CN111607635A - 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
- Publication number
- CN111607635A CN111607635A CN202010585509.2A CN202010585509A CN111607635A CN 111607635 A CN111607635 A CN 111607635A CN 202010585509 A CN202010585509 A CN 202010585509A CN 111607635 A CN111607635 A CN 111607635A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- blood
- acid binding
- functional body
- shaped functional
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000010146 3D printing Methods 0.000 title claims abstract description 80
- 210000004369 blood Anatomy 0.000 title claims abstract description 58
- 239000008280 blood Substances 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 48
- 238000007400 DNA extraction Methods 0.000 title claims abstract description 14
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 107
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 101
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 101
- 238000004140 cleaning Methods 0.000 claims abstract description 25
- 230000009089 cytolysis Effects 0.000 claims abstract description 22
- 238000005516 engineering process Methods 0.000 claims abstract description 14
- 239000003480 eluent Substances 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 71
- 239000006166 lysate Substances 0.000 claims description 25
- 108010067770 Endopeptidase K Proteins 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 238000005304 joining Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 7
- 229920005989 resin Polymers 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 7
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 6
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 6
- 238000005336 cracking Methods 0.000 claims description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 5
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 4
- NUJOXMJBOLGQSY-UHFFFAOYSA-N manganese dioxide Chemical compound O=[Mn]=O NUJOXMJBOLGQSY-UHFFFAOYSA-N 0.000 claims description 4
- 239000011236 particulate material Substances 0.000 claims description 4
- 229920001169 thermoplastic Polymers 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 239000002953 phosphate buffered saline Substances 0.000 claims description 3
- 239000004416 thermosoftening plastic Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 125000003277 amino group Chemical group 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 210000003743 erythrocyte Anatomy 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 125000000524 functional group Chemical group 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 2
- 230000001788 irregular Effects 0.000 claims description 2
- 229910052751 metal Inorganic materials 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- 235000012239 silicon dioxide Nutrition 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 239000006228 supernatant Substances 0.000 claims description 2
- 239000004408 titanium dioxide Substances 0.000 claims description 2
- 241000201297 Chelone Species 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 18
- 238000000605 extraction Methods 0.000 abstract description 14
- 238000000926 separation method Methods 0.000 abstract description 12
- 230000004907 flux Effects 0.000 abstract description 10
- 244000052769 pathogen Species 0.000 abstract description 10
- 230000001717 pathogenic effect Effects 0.000 abstract description 8
- 238000000338 in vitro Methods 0.000 abstract description 6
- 239000007788 liquid Substances 0.000 abstract description 6
- 238000012546 transfer Methods 0.000 abstract description 6
- 238000005119 centrifugation Methods 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 2
- 238000000746 purification Methods 0.000 abstract description 2
- 230000003321 amplification Effects 0.000 abstract 1
- 238000003199 nucleic acid amplification method Methods 0.000 abstract 1
- 238000003752 polymerase chain reaction Methods 0.000 description 19
- 230000008569 process Effects 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 239000000872 buffer Substances 0.000 description 9
- 238000003745 diagnosis Methods 0.000 description 8
- 238000002156 mixing Methods 0.000 description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 6
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 235000019441 ethanol Nutrition 0.000 description 5
- 238000007857 nested PCR Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 241000382353 Pupa Species 0.000 description 4
- 239000012149 elution buffer Substances 0.000 description 4
- 238000007885 magnetic separation Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 241000223810 Plasmodium vivax Species 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 238000005286 illumination Methods 0.000 description 3
- 239000002122 magnetic nanoparticle Substances 0.000 description 3
- 238000000386 microscopy Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 238000005191 phase separation Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 241000701447 unidentified baculovirus Species 0.000 description 3
- 239000011534 wash buffer Substances 0.000 description 3
- 229920000936 Agarose Polymers 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241000224016 Plasmodium Species 0.000 description 2
- 241000223960 Plasmodium falciparum Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 241000255978 Antheraea pernyi Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000002476 Falciparum Malaria Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010035500 Plasmodium falciparum infection Diseases 0.000 description 1
- 201000011336 Plasmodium falciparum malaria Diseases 0.000 description 1
- 206010035503 Plasmodium vivax infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000003317 immunochromatography Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 238000011022 operating instruction Methods 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
Abstract
The invention discloses a blood genome DNA extraction method based on a 3D printing special-shaped functional body and an application kit thereof, belonging to the field of nucleic acid extraction and separation. The invention uses lysis solution to perform lysis treatment on a sample, and uses a 3D printing special-shaped functional body to perform combination, separation and purification of nucleic acid. The method can be held in a lysis solution, a cleaning solution and an eluent by hands or machines to quickly transfer the 3D printing functional body, does not need time-consuming and tedious steps such as liquid transfer, centrifugation and the like, has low manual labor density, low cost, high flux, high quality of obtained nucleic acid, low requirements on equipment and operation space, and has automatic and high-flux downstream application prospects. Furthermore, the invention also provides an application kit for the nucleic acid extraction process. The invention solves the problems of complex preparation, time consumption, low flux and the like of target nucleic acid in the existing PCR in-vitro amplification technology, and has wide application prospect of rapid detection of blood pathogen.
