 55℃for 60 seconds, and 72℃for 60 seconds and then extending at 72℃for 5 minutes as a nucleic acid template.
Nested PCR: each 25. Mu.L of the system contained 0.5. Mu.L of each inner primer (10 pM) shown in SEQ ID NO. 3 and SEQ ID NO. 4, 0.25. Mu.L of the first-step PCR product 2. Mu. L, rTaq 0.25, 10 XBuffer 2.5. Mu.L, 2. Mu.L of dNTPs, and 17.25. Mu.L of deionized water. PCR conditions were 95℃for 4min; the PCR product obtained was used for gel electrophoresis by performing 35 cycles at 95℃for 30 seconds, 62℃for 30 seconds, and 72℃for 1 minute and then extending at 72℃for 5 minutes.
10. Mu.L of Nested PCR product was added with a nucleic acid stain, and gel electrophoresis was performed with 2% agarose, and after the electrophoresis was completed, the result was observed under ultraviolet light, as shown in FIG. 2, and 8 cases of the nucleic acid extracted according to the present invention were amplified to obtain the target band. In FIG. 2, lanes 1-4 are samples of 3D7 strain filter paper blood 1-4, lanes 5-8 are samples of filter paper blood 5-8 of patients infected with P.vivax, and lane M is DL2000 Marker.
The example shows that the concentration and purity of 8 cases of DNA provided by the invention are good, and the DNA extracted by the invention has high quality and can be used for downstream in vitro PCR diagnosis technology; the embodiment also shows that the 3D printing special functional body is simple to separate the nucleic acid from the lysate, and the functional body is quickly transferred in the lysate, the cleaning liquid and the eluent by hand without time-consuming and complicated steps such as pipetting and centrifuging, so that the labor intensity is low, the time-consuming process of separation and purification is less than 2min, and the method is quick and high in flux; and the whole operation process can be in 1m 2 Is completed in the operating environment, saves space and has high use flexibility.
Example 3
Photosensitive resin (PAA) is used as a raw material, and a light curing reaction is carried out under 400-800nm illumination by adopting a DLP digital image projection 3D printing technology, so that a 3D printing special-shaped functional body with a regular structure is prepared: the 3D printing special-shaped functional body is of an umbrella-shaped structure, the eight-connected 3D printing special-shaped functional body is composed of a nucleic acid binding area 1, a handle area 2 and a connecting area 3, the nucleic acid binding area 1 is a cone, the handle area 2 is a cylinder, the connecting area 3 is in a long strip shape, the center position of the cone bottom of the nucleic acid binding area 1 is connected with one bottom surface of the handle area 2, the other bottom surface of the handle area 2 is connected with the connecting area 3, and 8 single 3D printing special-shaped functional bodies are connected on the connecting area 3 side by side to form the eight-connected 3D printing special-shaped functional body. Specifically comprises an eight-linked functional body with 8 nucleic acid binding areas 1, the width of the nucleic acid binding area 1 is 3.8mm, the height is 10mm, the outer surface of the nucleic acid binding area 1 is provided with a thread and groove structure, and the height of the handle area 2 is 20mm. The octal 3D printing special function is shown in fig. 1 (b).
One of the tussah pupa infected with baculovirus is taken, 20 mu L of pupa blood is respectively taken and placed in each reaction hole of the 8-row tube, the serial numbers are 1-8 #, and 60 mu L of 1 XPBS is added for suspension and uniform mixing. Adding 80 mu L of 2 Xlysate (200 mmol Tris-HCl, 50mmol EDTA, 1000mmol NaCl and 2% SDS) into each tube, shaking and mixing 5 mu L of proteinase K solution, placing in a 55 ℃ water bath for 30 minutes, adding 60 mu L of absolute ethyl alcohol, placing in a 3D printing special-shaped functional body, enabling a nucleic acid binding area of the 3D printing special-shaped functional body to extend into the solution, holding a handle area, gently shaking the 3D printing special-shaped functional body and mixing the solution, transferring the 3D printing special-shaped functional body into an 8-row EP tube with 100 mu L preset with a cleaning buffer (75% alcohol) after 10 seconds, enabling the nucleic acid binding area to extend into the cleaning buffer, holding the handle area, gently shaking the 3D printing special-shaped functional body up and down for 5 seconds, and repeatedly transferring 2-3 times in the cleaning buffer; the 3D-printed special-shaped functional body was taken out from the washing Buffer, the residual liquid drop on the 3D-printed special-shaped functional body was thrown off, dried in a blast incubator at 37℃for 2 minutes, and the nucleic acid binding region was placed in 50. Mu.L of PCR reaction solution (containing 1. Mu. L, rTaq 0.5.5. Mu.L, 10 XBuffer 5. Mu.L, dNTPs 4. Mu.L, deionized water 38.5. Mu.L each of the outer primers (10 pM) shown in SEQ ID NO:5 and SEQ ID NO: 6) for 30 seconds, and then the reaction solution was used for PCR amplification by removing the 3D-printed special-shaped functional body. PCR conditions were 95℃for 4min; the PCR product obtained was subjected to gel electrophoresis with 2% agarose at 95℃for 30 seconds, 55℃for 60 seconds, and 72℃for 60 seconds for 30 cycles, and then extended at 72℃for 5 minutes, and after the electrophoresis was completed, the result was observed under ultraviolet light, as shown in FIG. 3, 8 samples each showed a target band at 406 bp. Lanes 1-8 in FIG. 3 are samples of blood 1-8 # of baculovirus infected tussah pupa, lane M is DL2000 Marker.
This example demonstrates that the method of the present invention can rapidly extract genomic DNA from insect (invertebrate) blood samples, and the extracted DNA has high purity, less pollution, and good quality, and can be used for identification and diagnosis of microorganisms such as downstream viruses.
