CN102834519A - 从复杂基质中提取核酸 - Google Patents
从复杂基质中提取核酸 Download PDFInfo
- Publication number
- CN102834519A CN102834519A CN2011800080195A CN201180008019A CN102834519A CN 102834519 A CN102834519 A CN 102834519A CN 2011800080195 A CN2011800080195 A CN 2011800080195A CN 201180008019 A CN201180008019 A CN 201180008019A CN 102834519 A CN102834519 A CN 102834519A
- Authority
- CN
- China
- Prior art keywords
- sample
- gac
- slurry
- test kit
- volume
- 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
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 15
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 15
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 15
- 238000000605 extraction Methods 0.000 title description 4
- 239000000463 material Substances 0.000 claims abstract description 22
- 239000002002 slurry Substances 0.000 claims abstract description 20
- 239000006228 supernatant Substances 0.000 claims abstract description 13
- 235000013162 Cocos nucifera Nutrition 0.000 claims abstract description 12
- 244000060011 Cocos nucifera Species 0.000 claims abstract description 12
- 108091005804 Peptidases Proteins 0.000 claims abstract description 9
- 238000003860 storage Methods 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000000034 method Methods 0.000 claims description 27
- 239000011248 coating agent Substances 0.000 claims description 17
- 238000000576 coating method Methods 0.000 claims description 17
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical group C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 claims description 15
- 229920000642 polymer Polymers 0.000 claims description 14
- 239000002594 sorbent Substances 0.000 claims description 13
- 239000000843 powder Substances 0.000 claims description 11
- 238000013016 damping Methods 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 9
- 102000035195 Peptidases Human genes 0.000 claims description 7
- 239000000284 extract Substances 0.000 claims description 7
- 241001465754 Metazoa Species 0.000 claims description 4
- 230000008021 deposition Effects 0.000 claims description 4
- 235000013305 food Nutrition 0.000 claims description 4
- 210000002700 urine Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000003463 adsorbent Substances 0.000 abstract description 5
- 239000000243 solution Substances 0.000 abstract description 5
- -1 DNA and RNA Chemical class 0.000 abstract description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract 4
- 239000004365 Protease Substances 0.000 abstract 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 2
- 229920002307 Dextran Polymers 0.000 abstract 1