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-shaped functional body and an application kit thereof.
Background
Diseases transmitted through the blood often cause significant infectious diseases, which raises global public health safety issues. Prevention of such infectious diseases is mainly based on three principles: the method is characterized by controlling the infection source, cutting off the transmission path and protecting susceptible people, wherein the most effective prevention means is to find the infection source as soon as possible by detecting the pathogen and cut off the transmission, and a convenient, quick, high-flux and high-sensitivity pathogen detection method is sought, which is the key problem of improving the detection efficiency and accuracy. Currently, the commonly used pathogen detection methods include: pathogenic detection, immunological detection and molecular biological detection.
The etiology detection is usually carried out by thick and thin blood film staining microscopy, and the method has the advantages of simple operation, low price, 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 microscopy method is time-consuming and labor-consuming, has high requirements on microscopy personnel and low sensitivity, and is easy to cause missed detection of pathogens to cause continuous spread of diseases. The immunochromatography technology is another common method for pathogen detection, and has the advantages of simple operation, portability and low equipment requirement. But the method has higher economic cost, obvious difference of detection results due to environment, humidity and sample state detection, high false negative or false positive rate and low detection sensitivity.
The PCR (polymerase chain reaction) technology is a commonly used 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 pathogenic detection and immunological diagnosis, and is widely applied to in vitro PCR diagnosis of disease pathogens at present. However, the problems of complicated process, time consumption, low throughput, etc. in extracting genomic DNA from blood for in vitro PCR diagnosis of disease pathogens have become bottlenecks that limit the application of molecular detection technology in clinical.
Nucleic acid is extracted from complex biological samples, and liquid phase extraction methods such as phenol chloroform extraction and trizol method are gradually eliminated because of heavy pollution and large sample demand. Solid phase separation, especially magnetic separation, can avoid complicated centrifugal operation and a plurality of manual operation errors in the traditional nucleic acid separation process by virtue of the performance that magnetic nanoparticles are combined with nucleic acid and can rapidly move in an external magnetic field, and is considered to have the prospect of downstream high-throughput and automatic application. However, the inherent aggregation and sedimentation effects of the nanoparticles are difficult to avoid, and the efficiency and stability of nucleic acid extraction are severely limited. The difficulty in desorbing nucleic acid by magnetic nanoparticles and the low efficiency of nucleic acid extraction are common problems, and although the problem is reported to be avoided by directly using the compound 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 inevitable. For non-magnetic solid phase separation techniques, such as traditional column extraction, etc., the separation of the solid phase carrier bound with nucleic acid from the lysate system still requires tedious operations such as multi-step centrifugation or repeated pipetting, resulting in labor intensity and low flux.
Based on this, it is the key point of research to develop a simple, high-throughput, fast nucleic acid extraction and detection method. The first problem to be solved is how to ensure the extraction quality and improve the flux to achieve the aims of high flux, rapidness, simplicity and convenience by limiting the control step of high flux in the whole process, namely nucleic acid extraction.
Disclosure of Invention
The invention provides a blood genome DNA extraction method based on a 3D printing special-shaped functional body, aiming at the problems of complex process, time consumption, low flux and the like of extracting genome DNA from blood at present.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a blood genome DNA extraction method based on 3D printing special-shaped functional bodies comprises the following steps:
(1) cracking the blood sample to be detected by using a cracking solution;
(2) extending the nucleic acid binding region of the 3D printing special-shaped functional body into the solution obtained in the step (1) through hand holding or machine holding to carry out nucleic acid binding;
(3) completing the 3D printing special-shaped functional body in the step (2) by hand-held or machine-held movement, and extending the nucleic acid binding region into a cleaning solution to clean the nucleic acid;
(4) taking out the 3D printing special-shaped functional body which is finished in the step (3) by hand holding or machine holding, and drying;
(5) and (3) placing the nucleic acid binding region of the 3D printing special-shaped functional body which is finished in the step (4) into an eluent by hand or machine, and eluting to obtain the eluent, namely the blood genome DNA.