While the extraction process of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made to the process described herein, or any suitable combination and modification, can be made to practice the inventive technique without departing from the spirit or scope of the invention. It is expressly noted that all like substitutions and alterations are: such as rational modification of the lysing agent, washing fluid, eluent composition, such as rational modification of the sample format and pretreatment, such as rational modification of the shape, size, surface and structure of the 3D printed special-shaped functionalities, etc., are considered to be included within the spirit, scope and content of the present invention as will be apparent to those skilled in the art.
SEQUENCE LISTING
<110> university of Dalian theory of engineering
<120> blood genome DNA extraction method based on 3D printing special-shaped functional body and application kit thereof
<130> 2020
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<170> PatentIn version 3.5
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<213> Artificial sequence (Artificial Sequence)
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<213> Artificial sequence (Artificial Sequence)
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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<212> DNA
<213> Artificial sequence (Artificial Sequence)
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Claims (8)
1. A blood genome DNA extraction method based on 3D printing of a specific functional body, characterized by comprising the steps of:
(1) Carrying out cracking treatment on a blood sample to be detected by using a cracking liquid;
(2) Stretching the nucleic acid binding area of the 3D printing special functional body into the solution obtained in the step (1) by hand or machine holding to bind nucleic acid;
(3) 3D printing the special functional body in the step (2) is completed through handheld or machine-held movement, and the nucleic acid binding area extends into the cleaning liquid to clean the nucleic acid;
(4) Taking out the 3D printing special-shaped functional body which is finished in the step (3) through hand holding or machine holding, and drying;
(5) Placing the nucleic acid binding region of the 3D printing special-shaped functional body which completes the step (4) into an eluent by hand or machine holding, and eluting to obtain the eluent, namely blood genome DNA;
the blood sample to be tested comprises blood whole blood separated from a human body or an animal body, red blood cells removed from supernatant and dry blood spots;
the 3D printing special-shaped functional body comprises a nucleic acid binding region and a handle region; the 3D printing special-shaped functional body comprises 1 or at least two nucleic acid binding areas, wherein the two nucleic acid binding areas are connected together side by virtue of a connecting area;
the 3D printing special-shaped functional body is a micro-component obtained by taking photosensitive resin PAA as a raw material and adopting a 3D printing technology;
the surface of the nucleic acid binding area is flat or has an uneven microstructure; the uneven microstructure is provided with a thread or groove structure;
the nucleic acid binding region is a cone; the handle area is a cylinder or a cuboid, and one end of the handle area is connected with the center of the cone bottom of the nucleic acid binding area to form an umbrella-shaped structure.
2. The method for extracting genomic DNA from blood according to claim 1, wherein the nucleic acid binding region, the handle region and the junction region are prepared integrally or separately, and are assembled by adhesion when the respective parts are prepared separately.
3. The blood genomic DNA extraction of claim 1The method is characterized in that the lysate in the step (1) comprises the following steps: CTAB lysate and NaHCO 3 Lysates, chelex lysates, proteinase K lysates or SDS lysates.
4. The method for extracting genomic DNA from blood according to claim 3, wherein the SDS lysate comprises the following components: 1-20% of SDS and 0-30 mug/mL of proteinase K.
5. The method of extracting genomic DNA from blood according to claim 1, wherein RNA digestive enzymes are added or not added to the lysate; the time of the cracking treatment is 1min-24h, and the temperature is 0-100 ℃.
6. The method for extracting genomic DNA from blood according to claim 1, wherein the nucleic acid is bound in the step (2), and an auxiliary binding solvent is added, wherein the auxiliary binding solvent comprises one or a mixed solution of isopropyl alcohol and absolute ethyl alcohol, and the volume of the auxiliary binding solvent is 0.6-0.8 times of the volume of the lysate; the bonding time is 5s-5min.
7. The method for extracting genomic DNA from blood according to claim 1, wherein the washing of nucleic acid in step (3) is performed 1 to 5 times for 2s to 1min each time; the cleaning liquid comprises 70-80% alcohol; the drying process in the step (4) can be carried out at room temperature or under heating, and the drying process time is 1min-2h; the eluent in the step (5) comprises water, PBS, TE buffer solution and downstream PCR reaction solution, and the elution time is 5s-5min.
8. An application kit of a blood genome DNA extraction method based on a 3D printing special-shaped functional body is characterized by comprising the following components:
(1) 3D printing of the contoured function;
(2) A lysate;
(3) Proteinase K;
(4) RNA digestive enzyme;
(5) Cleaning liquid;
(6) Eluting the eluent;
(7) A fixing frame;
(8) An operating instruction;
the 3D printing special-shaped functional body is a micro-component which is formed by a nucleic acid binding area and a handle area and is provided with an umbrella-shaped structure and is obtained by a 3D printing technology by taking photosensitive resin PAA as a raw material, and a plurality of micro-components with the umbrella-shaped structure are connected side by side through a connecting area; the outer surface of the nucleic acid binding area is provided with a thread or groove structure; the lysate comprises CTAB lysate and NaHCO 3 One of lysate, chelex lysate, proteinase K lysate or SDS lysate; the cleaning liquid comprises 70-80% alcohol; the eluent comprises one of water, PBS, TE buffer solution and downstream PCR reaction solution; the fixing frame is a structure capable of fixing and arranging a plurality of 3D printing special functional bodies.
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| CN110945124A (en) * | 2017-03-14 | 2020-03-31 | Aj耶拿检疫有限公司 | Method for enriching cells from a sample and subsequently isolating nucleic acids from these cells |
| CN111004719A (en) * | 2019-12-03 | 2020-04-14 | 中国科学院苏州纳米技术与纳米仿生研究所 | Nucleic acid detection module, detection unit and detection system |
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