- 239000000872 buffer Substances 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- 235000013312 flour Nutrition 0.000 abstract 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 abstract 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 abstract 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 abstract 1
- 238000003260 vortexing Methods 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 21
- 239000002512 suppressor factor Substances 0.000 description 8
- 238000007400 DNA extraction Methods 0.000 description 6
- 239000002689 soil Substances 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- 210000003608 fece Anatomy 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 241000223935 Cryptosporidium Species 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 241000224466 Giardia Species 0.000 description 4
- 230000002550 fecal effect Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- QJZYHAIUNVAGQP-UHFFFAOYSA-N 3-nitrobicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid Chemical compound C1C2C=CC1C(C(=O)O)C2(C(O)=O)[N+]([O-])=O QJZYHAIUNVAGQP-UHFFFAOYSA-N 0.000 description 3
- 241001263478 Norovirus Species 0.000 description 3
- 239000003610 charcoal Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000004021 humic acid Substances 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 241001504639 Alcedo atthis Species 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000224467 Giardia intestinalis Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000005352 clarification Methods 0.000 description 2
- 230000000593 degrading effect Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 229940085435 giardia lamblia Drugs 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000002203 pretreatment Methods 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 241000193163 Clostridioides difficile Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 241000223936 Cryptosporidium parvum Species 0.000 description 1
- 241000709661 Enterovirus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000002944 PCR assay Methods 0.000 description 1
- 206010037075 Protozoal infections Diseases 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 201000010989 colorectal carcinoma Diseases 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000013777 protein digestion Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000002352 surface water Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 239000002351 wastewater Substances 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/02—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material
- B01J20/20—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising inorganic material comprising free carbon; comprising carbon obtained by carbonising processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