Specifically, in the above technical solution, the blood sample to be tested in 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-mentioned technical solution, the 3D printing special-shaped functional body is a micro-element having an "umbrella" type structure obtained by 3D printing technology using photosensitive resin or thermoplastic as a raw material, and includes 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, and when the at least two nucleic acid binding regions are present, the handle regions are connected together side by means of the connecting region; the nucleic acid binding region is a cone with dimensions: 2-5mm (diameter of cone bottom) multiplied by 5-20mm (height); the handle area is a cylinder or a cuboid, one end of the handle area is connected with the center of the conical bottom of the nucleic acid binding area to form an umbrella-shaped structure; the surface of the nucleic acid binding region is flat or has uneven microstructures, and the uneven microstructures comprise a screw thread support structure, a groove structure, a porous structure and a protruding structure; the uneven microstructure can be in any shape and distributed at any position on the outer surface of the nucleic acid binding region; the porous structure can be in nanometer and micrometer sizes, and the shape of the porous structure can be a sphere, a cube or an irregular shape; the nucleic acid binding region is loaded or unloaded with a particulate material comprising inorganic salt particles comprising silicon dioxide, titanium dioxide, manganese dioxide and a metal particulate material; the nucleic acid binding region may be modified with or without functional groups including hydroxyl, carboxyl, amino groups. The handle area is cylindrical or cuboid, the diameter of the cylinder is 2-5mm, the length of the cuboid is 2-5mm, and the width of the cuboid is 1-5 mm; the connecting area is a cuboid, and the overall height h of the 3D printing special-shaped functional body is 2.1cm-10cm (length) multiplied by 2-10mm (width) multiplied by 1-5mm (height).
Furthermore, a special-shaped functional body is accurately prepared on a printer by utilizing a 3D printing technology, and the nucleic acid combining area, the handle area and the connecting area can be integrally prepared or separately prepared; when the parts are prepared separately, the assembly of the nucleic acid binding region and the handle region may be performed by adhesion.
Specifically, in the above technical solution, the lysis solution in step (1) refers to a buffer solution capable of releasing nucleic acid in a sample into a solution, and includes: CTAB lysate, NaHCO3Lysis solution, Chelex lysis solution, proteinase K lysis solution or SDS lysis solution.
Specifically, in the above-described technical solution, the CTAB lysate includes: 1-3% of CTAB, 0.5-5M NaCl, 0.01-0.05M EDTA, 0.05-0.5M Tris-HCl and 0.05-0.5% of mercaptoethanol, wherein the NaHCO is3The lysis solution comprises: 0.05-1.00M NaHCO30.5-10% SDS, the Chelex lysate comprises: 0.5-20% of Chelex-100 and 0.2-5M of DTT, wherein the proteinase K lysate comprises: 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 μ g/mL proteinase K.
Specifically, in the above-mentioned technical scheme, RNA digesting enzyme may or may not be added to the lysate. The treatment time of the cracking process is 1min-24h, and the treatment temperature is 0-100 ℃.
In the above technical solution, the step (2) of combining nucleic acids is performed, specifically, a nucleic acid combining region of the 3D printed special-shaped functional body is extended into the lysate obtained in the step (1), an auxiliary combining solvent is added or not added, and the lysate is agitated by shaking the 3D printed special-shaped functional body, wherein the auxiliary combining solvent may be one or a mixed solution of isopropanol and absolute ethyl alcohol, and the volume of the auxiliary combining solvent is 0.6 to 0.8 times of the volume of the lysate; and the 3D printing special-shaped functional body is used for combining nucleic acid, and the combination time is 5s-5 min.
In the above technical solution, the step (3) of cleaning nucleic acid is specifically to manually or mechanically move the handle region end of the 3D printed special functional body combined with the target nucleic acid into a container with a cleaning solution, so that the nucleic acid combining region extends into the cleaning solution to clean nucleic acid, and the 3D printed special functional body is stirred in the cleaning solution. The washing in the step (3) is generally 1 to 5 times; the cleaning time is 2s-1min each time; preferably, the cleaning solution comprises 70-80% alcohol.
In the above technical solution, the drying process in step (4) can be performed at room temperature or under heating, and the drying process time is 1min-2 h;
in the above-mentioned technical solution, the elution in step (5) is performed for 5s-5min, and the elution solution can be water with appropriate concentration or high concentration for convenient storage, PBS, TE buffer solution, downstream PCR reaction solution, etc., preferably, the TE buffer solution is used in the present invention, and the TE buffer solution comprises the following specific components: 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-shaped functional body. Specifically, the kit comprises the following components:
(1)3D printing special-shaped functional bodies;
(2) lysis solution
(3) Proteinase K;
(4) RNA digestive enzyme
(5) Cleaning fluid;
(6) eluting the solution;
(7) a fixed mount;
(8) and (5) operating instructions.
The 3D printing special-shaped functional body is a micro-element which is prepared from photosensitive resin or thermoplastic plastics as raw materials by a 3D printing technology, consists of a nucleic acid binding area and a handle area, has an umbrella-shaped structure, and is connected with the micro-element by a connecting areaConnecting a plurality of micro-elements with umbrella-shaped structures side by side; the lysis solution comprises CTAB lysis solution and NaHCO3One of a lysate, a Chelex lysate, a proteinase K lysate, or an SDS lysate; the cleaning solution comprises 70-80% of 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-shaped functional bodies.
