CN111778236A - Shellfish genome DNA extraction method based on 3D printing special-shaped functional body, kit and application thereof - Google Patents
Shellfish genome DNA extraction method based on 3D printing special-shaped functional body, kit and application thereof Download PDFInfo
- Publication number
- CN111778236A CN111778236A CN202010584270.7A CN202010584270A CN111778236A CN 111778236 A CN111778236 A CN 111778236A CN 202010584270 A CN202010584270 A CN 202010584270A CN 111778236 A CN111778236 A CN 111778236A
- Authority
- CN
- China
- Prior art keywords
- nucleic acid
- shellfish
- functional body
- acid binding
- 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.)
- Pending
Links
- 238000010146 3D printing Methods 0.000 title claims abstract description 90
- 235000015170 shellfish Nutrition 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 45
- 238000007400 DNA extraction Methods 0.000 title claims abstract description 19
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 117
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 105
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 105
- 238000004140 cleaning Methods 0.000 claims abstract description 26
- 238000005516 engineering process Methods 0.000 claims abstract description 24
- 230000009089 cytolysis Effects 0.000 claims abstract description 19
- 239000003480 eluent Substances 0.000 claims abstract description 13
- 244000000010 microbial pathogen Species 0.000 claims abstract description 8
- 230000008569 process Effects 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 60
- 239000006166 lysate Substances 0.000 claims description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 108010067770 Endopeptidase K Proteins 0.000 claims description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 11
- 238000012408 PCR amplification Methods 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 9
- 239000002994 raw material Substances 0.000 claims description 9
- 235000019441 ethanol Nutrition 0.000 claims description 8
- 229920005989 resin Polymers 0.000 claims description 8
- 239000011347 resin Substances 0.000 claims description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 6
- 238000005336 cracking Methods 0.000 claims description 6
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 230000003321 amplification Effects 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 5
- 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
- 239000002953 phosphate buffered saline Substances 0.000 claims description 4
- 229920001169 thermoplastic Polymers 0.000 claims description 4
- 238000007397 LAMP assay Methods 0.000 claims description 3
- 125000000524 functional group Chemical group 0.000 claims description 3
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000007857 nested PCR Methods 0.000 claims description 3
- 238000003753 real-time PCR Methods 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 108010091086 Recombinases Proteins 0.000 claims description 2
- 102000018120 Recombinases Human genes 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 102000038379 digestive enzymes Human genes 0.000 claims description 2
- 108091007734 digestive enzymes Proteins 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
- 230000001788 irregular Effects 0.000 claims description 2
- 239000002923 metal particle Substances 0.000 claims description 2
- 210000000056 organ Anatomy 0.000 claims description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 2
- 235000012239 silicon dioxide Nutrition 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 239000004416 thermosoftening plastic Substances 0.000 claims description 2
- 239000004408 titanium dioxide Substances 0.000 claims description 2
- 239000011236 particulate material Substances 0.000 claims 2
- 238000001514 detection method Methods 0.000 abstract description 18
- 239000007788 liquid Substances 0.000 abstract description 15
- 238000000605 extraction Methods 0.000 abstract description 11
- 238000000926 separation method Methods 0.000 abstract description 10
- 230000004907 flux Effects 0.000 abstract description 9
- 238000003745 diagnosis Methods 0.000 abstract description 8
- 238000012546 transfer Methods 0.000 abstract description 8
- 244000078673 foodborn pathogen Species 0.000 abstract description 7
- 238000005119 centrifugation Methods 0.000 abstract description 6
- 238000011161 development Methods 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000005304 joining Methods 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 14
- 241000193879 Corbicula fluminea Species 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
- 239000000047 product Substances 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- 239000003550 marker Substances 0.000 description 8
- 239000011534 wash buffer Substances 0.000 description 8
- 241001263478 Norovirus Species 0.000 description 7
- 238000001962 electrophoresis Methods 0.000 description 7
- 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
- 244000052769 pathogen Species 0.000 description 6
- 230000001717 pathogenic effect Effects 0.000 description 6
- 235000013305 food Nutrition 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 239000007790 solid phase Substances 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000000246 agarose gel electrophoresis Methods 0.000 description 4
- 239000012295 chemical reaction liquid Substances 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- 239000012149 elution buffer Substances 0.000 description 4
- 238000001502 gel electrophoresis Methods 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 238000007885 magnetic separation Methods 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 238000009360 aquaculture Methods 0.000 description 3
- 244000144974 aquaculture Species 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002156 mixing 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
- 239000011148 porous material Substances 0.000 description 3
- 238000007639 printing Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 238000002123 RNA extraction Methods 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000002122 magnetic nanoparticle Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 241000193882 Corbicula Species 0.000 description 1
- 241000224016 Plasmodium Species 0.000 description 1
- 241000620877 Ruditapes philippinarum Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 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
- 238000010586 diagram Methods 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
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 238000002414 normal-phase solid-phase extraction Methods 0.000 description 1
- 238000001821 nucleic acid purification Methods 0.000 description 1
- 238000002205 phenol-chloroform extraction Methods 0.000 description 1
- 239000002244 precipitate Substances 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
- 238000000746 purification Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 239000000238 shellfish toxin Substances 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000005406 washing Methods 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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
-
- 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
-
- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Immunology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a shellfish genome DNA extraction method based on a 3D printing special-shaped functional body, a kit and application thereof, belonging to the field of nucleic acid extraction and separation. The invention uses the lysis solution to perform lysis treatment on the sample, rapidly transfers the 3D printing special-shaped functional body in the lysis solution, the cleaning solution and the eluent by hands or machines so as to combine, clean and elute DNA, does not need time-consuming and tedious steps such as liquid transfer, centrifugation and the like in the process of DNA extraction, and has the advantages of low manual labor density, low cost, high flux, high quality of obtained nucleic acid, small requirements on equipment and operation space, and application prospect of automatic and high-flux downstream food-borne pathogen diagnosis. Furthermore, the invention also provides application of the nucleic acid extraction method in shellfish pathogenic microorganism diagnosis, solves the problems of complex preparation, time consumption, low flux and the like of the target nucleic acid of the existing shellfish food-borne pathogen detection technology, and is expected to promote development and application of molecular detection of shellfish food-borne pathogens.