- B01J20/3204—Inorganic carriers, supports or substrates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3234—Inorganic material layers
- B01J20/3236—Inorganic material layers containing metal, other than zeolites, e.g. oxides, hydroxides, sulphides or salts
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3244—Non-macromolecular compounds
- B01J20/3246—Non-macromolecular compounds having a well defined chemical structure
- B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
- B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
- B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
- B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
- B01J20/3268—Macromolecular compounds
- B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/50—Aspects relating to the use of sorbent or filter aid materials
- B01J2220/64—In a syringe, pipette, e.g. tip or in a tube, e.g. test-tube or u-shape tube
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Inorganic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
本公开描述了一种吸附剂以及从复杂基质(如粪便样品和水样品)中提取核酸(如DNA和RNA)的示例性方案。所述吸附剂是用如聚乙烯吡咯烷酮、葡聚糖或椰子粉的材料涂敷的活性炭。所述吸附剂可用于微量离心旋柱中，其中，其可以作为浆料在贮藏溶液中存在。所述样品可以如下制备：在缓冲溶液中涡旋，离心，将蛋白酶加入到上清液中及使上清液通过含有经涂敷的活性炭的微量离心旋柱。在试剂盒中可装入包括缓冲液、蛋白酶和旋柱的主要组成部分。
Description
政府权益的声明
按照no.1U01 AI075396-01基金的规定，美国政府可以拥有本公开的权利。
相关申请的交叉引用
根据35U.S.C.§119(e)，本申请要求享有2010年2月16日递交的美国临时专利申请第61/304,913号的优先权，其公开的全部内容在此通过引用的方式并入本申请。
本公开的背景
技术领域
本公开主要涉及一种从样本（例如，人类样本和环境样品）中纯化总核酸（包括DNA和RNA）的方法。人类样本的实例包括（仅仅出于举例的目的）：粪便、组织、尿和其他样本。环境样品的实例包括（仅仅出于举例的目的）：水、土壤和其他样品。本公开还适用于可以从中提取总核酸样品的农业、兽医医学、食品和其他样品。更具体地，本公开涉及一种新型材料收集法及其使用程序。
背景技术
人类粪便样本通常用于临床实验室中诊断包括结肠直肠癌以及病毒、细菌和原生动物感染在内的多种疾病。据估计，在美国每年总共对粪便样本进行超过610万例的体外诊断(IVD)测试。这些测试大多数使用如直接显微镜检查、培养或免疫测定的成熟技术。然而，日益增加的传染原（包括腺病毒、肠道病毒、诺如病毒、轮状病毒、大肠杆菌(E.coli)和艰难梭状芽孢杆菌(C.difficile)），现正在使用分子方法进行识别。
粪便是一种复杂基质，其包含许多在大多数分子测定的核心阶段抑制PCR测定的蛋白质、多糖和小分子。因此，实验室工作人员必须在分子分析之前分离粪便样本中包含的DNA和/或RNA。已有几种用于从粪便中提取DNA的商品。其中最受欢迎的是Qiagen制造的QIAamp粪便DNA袖珍试剂盒(QIAamp Stool DNA Mini Kit)。该产品要求使用者在使用微量离心旋柱(microcentrifuge spin column)收集和清洗最终DNA分离物之前通过细胞裂解、抑制剂吸附和蛋白质消化几个步骤对粪便样本进行处理。得到的DNA适用于大多数应用。然而，该提取过程漫长、复杂并且样品交叉污染的风险增加。另一种可选方案是使用自动化的DNA提取仪器，如Roche AppliedScience制造的MagNA Pure系列的仪器。然而，这些仪器需要大量的资金投资以及附加的对于粪便使用的预处理步骤。
因此，迫切需要具有至少一种下列特点的从样本中纯化DNA和/或RNA：更快、简便、资金投资少，并且需要更少的预处理步骤。
发明内容
本公开满足前述需要并且允许使用分子方法检测隐孢子虫属(Cryptosporidium)和贾第虫属(Giardia)物种等，这导致速度、灵敏性、再现性的显著提高以及从本文讨论中明显体现的其他优点。
本公开包括一种从人类样本中纯化总核酸(包括DNA和RNA)的方法。其他样本类型也被考虑在内，包括，例如，土壤样品、水样品、兽医医学样品、农业样品和食品样品。本公开包括一种新型材料收集法及其使用程序。所述核酸提取程序可以包括两个阶段：
在第一阶段中，将样品在缓冲液中均质化并通过短暂离心澄清。然后使用蛋白酶溶解悬浮细胞并降解样品中存在的任何蛋白质PCR抑制剂。
在第二阶段中，使用酶分解过的样品通过含有新型吸附剂的微量离心柱。该吸附剂包括涂敷有聚乙烯吡咯烷酮的活性炭，其从样品中除去小分子PCR抑制剂同时使纯化的DNA流过所述柱进入收集管。
根据本公开的一个方面，从样品中提取核酸的方法包括：将一部分样本在缓冲液中均质化，离心该均质化的样品以得到沉淀(pellet)和上清液，分开上清液和沉淀，将蛋白酶加入到上清液中，以及使上清液通过包含经涂敷的活性炭的吸附剂。