The 3D printing special-shaped functional body is in any structure as described above, preferably a structure which is used by matching 8-row tubes and contains 8 nucleic acid binding regions as shown in FIG. 1(b), and can be broken off for use when being used singly; the mount is for can fixing, arrange the structure that the special functional body was printed to several 3D for a plurality of 3D print special functional body and can remove along with the mount simultaneously, match perforated plate or a plurality of centrifuging tube and use.
Has the advantages that: the method for obtaining the blood DNA provided by the invention can extract and separate the target DNA with high efficiency, simplicity, convenience, rapidness and high flux, and has the advantages obviously advanced to 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 the nucleic acid, and the 3D printing special-shaped functional body is quickly transferred by mechanical force to complete the separation, so that the bottleneck restriction that the traditional non-magnetic nucleic acid solid-phase separation depends on multi-step centrifugation and liquid transfer 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 pipe, a 96-pore plate or a 384-pore plate for use, high-flux operation is realized, which is an important breakthrough of the low general flux of the existing extraction technology;
2) the blood DNA extraction method provided by the invention adopts a 3D printing special-shaped functional body to separate and purify 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 throughput, and the method of the present invention can achieve a faster separation speed, a simpler operation process and a more stable separation effect than magnetic separation. The 3D printing special-shaped functional body is used for separating nucleic acid, the nucleic acid can be separated and purified from the lysate within 1-5min, and complicated and time-consuming operations such as liquid transfer, centrifugation and the like are not needed; magnetic separation usually takes about 14.5min and requires 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 30seconds. PLoS Biol 15(11): E2003916.). Meanwhile, the problems of insufficient nucleic acid combination, impurity wrapping 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 the quality of nucleic acid extraction are effectively guaranteed;
3) the raw materials of the 3D printing special-shaped functional body provided by the invention are cheap and easy to obtain; 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, liquid transfer and the like, can realize artificial low-labor-density high-throughput nucleic acid separation, and the whole process can be within 1m2The operation is finished in the operating environment, so that 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 transfer the 3D printing special-shaped functional body among lysis solution, cleaning solution and eluent through designing programmed equipment, so that automation is realized, the labor cost is further reduced, and the method is favorable for promoting the downstream molecular technology, particularly the accurate and rapid diagnosis of pathogenic pathogens in blood, such as the clinical application of plasmodium PCR in-vitro diagnosis.
Drawings
Fig. 1 is a schematic structural diagram of a 3D printing special-shaped functional body according to the present invention, wherein (a) is a single 3D printing special-shaped functional body, and (b) is an octal 3D printing special-shaped functional body.
FIG. 2 is an agarose gel electrophoresis image of example 2 of the present invention, wherein lanes 1-4 are filter blood No. 1-4 samples of 3D7 strain Plasmodium falciparum, lanes 5-8 are filter blood No. 5-8 samples of P.vivax infected patients, and lane M is DL2000 Marker.
FIG. 3 is an agarose gel electrophoresis image of example 3 of the present invention, wherein lanes 1-8 are samples of tussah pupa blood 1-8 infected with baculovirus, and lane M is DL2000 Marker.
In the figure, 1, nucleic acid binding region; 2. a handle region; 3. a connecting region.
Detailed Description
The invention is further described below with reference to the 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 scientific and technical 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 equipment and experimental equipment related to the embodiment of the invention are all in accordance with the commercial products in the biotechnology field if no special description is given.
The primers related to the embodiment of the invention are synthesized by entrusted bioengineering company, 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 the illumination of 400-800nm by adopting a DLP digital image projection 3D printing technology, so that the 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 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 of the cone bottom of the nucleic acid binding area 1 is connected with one bottom surface of the handle area 2. Specifically, the kit comprises 1 monomer functional body with a single nucleic acid binding region 1, wherein the width of the nucleic acid binding region 1 is 5mm, the height of the nucleic acid binding region 1 is 10mm, the outer surface of the nucleic acid binding region 1 is provided with a thread and groove structure, and the height of a handle region 2 is 35 mm. The single 3D print special feature function is shown in fig. 1 (a).
20 mu L of whole blood from a human source and pig blood source which are frozen in a laboratory are respectively placed in two EP tubes, and 1 XPBS 60 mu L of whole blood is added for suspension and even mixing. Adding 80 microliter of 2 multiplied lysis solution (200mmol of Tris-HCl, 50mmol of EDTA, 1000mmol of NaCl and 2 percent of SDS) and 5 microliter of proteinase K solution into an EP tube, shaking and mixing uniformly, placing in a water bath at 55 ℃ for 30 minutes, adding 60 microliter of absolute ethyl alcohol and 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 by hand, shaking the 3D printing special-shaped functional body mixing solution slightly, transferring the 3D printing special-shaped functional body into an 8-row EP tube preset with 100 microliter of a cleaning buffer (75 percent alcohol) after 10 seconds, enabling the nucleic acid binding area to extend into the cleaning buffer, holding the handle area by hand, shaking the 3D printing special-shaped functional body up and down for 5 seconds, and repeatedly transferring in the cleaning buffer for 2 to 3 times; the 3D printing special-shaped functional body is taken out from the washing Buffer, residual liquid drops on the 3D printing special-shaped functional body are thrown off, the 3D printing special-shaped functional body is dried in a blast incubator at 37 ℃ for 2 minutes and is placed in 40 mu L of elution Buffer (TE), the nucleic acid binding area extends into the elution Buffer, the handle area is held by hands, and elution is carried out by gently shaking for 30 s. The obtained eluate was measured by UV spectrophotometer to have DNA concentrations of 45.060 ng/. mu.L and 53.273 ng/. mu.L, 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 DNA purity, low contamination, and good quality.
