WO2024017155A1 - Lysis composition - Google Patents

Lysis composition Download PDF

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WO2024017155A1
WO2024017155A1 PCT/CN2023/107404 CN2023107404W WO2024017155A1 WO 2024017155 A1 WO2024017155 A1 WO 2024017155A1 CN 2023107404 W CN2023107404 W CN 2023107404W WO 2024017155 A1 WO2024017155 A1 WO 2024017155A1
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lysis
nucleic acid
composition
buffer
edta
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PCT/CN2023/107404
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French (fr)
Chinese (zh)
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王少坤
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友康生物科技(北京)股份有限公司
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Publication of WO2024017155A1 publication Critical patent/WO2024017155A1/en

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    • 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
    • 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
    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the invention belongs to the technical field of molecular biology, and specifically relates to a bubble-free lysis composition that can rapidly lyse cells and the like to extract viral nucleic acid.
  • the extraction and purification of viral nucleic acid must first release the viral nucleic acid and separate it from other biological macromolecules.
  • the traditional spin column method or magnetic bead method to extract nucleic acids generally requires steps such as lysis, binding, rinsing, and elution.
  • the first critical step is lysis. If the lysis is insufficient, the amount of target nucleic acid that can be extracted will be lower, which will affect the subsequent detection process.
  • the main methods of cleavage include mechanical action, enzymatic action and chemical action.
  • Mechanical action means the release of nucleic acids through mechanical force, and may cause nucleic acid chain breakage, so it is not suitable for the separation of high molecular weight long-chain nucleic acids;
  • enzymatic action mainly uses lysozyme or protease;
  • chemical action requires certain pH and denaturation conditions. Denaturation conditions Generally, it is heated and added with surfactants (SDS, TritonX2100, Tween 20, NP240, CTAB, etc.) or strong ionic agents (guanidine isothiocyanate, guanidine hydrochloride, guanidine creatine).
  • SDS surfactants
  • TritonX2100 TritonX2100
  • Tween 20 NP240
  • CTAB guanidine hydrochloride
  • guanidine creatine guanidine creatine
  • lysis reagents often contain ingredients such as surfactants and strong ionic agents (guanidinium salts).
  • surfactants can easily generate aerosols, thus causing sample contamination; and guanidinium salts will lower the pH value of the solution in actual operations.
  • guanidinium salts tend to precipitate when near neutral or acidic, affecting the lysis effect and reducing the nucleic acid extraction yield.
  • these components usually have a strong inhibitory effect on downstream reactions such as polymerase chain reaction (PCR) technology, and need to be removed through multiple cleaning and purification steps, which not only generates a large amount of waste liquid and consumes a large number of pipette tips, but also At the same time, it also greatly increases the extraction time and reduces the work efficiency of the experimental personnel. Therefore, there is an urgent need to develop a foam-free lysis composition that can quickly cleave and release viral nucleic acids and does not contain surfactants and guanidinium salts, which can reduce sample contamination while eliminating multiple cleaning and purification steps, thereby saving money. Save time on nucleic acid extraction.
  • PCR polymerase chain reaction
  • the present invention provides a bubble-free lysis composition that can rapidly cleave and release nucleic acids such as viruses. It is acidic, does not contain surfactant and guanidinium salt components, and has almost no inhibitory effect on downstream detection reactions at a certain residual amount, thereby eliminating the need for After multiple washes and purifications, nucleic acid can be obtained directly by elution.
  • the invention relates to a lysis composition
  • a lysis composition comprising the following components:
  • Arginine concentration is 0.5-1.5M
  • the lysis composition also contains a metal chelating agent at a concentration of 5-10mM.
  • the inorganic salt is selected from sodium citrate, sodium chloride, potassium chloride and any mixture thereof.
  • the buffer is Tris buffer or MES buffer. More preferably, the concentration of the buffer is 10-30mM.
  • the lysis composition of the present invention also contains a pH adjuster.
  • the metal chelating agent is EDTA and/or a salt of EDTA (such as disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.).
  • EDTA EDTA and/or a salt of EDTA (such as disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.).
  • the pH adjuster is an aqueous sodium hydroxide solution and/or an aqueous hydrochloric acid solution, more preferably a 1M aqueous sodium hydroxide solution and/or a 1M aqueous hydrochloric acid solution.
  • the pH is adjusted to about 4.
  • the lysis composition consists of the above components.
  • cleavage composition of the present invention can be prepared by methods known in the art.
  • the invention relates to a kit containing a lysis composition of the invention.
  • the volume of the lysis composition is 1-10 ml, more preferably 2-3 ml.
  • the lysis composition and kit of the present invention can be used to cleave and release microbial nucleic acids, and can further be used to detect microorganisms.