所述样品可为粪便样品、组织样品、尿样品、血液样品、水样品、土壤样品、农业样品、兽医医学样品或食品样品中的一种。所述样品的质量可为0.2g，或者体积可为0.2μL。所述上清液可被孵育。所述活性炭可以用聚乙烯吡咯烷酮、葡聚糖或椰子粉涂敷。经涂敷的活性炭可以以活性炭浆料的形式存在。所述浆料的重量/体积比可为约5%~约20%，并且所述浆料可以包括约1%~约10%重量/体积的涂敷材料，所述涂敷材料可为聚乙烯吡咯烷酮、葡聚糖或椰子粉中的一种。
根据本公开的另一个方面，一种用于从样品中提取核酸的试剂盒包括：缓冲溶液的容器，蛋白酶的容器和一个或多个旋柱。所述旋柱包含经涂敷的活性炭。
所述经涂敷的活性炭可以包括活性炭浆料。所述活性炭可以用聚乙烯吡咯烷酮、葡聚糖或椰子粉涂敷。所述浆料的重量/体积比可为约5%~约20%，并且所述浆料可以包括约1%~约10%重量/体积的涂敷材料，所述涂敷材料可为聚乙烯吡咯烷酮、葡聚糖或椰子粉中的一种。所述缓冲溶液可以用于在将一部分样品置于缓冲液中并涡旋时使该样品均质化。所述经涂敷的活性炭可以用于结合污染物，同时使核酸保持游离。
根据本公开的其他方面，吸附剂包括用涂敷材料涂敷的活性炭的浆料。所述浆料的重量/体积比可为约5%~约20%，并且所述浆料可以包括约1%~约10%重量/体积的涂敷材料，所述涂敷材料可为聚乙烯吡咯烷酮、葡聚糖或椰子粉中的一种。微量离心柱可以包括贮藏溶液中的吸附剂。所述贮藏溶液可以包括约0.1%~约10%重量/体积比的涂敷材料。
考虑到下列详细描述的说明和权利要求书，可以提出或显而易见的是本公开的其他特征、优点和实施方式。而且，将要理解的是，本公开的前述概述及下列详细描述的说明是示例性的并用来提供进一步的解释，而不会限制如权利要求所述的本公开的范围。
附图说明
为了进一步理解本发明，本申请包括附图，所述附图被引入并构成本说明书的一部分，图示说明本发明的实施方式，并且连同详细的说明一起用于解释本发明的原理。没有试图显示本发明的比基本理解本发明和实施本发明的各种方式所必需的结构更详细的结构细节。图中：
图1显示根据本公开的提取核酸的方法的示例性工作流程；以及
图2显示DNA和腐殖酸对经涂敷的和未经涂敷的活性炭的吸附性。
具体实施方式
参照在以下说明书中详细描述的非限定性的实施方式和实施例，更加全面地解释本公开的实施方式及其各种特征和有利细节。应当注意的是，即使在此没有明确地阐述，技术人员也会认可一个实施方式的特征可以与其它实施方式一起使用。可以省略众所周知的成分和处理技术的说明，从而不会不必要地使本公开的实施方式变得含混不清。在此所用的实施例仅仅用来方便理解本公开可以实施的方式并进一步使本领域技术人员能够实施本公开的实施方式。因此，本文中的实施例和实施方式不应构成对本公开的范围的限制，本公开的范围仅由所附权利要求和适用法律所限定。
所述核酸提取程序可以包括下列步骤：
样品可以在缓冲液中进行均质化并通过短暂离心澄清。然后可以使用蛋白酶溶解悬浮细胞和降解样品中存在的任何蛋白质PCR抑制剂。然而，也可以考虑在本公开中使用其他的酶。
使用酶消化过的样品通过含有新型吸附剂的微量离心柱。该吸附剂可包括涂敷有聚乙烯吡咯烷酮的活性炭，其从样品中除去小分子PCR抑制剂同时使纯化的DNA流过所述柱进入收集管。其他的吸附剂也被考虑并且在本公开的实质和范围内。
一种相关的提取产品可以推向市场待售，其包含下列组成部分：
·一个(1)含有25~50mL缓冲液的60-mL瓶
·一个(1)含有100~260μL的酶的微量离心管
·一个(1)含有五十(50)个微量离心旋柱的可重复密封的塑料袋
这些组成部分可以用于下列示例性的程序中，以从多个0.2g或0.2μL的人类粪便样本中提取DNA：
1.将0.2g或0.2μL粪便样品置于微量离心管中。
2.将0.5-mL的缓冲液加入到所述管中。涡旋所述管2分钟或直到所述样品完全均质化。
3.将管在200xg下离心30秒以使固体物质沉淀。
4.将45μL的各样品上清液转移到干净的微量离心管中。
5.将5μL的酶加入到各样品中。短暂混合。
6.在75°C孵育样品15分钟以溶解细胞和消化蛋白质抑制剂。
7.在样品孵育过程中，将适当数量的旋柱放入新的微量离心管中。在8,000xg下离心所述柱3分钟以除去贮藏溶液。将所述柱放入新的微量离心管。
8.孵育后，将用酶消化过的样品转移到所处理好的旋柱的顶部。
9.在8,000xg离心所述旋柱1分钟，使DNA提取物流通到所述微量离心管的底部。
上述时间和量是示例性的。如本公开考虑的，也可以使用其他时间或量以及或多或少的上述步骤。
所述缓冲液和蛋白酶可以用于溶解悬浮细胞和降解存在于样品中的任何蛋白质PCR抑制剂。空的微量离心柱和盖可由第三方卖主提供。
包含在微量离心柱内的吸附剂可以包括5~20%重量/体积(w/v)的在贮藏溶液中的活性炭(100-400目)的浆料。所述浆料可以在含有0.1~10%w/v的HEPES和/或涂敷材料的蒸馏水中制备。合适的涂敷材料包括聚乙烯吡咯烷酮、葡聚糖、椰子粉等。
实施例1
以1~10%(v/v)用聚乙烯吡咯烷酮涂敷活性炭并且在7.0~8.0的适当的pH下制备。经涂敷的活性炭有效地结合污染物和其他PCR抑制剂，同时使核酸保持完整和游离。如图2中所示，随着炭量的增加，未经涂敷的炭对DNA和腐殖酸的吸附增加。然而，当用PVP涂敷活性炭时，腐殖酸吸附与未经涂敷的炭的吸附保持相似，而DNA吸附下降。
实施例2
本公开使用制造商推荐的微生物DNA提取程序以QIAamp粪便DNA袖珍试剂盒作为基准。将6个0.2g人类粪便样品掺入大约1x105个小球隐孢子虫(Cyptosporidium parvum)和兰伯贾第虫(Giardia lamblia)生物体。在-20°C保藏样品5个月。使用本公开处理三个样品，而使用QIAamp方法处理余下的三个样品。使用特异性针对隐孢子虫属或贾第虫属基因组DNA的自建(in-house)PCR测定法分析纯化的DNA提取物。得到下列结果：
| 本公开 | QIAamp | |
| 隐孢子虫属产量(yield)(Ct) | 30.55±0.50 | 29.96±0.52 |
| 贾第虫属产量(Ct) | 26.84±0.62 | 26.00±0.51 |
| 提取物稳定性(ΔCt) | 0.79 | 0.42 |
| 6个样品的时间(分钟) | 30 | 94 |
| 处理步骤 | 8-10 | 32 |
| 样品转移的数目 | 2 | 4 |
本公开的再现性可与QIAamp相媲美，6x Ct值间的%CV小于10%。通过在-20°C保藏2周后获得ΔCt来测试本公开和QIAamp提取物的提取物稳定性。
需要注意的是，以PCR循环阈(Ct)值给出了DNA的产量，Ct值表示荧光信号上升超过基线的估计反应循环。Ct值越低，说明样品中存在的靶DNA越多。
与QIAamp方法相比，本公开提供了相同或更好的DNA质量和数量并且需要更少的步骤和更短的时间。
实施例3
本公开还对照Kingfisher自动化平台上的Ambion MagMax-96病毒RNA试剂盒进行了测试。两种方案均用于提取十个临床粪便样本中的诺如病毒(Norovirus)RNA，并且在LightCycler平台上通过PCR扩增分析提取物。
得到下列结果：
本公开制得了高质量和数量的RNA。在一些情况下，本公开胜过自动化Kingfisher提取方法。
实施例4
本公开对照MoBio Laboratories Inc.制造的
土壤DNA分离试剂盒对地表水和废水进行测试。通过Envirocheck和离心技术浓缩10L和1L水。使用本公开和MoBio产品分别对各样品重复进行DNA提取。在iCycler(Biorad)平台上使用SYBR Green对每份DNA提取物进行三次重复扩增。
得到下列结果：
在DNA的质量和数量方面，本公开均胜过MoBio产品。
虽然已经根据示例性的实施方式描述了本公开，但是本领域技术人员将认识到，在所附权利要求的实质和范围内进行改变可以实施本公开。上述给出的这些实施例仅仅出于说明目的，并不意味着穷尽列出了本公开的所有可能的设计、实施方式、应用或改进。
Claims (22)
1.一种从样品中提取核酸的方法，所述方法包括：
在缓冲液中使至少一部分样品均质化；
离心该均质化的样品部分，所述离心产生沉淀和上清液；
分开所述上清液和沉淀；
将蛋白酶加入到所述上清液中；和
使所述上清液通过吸附剂，所述吸附剂包含经涂敷的活性炭。
2.权利要求1所述的方法，其中，所述样品选自粪便样品、组织样品、尿样品、血液样品、水样品、土壤样品、农业样品、兽医医学样品和食品样品。
3.权利要求1所述的方法，其中，所述部分样品至少具有0.2g的质量或0.2μL的体积。
4.权利要求1所述的方法，其进一步包括孵育所述上清液。
5.权利要求1所述的方法，其中，所述活性炭用选自聚乙烯吡咯烷酮、葡聚糖和椰子粉中的材料涂敷。
6.权利要求1所述的方法，其中，该经涂敷的活性炭包括活性炭浆料。
7.权利要求6所述的方法，其中，所述浆料的重量/体积比为约5%~约20%。
8.权利要求6所述的方法，其中，所述浆料包含约1%~约10%重量/体积的涂敷材料。
9.权利要求8所述的方法，其中，所述涂敷材料选自聚乙烯吡咯烷酮、葡聚糖和椰子粉。
10.一种从样品中提取核酸的试剂盒，所述试剂盒包括：
缓冲溶液的容器；
蛋白酶的容器；和
至少一个含有经涂敷的活性炭的旋柱。
11.权利要求10所述的试剂盒，其中，所述经涂敷的活性炭包含活性炭浆料。
12.权利要求11所述的试剂盒，其中，所述浆料的重量/体积比为约5%~约20%。
13.权利要求1所述的试剂盒，其中，所述浆料包含约1%~约10%重量/体积的涂敷材料。
14.权利要求13所述的方法，其中，所述涂敷材料选自聚乙烯吡咯烷酮、葡聚糖和椰子粉。
15.权利要求10所述的试剂盒，其中，所述活性炭用选自聚乙烯吡咯烷酮、葡聚糖和椰子粉中的材料涂敷。
16.权利要求10所述的试剂盒，其中，在将一部分样品置于缓冲液中并涡旋时，所述缓冲液用于使所述样品均质化。
17.权利要求10所述的试剂盒，其中，所述经涂敷的活性炭用于结合污染物，同时使核酸保持游离。
18.一种吸附剂，其包含用涂敷材料涂敷的活性炭的浆料。
19.权利要求18所述的吸附剂，其中，所述浆料的重量/体积比为约5%~约20%。
20.权利要求18所述的吸附剂，其中，所述涂敷材料选自聚乙烯吡咯烷酮、葡聚糖和椰子粉。
21.一种微量离心柱，其包含在贮藏溶液中的权利要求18所述的吸附剂。
22.权利要求21所述的微量离心柱，其中，所述贮藏溶液包含约0.1%~约10%的重量/体积比的涂敷材料。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US30491310P | 2010-02-16 | 2010-02-16 | |
| US61/304,913 | 2010-02-16 | ||
| PCT/US2011/025051 WO2011103163A2 (en) | 2010-02-16 | 2011-02-16 | Nucleic acid extraction from complex matrices |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102834519A true CN102834519A (zh) | 2012-12-19 |
Family
ID=44369908
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011800080195A Pending CN102834519A (zh) | 2010-02-16 | 2011-02-16 | 从复杂基质中提取核酸 |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US8574890B2 (zh) |
| EP (1) | EP2536837A2 (zh) |
| JP (1) | JP2013519396A (zh) |
| KR (1) | KR20120138766A (zh) |
| CN (1) | CN102834519A (zh) |
| AU (1) | AU2011218219A1 (zh) |
| CA (1) | CA2787950A1 (zh) |
| IL (1) | IL221028A0 (zh) |
| WO (1) | WO2011103163A2 (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105717294A (zh) * | 2014-12-16 | 2016-06-29 | 生物梅里埃公司 | 使用粪便或来自粪便的液体样品采用活性炭预处理而确定微生物存在的方法及相关装置 |
| CN114085830A (zh) * | 2022-01-12 | 2022-02-25 | 济凡生物科技（北京）有限公司 | 一种复杂样本中微生物基因组dna的提取方法 |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8993341B2 (en) | 2011-05-12 | 2015-03-31 | Exact Sciences Corporation | Removal of PCR inhibitors |
| US8808990B2 (en) | 2011-05-12 | 2014-08-19 | Exact Sciences Corporation | Serial isolation of multiple DNA targets from stool |
| US8980107B2 (en) | 2011-05-12 | 2015-03-17 | Exact Sciences Corporation | Spin filter |
| CN104450680A (zh) * | 2011-05-12 | 2015-03-25 | 精密科学公司 | 核酸的分离 |
| WO2020116955A1 (ko) * | 2018-12-05 | 2020-06-11 | 가톨릭대학교 산학협력단 | 대변 또는 직장 면봉 검체에서 직접 카바페넴아제 생성 유전자의 분석적 진단을 위한 핵산 추출 방법 |
| GB202020576D0 (en) * | 2020-12-24 | 2021-02-10 | Univ Dundee | Method of eluting nucleic acid |
| EP4163369A1 (en) | 2021-10-08 | 2023-04-12 | AXAGARIUS GmbH & Co. KG | Use of mixtures of polyvinylpyrrolidone and ammonium sulfate in the isolation of nucleic acids |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1824809A (zh) * | 2005-12-28 | 2006-08-30 | 中国科学院南海海洋研究所 | 水生动物粪样细菌dna提取试剂盒及方法 |
| CN101307311A (zh) * | 2008-07-14 | 2008-11-19 | 四川大学 | 一种猪粪样总dna的提取方法 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6129892A (en) * | 1998-07-10 | 2000-10-10 | Garrett; John W. | Method and application unit for eliminating human urine odor |
| US7464694B2 (en) * | 2006-06-23 | 2008-12-16 | Chun-Hsiung Chang | Variable flow control method and device between air intake and throttle |
| US7919251B2 (en) * | 2007-10-12 | 2011-04-05 | Guy's And St. Thomas' Nhs Foundation Trust | Non-thiopurine methyltransferase related effects in 6-mercaptopurine therapy |
| KR101106969B1 (ko) * | 2008-05-30 | 2012-01-20 | 가톨릭대학교 산학협력단 | 활성탄을 이용한 고순도 플라스미드 ｄｎａ의 분리정제방법 |
-
2011
- 2011-02-16 WO PCT/US2011/025051 patent/WO2011103163A2/en active Application Filing
- 2011-02-16 CN CN2011800080195A patent/CN102834519A/zh active Pending
- 2011-02-16 JP JP2012553990A patent/JP2013519396A/ja not_active Withdrawn
- 2011-02-16 AU AU2011218219A patent/AU2011218219A1/en not_active Abandoned
- 2011-02-16 KR KR1020127024050A patent/KR20120138766A/ko not_active Application Discontinuation
- 2011-02-16 US US13/028,714 patent/US8574890B2/en not_active Expired - Fee Related
- 2011-02-16 EP EP11745160A patent/EP2536837A2/en not_active Withdrawn
- 2011-02-16 CA CA2787950A patent/CA2787950A1/en not_active Abandoned
-
2012
- 2012-07-19 IL IL221028A patent/IL221028A0/en unknown
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1824809A (zh) * | 2005-12-28 | 2006-08-30 | 中国科学院南海海洋研究所 | 水生动物粪样细菌dna提取试剂盒及方法 |
| CN101307311A (zh) * | 2008-07-14 | 2008-11-19 | 四川大学 | 一种猪粪样总dna的提取方法 |
Non-Patent Citations (3)
| Title |
|---|
| DIANA SARA LEAL-KLEVEZAS ET AL: "Antifreeze Solution Improves DNA Recovery by Preserving the Integrity of Pathogen-Infected Blood and Other Tissues", 《CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY》 * |
| MITJA KRIŽMAN ET AL.: "Robust CTAB-activated charcoal protocol for plant DNA extraction", 《ACTA AGRICULTURAE SLOVENICA》 * |
| 丁晓东 吕柳新: "从顽拗植物荔枝中提取基因组DNA技术的研究", 《应用与环境生物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105717294A (zh) * | 2014-12-16 | 2016-06-29 | 生物梅里埃公司 | 使用粪便或来自粪便的液体样品采用活性炭预处理而确定微生物存在的方法及相关装置 |
| CN114085830A (zh) * | 2022-01-12 | 2022-02-25 | 济凡生物科技（北京）有限公司 | 一种复杂样本中微生物基因组dna的提取方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| IL221028A0 (en) | 2012-09-24 |
| CA2787950A1 (en) | 2011-08-25 |
| KR20120138766A (ko) | 2012-12-26 |
| US8574890B2 (en) | 2013-11-05 |
| WO2011103163A3 (en) | 2012-01-19 |
| AU2011218219A1 (en) | 2012-08-16 |
| JP2013519396A (ja) | 2013-05-30 |
| US20110201085A1 (en) | 2011-08-18 |
| WO2011103163A2 (en) | 2011-08-25 |
| EP2536837A2 (en) | 2012-12-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102834519A (zh) | 从复杂基质中提取核酸 | |
| US10731146B2 (en) | Purification of nucleic acid from environmental or biological samples | |
| US9856515B2 (en) | Removal of PCR inhibitors | |
| Dhaliwal | DNA extraction and purification | |
| Bordelon et al. | Development of a low-resource RNA extraction cassette based on surface tension valves | |
| CN105592925B (zh) | 用于收集核酸的样品的系统和方法 | |
| EP2081442A2 (en) | Compositions, methods and kits for isolating nucleic acids from body fluids using anion exchange media | |
| CN107249746A (zh) | 用于收集核酸样本的系统和方法 | |
| Pandit et al. | Capture and direct amplification of DNA on chitosan microparticles in a single PCR-optimal solution | |
| EP2854981B1 (en) | Methods of nucleic acid fractionation and detection | |
| KR20210005648A (ko) | 복합 샘플로부터의 핵산 단리 및 억제제 제거 | |
| CN106715717A (zh) | 使用有机溶剂除去抑制剂的核酸提取 | |
| CN110272897B (zh) | 肠道内容物样本保存液及制备方法 | |
| JP2007124926A (ja) | Ｄｎａ抽出剤およびそれを用いるｄｎａ抽出方法 | |
| CN108603221A (zh) | 综合样本处理系统 | |
| Weis et al. | RNA-based stable isotope probing (RNA-SIP) in the gut environment | |
| EP3408388B1 (en) | Method for producing a lysate from cells contained in a liquid sample | |
| CN107988206A (zh) | 一种高效提取土壤微生物dna的方法 | |
| Arcuri et al. | Polyethylene glycol and polyvinylpirrolidone effect on bacterial rRNA extraction and hybridization from cells exposed to tannins | |
| EP3971290A1 (en) | Method for producing a lysate from cells contained in a liquid sample | |
| Ajayi | Modified Methods for Quick and Safe Extraction of DNA from Microbiological Samples | |
| CN114317524A (zh) | 用于粮食籽粒附着真菌dna提取的试剂、试剂盒及提取方法 | |
| Jarallah et al. | Design of a new rapid and efficient kit for extracting DNA from blood sample | |
| Rogers | Nucleic Acid Extraction from Diverse Samples | |
| Hahnel | Extraction of DNA from difficult samples with an automatable portable system for early detection of intestinal cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121219 |