Example 2
(1) Preparation of special-shaped functional body
Photosensitive resin (PAA) is used as a raw material, and a light curing reaction is carried out under the illumination of 400-800nm by adopting a DLP digital image projection 3D printing technology, so that the 3D printing special-shaped functional body with a regular structure is prepared: the 3D prints special shape function body is the structure similar to the umbelliform, eight ally oneself with 3D and prints special shape function body and comprises nucleic acid bonding region 1, handle district 2 and joining region 3, nucleic acid bonding region 1 is the cone, handle district 2 is the cylinder, joining region 3 is rectangular shape, 1 awl end central point in nucleic acid bonding region puts and is connected with a bottom surface in handle district 2, another bottom surface in handle district 2 is connected with joining region 3, connect 8 single 3D side by side on joining region 3 and print special shape function bodies, form eight ally oneself with 3D and print special shape function body. The kit specifically comprises an octa-linked functional body with 8 nucleic acid binding regions 1, wherein the width of the nucleic acid binding regions 1 is 3.8mm, the height of the nucleic acid binding regions 1 is 10mm, the outer surface of the nucleic acid binding regions 1 is provided with a thread and groove structure, and the height of a handle region 2 is 20 mm. The functional body for eight-link 3D printing special shapes is shown in fig. 1 (b).
(2) Genomic DNA extraction
Taking an artificially cultured filter paper blood sample of 3D7 strains of falciparum malaria and a filter paper blood sample of a patient infected with plasmodium vivax, respectively taking 4 disc dry blood spot blood samples with the diameter of 3mm from the two samples by using a puncher, placing the samples in 8 sample holes of an 8-row EP tube, and respectively numbering 1-4 # and 5-8 #. Adding 80 microliter of lysis solution (100mmol of Tris-HCl, 25mmol of EDTA, 500mmol of NaCl and 1% of SDS) and 5 microliter of proteinase K solution into an EP tube, shaking and uniformly mixing, standing at room temperature for 30 minutes, adding 60mL of absolute ethyl alcohol and putting 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 by hand or machinery, slightly shaking the 3D printing special-shaped functional body uniformly-mixing solution, transferring the 3D printing special-shaped functional body into an 8-row EP tube which is preset with 100 microliter of cleaning buffer (75% alcohol) after 10 seconds, enabling the nucleic acid binding area to extend into the cleaning buffer, holding the handle area by hand or machinery, slightly shaking the 3D printing special-shaped functional body up and down for 5 seconds, and repeatedly transferring in the cleaning buffer for 2-3 times; and taking the 3D printing special-shaped functional body out of the washing 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 of elution Buffer (TE), extending the nucleic acid binding area into the elution Buffer, holding the handle area by hand or machinery, and slightly shaking for 30s for elution. And quantifying 1 mu L of the obtained eluent by using an ultraviolet spectrophotometer, wherein the concentration is 19.882-25.734 ng/mu L, the A260/280 is 1.775-1.893, and the A260/230 is 1.792-1.945. And freezing the rest nucleic acid at-20 ℃ for later use.
(3) The DNA is used for in vitro PCR diagnosis
The extracted blood genome DNA is used as a template, nested PCR (nested PCR) is selected, and the target plasmodium genome gene is amplified.
The first step of PCR: each 25. mu.L system contains 0.5. mu.L of each of the outer primers (10pM) shown in SEQ ID NO. 1 and SEQ ID NO. 2, 2. mu. L, rTaq 0.25 of the template DNA, 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 4 min; 30 cycles of 95 ℃ for 30seconds, 55 ℃ for 60 seconds and 72 ℃ for 60 seconds, and extension at 72 ℃ for 5min, and performing Nested PCR by using the obtained PCR product as a nucleic acid template.
Nested PCR: each 25. mu.L system contains 0.5. mu.L of each of the inner primers (10pM) shown as SEQ ID NO. 3 and SEQ ID NO. 4, 2. mu. L, rTaq 0.25.25. mu.L of the first-step PCR product, 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 4 min; 35 cycles of 95 ℃ for 30seconds, 62 ℃ for 30seconds and 72 ℃ for 1 minute, and extension at 72 ℃ for 5 minutes, the PCR product obtained was used for gel electrophoresis.