  • the invention relates to a method of lysing to release microbial nucleic acids, wherein a sample is treated using a lysis composition of the invention.
  • a sample is treated using a lysis composition of the invention.
  • the invention relates to a method for detecting microorganisms, wherein the sample is treated with the lysis composition of the invention to lyse the microorganism nucleic acid.
  • the method of the present invention is not For the purpose of disease diagnosis.
  • the invention relates to the use of the lysis composition of the invention for the preparation of reagents for lysing to release microbial nucleic acids or detecting microorganisms.
  • the microorganism is a virus, bacteria, fungus, actinomycete, rickettsia, mycoplasma, chlamydia or spirochete.
  • the nucleic acid is DNA and/or RNA.
  • the microorganism is a virus, more preferably a coronavirus or an influenza virus, and more preferably a novel coronavirus.
  • the sample is blood, serum, plasma, lymph, saliva, exfoliated cells or their supernatants (for example, from throat swabs or nasopharyngeal swabs, etc.), etc. It can also be an article or food swab sample.
  • the lysis composition of the present invention is acidic and does not contain surfactant and guanidinium salt components, which greatly reduces the risk of sample contamination during the nucleic acid extraction process; it has almost no inhibition on downstream detection under a certain residual amount, thereby eliminating the need for multiple cleanings.
  • Purification can be directly eluted to obtain nucleic acids; it can be used at room temperature without additional steps such as heating or protease treatment. It does not contain any toxic components or organic solvents. It is green and environmentally friendly, easy to operate, and the lysis and extraction effects are comparable to those of mainstream products on the market. Lysis reagent products were flat.
  • Figure 1 is a comparison chart of the viral nucleic acid released by cleavage compositions A-D of Example 1 of the present invention and the positive control and negative control through RT-qPCR amplification and detection.
  • Figure 2 is a comparison chart of RT-qPCR amplification detection after adding different amounts of cleavage compositions A and D of Example 1 of the present invention and positive controls and negative controls to the eluate in Example 3 of the present invention.
  • the instruments, reagents, materials, etc. involved in the following examples are conventional instruments, reagents, materials, etc. that are already in the prior art and can be obtained through regular commercial channels.
  • the experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. existing in the prior art.
  • arginine 0.5M KCl, 25mM Tris buffer, 10mM EDTA, adjust pH to approximately 3.0 using 1M sodium hydroxide or aqueous hydrochloric acid.
  • Example 2 Four lysis compositions A, B, C, and D prepared in Example 1 were used to cleave and release PEDV virus nucleic acid, and a commercially available magnetic bead method nucleic acid extraction kit (Youkang Biotechnology (Beijing) Co., Ltd., MK0102-100) was used. Extract viral nucleic acid and use commercially available lysis reagent (Youkang Biotechnology (Beijing) Co., Ltd., inactivated sampling tube MT0501-7-3) as a positive control.
  • a commercially available magnetic bead method nucleic acid extraction kit Youkang Biotechnology (Beijing) Co., Ltd., MK0102-100
  • Extract viral nucleic acid and use commercially available lysis reagent (Youkang Biotechnology (Beijing) Co., Ltd., inactivated sampling tube MT0501-7-3) as a positive control.
  • Tris-HCl buffer Tris-HCl buffer, isothiocyanate Guanidine, urea, phenol red sodium salt
  • ultrapure water was used as the lysis solution as a negative control, and the effect of lysis and release of viral nucleic acid was evaluated through RT-qPCR amplification detection.
  • Example 1 Take 900 ⁇ L of the four lysis compositions, positive control and ultrapure water prepared in Example 1 and place them in 1.5 mL centrifuge tubes respectively. Add 100 ⁇ L of laboratory-cultured PEDV virus liquid of equal concentration and mix evenly. Place it on a shaker at 37 °C, 20min at 150rmp.
  • RT-qPCR the more target nucleic acids released by cleavage and the higher the initial copy number, the faster a significant increase in fluorescence signal will be observed, and the corresponding Ct value will be smaller.
  • the RT-qPCR amplification detection results are shown in Figure 1. According to Figure 1, the Ct values of the cleavage compositions A, B, C, and D of the present invention are comparable to the positive control, while the negative control has no Ct value.
  • nucleic acid extraction kit of Example 2 Use the nucleic acid extraction kit of Example 2 to perform nucleic acid extraction, obtain twelve tubes of eluate, each tube is 100 ⁇ L, and add 4 ⁇ L, 6 ⁇ L, 8 ⁇ L, and 10 ⁇ L of lysis compositions A and D of Example 1, and commercially available products of Example 2, respectively. Sell lysis reagent (positive control) and Depc water (negative control), which are then used for RT-qPCR amplification detection.
  • lysis compositions A and D have almost no inhibitory effect on subsequent detection.