Description
Technical Field
The invention belongs to the technical field of nucleic acid extraction and separation and molecular biology, and particularly relates to a shellfish genome DNA extraction method based on a 3D printing special-shaped functional body, a kit and application of the shellfish genome DNA extraction method and the kit in detection of shellfish pathogenic microorganisms.
Background
China is the first major country of aquaculture in the world. As an important component of Chinese ocean economy, aquaculture makes outstanding contributions in the aspects of promoting the prosperity of fishery industry and the abundance of fisherman life, guaranteeing the food safety of China, improving the international market competitiveness and the like.
Shellfish belongs to filter feeding animals, can enrich various pathogenic microorganisms in the surrounding environment, and spread to human through food chain to cause high pathogenic risk, is widely regarded as a main spreading carrier of food-borne pathogenic microorganisms, mainly uses highly pathogenic bacteria, viruses and shellfish poison to be most common, and seriously harms human health. However, current detection techniques for these high-risk pathogenic pathogens lag behind and present serious limitations. The traditional morphological and ecological detection method is tedious, time-consuming and highly professional, and the immunological means has insufficient sensitivity, so that the epidemic prevention and early warning requirements for quickly monitoring the pathogen cannot be met. Although the molecular detection means with high sensitivity and high specificity has been reported, the technology has the limitations of high pollution risk, high detection cost and complicated template preparation process. The sensitivity limitation and the hysteresis of the detection technology cause that high-risk pathogenic pathogens cannot be detected in time, great potential safety hazard is brought to food safety, and meanwhile, the establishment, the soundness and the development of an early warning and risk prevention and control technology system are retarded, and the public health safety and the healthy development of the shellfish aquaculture industry are seriously harmed.
The molecular detection technology (PCR diagnosis) is based on nucleic acid as a target for amplification, has the advantages of high sensitivity, good specificity and rapid detection compared with the pathogenic detection and the immunological diagnosis, and is widely applied to the high-sensitivity detection of pathogens at present. However, the problems of complicated process, time consumption, low flux and the like of target nucleic acid extraction in the key technology in molecular diagnosis have become bottlenecks that limit the application of molecular detection technology to clinic. 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. In solid phase separation, especially magnetic separation, by virtue of the performance that magnetic nanoparticles are combined with nucleic acid and can rapidly move in an external magnetic field, complicated centrifugal operation and a plurality of manual operation errors in the traditional nucleic acid separation process can be avoided, and the magnetic nanoparticles are considered to have the prospect of downstream high-flux and automatic application.
Based on the method, a novel nucleic acid solid phase extraction technology which does not depend on complex operations such as centrifugation, liquid transfer and the like and has an automatic prospect is developed, the promotion effect on the improvement of the molecular detection technology of pathogens in the shellfish is achieved, the development of the molecular detection technology of pathogenic microorganisms in the shellfish is promoted, and the safety and the national health of shellfish food are guaranteed.
Disclosure of Invention
Aiming at the problems of complex preparation, time consumption, low flux and the like of target nucleic acid in the existing shellfish pathogenic molecule detection technology, the invention provides a shellfish genome DNA extraction method based on a 3D printing special-shaped functional body and application thereof in shellfish pathogenic microorganism diagnosis
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a shellfish genome DNA extraction method based on 3D printing special-shaped functional bodies comprises the following steps:
(1) cracking the shellfish 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) by hand or machine to bind nucleic acid;
(3) putting the nucleic acid binding region of the 3D printing special-shaped functional body which is finished in the step (2) into a cleaning solution in a hand-held or machine-held mode for cleaning;
(4) taking out the 3D printing special-shaped functional body after the step (3) is completed, 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 genome DNA.
Specifically, in the above technical solution, the shellfish sample to be tested in step (1) may be the whole shellfish or any component of any part of the shellfish, including shellfish tissue or organs.
Specifically, in the above technical solution, the lysis solution in step (1) is a buffer solution capable of releasing nucleic acid in a tissue into a solution, and includes: CTAB lysate, NaHCO3Lysis solution, Chelex lysis solution, proteinase K lysis solution, SDS lysis solution or Trizol lysis solution.
Specifically, in the above technical scheme, the CTAB lysate includes: 1-3 wt% 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 NaHCO3 lysate comprises: 0.05-1.00M NaHCO3 and 0.5-10% SDS, wherein the Chelex lysate comprises: 0.5-20 wt% 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: 1-10% SDS, 0-30 μ g/mL proteinase K. The lysate may or may not contain RNA digesting enzymes. The treatment time of the cracking process is 1min-24h, and the treatment temperature is 0-100 ℃.
Specifically, in the above technical solution, the 3D printing special-shaped functional body in step (2) is a micro-element with an umbrella-shaped structure obtained by 3D printing technology using photosensitive resin or thermoplastic as raw material, and includes a nucleic acid binding region and a handle region; the nucleic acid binding area 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; the 3D printing special-shaped functional body comprises 1 or at least two nucleic acid binding regions, and when the number of the nucleic acid binding regions is at least two, the handle regions are connected together side by virtue of the connecting regions; 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 uneven microstructure, the uneven microstructure can be in any shape and is distributed at any position of the outer surface of the nucleic acid binding region, and the uneven microstructure comprises a threaded support structure, a groove structure, a porous structure, a protruding structure and the like; the porous structure can be in the size of nanometer, micrometer and the like, and the shape can be a sphere, a cube or an irregular shape. The nucleic acid binding region is loaded or unloaded with a particle material, and the particle material comprises inorganic salt particles such as silicon dioxide, titanium dioxide and manganese dioxide and metal particles; the nucleic acid binding region is modified or not by surface functional groups, and the surface functional groups comprise hydroxyl, carboxyl, amino and the like; 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 size of the connecting area is 6.5-10cm (length) multiplied by 2-10mm (width) multiplied by 1-5mm (height); the overall height h of the 3D printing special-shaped functional body is 20mm-100 mm.
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.
In the technical scheme, the operation of adding or not adding an auxiliary binding solvent for binding the nucleic acid and stirring the lysate by the 3D printing special-shaped functional body is performed in the step (2), wherein the auxiliary binding solvent can be one or a mixed solution of isopropanol 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 3D special-shaped printing functional body is used for combining nucleic acid, and the combination time of shaking the 3D special-shaped printing functional body is 5s-5 min.
In the above-mentioned technical solution, the cleaning in step (3) is usually accompanied by the operation of stirring the 3D printing special shape function in the cleaning liquid. Cleaning is generally 1-5 times; the cleaning time is 2s-1min each time; preferably, the cleaning solution comprises 70-80% alcohol;
in the technical scheme, the drying in the step (4) can be carried out at room temperature or under heating, and the drying time is 1min-2 h.
In the above-mentioned technical scheme, the elution in step (5) is performed for 5s-5min, and the elution solution can be high-concentration water, PBS, TE buffer solution, downstream PCR reaction solution, etc. with appropriate concentration or convenient storage, preferably, the TE buffer solution is used in the present invention, and the specific components of the TE buffer solution are: 10mM Tris-HCl, 1mM EDTA (pH 8.0).
The invention also provides an extraction kit of the shellfish genome DNA, which specifically comprises a 3D printing special-shaped functional body, a lysate, a cleaning solution, an eluent, a protease K, RNA digestive enzyme, a fixing frame and an instruction book, wherein the 3D printing special-shaped functional body is a micro-element which is prepared from photosensitive resin or thermoplastic plastic serving as a raw material by 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 the umbrella-shaped structure are connected in parallel by connecting areas; 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 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-shaped functional bodies.
Preferably, the 3D printing special-shaped functional body has a structure which contains 8 nucleic acid binding regions and is matched with 8 parallel tubes for use; the mount is can be fixed, 12 8 rigid structure that ally oneself with row 3D that arrange print special shape functional body, match 96 orifice plates and use for 12 3D prints special shape functional body and can remove simultaneously along with the mount removes, thereby when matching 96 orifice plates and using, draws the flux once and improves to 96 samples.
The invention also provides application of the method for extracting shellfish genome DNA by using the 3D printing special-shaped functional body, wherein the application is to obtain template DNA according to the method for extracting shellfish genome DNA by using the 3D printing special-shaped functional body, and detect shellfish pathogenic microorganisms by combining with a PCR amplification technology. The method specifically comprises the following steps: non-disease based diagnostic and therapeutic objectives, namely: not to a living human or animal body, while not having the direct objective of obtaining a disease diagnosis or a health condition; the template nucleic acid obtained by the method for obtaining the template nucleic acid is amplified by a nucleic acid amplification technology, and various pathogens in the shellfish are detected, so that the control and early warning of the shellfish food-borne pathogens are controlled, and the safety of shellfish food is guaranteed.
Specifically, the PCR amplification techniques described above include: conventional PCR, Nested PCR (Nested-PCR), Recombinase Polymerase Amplification (RPA), Loop-mediated isothermal Amplification (LAMP), Real-time fluorescent quantitative PCR (Real-time PCR, RT-PCR), and the like.
Has the advantages that:
the method for quickly extracting nucleic acid from shellfish provided by the invention can efficiently, simply, quickly and high-flux extract and separate target nucleic acid, and has the advantages of being obviously advanced in the prior art:
1) according to the method for rapidly extracting the genome DNA from the shellfish, the 3D printing special-shaped functional body is adopted for separating and purifying the nucleic acid, and the functional body is rapidly transferred by mechanical force, 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 low general flux of the existing extraction technology;
2) the method for rapidly extracting nucleic acid from shellfish provided by the invention adopts 3D printing special-shaped functional bodies 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; whereas magnetic separation usually requires 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 connectors 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 method for rapidly extracting nucleic acid from shellfish provided by the invention does not depend on complex operations such as centrifugation, liquid transfer and the like, can realize manual low-labor-density high-throughput nucleic acid separation, and the whole process can be performed at 1m2The operation is finished in the operating environment, so that the space is saved, and the use flexibility is high; meanwhile, the 3D printing special-shaped functional body-based plasmodium nucleic acid extraction method can provide mechanical driving force through a programmed device, realizes automatic application, further reduces the labor cost, and is favorable forPromote the development and clinical application of the PCR in vitro diagnosis technology of shellfish food-borne pathogens
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 of example 1 of the present invention, wherein lanes 1-3 are frozen corbicula fluminea tissue samples # 1, # 2 and # 3, respectively, lane 4 is a negative control, and lane M is DL2000 Marker.
FIG. 3 is an agarose gel electrophoresis of example 2 of the present invention, wherein lane M is DL2000 Marker, lane 1 is a negative reference, and lanes 2-6 are frozen corbicula fluminea tissue samples 1#, 2#, 3#, 4#, and 5# suspected of containing norovirus.
FIG. 4 is an agarose gel electrophoresis image of example 3 of the present invention, wherein lane M is DL2000 Marker, and lanes 1-8 are frozen corbicula fluminea 1# -8# suspected of containing norovirus, respectively.
FIG. 5 is an agarose gel electrophoresis of example 4 of the present invention, wherein lane 1 is a negative control, lane M is DL2000 Marker, and lanes 2-9 are frozen and thawed clam tissue samples # 1- # 8.
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, practical 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’-actgggataatacgatagaag-3’
SEQ ID NO:2：
5’-gtgcgttaggattagttatgt-3’
SEQ ID NO:3：
5’-actgggataatacgatagaag-3’
SEQ ID NO:4：
5’-gtgcgttaggattagttatgt-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 prints special shape function body for being similar to umbrella structure, two 3D print special shape function body by nucleic acid bonding region 1, handle area 2 and joining region 3 constitute, nucleic acid bonding region 1 is the cone, handle area 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 of handle area 2, another bottom surface and joining region 3 of handle area 2 are connected, connect two single 3D print special shape function bodies on joining region 3 side by side, form the duplex 3D and print special shape function body. The kit specifically comprises two monomer functional bodies 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 the handle region 2 is 35 mm.
1 frozen corbicula fluminea is taken, 3 parts of tissue are picked and respectively placed in 3 EP tubes with the number of 1.5mL, 200 muL of lysis solution (100mmol of Tris-HCl, 25mmol of EDTA, 500mmol of NaCl and 1% SDS) and 5 muL of proteinase K solution are added into the EP tubes with the numbers of 1#, 2#, 3#, 1# and 2#, uniformly mixed by shaking, placed in a water bath with the temperature of 55 ℃ for 2h, taken out, added with 150mL of absolute ethyl alcohol into the EP tubes and placed into a 3D printing special-shaped functional body, a nucleic acid binding area of the 3D printing special-shaped functional body is stretched into the solution, a handle area is held by hand or machinery, the 3D printing special-shaped functional body is gently shaken and uniformly mixed by shaking, the 3D printing special-shaped functional body is transferred into an EP tube with the volume of 300 muL and provided with a washing buffer (75% alcohol) after 10 seconds, the nucleic acid binding area is stretched into a washing buffer, a handle area is held by hand or machinery, the functional body is shaken up and down for 5s, transferring in the washing buffer for 2-3 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 blowing incubator at 37 ℃ for 1 minute and is placed in 40 mu L of elution Buffer (1 XTE), the nucleic acid binding area is stretched into the elution Buffer, the handle area is held by hands or machinery, and the elution is carried out by gently shaking for 30 s. The obtained eluent is shellfish genome DNA, and the extracted nucleic acid is frozen at-20 ℃ for standby.
The sample # 3 was extracted using a commercial kit (Biomiga, GD2211 tissue genomic DNA extraction kit) and the procedure was performed according to the kit instructions. The extracted nucleic acid is used as a positive reference and is frozen at-20 ℃ for standby.
The DNA is used for PCR amplification, each 25 mu L system contains corbicula Ruditapes philippinarum tyr9 gene shown as SEQ ID NO:1 and SEQ ID NO:2, each 0.5 mu L of primer (10pM), 2 mu L, rTaq 0.25.25 mu L of template nucleic acid (shellfish nucleic acid), 10 xBuffer 2.5 mu L, 2 mu L of dNTPs, 17.25 mu L of deionized water, and sterile distilled water as negative reference template. PCR conditions were 95 ℃ for 4 min; 35 cycles of 95 ℃ for 30seconds, 55 ℃ for 30seconds and 72 ℃ for 60 seconds, extension at 72 ℃ for 5 minutes, gel electrophoresis of the obtained PCR product with 2% agarose, and observation under ultraviolet light after electrophoresis, as shown in FIG. 2.
The results are illustrated below: the electrophoresis pore channels from left to right are respectively 1#, 2#, 3#, negative water reference and marker, as shown in fig. 2, the negative reference has no specific band, the DNA obtained by the extraction method of the invention of 1# and 2# is the same as the PCR amplification result of the DNA of the sample 3# extracted by a commercial kit, the specific band is arranged at the same position, and the brightness of 1# and 2# is higher than 3#, which indicates that the nucleic acid sample extracted by the method of the invention has higher quality.
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 separation and purification process is less than 2min, and the operation is quick; and the whole operation process can be within 1m2The operation environment is finished, the space is saved, and the use flexibility is high.
Example 2
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, quintuplet 3D 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 5 single 3D side by side on joining region 3 and print special shape function body, form quintuplet 3D and print special shape function body. The kit specifically comprises a quintuplet functional body with 5 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 35 mm.
1 frozen corbicula fluminea suspected of containing norovirus is taken, 5 parts of tissues are respectively placed in 5 EP tubes with the volume being 1.5mL, the numbers being 1#, 2#, 3#, 4# and 5#, 1#, 2#, 3#, 4# and 5# are respectively added with 200 muL of lysis solution (100mmol of Tris-HCl, 25mmol of EDTA, 500mmol of NaCl and 1% of SDS), 5 muL of protease K solution is shaken and mixed evenly, placed in a water bath with the temperature being 55 ℃ for 1h, taken out, 150mL of absolute ethyl alcohol is added into the EP tubes and is placed into a 3D printing special-shaped functional body, the nucleic acid binding area of the 3D printing special-shaped functional body is stretched into the solution, a handle area is held by hand, the 3D printing special-shaped functional body mixed solution is shaken evenly, the 3D printing special-shaped functional body is transferred into an EP tube with the volume being 300 muL of a pre-washing buffer (75% alcohol) after 20 seconds, the nucleic acid binding area is stretched into a washing buffer, holding the handle area by hand, slightly shaking the functional body up and down for 10s, and repeatedly transferring 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, extending a nucleic acid binding region of the 3D printing special-shaped functional body into a 0.2mL PCR reaction tube which is preset with 50 μ L of PCR reaction liquid (containing 1 μ L, rTaq 0.5.5 μ L, 10 XBuffer 5.0 μ L, dNTPs 4.0 μ L and deionized water 38.5 μ L of amplification primers (10pM) shown as SEQ ID NO:3 and SEQ ID NO: 4), slightly shaking for 30s to remove the 3D printing special-shaped functional body, using the eluted PCR reaction liquid for PCR amplification, wherein the PCR condition is 95 ℃ for 4 min; 35 cycles of 95 ℃ for 30seconds, 55 ℃ for 30seconds and 72 ℃ for 60 seconds, extension at 72 ℃ for 5 minutes, gel electrophoresis of the obtained PCR product with 2% agarose, and observation under ultraviolet light after electrophoresis, as shown in FIG. 3.
The results show that from left to right electrophoresis channels are marker, negative water reference, samples # 1#, 2#, 3#, 4# and 5#, respectively. Negative reference is a non-specific band, and a 85bp product band is amplified from the sample No. 1-5, which indicates that the corbicula fluminea carries norovirus particles. Meanwhile, the embodiment also shows that the method provided by the invention has a wide application prospect in the detection of shellfish food-borne pathogens.
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 eight-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 8 frozen corbicula fluminea 1-8# suspected to contain norovirus, picking a small amount of tissue and placing the tissue in 8 sample holes of 8 parallel EP tubes. Adding 80 microliter of lysis solution (100mmol of Tris-HCl, 25mmol of EDTA, 500mmol of NaCl and 1% of SDS) into an EP tube, blowing and uniformly mixing 5 microliter of proteinase K solution with an 8-row liquid transfer gun, standing at room temperature for 60 minutes, adding 60 milliliter of absolute ethyl alcohol and putting into a 3D printing special-shaped functional body, enabling a nucleic acid binding region of the 3D printing special-shaped functional body to extend into the solution, holding a handle region 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 which is preset with 100 microliter of cleaning buffer (75% alcohol) after 10 seconds, slightly extending the nucleic acid binding region into the cleaning buffer, holding the handle region by hand, shaking the 3D printing special-shaped functional body up and down for 5-10 seconds, and repeatedly transferring in the cleaning buffer for 2-3 times; taking out the 3D printing special-shaped functional body from the washing Buffer, throwing away residual liquid drops on the 3D printing special-shaped functional body, drying in a blast incubator at 37 ℃ for 2 minutes, extending a nucleic acid binding region of the 3D printing special-shaped functional body into 50 mu L of PCR reaction liquid (containing 1 mu L, rTaq 0.5 mu L, 10 XBuffer 5 mu L, dNTPs 4 mu L and deionized water 38.5 mu L of each of primers (10pM) shown as SEQ ID NO:3 and SEQ ID NO: 4), slightly shaking for 30s, then removing the 3D printing special-shaped functional body, and carrying out PCR on the obtained reaction liquid: 4min at 95 ℃; 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 used for 2% agarose for gel electrophoresis, and the result is observed under ultraviolet light after the electrophoresis is finished, as shown in figure 4.
The results are illustrated below: from left to right, electrophoresis channels are marker and sample 1-8# respectively. Specific product bands appear in No. 1#, No. 2#, No. 4#, No. 5# and No. 8# corbicula fluminea at 85bp, which indicates that No. 1#, No. 2#, No. 4#, No. 5# and No. 8 corbicula fluminea carry the Noro virus particles, and No. 3#, No. 6# and No. 7 corbicula fluminea carry no noro virus particles.
The embodiment shows that the method provided by the invention has wide application prospect in the detection of shellfish food-borne pathogens, is simple to operate, does not need time-consuming and tedious steps such as pipetting, centrifuging and the like, and has low manual labor density and high flux.
Example 4
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 eight-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.
Taking 1 case of frozen corbicula fluminea, directly putting tissue blocks with the weight of about 500mg into a mortar sterilized at high temperature and high pressure, adding liquid nitrogen for quick grinding until the tissue becomes soft, adding a small amount of liquid nitrogen for grinding again, and repeating the steps for three times. Then, a tissue sample of about 150-. Centrifuging at 12000r/min for five minutes, discarding the precipitate, adding 400 μ L chloroform, covering the centrifuge tube, shaking by hand, mixing for 15s, and standing at room temperature for 10 min. Then, the mixture was centrifuged at 12000g at 4 ℃ for 15min, and the aqueous phase was transferred to a new EP tube to obtain a solution containing RNA.
Putting 80 mu L of RNA solution into each reaction hole in an 8-linked calandria, putting an eight-linked function body, enabling a nucleic acid binding region of a 3D printing special-shaped function body to stretch into the solution, adding 55 mu L of isopropanol, slightly shaking the 3D printing special-shaped function body to mix the solution uniformly, transferring the eight-linked function body into an EP (ultraviolet) tube preset with 200 mu L of cleaning buffer (75% alcohol) after 5-10 seconds, enabling the nucleic acid binding region of the 3D printing special-shaped function body to stretch into the cleaning buffer, slightly shaking the eight-linked function body for 5-10s, and repeatedly transferring in the cleaning buffer for 1 time; and taking the octameric functional bodies out of the washing Buffer, throwing away residual liquid drops on the octameric functional bodies, drying at room temperature for 5min, placing in a 40 mu L elution Buffer, extending the nucleic acid binding area of the 3D printing special-shaped functional bodies into the elution Buffer, and slightly shaking for 30s for elution.
The eluate was subjected to reverse transcription using a reverse transcription kit (Takara, Code No. RR047A) to prepare cDNAs according to the protocol, and 1. mu.L each of the obtained products was used as a template for PCR amplification to prepare a PCR reaction solution: each 25. mu.L system contained 0.5. mu.L each of the primers shown in SEQ ID NO:1 and SEQ ID NO:2 (10pM), 1. mu. L, ExTaq 0.25.25. mu.L of template, 2.5. mu.L of 10 XExBuffer, 2. mu.L of dNTPs, 17.25. mu.L of deionized water, and sterile distilled water as a negative reference template. PCR conditions were 95 ℃ for 4 min; 35 cycles of 95 ℃ for 30seconds, 55 ℃ for 30seconds and 72 ℃ for 60 seconds, extension at 72 ℃ for 5 minutes, gel electrophoresis of the obtained PCR product with 2% agarose, and observation under ultraviolet light after electrophoresis, as shown in FIG. 5.
The results are illustrated in FIG. 5, from left to right, which shows the PCR amplification products of the negative reference, marker and 8 samples, wherein 8 samples show the target band, the negative reference shows no target band, and 8 solutions used as templates all contain the target nucleic acid.
This example demonstrates that the octant functionality via 3D printing enables high throughput RNA extraction with RNA extraction quality that is sufficient for downstream PCR diagnostic applications.
The embodiment also shows that the nucleic acid extraction by using the octa-linkage functional body can simultaneously extract target nucleic acid, has high flux, effectively breaks through the bottleneck that the non-magnetic solid-phase separation depends on complicated operations such as multi-step centrifugation and liquid transfer and has low separation flux, and is beneficial to realizing automation.
While the 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 practiced with modification of, or with appropriate modification and combination of, the methods described herein without departing from the spirit and scope of the invention. It is specifically intended to point out all such substitutions and modifications: such as the splitting agent, the washing solution, the reasonable changes in the composition of the eluent, such as the reasonable shape, size, surface, internal structure adjustments of the 3D printing shaped features, and the like, will be apparent to those skilled in the art and are considered to be included within the spirit, scope and content of the present invention.
SEQUENCE LISTING
<110> research institute of marine aquatic science in Liaoning province
<120> shellfish genome DNA extraction method based on 3D printing special-shaped functional body, kit and application thereof
<130>2020
<160>4
<170>PatentIn version 3.5
<210>1
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
actgggataa tacgatagaa g 21
<210>2
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
gtgcgttagg attagttatg t 21
<210>3
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
actgggataa tacgatagaa g 21
<210>4
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
gtgcgttagg attagttatg t 21
Claims (10)
1. A shellfish genome DNA extraction method based on 3D printing special-shaped functional bodies is characterized by comprising the following steps:
(1) cracking the shellfish 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) by hand or machine to bind nucleic acid;
(3) putting the nucleic acid binding region of the 3D printing special-shaped functional body which is finished in the step (2) into a cleaning solution in a hand-held or machine-held mode for cleaning;
(4) taking out the 3D printing special-shaped functional body after the step (3) is completed, and drying;
(5) placing the nucleic acid binding region of the 3D printing special-shaped functional body which is subjected to the step (4) into an eluent in a handheld manner or a machine-held manner, and eluting to obtain the eluent, namely the genome DNA;
the shellfish sample to be detected comprises the whole shellfish, any components of any part of the shellfish, shellfish tissues or each organ.
2. The shellfish genomic DNA extraction method as claimed in claim 1, wherein the lysate in step (1) comprises: CTAB lysate, NaHCO3Lysis solution, Chelex lysis solution, proteinase K lysis solution, SDS lysis solution or Trizol lysis solution.
3. The shellfish genomic DNA extraction method of claim 2, wherein the SDS lysate comprises the following components: 1-10% SDS, 0-30 μ g/mL proteinase K.
4. The shellfish genomic DNA extraction method of claim 1, wherein the lysate may or may not include RNA digesting enzymes; the treatment time of the cracking process is 1min-24h, and the treatment temperature is 0-100 ℃.
5. The shellfish genomic DNA extraction method as claimed in claim 1, wherein the 3D printing special-shaped functional body in step (2) is a micro-element with an umbrella-shaped structure obtained by 3D printing technology using photosensitive resin or thermoplastic as raw material, and comprising a nucleic acid binding region and a handle region;
the nucleic acid binding area 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; the 3D printing special-shaped functional body comprises 1 or at least two nucleic acid binding regions, and when the number of the nucleic acid binding regions is at least two, the handle regions are connected together side by virtue of the connecting regions; the surface of the nucleic acid binding region is a flat or uneven microstructure, and the uneven microstructure comprises a screw thread susceptor structure, a groove structure, a porous structure and a convex structure; the porous structure comprises nanometer and micrometer sizes, and the shape of the porous structure comprises a sphere, a cube or an irregular shape; the nucleic acid binding region is loaded or unloaded with a particulate material, the particulate material comprises inorganic salt particles and metal particles, and the inorganic salt particles comprise silicon dioxide, titanium dioxide and manganese dioxide; the nucleic acid binding region is modified with or without surface functional groups, including hydroxyl, carboxyl, amino.
6. The shellfish genomic DNA extraction method as claimed in claim 5, wherein the nucleic acid binding region, the handle region and the connecting region are prepared integrally or separately; when the parts are prepared separately, they are assembled by bonding.
7. The shellfish genome DNA extraction method according to claim 1, characterized in that, in the step (2), the nucleic acid is combined with or without an auxiliary combination solvent, wherein the auxiliary combination solvent comprises one or a mixture of isopropanol and absolute ethyl alcohol, and the volume of the auxiliary combination solvent is 0.6-0.8 times of the volume of the lysate; the binding time is 5s-5 min;
the cleaning in the step (3) is carried out for 1 to 5 times; the cleaning time is 2s-1min each time; the cleaning solution comprises 70-80% of alcohol;
the drying in the step (4) can be carried out at room temperature or under heating, and the drying time is 1min-2 h.
The eluent in the step (5) comprises water, PBS, TE buffer solution and downstream PCR reaction solution; the elution time is 5s-5 min.
8. The shellfish genome DNA extraction kit based on the 3D printing special-shaped functional body is characterized by comprising the 3D printing special-shaped functional body, lysate, cleaning solution, eluent, protease K, RNA digestive enzyme, a fixing frame and an instruction book, wherein the 3D printing special-shaped functional body is a micro-element which is prepared from photosensitive resin or thermoplastic plastic serving as a raw material and is provided with an umbrella-shaped structure and composed of a nucleic acid binding area and a handle area by a 3D printing technology, and a plurality of micro-elements with the umbrella-shaped structure are connected in parallel by connecting areas;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.
9. The application of the shellfish genome DNA extraction method based on the 3D printing special-shaped functional body is characterized in that the application is that shellfish genome DNA is extracted according to the method of any one of claims 1-7, and shellfish pathogenic microorganisms are detected by combining with a PCR amplification technology. .
10. The use of claim 9, wherein the PCR amplification technique comprises: conventional PCR, nested PCR, recombinase polymerase amplification, loop-mediated isothermal amplification and real-time fluorescent quantitative PCR.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010584270.7A CN111778236A (en) | 2020-06-23 | 2020-06-23 | Shellfish genome DNA extraction method based on 3D printing special-shaped functional body, kit and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010584270.7A CN111778236A (en) | 2020-06-23 | 2020-06-23 | Shellfish genome DNA extraction method based on 3D printing special-shaped functional body, kit and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111778236A true CN111778236A (en) | 2020-10-16 |
Family
ID=72757172
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010584270.7A Pending CN111778236A (en) | 2020-06-23 | 2020-06-23 | Shellfish genome DNA extraction method based on 3D printing special-shaped functional body, kit and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111778236A (en) |
Cited By (1)
| 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 |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110092687A1 (en) * | 2008-04-22 | 2011-04-21 | Peter Bendzko | Stable lysis buffer mixture for extracting nucleic acids |
| US20140120544A1 (en) * | 2012-10-25 | 2014-05-01 | Neumodx Molecular, Inc. | Method and materials for isolation of nucleic acid materials |
| TW201923076A (en) * | 2017-07-27 | 2019-06-16 | 澳大利亞商行科技有限公司 | Method of isolating nucleic acid |
| CN110343742A (en) * | 2019-07-23 | 2019-10-18 | 中国海洋大学 | A kind of micro shellfish DNA extraction method for high-throughput sequencing library preparation |
| 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 |
| 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 |
-
2020
- 2020-06-23 CN CN202010584270.7A patent/CN111778236A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110092687A1 (en) * | 2008-04-22 | 2011-04-21 | Peter Bendzko | Stable lysis buffer mixture for extracting nucleic acids |
| US20140120544A1 (en) * | 2012-10-25 | 2014-05-01 | Neumodx Molecular, Inc. | Method and materials for isolation of nucleic acid materials |
| 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 |
| TW201923076A (en) * | 2017-07-27 | 2019-06-16 | 澳大利亞商行科技有限公司 | Method of isolating nucleic acid |
| CN110343742A (en) * | 2019-07-23 | 2019-10-18 | 中国海洋大学 | A kind of micro shellfish DNA extraction method for high-throughput sequencing library preparation |
| 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 |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| "3D printed integrated separator with hybrid micro-structures for high throughput and magnetic-free nucleic acid separation from organism samples", SEPARATION ANDPURIFICATIONTECHNOLOGY, vol. 271, pages 1 - 11 * |
| 李蒙航: "基于微尺度载体 的高通量核酸固相分离 过程研究", 《中国优秀硕士学位论文全 文数据库工程科技I 辑》, pages 014 - 181 * |
Cited By (1)
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3787354B2 (en) | Nucleic acid isolation method | |
| CN1187362C (en) | Solid-phase nucleic acid isolation | |
| CN111607489B (en) | Nucleic acid extraction system and method based on 3D printing micro-component | |
| US20190225958A1 (en) | Method for enrichment and purification of cell-free dna from body fluid for high-throughput processing | |
| KR20120138766A (en) | Nucleic acid extraction from complex matrices | |
| CN111154835A (en) | Method for constructing ATAC-seq sequencing library | |
| CN111607635B (en) | Blood genome DNA extraction method based on 3D printing special-shaped functional body and application kit thereof | |
| WO2012155577A1 (en) | Method for separating and purifying rna from biomaterial | |
| CN111778236A (en) | Shellfish genome DNA extraction method based on 3D printing special-shaped functional body, kit and application thereof | |
| JP2020536491A5 (en) | ||
| CN111647596A (en) | Extraction method of microsporidian DNA based on 3D printing functional body, kit and application thereof | |
| CN110923338A (en) | Microarray chip capable of detecting various bacterial genome DNAs and preparation method thereof | |
| CN110863037A (en) | Microarray chip capable of detecting various fungal genome DNAs (deoxyribonucleic acids) and preparation method thereof | |
| CN107365766B (en) | Method for extracting RNA of mould spore by mechanical crushing method | |
| CN107400719B (en) | Tussah microsporidian detection primers and application thereof | |
| CN110616254A (en) | PET (polyethylene terephthalate) series carrier forward sequencing primer and sequencing method | |
| JP2005253464A (en) | Simple method for extracting nucleic acid | |
| CN114874998B (en) | In-vitro separation culture method for carp edema virus | |
| CN108929875B (en) | Method for separating embryonic stem cells and fixing embryonic stem cells on surface based on DNA aptamer | |
| Anant et al. | [3] Isolation of low molecular weight DNA from bacteria and animal cells | |
| US20210198807A1 (en) | Methods and systems for production of dna libraries directly from a stool sample for 16s metagenomics next generation sequencing | |
| CN100340670C (en) | Application of Nano carbon tubes in biology | |
| CN110846307A (en) | Use method of magnetic bead method blood genome DNA extraction kit | |
| CN112979778A (en) | Polypeptide and nucleic acid for mediating endocytosis of foreign protein, application thereof and method for gene editing in allopodic new shrimps | |
| JP2014060965A (en) | Method of crushing cell and method of separating nucleic acid of cell |
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 |