10 mu L of Nested PCR product is added with nucleic acid stain, 2% agarose is used for gel electrophoresis, and the result is observed under ultraviolet light after the electrophoresis is finished, as shown in figure 2, 8 cases of nucleic acid extracted by the invention all amplify target bands. In FIG. 2, lanes 1-4 are filter paper blood samples 1-4 of 3D7 Plasmodium falciparum strain, lanes 5-8 are filter paper blood samples 5-8 of patients infected with Plasmodium vivax, and lane M is DL2000 Marker.
This example demonstrates that the concentration and purity of 8 DNA samples proposed by the present invention are better, which demonstrates that the DNA extracted by the present invention has high quality and can be used for downstream in vitro PCR diagnostic techniques; the embodiment also shows that the operation of separating the nucleic acid from the lysate by the 3D printing special-shaped functional body is simple, the functional body is quickly transferred in the lysate, the cleaning solution and the eluent by hand without the time-consuming and tedious steps of pipetting, centrifuging and the like, the manual labor density is low, the time-consuming process of separation and purification is less than 2min, and the operation is quick and the flux is high; and the whole operation process can be within 1m2The operation environment is completed, the space is saved, and the use flexibility is high.
Example 3
Photosensitive resin (PAA) is used as a raw material, and a light curing reaction is carried out under the illumination of 400-800nm by adopting a DLP digital image projection 3D printing technology, so that the 3D printing special-shaped functional body with a regular structure is prepared: the 3D prints special shape function body is the structure similar to the umbelliform, eight ally oneself with 3D and prints special shape function body and comprises nucleic acid bonding region 1, handle district 2 and joining region 3, nucleic acid bonding region 1 is the cone, handle district 2 is the cylinder, joining region 3 is rectangular shape, 1 awl end central point in nucleic acid bonding region puts and is connected with a bottom surface in handle district 2, another bottom surface in handle district 2 is connected with joining region 3, connect 8 single 3D side by side on joining region 3 and print special shape function bodies, form eight ally oneself with 3D and print special shape function body. The kit specifically comprises an octa-linked functional body with 8 nucleic acid binding regions 1, wherein the width of the nucleic acid binding regions 1 is 3.8mm, the height of the nucleic acid binding regions 1 is 10mm, the outer surface of the nucleic acid binding regions 1 is provided with a thread and groove structure, and the height of a handle region 2 is 20 mm. The functional body for eight-link 3D printing special shapes is shown in fig. 1 (b).
Taking one infected baculovirus tussah pupa, respectively taking 20 mu L of pupa blood, placing the pupa blood into each reaction hole of an 8-linked calandria, numbering 1-8 #, adding 1 multiplied by PBS 60 mu L, suspending and mixing uniformly. Adding 80 microliter of 2 multiplied lysis solution (200mmol of Tris-HCl, 50mmol of EDTA, 1000mmol of NaCl and 2 percent of SDS) and 5 microliter of proteinase K solution into each tube, shaking and uniformly mixing, placing in a water bath at 55 ℃ for 30 minutes, adding 60 microliter of absolute ethyl alcohol and 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 by hand, slightly shaking the 3D printing special-shaped functional body uniformly-mixing solution, transferring the 3D printing special-shaped functional body into an 8-row EP tube preset with 100 microliter of a cleaning buffer (75 percent alcohol) after 10 seconds, enabling the nucleic acid binding area to extend into the cleaning buffer, holding the handle area by hand, slightly shaking the 3D printing special-shaped functional body up and down for 5 seconds, and repeatedly transferring in the cleaning buffer for 2-3 times; the 3D printed shaped function was removed from the wash Buffer, the remaining droplets on the 3D printed shaped function were spun off, dried in a forced air incubator at 37 ℃ for 2 minutes, the nucleic acid binding region was immersed in 50. mu.L of PCR reaction solution (containing 1. mu. L, rTaq 0.5, 0.5. mu.L each of the outer primers (10pM) as shown in SEQ ID NO:5 and SEQ ID NO:6, 10 Xbuffer 5. mu.L, dNTPs 4. mu.L, and deionized water 38.5. mu.L) for 30seconds, and then the 3D printed shaped function was removed and the reaction solution was used for PCR amplification. PCR conditions were 95 ℃ for 4 min; 30 cycles of 95 ℃ for 30seconds, 55 ℃ for 60 seconds and 72 ℃ for 60 seconds, extension is carried out for 5 minutes at 72 ℃, the obtained PCR product is subjected to gel electrophoresis by using 2% agarose, and the result is observed under ultraviolet light after the electrophoresis is finished, as shown in figure 3, target bands appear at 406bp positions in 8 samples. In FIG. 3, lanes 1-8 are samples of the blood of Antheraea pernyi pupae infected with baculovirus 1-8 # and 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, the extracted DNA has high purity, low contamination, and good quality, and can be used for identification and diagnosis of microorganisms such as downstream viruses.