  • commercially available lysis reagents contain surfactants and high concentrations of guanidinium salts, which have little inhibitory effect on subsequent detection.
  • the inhibitory effect of subsequent detection was much greater than that of lysis compositions A and D.
  • MDCK cell recovery and passage Use high-glucose DMEM medium containing 10% FBS to recover and passage MDCK cells, and culture them continuously for 3 generations.
  • lysis composition C of the present invention lyses MDCK cells infected with H1N1 virus to extract viral nucleic acid,
  • the Ct value obtained after testing is the same as that of the commercially available cleavage reagent in Example 2.

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Abstract

Provided is a bubble-free lysis composition capable of rapidly lysing and releasing a nucleic acid of a virus and the like, wherein the lysis composition is acidic and does not contain a surfactant and a guanidine salt, and thus has almost no inhibitory effect on downstream detection reaction at a certain residual amount, so that a nucleic acid can be obtained by direct elution without undergoing multiple times of cleaning and purification.

Description

一种裂解组合物A lysis composition
本申请要求2022年7月22日向中国国家知识产权局提交的专利申请号为202210869302.7，发明名称为“一种裂解组合物”的在先申请的优先权。该在先申请的全文通过引用的方式结合于本申请中。This application claims the priority of the earlier application with the patent application number 202210869302.7 submitted to the State Intellectual Property Office of China on July 22, 2022, and the invention name is "A pyrolysis composition". The entirety of this prior application is incorporated by reference into this application.
技术领域Technical field
本发明属于分子生物学技术领域，具体涉及一种能够快速裂解细胞等以提取病毒核酸的无泡裂解组合物。The invention belongs to the technical field of molecular biology, and specifically relates to a bubble-free lysis composition that can rapidly lyse cells and the like to extract viral nucleic acid.
背景技术Background technique
在病毒核酸检测过程中，提取纯化病毒核酸首先需将病毒核酸释放出来，并与其他生物大分子分开。传统的离心柱法或磁珠法提取核酸一般需要裂解、结合、漂洗、洗脱等步骤。第一个关键步骤就是裂解，如果裂解不充分，可提取的目标核酸量便会较低，进而影响后续检测流程。In the process of viral nucleic acid detection, the extraction and purification of viral nucleic acid must first release the viral nucleic acid and separate it from other biological macromolecules. The traditional spin column method or magnetic bead method to extract nucleic acids generally requires steps such as lysis, binding, rinsing, and elution. The first critical step is lysis. If the lysis is insufficient, the amount of target nucleic acid that can be extracted will be lower, which will affect the subsequent detection process.
裂解的方式主要有机械作用、酶作用和化学作用。机械作用即通过机械力释放核酸，同时可能会引起核酸链断裂，因此不适于高分子量长链核酸的分离；酶作用主要是使用溶菌酶或蛋白酶；化学作用需要一定的pH和变性条件，变性条件一般为加热、加入表面活性剂(SDS、TritonX2100、Tween 20、NP240、CTAB等)或强离子剂(异硫氰酸胍、盐酸胍、肌酸胍)。基因组核酸提取试剂盒主要用CTAB、SDS和异硫氰酸胍即裂解试剂进行裂解。The main methods of cleavage include mechanical action, enzymatic action and chemical action. Mechanical action means the release of nucleic acids through mechanical force, and may cause nucleic acid chain breakage, so it is not suitable for the separation of high molecular weight long-chain nucleic acids; enzymatic action mainly uses lysozyme or protease; chemical action requires certain pH and denaturation conditions. Denaturation conditions Generally, it is heated and added with surfactants (SDS, TritonX2100, Tween 20, NP240, CTAB, etc.) or strong ionic agents (guanidine isothiocyanate, guanidine hydrochloride, guanidine creatine). The genomic nucleic acid extraction kit mainly uses CTAB, SDS and guanidinium isothiocyanate, the cleavage reagent, for lysis.
目前的裂解试剂中往往含有表面活性剂和强离子剂(胍盐)等成分，表面活性剂的存在极易产生气溶胶，从而造成样本的污染；而胍盐在实际操作中会降低溶液pH值，近中性或偏酸性时胍盐易产生沉淀，影响裂解效果，降低核酸提取得率。此外，这些成分通常对下游反应如聚合酶链式反应(PCR)技术具有较强的抑制作用，需经过多次清洗纯化步骤去除，不仅产生大量的废液和消耗大量的移液枪枪头，同时也大大增加了提取时间，降低了实验人员的工作效率。因此，亟需开发一种可快速裂解释放病毒核酸且不含表面活性剂和胍盐的无泡裂解组合物，在降低样本被污染的同时省去多次清洗纯化步骤，从而可以节 省核酸提取的时间。Current lysis reagents often contain ingredients such as surfactants and strong ionic agents (guanidinium salts). The presence of surfactants can easily generate aerosols, thus causing sample contamination; and guanidinium salts will lower the pH value of the solution in actual operations. , Guanidine salts tend to precipitate when near neutral or acidic, affecting the lysis effect and reducing the nucleic acid extraction yield. In addition, these components usually have a strong inhibitory effect on downstream reactions such as polymerase chain reaction (PCR) technology, and need to be removed through multiple cleaning and purification steps, which not only generates a large amount of waste liquid and consumes a large number of pipette tips, but also At the same time, it also greatly increases the extraction time and reduces the work efficiency of the experimental personnel. Therefore, there is an urgent need to develop a foam-free lysis composition that can quickly cleave and release viral nucleic acids and does not contain surfactants and guanidinium salts, which can reduce sample contamination while eliminating multiple cleaning and purification steps, thereby saving money. Save time on nucleic acid extraction.
发明内容Contents of the invention
本发明中提供一种能快速裂解释放病毒等核酸的无泡裂解组合物，呈酸性，不含表面活性剂和胍盐成分，在一定残留量下对下游检测反应几乎无抑制作用，从而无需经多次清洗纯化即可直接进行洗脱而获取核酸。The present invention provides a bubble-free lysis composition that can rapidly cleave and release nucleic acids such as viruses. It is acidic, does not contain surfactant and guanidinium salt components, and has almost no inhibitory effect on downstream detection reactions at a certain residual amount, thereby eliminating the need for After multiple washes and purifications, nucleic acid can be obtained directly by elution.
根据本发明的一个方面，本发明涉及一种裂解组合物，含有如下组分：According to one aspect of the invention, the invention relates to a lysis composition comprising the following components:
(1)精氨酸，浓度为0.5-1.5M；(1) Arginine, concentration is 0.5-1.5M;
(2)无机盐，浓度为0.2-1M；(2) Inorganic salt, concentration is 0.2-1M;
(3)缓冲液；(3) Buffer;
(4)pH为2-6。(4) pH is 2-6.
优选地，裂解组合物中还含有金属螯合剂，浓度为5-10mM。Preferably, the lysis composition also contains a metal chelating agent at a concentration of 5-10mM.
优选地，无机盐选自柠檬酸钠、氯化钠、氯化钾及其任意混合物。Preferably, the inorganic salt is selected from sodium citrate, sodium chloride, potassium chloride and any mixture thereof.
优选地，缓冲液为Tris缓冲液或MES缓冲液。更优选地，缓冲液的浓度为10-30mM。Preferably, the buffer is Tris buffer or MES buffer. More preferably, the concentration of the buffer is 10-30mM.
优选地，本发明的裂解组合物还含有pH调节剂。Preferably, the lysis composition of the present invention also contains a pH adjuster.
优选地，金属螯合剂为EDTA和/或EDTA的盐(例如EDTA二钠、EDTA四钠、EDTA二钠钙等)。Preferably, the metal chelating agent is EDTA and/or a salt of EDTA (such as disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.).
优选地，pH调节剂为氢氧化钠水溶液和/或盐酸水溶液，更优选为1M氢氧化钠水溶液和/或1M盐酸水溶液。Preferably, the pH adjuster is an aqueous sodium hydroxide solution and/or an aqueous hydrochloric acid solution, more preferably a 1M aqueous sodium hydroxide solution and/or a 1M aqueous hydrochloric acid solution.
优选地，将pH值调节至约4。Preferably, the pH is adjusted to about 4.
优选地，裂解组合物由以上组分组成。Preferably, the lysis composition consists of the above components.
本领域技术人员可以理解，可以通过本领域已知的方法制备本发明的上述裂解组合物。Those skilled in the art will understand that the above-mentioned cleavage composition of the present invention can be prepared by methods known in the art.
根据本发明的另一个方面，本发明涉及一种试剂盒，含有本发明的裂解组合物。优选地，裂解组合物的体积为1-10ml，更优选2-3ml。According to another aspect of the invention, the invention relates to a kit containing a lysis composition of the invention. Preferably, the volume of the lysis composition is 1-10 ml, more preferably 2-3 ml.
本发明的裂解组合物和试剂盒可用于裂解释放微生物核酸，进而可用于检测微生物。The lysis composition and kit of the present invention can be used to cleave and release microbial nucleic acids, and can further be used to detect microorganisms.
根据本发明的另一个方面，本发明涉及一种裂解释放微生物核酸的方法，其中，使用本发明的裂解组合物处理样本。本领域技术人员可以理解，本发明的方法并非以疾病的诊断为目的。According to another aspect of the invention, the invention relates to a method of lysing to release microbial nucleic acids, wherein a sample is treated using a lysis composition of the invention. Those skilled in the art will understand that the method of the present invention is not aimed at diagnosing disease.
根据本发明的另一个方面，本发明涉及一种检测微生物的方法，其中，使用本发明的裂解组合物处理样本裂解释放微生物核酸。本领域技术人员可以理解，本发明的方法并非 以疾病的诊断为目的。According to another aspect of the invention, the invention relates to a method for detecting microorganisms, wherein the sample is treated with the lysis composition of the invention to lyse the microorganism nucleic acid. Those skilled in the art can understand that the method of the present invention is not For the purpose of disease diagnosis.
根据本发明的另一个方面，本发明涉及本发明的裂解组合物在制备用于裂解释放微生物核酸或检测微生物的试剂中的用途。According to another aspect of the invention, the invention relates to the use of the lysis composition of the invention for the preparation of reagents for lysing to release microbial nucleic acids or detecting microorganisms.
优选地，微生物为病毒、细菌、真菌、放线菌、立克次氏体、支原体、衣原体或螺旋体。Preferably, the microorganism is a virus, bacteria, fungus, actinomycete, rickettsia, mycoplasma, chlamydia or spirochete.
优选地，核酸为DNA和/或RNA。Preferably, the nucleic acid is DNA and/or RNA.
优选地，所述微生物为病毒，进一步优选为冠状病毒或流感病毒，更优选为新型冠状病毒。Preferably, the microorganism is a virus, more preferably a coronavirus or an influenza virus, and more preferably a novel coronavirus.
优选地，样本为血液、血清、血浆、淋巴液、唾液、脱落细胞或其上清(例如来自于咽拭子或鼻咽拭子等)等，也可以为物品或食品擦拭样本。Preferably, the sample is blood, serum, plasma, lymph, saliva, exfoliated cells or their supernatants (for example, from throat swabs or nasopharyngeal swabs, etc.), etc. It can also be an article or food swab sample.
本发明的裂解组合物呈酸性，不含表面活性剂和胍盐成分，在核酸提取过程中大大降低了样本污染的风险；在一定残留量下对下游检测几乎无抑制，从而无需经多次清洗纯化即可直接进行洗脱而获取核酸；可在室温条件下使用，无需加热或蛋白酶处理等额外步骤，无任何有毒成分或有机溶剂，绿色环保，操作简便，且裂解提取效果可与市场上主流裂解试剂产品持平。The lysis composition of the present invention is acidic and does not contain surfactant and guanidinium salt components, which greatly reduces the risk of sample contamination during the nucleic acid extraction process; it has almost no inhibition on downstream detection under a certain residual amount, thereby eliminating the need for multiple cleanings. Purification can be directly eluted to obtain nucleic acids; it can be used at room temperature without additional steps such as heating or protease treatment. It does not contain any toxic components or organic solvents. It is green and environmentally friendly, easy to operate, and the lysis and extraction effects are comparable to those of mainstream products on the market. Lysis reagent products were flat.
附图说明Description of drawings
图1为本发明实施例1的裂解组合物A-D与阳性对照、阴性对照裂解释放的病毒核酸经RT-qPCR扩增检测的对比图。Figure 1 is a comparison chart of the viral nucleic acid released by cleavage compositions A-D of Example 1 of the present invention and the positive control and negative control through RT-qPCR amplification and detection.
图2为本发明实施例3在洗脱液中添加不同量的本发明实施例1的裂解组合物A、D和阳性对照、阴性对照后进行RT-qPCR扩增检测的对比图。Figure 2 is a comparison chart of RT-qPCR amplification detection after adding different amounts of cleavage compositions A and D of Example 1 of the present invention and positive controls and negative controls to the eluate in Example 3 of the present invention.
具体实施方式Detailed ways
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外，应理解，在阅读了本发明所记载的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本发明所限定的范围。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. In addition, it should be understood that after reading the contents described in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope of the present invention.
下述实施例中所涉及的仪器、试剂、材料等，若无特别说明，均为现有技术中已有的常规仪器、试剂、材料等，可通过正规商业途径获得。下述实施例中所涉及的实验方法，检测方法等，若无特别说明，均为现有技术中已有的常规实验方法，检测方法等。Unless otherwise specified, the instruments, reagents, materials, etc. involved in the following examples are conventional instruments, reagents, materials, etc. that are already in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods, detection methods, etc. existing in the prior art.
实施例1裂解组合物的制备 Example 1 Preparation of lysis composition
首先配制Tris缓冲液或MES缓冲液，然后加入其它组分，获得如下裂解组合物：First prepare Tris buffer or MES buffer, then add other components to obtain the following lysis composition:
(1)裂解组合物A，含有：(1) Cleavage composition A, containing:
0.6M精氨酸，0.3M柠檬酸钠，10mM Tris缓冲液，使用1M氢氧化钠或盐酸水溶液，将pH调节至约3.0。0.6M arginine, 0.3M sodium citrate, 10mM Tris buffer, adjust pH to approximately 3.0 using 1M sodium hydroxide or aqueous hydrochloric acid.
(2)裂解组合物B，含有：(2) Cleavage composition B, containing:
1M精氨酸，0.5M NaCl，20mM Tris缓冲液，5mM EDTA，使用1M氢氧化钠或盐酸水溶液，将pH调节至约6.0。1M arginine, 0.5M NaCl, 20mM Tris buffer, 5mM EDTA, adjust pH to approximately 6.0 using 1M sodium hydroxide or aqueous hydrochloric acid.
(3)裂解组合物C，含有：(3) Cleavage composition C, containing:
1.3M精氨酸，0.5M KCl，25mM Tris缓冲液，10mM EDTA，使用1M氢氧化钠或盐酸水溶液，将pH调节至约3.0。1.3M arginine, 0.5M KCl, 25mM Tris buffer, 10mM EDTA, adjust pH to approximately 3.0 using 1M sodium hydroxide or aqueous hydrochloric acid.
(4)裂解组合物D，含有：(4) Cleavage composition D, containing:
1M精氨酸，1M NaCl，10mM MES缓冲液，10mM EDTA，使用1M氢氧化钠或盐酸水溶液，将pH值调节至约4.0。1M arginine, 1M NaCl, 10mM MES buffer, 10mM EDTA, adjust pH to approximately 4.0 using 1M sodium hydroxide or aqueous hydrochloric acid.
(5)裂解组合物E，含有：(5) Cleavage composition E, containing:
1.5M精氨酸，0.8M KCl，20mM MES缓冲液，6mM EDTA，使用1M氢氧化钠或盐酸水溶液，将pH值调节至约4.0。1.5M arginine, 0.8M KCl, 20mM MES buffer, 6mM EDTA, adjust pH to approximately 4.0 using 1M sodium hydroxide or aqueous hydrochloric acid.
(6)裂解组合物F，含有：(6) Cleavage composition F, containing:
0.5M精氨酸，1M柠檬酸钠，25mM MES缓冲液，5mM EDTA，使用1M氢氧化钠或盐酸水溶液，将pH值调节至约2.0。0.5M arginine, 1M sodium citrate, 25mM MES buffer, 5mM EDTA, adjust the pH to approximately 2.0 using 1M sodium hydroxide or aqueous hydrochloric acid.
实施例2裂解释放PEDV病毒核酸Example 2 Cleavage to release PEDV viral nucleic acid
采用实施例1制备的A、B、C、D四种裂解组合物裂解释放PEDV病毒核酸，并采用市售磁珠法核酸提取试剂盒(友康生物科技(北京)股份有限公司，MK0102-100)提取病毒核酸，以市售裂解试剂(友康生物科技(北京)股份有限公司，灭活型采样管MT0501-7-3)作为阳性对照，其主要成分为：Tris-HCl缓冲液，异硫氰酸胍，尿素，酚红钠盐；另，以超纯水为裂解液作为阴性对照，通过RT-qPCR扩增检测评估裂解释放病毒核酸的效果。Four lysis compositions A, B, C, and D prepared in Example 1 were used to cleave and release PEDV virus nucleic acid, and a commercially available magnetic bead method nucleic acid extraction kit (Youkang Biotechnology (Beijing) Co., Ltd., MK0102-100) was used. Extract viral nucleic acid and use commercially available lysis reagent (Youkang Biotechnology (Beijing) Co., Ltd., inactivated sampling tube MT0501-7-3) as a positive control. Its main components are: Tris-HCl buffer, isothiocyanate Guanidine, urea, phenol red sodium salt; in addition, ultrapure water was used as the lysis solution as a negative control, and the effect of lysis and release of viral nucleic acid was evaluated through RT-qPCR amplification detection.
具体步骤如下：Specific steps are as follows:
1.取900μL实施例1制备的四种裂解组合物、阳性对照及超纯水分别置于1.5mL离心管中，加入100μL等浓度的实验室培养的PEDV病毒液混合均匀，置于摇床37℃，150rmp条件下20min。1. Take 900 μL of the four lysis compositions, positive control and ultrapure water prepared in Example 1 and place them in 1.5 mL centrifuge tubes respectively. Add 100 μL of laboratory-cultured PEDV virus liquid of equal concentration and mix evenly. Place it on a shaker at 37 ℃, 20min at 150rmp.
2.取300μL上述混合液，加入300μL异丙醇和20μL磁珠，混合均匀并静置后进行磁分 离。2. Take 300 μL of the above mixture, add 300 μL of isopropyl alcohol and 20 μL of magnetic beads, mix evenly and let stand before magnetic separation. Leave.
3.加入800μL纯化液进行清洗，而后进行磁分离，将残留液滴吸取干净并晾干。3. Add 800 μL purification solution for cleaning, then perform magnetic separation, absorb the remaining droplets and dry them.
4.加入100μLdepc水洗脱，室温静置5min，进行磁分离，洗脱液即为提取的核酸，用于RT-qPCR扩增检测。4. Add 100 μL depc water for elution, let it stand at room temperature for 5 minutes, and perform magnetic separation. The eluent is the extracted nucleic acid, which is used for RT-qPCR amplification detection.
在RT-qPCR中，裂解释放的目标核酸越多，起始拷贝数越高，会越快观察到荧光信号显著增加，则对应的Ct值越小。RT-qPCR扩增检测结果见图1。根据图1，本发明的裂解组合物A、B、C、D的Ct值与阳性对照相比裂解效果相当，而阴性对照并未出Ct值。In RT-qPCR, the more target nucleic acids released by cleavage and the higher the initial copy number, the faster a significant increase in fluorescence signal will be observed, and the corresponding Ct value will be smaller. The RT-qPCR amplification detection results are shown in Figure 1. According to Figure 1, the Ct values of the cleavage compositions A, B, C, and D of the present invention are comparable to the positive control, while the negative control has no Ct value.
实施例3Example 3
裂解试剂的残留量对RT-qPCR扩增的影响。Effect of residual lysis reagent on RT-qPCR amplification.
使用实施例2的核酸提取试剂盒进行核酸提取，获取十二管洗脱液，每管100μL，分别加入4μL、6μL、8μL和10μL实施例1的裂解组合物A、D、实施例2的市售裂解试剂(阳性对照)和Depc水(阴性对照)，而后用于RT-qPCR扩增检测。Use the nucleic acid extraction kit of Example 2 to perform nucleic acid extraction, obtain twelve tubes of eluate, each tube is 100 μL, and add 4 μL, 6 μL, 8 μL, and 10 μL of lysis compositions A and D of Example 1, and commercially available products of Example 2, respectively. Sell lysis reagent (positive control) and Depc water (negative control), which are then used for RT-qPCR amplification detection.
如图2所示，随着添加量的增大，裂解组合物A和D对后续检测几乎无抑制作用，同等添加量下，市售裂解试剂由于含有表面活性剂和高浓度的胍盐，对后续检测的抑制作用要远大于裂解组合物A和D。As shown in Figure 2, as the addition amount increases, lysis compositions A and D have almost no inhibitory effect on subsequent detection. At the same addition amount, commercially available lysis reagents contain surfactants and high concentrations of guanidinium salts, which have little inhibitory effect on subsequent detection. The inhibitory effect of subsequent detection was much greater than that of lysis compositions A and D.
实施例4 H1N1病毒感染的MDCK细胞的裂解和病毒核酸提取Example 4 Lysis of MDCK cells infected with H1N1 virus and extraction of viral nucleic acid
具体步骤如下：Specific steps are as follows:
1、MDCK细胞复苏和传代，采用含有10％FBS的高糖DMEM培养基对MDCK细胞进行复苏和传代，连续培养3代。1. MDCK cell recovery and passage. Use high-glucose DMEM medium containing 10% FBS to recover and passage MDCK cells, and culture them continuously for 3 generations.
2、第三代细胞消化后离心，并进行细胞铺板，将细胞接种至6孔板中，细胞的接种密度控制为30％融合度。2. Centrifuge the third-generation cells after digestion, and plate the cells. The cells are seeded into a 6-well plate. The seeding density of the cells is controlled to 30% confluency.
3、24小时后，采用H1N1病毒感染MDCK细胞。3. After 24 hours, MDCK cells were infected with H1N1 virus.
4、病毒孵育完成后，采用PBS清洗细胞5遍，然后更换为含有2％FBS的高糖DMEM培养基维持细胞生长状态。4. After the virus incubation is completed, wash the cells 5 times with PBS, and then replace them with high-sugar DMEM medium containing 2% FBS to maintain cell growth.
5、感染24小时后，细胞尚未发生病变效应。消化细胞，并收集后进行离心，800rpm离心5min，弃去上清，得到细胞沉淀，加1mL实施例1中的裂解组合物C重悬细胞。5. After 24 hours of infection, the cells have not yet experienced any pathological changes. The cells were digested, collected and centrifuged at 800 rpm for 5 min. The supernatant was discarded to obtain a cell pellet. 1 mL of lysis composition C in Example 1 was added to resuspend the cells.
6、取300μL细胞悬液，使用实施例2的核酸提取试剂盒进行核酸提取，提取的核酸用于RT-qPCR扩增检测。6. Take 300 μL of cell suspension and use the nucleic acid extraction kit of Example 2 to extract nucleic acid. The extracted nucleic acid is used for RT-qPCR amplification detection.
如下表1所示，本发明的裂解组合物C裂解H1N1病毒感染的MDCK细胞提取病毒核酸， 测试后所得Ct值与实施例2市售裂解试剂相持平。As shown in Table 1 below, lysis composition C of the present invention lyses MDCK cells infected with H1N1 virus to extract viral nucleic acid, The Ct value obtained after testing is the same as that of the commercially available cleavage reagent in Example 2.
表1 H1N1病毒感染的MDCK细胞的裂解和核酸提取
Table 1 Lysis and nucleic acid extraction of MDCK cells infected with H1N1 virus
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。 The embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiment. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (9)

  1. 一种裂解组合物，其特征在于，含有如下组分：A cracking composition, characterized by containing the following components:
    (1)精氨酸，浓度为0.5-1.5M；(1) Arginine, concentration is 0.5-1.5M;
    (2)无机盐，浓度为0.2-1M；(2) Inorganic salt, concentration is 0.2-1M;
    (3)缓冲液；(3) Buffer;
    (4)pH为2-6。(4) pH is 2-6.
    优选地，无机盐选自柠檬酸钠、氯化钠、氯化钾及其任意混合物。Preferably, the inorganic salt is selected from sodium citrate, sodium chloride, potassium chloride and any mixture thereof.
    优选地，缓冲液为Tris缓冲液或MES缓冲液。Preferably, the buffer is Tris buffer or MES buffer.
    优选地，缓冲液的浓度为10-30mM。Preferably, the concentration of the buffer is 10-30mM.
  2. 如权利要求1所述的裂解组合物，其特征在于，还含有金属螯合剂，浓度为5-10mM。The cleavage composition of claim 1, further comprising a metal chelating agent at a concentration of 5-10mM.
    优选地，金属螯合剂为EDTA和/或EDTA的盐(例如EDTA二钠、EDTA四钠、EDTA二钠钙等)。Preferably, the metal chelating agent is EDTA and/or a salt of EDTA (such as disodium EDTA, tetrasodium EDTA, calcium disodium EDTA, etc.).
  3. 如权利要求1所述的裂解组合物，其特征在于，还含有pH调节剂。The cleavage composition according to claim 1, further comprising a pH adjuster.
    优选地，pH调节剂为氢氧化钠水溶液和/或盐酸水溶液。Preferably, the pH adjuster is sodium hydroxide aqueous solution and/or hydrochloric acid aqueous solution.
  4. 如权利要求1-3中任一项所述的裂解组合物，其特征在于，所述裂解组合物由所述组分组成。The cleavage composition of any one of claims 1-3, wherein the cleavage composition consists of the components.
  5. 一种试剂盒，其特征在于，含有如权利要求1-4中任一项所述的裂解组合物。A kit, characterized in that it contains the lysis composition according to any one of claims 1-4.
    优选地，裂解组合物的体积为1-10ml，更优选2-3ml。Preferably, the volume of the lysis composition is 1-10 ml, more preferably 2-3 ml.
  6. 一种裂解释放微生物核酸的方法，其特征在于，使用如权利要求1-4中任一项所述的裂解组合物处理样本。A method for lysing and releasing microbial nucleic acid, characterized by using the lysis composition as claimed in any one of claims 1 to 4 to treat the sample.
  7. 一种检测微生物的方法，其特征在于，使用如权利要求1-4中任一项所述的裂解组合物处理样本裂解释放微生物核酸。A method for detecting microorganisms, characterized by using the lysis composition according to any one of claims 1 to 4 to treat a sample to lyse and release microbial nucleic acid.
  8. 如权利要求1-4中任一项所述的裂解组合物在制备用于裂解释放微生物核酸或检测 微生物的试剂中的用途。The lysis composition according to any one of claims 1 to 4 is used for lysis to release microbial nucleic acid or detection. Use in microbial reagents.
  9. 如权利要求6-8中任一项所述的方法或用途，其特征在于，所述微生物为病毒、细菌、真菌、放线菌、立克次氏体、支原体、衣原体或螺旋体。The method or use according to any one of claims 6 to 8, wherein the microorganism is a virus, a bacterium, a fungus, an actinomycete, a rickettsia, a mycoplasma, a chlamydia or a spirochete.
    优选地，所述微生物为病毒，进一步优选为冠状病毒或流感病毒，更优选为新型冠状病毒。Preferably, the microorganism is a virus, more preferably a coronavirus or an influenza virus, and more preferably a novel coronavirus.
    优选地，所述核酸为DNA和/或RNA。 Preferably, the nucleic acid is DNA and/or RNA.
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