While the extraction method of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the inventive technique can be implemented by modifying or appropriately changing or combining the methods described herein without departing from the spirit and scope of the present invention. It is specifically noted that all similar substitutions and alterations: such as the rational modification of the components of the lysing agent, the washing solution, and the eluent, such as rational modification and pretreatment of the sample form, such as rational modification of the shape, size, surface, and structure of the 3D printed shaped feature, etc., will be apparent to those skilled in the art and are considered to be included in the spirit, scope, and content of the present invention.
SEQUENCE LISTING
<110> university of Large Community
<120> blood genome DNA extraction method based on 3D printing special-shaped functional body and application kit thereof
<130>2020
<160>4
<170>PatentIn version 3.5
<210>1
<211>24
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
tcaaagatta agccatgcaa gtga 24
<210>2
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
cctgttgttg ccttaaactt c 21
<210>3
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
tttttataag gataactacg gaaaagctgt 30
<210>4
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
tacccgtcat agccatgtta ggccaatacc 30
Claims (10)
1. A blood genome DNA extraction method based on 3D printing special-shaped functional bodies is characterized by comprising the following steps:
(1) cracking the blood sample to be detected by using a cracking solution;
(2) extending the nucleic acid binding region of the 3D printing special-shaped functional body into the solution obtained in the step (1) through hand holding or machine holding to carry out nucleic acid binding;
(3) completing the 3D printing special-shaped functional body in the step (2) by hand-held or machine-held movement, and extending the nucleic acid binding region into a cleaning solution to clean the nucleic acid;
(4) taking out the 3D printing special-shaped functional body which is finished in the step (3) by hand holding or machine holding, and drying;
(5) placing the nucleic acid binding region of the 3D printing special-shaped functional body which is finished in the step (4) into an eluent by hand or machine, and eluting to obtain an eluent, namely the blood genome DNA;
the blood sample to be detected comprises whole blood separated from a human body or an animal body, red blood cells removed from supernatant and dried blood spots.
2. The method for extracting genomic DNA from blood according to claim 1, wherein the 3D printing special-shaped functional body is a micro-element having an umbrella-shaped structure obtained by 3D printing technology using photosensitive resin or thermoplastic as a raw material, and comprises a nucleic acid binding region and a handle region.
3. The method for extracting genomic DNA from blood according to claim 2, wherein the 3D-printed special-shaped functional body comprises 1 or at least two nucleic acid binding regions, and when the at least two nucleic acid binding regions are formed, the handle regions are connected together side by virtue of the connecting regions; the nucleic acid binding region is a cone; the handle area is a cylinder or a cuboid, one end of the handle area is connected with the center of the conical bottom of the nucleic acid binding area to form an umbrella-shaped structure; the surface of the nucleic acid binding region is flat or has an uneven microstructure which comprises a screw thread susceptor structure, a groove structure, a porous structure and a convex structure; the uneven microstructure can be in any shape and distributed at any position on the outer surface of the nucleic acid binding region; the porous structure can be in nanometer and micrometer sizes, and the shape of the porous structure can be a sphere, a cube or an irregular shape; the nucleic acid binding region is loaded or unloaded with a particulate material comprising inorganic salt particles comprising silicon dioxide, titanium dioxide, manganese dioxide and a metal particulate material; the nucleic acid binding region may be modified with or without functional groups including hydroxyl, carboxyl, amino groups.
4. The method for extracting genomic DNA from blood according to claim 3, wherein the nucleic acid binding region, the handle region and the joining region are prepared integrally or separately, and when the parts are prepared separately, they are assembled by adhesion.
5. The method for extracting genomic DNA from blood according to claim 1, wherein the lysis solution in the step (1) comprises: CTAB lysate, NaHCO3Lysis solution, Chelex lysis solution, proteinase K lysis solution or SDS lysis solution.
6. The method for extracting genomic DNA from blood according to claim 5, wherein the SDS lysate comprises the following components: 1-20% SDS, 0-30 μ g/mL proteinase K.
7. The method for extracting genomic DNA from blood according to claim 1, wherein an RNA digesting enzyme is optionally added to the lysate; the time of the cracking treatment is 1min-24h, and the temperature is 0-100 ℃.
8. The method for extracting genomic DNA from blood according to claim 1, wherein the nucleic acid is bound in step (2) with or without an auxiliary binding solvent comprising one or a mixture of isopropanol and absolute ethanol, wherein the volume of the auxiliary binding solvent is 0.6-0.8 times that of the lysate; the binding time is 5s-5 min.
9. The method for extracting genomic DNA from blood according to claim 1, wherein the nucleic acid washing in step (3) is performed 1 to 5 times for 2s to 1min each time; the cleaning solution comprises 70-80% of 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-2 h; and (5) eluting the eluent in the step (5) by using water, PBS, TE buffer solution and downstream PCR reaction solution for 5s-5 min.
10. 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 special-shaped functional bodies;
(2) a lysis solution;
(3) proteinase K;
(4) an RNA digesting enzyme;
(5) cleaning fluid;
(6) eluting the solution;
(7) a fixed mount;
(8) instructions for operating;
the 3D printing special-shaped functional body is a micro-element which is obtained by taking photosensitive resin or thermoplastic as a raw material through a 3D printing technology, consists of a nucleic acid binding area and a handle area and has an umbrella-shaped structure, and a plurality of micro-elements with umbrella-shaped structures are connected in parallel through connecting areas; the lysis solution comprises CTAB lysis solution and NaHCO3Lysate, ChelOne of ex lysate, proteinase K lysate or SDS lysate; the cleaning solution comprises 70-80% of 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-shaped functional bodies.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010585509.2A CN111607635B (en) | 2020-06-23 | 2020-06-23 | Blood genome DNA extraction method based on 3D printing special-shaped functional body and application kit thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010585509.2A CN111607635B (en) | 2020-06-23 | 2020-06-23 | Blood genome DNA extraction method based on 3D printing special-shaped functional body and application kit thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111607635A true CN111607635A (en) | 2020-09-01 |
| CN111607635B CN111607635B (en) | 2024-03-22 |
Family
ID=72203844
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010585509.2A Active CN111607635B (en) | 2020-06-23 | 2020-06-23 | Blood genome DNA extraction method based on 3D printing special-shaped functional body and application kit thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111607635B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021259141A1 (en) * | 2020-06-23 | 2021-12-30 | 大连理工大学 | Nucleic acid extraction system and method based on 3d printed micro-element |
| WO2022088511A1 (en) * | 2020-10-29 | 2022-05-05 | 杭州微策生物技术股份有限公司 | Reagent for lysing virus sample |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040029166A1 (en) * | 2002-07-29 | 2004-02-12 | Jsr Corporation | Nucleic acid-separating method and nucleic acid-extracting reagent |
| US20150275269A1 (en) * | 2012-10-26 | 2015-10-01 | The Emerther Company | Method for purifying nucleic acid and kit |
| 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 |
-
2020
- 2020-06-23 CN CN202010585509.2A patent/CN111607635B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040029166A1 (en) * | 2002-07-29 | 2004-02-12 | Jsr Corporation | Nucleic acid-separating method and nucleic acid-extracting reagent |
| US20150275269A1 (en) * | 2012-10-26 | 2015-10-01 | The Emerther Company | Method for purifying nucleic acid and kit |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| ZOU Y ET AL.: "Nucleic acid purification from plants, animals and microbes in under 30 seconds" * |
| 李蒙航: "基于微尺度载体的高通量核酸固相分离过程研究" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021259141A1 (en) * | 2020-06-23 | 2021-12-30 | 大连理工大学 | Nucleic acid extraction system and method based on 3d printed micro-element |
| WO2022088511A1 (en) * | 2020-10-29 | 2022-05-05 | 杭州微策生物技术股份有限公司 | Reagent for lysing virus sample |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111607635B (en) | 2024-03-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7408229B2 (en) | Polynucleotide capture materials and methods for their use | |
| JP6242896B2 (en) | Methods and kits for purifying nucleic acids | |
| US7560228B2 (en) | Solid-phase nucleic acid isolation | |
| US20100009351A1 (en) | Polynucleotide Capture Materials, and Method of Using Same | |
| CN108410951B (en) | Novel nucleic acid extraction reagent and application thereof | |
| US8202693B2 (en) | Method of isolation of nucleic acids | |
| CN111607489B (en) | Nucleic acid extraction system and method based on 3D printing micro-component | |
| CN111607635B (en) | Blood genome DNA extraction method based on 3D printing special-shaped functional body and application kit thereof | |
| US10125388B2 (en) | Integrated sample processing system | |
| KR20150127917A (en) | Method of extracting and amplifying nucleic acids by using magnetic bead | |
| CN111647596A (en) | Extraction method of microsporidian DNA based on 3D printing functional body, kit and application thereof | |
| CN111778236A (en) | Shellfish genome DNA extraction method based on 3D printing special-shaped functional body, kit and application thereof | |
| CN110923338A (en) | Microarray chip capable of detecting various bacterial genome DNAs and preparation method thereof | |
| JP2018537676A (en) | Integrated sample processing system | |
| CN110863037A (en) | Microarray chip capable of detecting various fungal genome DNAs (deoxyribonucleic acids) and preparation method thereof | |
| US20150219637A1 (en) | Analytical processing and detection device | |
| WO2010005444A1 (en) | Polynucleotide capture materials, and methods of using same | |
| KR20210132375A (en) | Method for isolating nucleic acid using integrated magnetite nanoparticles on graphene oxide | |
| AU2016203610A1 (en) | Polynucleotide capture materials, and methods of using same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |