WO2020200201A1

WO2020200201A1 - Preparation method for nano magnetic bead, free dna extraction kit and extraction method

Info

Publication number: WO2020200201A1
Application number: PCT/CN2020/082451
Authority: WO
Inventors: 万昊; 张博; 赵舒婷; 葛东亮; 张飞; 张海燕
Original assignee: 深圳市南科征途有限公司
Priority date: 2019-04-04
Filing date: 2020-03-31
Publication date: 2020-10-08

Abstract

Disclosed by the present application are a magnetic bead for extracting free DNA and a preparation method therefor, on the basis of which, further disclosed is a free DNA extraction kit based on the magnetic bead, comprising sample lysate, binding solution, primary washing solution, secondary washing solution and eluent. The sample lysate comprises nuclease-free water, reagent A and reagent B. The reagent A is at least one of guanidine isothiocyanate, guanidine hydrochloride, sodium iodide, lithium acetate, lithium perchlorate and sodium perchlorate. The reagent B is at least one of sodium dodecyl benzene sulfonate, fatty acid glyceride, Tween20, Triton-X100, SDS, NP40 and polyoxyethylene nonionic surfactant. In addition to the sample lysate, the binding solution comprises reagent C, and the reagent C is at least one of isopropanol, butanol, methanol and absolute ethanol. The primary washing solution is the same as the binding solution in terms of components. The secondary washing solution is one of nuclease-free water, sodium chloride solution with a molar concentration of 0.1-1M, Tris buffer or TE buffer. The eluent is one of nuclease-free water, Tris buffer or TE buffer. Also disclosed by the present application is a method for extracting the free DNA. The present application may greatly improve the extraction efficiency and purity of free DNA.

Description

纳米磁珠的制备方法、游离DNA提取试剂盒及提取方法Preparation method of nano magnetic beads, free DNA extraction kit and extraction method

技术领域Technical field

本申请涉及DNA检测领域，具体涉及一种用于游离DNA提取的纳米磁珠的制备方法、游离DNA提取试剂盒及游离DNA提取方法。This application relates to the field of DNA detection, in particular to a method for preparing nano magnetic beads for free DNA extraction, a free DNA extraction kit and a free DNA extraction method.

背景技术Background technique

核酸包括脱氧核糖核酸(DNA)和核糖核酸(RNA)两类，是一类带有遗传信息的生物大分子。血中游离DNA(cell free DNA,cfDNA)简称循环脱氧核糖核酸，是指循环血中游离于细胞外的部分降解了的机体内源性DNA；研究发现，游离DNA也含有非常重要的遗传信息。近年来，游离DNA研究成为基因组分子诊断研究的一个热门领域，目前已被临床逐渐运用到了从肿瘤的最初诊断到治疗、发展的各个阶段，包括：早期筛查，评估肿瘤的异质性，转移复发风险和预后评估、实时监控治疗反应和耐药，肿瘤分期分级和指导治疗方案选择等。Nucleic acid includes two types of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are a class of biological macromolecules with genetic information. Cell free DNA (cfDNA) in the blood is abbreviated as circulating deoxyribonucleic acid, which refers to the partially degraded endogenous DNA in the circulating blood that is free from cells. Studies have found that free DNA also contains very important genetic information. In recent years, cell-free DNA research has become a hot field of genomic molecular diagnostic research. It has been gradually used clinically in all stages from the initial diagnosis to treatment and development of tumors, including: early screening, evaluation of tumor heterogeneity, and metastasis Evaluation of recurrence risk and prognosis, real-time monitoring of treatment response and drug resistance, tumor staging and classification, and guidance of treatment options.

cfDNA在血循环中的含量很低，且极易被丰度较高的非cfDNA污染和稀释，尤其是早期癌症患者。而且，原发性脑瘤患者的cfDNA可能由于血脑屏障的阻隔作用难以在外周血中被检测到，这给cfDNA的检测带来了极大挑战。使用cfDNA进行检测的第一步就是要对人类血清血浆中的cfDNA进行提取纯化，提取产物的质量直接影响到后续检测结果的准确性和可靠性。而游离DNA的片段小、在血清和血浆中含量低等特性给cfDNA提取的得率提出了更高的要求。目前核酸提取方法主要有酚/氯仿抽提、离心柱法和磁珠法。酚/氯仿抽提因大量使用有毒溶剂，对操作者毒性较大，操作步骤繁琐，难于实现自动化操作。离心柱法因其游离核酸提取效率低、成本高，且需要高速离心机，也难于实现自动化，均不能广泛适用于大规模的临床检测。近年发展起来的磁珠法提取核酸技术，其采用超顺磁性纳米颗粒，利用在高盐条件低pH下吸附核酸，然后在低盐高pH下分离核酸的原理进行样本核酸的分离纯化，因其只需要在磁场作用下就高效简易地将核酸分离纯化出来，故其能实现高通量自动化标准化操作。The content of cfDNA in the blood circulation is very low, and it is easily contaminated and diluted by non-cfDNA with higher abundance, especially in patients with early cancer. Moreover, the cfDNA of patients with primary brain tumors may be difficult to detect in the peripheral blood due to the blocking effect of the blood-brain barrier, which brings great challenges to the detection of cfDNA. The first step in using cfDNA for detection is to extract and purify cfDNA in human serum and plasma. The quality of the extracted product directly affects the accuracy and reliability of subsequent detection results. The small fragments of free DNA and low content in serum and plasma put forward higher requirements for the extraction rate of cfDNA. At present, nucleic acid extraction methods mainly include phenol/chloroform extraction, spin column method and magnetic bead method. Phenol/chloroform extraction uses a large amount of toxic solvents, which is more toxic to operators, and the operation steps are cumbersome, and it is difficult to realize automatic operation. The spin column method has low extraction efficiency of free nucleic acid, high cost, and requires a high-speed centrifuge, and it is difficult to realize automation, and it is not widely applicable to large-scale clinical testing. The magnetic bead method for nucleic acid extraction technology developed in recent years uses superparamagnetic nanoparticles, which uses the principle of adsorbing nucleic acids under high salt conditions and low pH, and then separating nucleic acids under low salt and high pH to separate and purify sample nucleic acids. The nucleic acid can be separated and purified efficiently and simply under the action of a magnetic field, so it can realize high-throughput automated standardized operations.

目前市场上游离DNA提取试剂盒中的溶液是根据不同的提取方法相应设计，比如基于酚/氯仿抽提的试剂盒，大多通过酚、氯仿和异戊醇来变性蛋白质使其沉降，并使液体分层，再用乙醇等沉淀离心，达到提取目的；但上述有机溶剂有一定的毒性，不利于技术人员操作。而离心柱法和磁珠法则多采用高盐低pH的原理制备结合液，使得核酸在该条件下与离心柱的硅胶膜或磁珠相结合；再采用低盐高pH的条件制备洗脱液，便于核酸洗脱。这一原理虽广泛应用于基因组核酸的提取，但并不完全适用于游离核酸的提取，主要体现在：1.游离核酸的片段长度相对于基因组核酸短，游离DNA主峰长度在170bp左右,游离RNA小于1000nt，在一般高盐溶液中难以与磁珠高效结合；2.游离核酸的含量少，残留的杂质和盐将会严重影响后续测量和下游应用。At present, the solutions in free DNA extraction kits on the market are designed according to different extraction methods. For example, kits based on phenol/chloroform extraction mostly use phenol, chloroform, and isoamyl alcohol to denature the protein and make it settle and make the liquid Layering, and then precipitation and centrifugation with ethanol, etc., to achieve the purpose of extraction; but the above-mentioned organic solvents have certain toxicity, which is not conducive to the operation of technicians. The spin column method and the magnetic bead method mostly use the principle of high salt and low pH to prepare the binding solution, so that the nucleic acid is combined with the silica membrane or magnetic beads of the spin column under this condition; then the eluent is prepared under the conditions of low salt and high pH. , Facilitate the elution of nucleic acid. Although this principle is widely used in the extraction of genomic nucleic acid, it is not completely applicable to the extraction of free nucleic acid. It is mainly reflected in: 1. The fragment length of free nucleic acid is shorter than that of genomic nucleic acid. The main peak length of free DNA is about 170bp, free RNA Less than 1000nt, it is difficult to efficiently combine with magnetic beads in general high-salt solutions; 2. The content of free nucleic acid is small, and residual impurities and salts will seriously affect subsequent measurements and downstream applications.

发明内容Summary of the invention

本申请的主要目的是提出一种用于游离DNA提取的纳米磁珠的制备方法、游离DNA提取试剂盒及游离DNA的提取方法，旨在解决现有游离核酸提取法中，提取效率和纯度不高等技术问题。The main purpose of this application is to propose a method for preparing magnetic nanobeads for free DNA extraction, a free DNA extraction kit, and a free DNA extraction method, aiming to solve the problems in the existing free nucleic acid extraction methods in terms of extraction efficiency and purity. Advanced technical issues.

为实现上述目的，本申请提出一种纳米磁珠的制备方法，其包括以下步骤：In order to achieve the above objective, this application proposes a method for preparing nano magnetic beads, which includes the following steps:

将氯化铁、氯化钴和氯化镍等比混合，得到金属盐混合物；Mix ferric chloride, cobalt chloride and nickel chloride in equal ratios to obtain a metal salt mixture;

将乙二醇与所述金属盐混合物在机械搅拌条件下充分混合，得到乙二醇与金属盐混合物；Fully mixing ethylene glycol and the metal salt mixture under mechanical stirring conditions to obtain a ethylene glycol and metal salt mixture;

在乙二醇与金属盐混合物中加入乙酸钠，并继续进行搅拌，得到混合溶液；Add sodium acetate to the mixture of ethylene glycol and metal salt, and continue to stir to obtain a mixed solution;

将混合溶液倒入非金属材质反应釜中；Pour the mixed solution into the non-metallic reactor;

将反应釜放入控温烘箱中，控制烘箱按预设程序工作，使混合溶液热反应，形成铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒； Put the reaction kettle into a temperature-controlled oven, and control the oven to work according to a preset program to thermally react the mixed solution to form Fe-Co-NiO _X magnetic nanoparticles;

磁性分离所述铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒，并对其进行清洗和干燥。 Magnetically separate the Fe-Co-NiO _X magnetic nanoparticles of Fe-Co-Ni alloy, and clean and dry them.

本申请还提出一种基于上述纳米磁珠的游离DNA提取试剂盒，其包括样本裂解液、结合液、一次洗涤液、二次洗涤液、洗脱液和纳米磁珠，所述样本裂解液成份包括无核酸酶水、试剂A和试剂B，所述试剂A为异硫氰酸胍、盐酸胍、碘化钠、乙酸锂、高氯酸锂和高氯酸钠中至少一种，试剂B为十二烷基苯磺酸钠、脂肪酸甘油酯、Tween20、Triton-X100、SDS、NP40和聚氧乙烯型非离子表面活性剂中至少一种；所述结合液中除样本裂解液的成份外还包括试剂C，所述试剂C为异丙醇、丁醇、甲醇和无水乙醇中至少一种；所述一次洗涤液的成份与结合液相同；所述二次洗涤液为无核酸酶水、摩尔浓度为0.1-1M氯化钠溶液、Tris缓冲液或者TE缓冲液中的一种；所述洗脱液为无核酸酶水，Tris缓冲液或者TE缓冲液中的一种；所述纳米磁珠其磁核为铁钴镍合金Fe-Co-NiO _X，粒径分布在300-400nm之间，表层由内到外依次至少包覆有碳层和无机二氧化硅层，且在最外层共价接枝有聚乙二醇。 This application also proposes a free DNA extraction kit based on the above-mentioned nano magnetic beads, which includes a sample lysis solution, a binding solution, a primary washing solution, a secondary washing solution, an eluent, and nano magnetic beads. Including nuclease-free water, reagent A and reagent B, the reagent A is at least one of guanidine isothiocyanate, guanidine hydrochloride, sodium iodide, lithium acetate, lithium perchlorate and sodium perchlorate, and reagent B is At least one of sodium dodecylbenzene sulfonate, fatty acid glyceride, Tween20, Triton-X100, SDS, NP40, and polyoxyethylene type non-ionic surfactant; in addition to the components of the sample lysate, the binding solution Including reagent C, said reagent C is at least one of isopropanol, butanol, methanol and absolute ethanol; the composition of the primary washing liquid is the same as the binding liquid; the secondary washing liquid is nuclease-free water, The molar concentration is one of 0.1-1M sodium chloride solution, Tris buffer or TE buffer; the eluent is one of nuclease-free water, Tris buffer or TE buffer; the nano magnetic The magnetic core of the bead is Fe-Co-NiO _X , with a particle size distribution between 300-400nm. The surface layer is covered with at least a carbon layer and an inorganic silica layer from the inside to the outside, and the outermost layer Covalently grafted with polyethylene glycol.

本申请还提供一种游离DNA的提取方法，其采用前述的游离DNA提取试剂盒完成，所述方法包括以下步骤：This application also provides a method for extracting free DNA, which is completed by using the aforementioned free DNA extraction kit, and the method includes the following steps:

按照先添加蛋白酶K，然后添加样本，最后加入裂解液的顺序，在离心管中依次添加各个组分，震荡混匀各组分并孵育，以进行样品裂解；In the order of adding proteinase K first, then adding the sample, and finally adding the lysis solution, add each component in the centrifuge tube, shake and mix each component and incubate to perform sample lysis;

将纳米磁珠和结合液按比例配制成纳米磁珠结合液；Prepare the nano magnetic beads and the binding liquid in proportion to make the nano magnetic bead binding liquid;

将制备好的纳米磁珠结合溶液加入样本中，震荡混匀后静置，并弃掉上清；Add the prepared nanomagnetic bead binding solution to the sample, shake and mix, and then stand still, and discard the supernatant;

使用一次洗涤液清洗游离DNA；Use a washing solution to clean free DNA;

使用二次洗涤工作液清洗游离DNA；所述二次洗涤工作液由二次洗涤液用无水乙醇稀释得到；Use a secondary washing working solution to clean the free DNA; the secondary washing working solution is obtained by diluting the secondary washing solution with absolute ethanol;

加入洗脱液，震荡以使纳米磁珠重悬，并进一步震荡以使纳米磁珠上的游离DNA洗脱。Add the eluent, shake to resuspend the nano magnetic beads, and further shake to elute the free DNA on the nano magnetic beads.

本申请中，通过将氯化铁、氯化钴和氯化镍等比混合并进行机械搅拌得到铁钴镍合金纳米磁珠磁核，具有强磁响应能力，能在外界磁场条件下从溶液环境中有效快速分离。另外，磁珠粒径小且均一，粒径分布在500-600nm之间，在溶液环境中具备良好的悬浮性，有利于磁珠和溶液中DNA的相互接触，促进分离富集过程。另外，本申请采用无核酸酶水、异硫氰酸胍、盐酸胍、碘化钠、高氯酸钠、Tween20、Triton-X100、SDS、NP40和聚氧乙烯型非离子表面活性剂等环保材料作为主要溶液，不使用传统方法中的苯、氯仿等有毒试剂，对实验操作人员的伤害减少到最少，完全符合现代环保理念，能更好地满足临床使用条件。这些溶液于磁珠周围营造出特定化学环境，使得磁珠选择性高效吸附血清或血浆中游离DNA。具体效果如下：1、所有试剂无毒安全，不使用传统方法中的苯、氯仿等有毒试剂，对实验操作人员的伤害减少到最少。2、针对低浓度、小片段的游离核酸富集效率和纯度高，无需引入Carrier RNA等外源干扰。3、所有结合、洗涤和洗脱过程均在常温下进行，耗时短，操作简单。In this application, the iron-cobalt-nickel alloy nano-magnetic bead core is obtained by mixing ferric chloride, cobalt chloride and nickel chloride in equal ratios and performing mechanical stirring. It has strong magnetic response capability and can be removed from the solution environment under external magnetic field conditions. Effective and rapid separation. In addition, the particle size of the magnetic beads is small and uniform, with a particle size distribution between 500-600nm, and has good suspension in the solution environment, which is beneficial to the mutual contact of the magnetic beads and the DNA in the solution, and promotes the separation and enrichment process. In addition, this application uses environmentally friendly materials such as nuclease-free water, guanidine isothiocyanate, guanidine hydrochloride, sodium iodide, sodium perchlorate, Tween20, Triton-X100, SDS, NP40 and polyoxyethylene type non-ionic surfactants As the main solution, benzene, chloroform and other toxic reagents in the traditional method are not used, and the harm to the experimental operators is minimized. It is fully in line with modern environmental protection concepts and can better meet the conditions of clinical use. These solutions create a specific chemical environment around the magnetic beads, allowing the magnetic beads to selectively and efficiently adsorb free DNA in serum or plasma. The specific effects are as follows: 1. All reagents are non-toxic and safe, and toxic reagents such as benzene and chloroform in the traditional method are not used, and the harm to the experimental operators is minimized. 2. The enrichment efficiency and purity of free nucleic acid with low concentration and small fragments are high, and there is no need to introduce foreign interference such as carrier RNA. 3. All binding, washing and elution processes are carried out at room temperature, which is time-consuming and simple to operate.

附图说明Description of the drawings

图1为本申请高磁性微粒径纳米磁珠的制备方法一实施例的流程示意图；FIG. 1 is a schematic flow chart of an embodiment of a method for preparing high magnetic micro-particle size nano magnetic beads according to the present application;

图2为本申请一实施例中游离DNA提取试剂盒的操作流程示意图；2 is a schematic diagram of the operation flow of the free DNA extraction kit in an embodiment of the application;

图3A展示了使用本申请的游离DNA提取试剂盒抽提血浆中cfDNA和其他同类主流抽提试剂抽提游离DNA的结果；Figure 3A shows the results of using the free DNA extraction kit of the application to extract cfDNA from plasma and other similar mainstream extraction reagents to extract free DNA;

图3B展示了使用本申请的游离DNA提取试剂盒抽提尿液中cfDNA和其他同类主流抽提试剂抽提游离DNA的结果；Figure 3B shows the results of extracting free DNA from urine using the free DNA extraction kit of this application and other similar mainstream extraction reagents;

图4A展示了使用游离DNA提取试剂盒抽提血浆中的cfDNA，通过荧光定量PCR检测β-球蛋白的水平，得到的扩增曲线和C _T值的结果； Figure 4A shows the results of the amplification curve and _CT value obtained by extracting cfDNA from plasma using a free DNA extraction kit and detecting the level of β-globulin by fluorescent quantitative PCR;

图4B展示了上述荧光定量PCR 3复孔扩增曲线对应的C _T值； Figure 4B shows the _CT value corresponding to the above-mentioned fluorescence quantitative PCR 3 multiple-well amplification curve;

图5A展示了使用本申请游离DNA提取试剂盒抽提DNA的验证结果；Figure 5A shows the verification result of DNA extraction using the free DNA extraction kit of this application;

图5B展示了使用安捷伦2100生物分析仪分析不同实验批次间的抽提产物的结果；Figure 5B shows the results of using Agilent 2100 Bioanalyzer to analyze the extracted products between different experimental batches;

图5C展示了血清中人工合成并含有突变的EGFR片段的标准品经由游离DNA提取试剂盒抽提产物经过荧光定量PCR探针法检测c.2573T>G(L858R)突变水平，得到的扩增曲线；Figure 5C shows the amplification curve obtained by detecting the mutation level of c.2573T>G (L858R) by the fluorescent quantitative PCR probe method of the standard product of artificially synthesized and mutated EGFR fragment in the serum through the free DNA extraction kit. ；

图5D展示了图5C所示荧光定量PCR 3复孔扩增曲线对应的C _T值。 Figure 5D shows the _CT value corresponding to the fluorescence quantitative PCR 3-well amplification curve shown in Figure 5C.

具体实施方式detailed description

下面将详细描述本申请的实施例，实施例的示例在附图中示出，其中自始至终相同标号表示相同的元件或具有相同功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本申请，而不能理解为对本申请的限制，基于本申请中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。The embodiments of the present application will be described in detail below. Examples of the embodiments are shown in the drawings, in which the same reference numerals denote the same elements or elements with the same functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the application, and should not be understood as a limitation to the application. Based on the embodiments in the application, those of ordinary skill in the art have not made creative work premises. All other embodiments obtained below belong to the protection scope of this application.

为解决上述技术问题，本申请提出一种纳米磁珠的制备方法，该纳米磁珠用于游离DNA提取。参照图1所示，该方法包括以下步骤：In order to solve the above technical problems, the present application proposes a method for preparing nano magnetic beads, which are used for free DNA extraction. Referring to Figure 1, the method includes the following steps:

步骤S10，将氯化铁、氯化钴和氯化镍等比混合，得到金属盐混合物；本申请一实施例中，可将氯化铁、氯化钴和氯化镍以总量5.4g投入反应，三种盐质量比为1:1:1。Step S10, mixing ferric chloride, cobalt chloride and nickel chloride in equal ratios to obtain a metal salt mixture; in an embodiment of the present application, ferric chloride, cobalt chloride and nickel chloride can be added in a total amount of 5.4g In the reaction, the mass ratio of the three salts is 1:1:1.

步骤S20，将乙二醇与所述金属盐混合物在机械搅拌条件下充分混合，得到乙二醇与金属盐混合物；本申请实施例中，乙二醇的剂量可根据氯化铁、氯化钴和氯化镍以及搅拌容器的大小进行选择，例如，前述实施例中，可取170毫升乙二醇，和5.4g三种金属盐混合物在机械搅拌条件下充分混合均匀，搅拌速度可视混合物的多少选择200～400rpm(转/分)，搅拌时间可选择为20～40分钟(分钟)。前述实施例中，可选择转速为300rpm，搅拌时间为30 分钟。In step S20, the ethylene glycol and the metal salt mixture are fully mixed under mechanical stirring conditions to obtain a ethylene glycol and metal salt mixture; in the embodiment of the present application, the dosage of ethylene glycol can be based on ferric chloride and cobalt chloride. Choose between nickel chloride and the size of the stirring container. For example, in the foregoing example, 170 ml of ethylene glycol and 5.4 g of the three metal salt mixtures can be fully mixed under mechanical stirring conditions. The stirring speed may vary depending on the mixture. Choose 200～400rpm (revolution/min), and the stirring time can choose 20～40 minutes (minutes). In the foregoing embodiment, the rotation speed may be 300 rpm and the stirring time may be 30 minutes.

步骤S30，在乙二醇与金属盐混合物中加入乙酸钠，并继续进行搅拌，得到混合溶液；例如，前述实施例中，可向混合均匀后的液体中加入10g乙酸钠，并继续搅拌30分钟。Step S30, add sodium acetate to the mixture of ethylene glycol and metal salt, and continue to stir to obtain a mixed solution; for example, in the foregoing embodiment, 10g of sodium acetate can be added to the uniformly mixed liquid, and the stirring is continued for 30 minutes .

步骤S40，将混合溶液倒入非金属材质反应釜中；例如，可将混合后的溶液倒入300毫升容量的特氟龙反应釜中。In step S40, the mixed solution is poured into a non-metallic material reactor; for example, the mixed solution can be poured into a Teflon reactor with a capacity of 300 ml.

步骤S50，将反应釜放入控温烘箱中，控制烘箱按预设程序工作，使混合溶液热反应，形成铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒；本申请实施例中，温度可控制在150℃左右，具体温度程序可以为：2小时内将温度从30℃提升至150℃，并维持在150℃进行6小时反应，最后在2小时内将温度降至室温。 In step S50, the reaction kettle is put into a temperature-controlled oven, and the oven is controlled to work according to a preset program to thermally react the mixed solution to form Fe-Co-NiO _X magnetic nanoparticles; in the embodiment of the application, the temperature can be The temperature is controlled at about 150°C, and the specific temperature program can be: raise the temperature from 30°C to 150°C within 2 hours, maintain it at 150°C for 6 hours, and finally lower the temperature to room temperature within 2 hours.

步骤S60，磁性分离所述铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒，并对其进行清洗和干燥。本申请实施例中，可用超纯水和乙醇依次清洗磁性纳米颗粒，用于洗去多余未反应的盐和有机溶剂，然后在60℃真空条件下干燥清洗后的磁性纳米颗粒。 Step S60, magnetically separating the Fe-Co-NiO _X magnetic nanoparticles of Fe-Co-Ni alloy, and cleaning and drying them. In the embodiments of the present application, the magnetic nanoparticles can be washed sequentially with ultrapure water and ethanol to remove excess unreacted salt and organic solvent, and then the washed magnetic nanoparticles can be dried under vacuum at 60°C.

本申请通过将氯化铁、氯化钴和氯化镍等比混合并进行机械搅拌得到铁钴镍合金纳米磁珠磁核，具有强磁响应能力，能在外界磁场条件下从溶液环境中有效快速分离。另外，磁核粒径小且均一，粒径分布在300-400nm之间，在溶液环境中具备良好的悬浮性，有利于磁珠和溶液中DNA的相互接触，促进分离富集过程。进一步的，本申请磁珠制备过程中用到的反应步骤，属于溶剂热法，都是高度可控的材料制备技术，保证了磁核生产的重复性。通过验证，此磁核制备方法产量较大，一次制备能获得1～10克磁核，可用将近200个核酸提取试剂盒的制备，能较好产业化。In this application, the iron-cobalt-nickel alloy nano magnetic bead core is obtained by mixing ferric chloride, cobalt chloride and nickel chloride in equal ratios and performing mechanical stirring. It has strong magnetic response capability and can be effectively removed from the solution environment under external magnetic field conditions. Quick separation. In addition, the magnetic core has a small and uniform particle size, with a particle size distribution between 300-400nm, and has good suspension in the solution environment, which is beneficial to the mutual contact of the magnetic beads and the DNA in the solution, and promotes the separation and enrichment process. Further, the reaction steps used in the preparation process of the magnetic beads of the present application belong to the solvothermal method, which are all highly controllable material preparation techniques, which ensure the reproducibility of magnetic core production. It has been verified that this magnetic core preparation method has a relatively large yield. It can obtain 1-10 grams of magnetic cores in one preparation, and can be prepared with nearly 200 nucleic acid extraction kits, which can be better industrialized.

为了得到性能较好的纳米磁珠，在本申请实施例中，除上述步骤外，还包括以下步骤：In order to obtain nano magnetic beads with better performance, in the embodiments of the present application, in addition to the above steps, the following steps are further included:

步骤S70，称量1份干燥后的铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒，以重量计按1:18的比例配比葡萄糖，加入质量为200倍磁性纳米颗粒的超纯水并在机械搅拌条件下混合均匀；本申请实施例中，1份铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒可以为1克，可加入18克葡萄糖和200克(200毫升)超纯水在机械搅拌条件下混合均匀，搅拌速度可按前述步骤S30中的搅拌速度。 Step S70, weighing 1 part of the dried Fe-Co-Ni alloy Fe-Co-NiO _X magnetic nanoparticles, mixing glucose in a ratio of 1:18 by weight, adding ultrapure water 200 times the mass of the magnetic nanoparticles and adding Mix uniformly under mechanical stirring conditions; in the examples of this application, 1 part of Fe-Co-NiO _X magnetic nanoparticles can be 1 gram, and 18 grams of glucose and 200 grams (200 ml) of ultrapure water can be added to The mixing is uniform under mechanical stirring conditions, and the stirring speed can be the same as the stirring speed in step S30.

步骤S80，将混和均匀的溶液倒入非金属材质反应釜中；Step S80, pour the uniformly mixed solution into the non-metallic material reaction kettle;

步骤S90，将非金属材质反应釜放入控温烘箱中，控制烘箱按预设程序工作，开始溶剂热反应，得到表层包覆有碳层的铁钴镍合金Fe-Co-NiO _X@C磁性纳米颗粒。程序升温条件可以为：2小时将温度从30℃提升至160℃，并维持在160℃进行4小时反应，最后在2小时内将温度降至室温。 Step S90: Put the non-metallic material reactor into a temperature-controlled oven, control the oven to work according to the preset program, start the solvothermal reaction, and obtain an iron-cobalt-nickel alloy Fe-Co-NiO _X @C magnetism with a carbon layer on the surface Nano particles. The temperature program can be as follows: increase the temperature from 30°C to 160°C in 2 hours, maintain the temperature at 160°C for 4 hours, and finally lower the temperature to room temperature within 2 hours.

本申请实施例中，通过在磁核-铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒的表层包覆一层惰性碳层，可以将磁核与外界环境分隔，避免外界化学环境对磁核的损坏，影响其磁响应能力。 In the embodiment of the present application, by coating an inert carbon layer on the surface of the magnetic core-iron-cobalt-nickel alloy Fe-Co-NiO _X magnetic nanoparticles, the magnetic core can be separated from the external environment and prevent the external chemical environment from affecting the magnetic core. The damage affects its magnetic response capability.

步骤S100，用乙醇和氨水进行超声分散，对超声分散后形成的溶液进行机械搅拌，并在机械搅拌过程中加入正硅酸乙酯；本申请实施例中，可称量1克Fe-Co-NiO _X@C磁性纳米颗粒，超声均匀分散在40毫升水中；往上述溶液中加入160毫升乙醇，4毫升氨水(28-30％重量百分含量)，超声分散10分钟；机械搅拌条件下，逐滴加入3毫升正硅酸乙酯，室温反应5小时； In step S100, ultrasonic dispersion is performed with ethanol and ammonia water, and the solution formed after ultrasonic dispersion is mechanically stirred, and ethyl orthosilicate is added during the mechanical stirring; in the embodiment of the present application, 1 gram of Fe-Co- NiO _X @C magnetic nanoparticles are uniformly dispersed in 40 ml of water by ultrasonic; add 160 ml of ethanol and 4 ml of ammonia (28-30% by weight) to the above solution, and disperse ultrasonically for 10 minutes; under mechanical stirring conditions, gradually Add 3 ml of ethyl orthosilicate dropwise, and react at room temperature for 5 hours;

步骤S101，磁性分离所生成的产物，得到表层包覆有无机二氧化硅的铁钴镍合金Fe-Co-NiO _X@C@SiO ₂磁性纳米颗粒。磁性分离生成产物，用超纯水和乙醇依次清洗产物，用于洗去多余未反应的正硅酸乙酯和有机溶剂。然后，于60℃真空干燥清洗后的产物(Fe-Co-NiO _X@C@SiO ₂)。 In step S101, the generated products are magnetically separated to obtain Fe-Co-NiO _X @C@SiO ₂ magnetic nanoparticles coated with inorganic silicon dioxide on the surface. Magnetic separation produces the product, and the product is washed with ultrapure water and ethanol in turn to wash away excess unreacted ethyl orthosilicate and organic solvent. Then, the cleaned product (Fe-Co-NiO _X @C@SiO ₂ ) was vacuum dried at 60°C.

本申请实施例中，由于在磁珠表层包覆有无机二氧化硅，磁珠比表面积大，无机二氧化硅层能提供大量羟基活性位点，用于抓取样品中游离DNA组分，最大程度提高分离提取效率。In the examples of this application, since the surface layer of the magnetic beads is covered with inorganic silica, the magnetic beads have a large specific surface area. The inorganic silica layer can provide a large number of hydroxyl active sites for grabbing free DNA components in the sample. To improve the separation and extraction efficiency.

进一步地，本申请实施例中，还可在表层包覆有无机二氧化硅的铁钴镍合金Fe-Co-NiO _X@C@SiO ₂磁性纳米颗粒表层共价接枝聚乙二醇。聚乙二醇的引入可以增加磁珠的防聚集性，使得磁珠即使在极端盐浓度和pH环境中都能保持良好的悬浮性。同时，防非特异性吸附分子链聚乙二醇的引入，能有效减小其它分子杂质(例如：蛋白质，无机盐和糖等)非特异吸附干扰，提高分离提取后游离DNA纯度，可直接用于下游检测。 Further, in the embodiments of the present application, the surface layer of the Fe-Co-NiO _X @C@SiO ₂ magnetic nano-particles of Fe-Co-NiO _X @C@SiO ₂ magnetic nanoparticles coated with inorganic silica can also be covalently grafted with polyethylene glycol. The introduction of polyethylene glycol can increase the anti-aggregation properties of the magnetic beads, so that the magnetic beads can maintain good suspension even in extreme salt concentrations and pH environments. At the same time, it prevents the introduction of non-specific adsorption molecular chain polyethylene glycol, which can effectively reduce the non-specific adsorption interference of other molecular impurities (such as protein, inorganic salt and sugar, etc.), improve the purity of free DNA after separation and extraction, and can be used directly Downstream testing.

前述共价接枝聚乙二醇的步骤中可包括：The aforementioned step of covalently grafting polyethylene glycol may include:

步骤S110，将所述铁钴镍合金Fe-Co-NiO _X@C@SiO ₂磁性纳米颗粒用异丙醇进行超声分散；本申请实施例中，可称量0.5克Fe-Co-NiO _X@C@SiO ₂，加入40毫升异丙醇，超声分散10分钟； Step S110, the iron-cobalt-nickel alloy Fe-Co-NiO _X @C@SiO ₂ magnetic nanoparticles are ultrasonically dispersed with isopropanol; in the embodiment of the present application, 0.5 g of Fe-Co-NiO _X @ can be weighed C@SiO ₂ , add 40 ml of isopropanol, and ultrasonically disperse for 10 minutes;

步骤S120，在氮气保护下，对超声分散后的产物进行机械搅拌，并在搅拌过程中加入氨丙基三乙氧基硅烷；可逐滴加入1毫升3-氨丙基三乙氧基硅烷。氮气保护下，以300转/分速度机械搅拌溶液，反应24小时；Step S120, under the protection of nitrogen, mechanically stir the ultrasonically dispersed product, and add aminopropyltriethoxysilane during the stirring process; 1 ml of 3-aminopropyltriethoxysilane can be added dropwise. Under nitrogen protection, mechanically stir the solution at a speed of 300 rpm and react for 24 hours;

步骤S130，磁性分离搅拌后的产物并用异丙醇和乙醇进行清洗，得到氨基铁钴镍合金Fe-Co-NiO _X@C@SiO ₂-NH ₂磁性纳米颗粒；磁性分离产物Fe-Co-NiO _X@C@SiO ₂-NH ₂，异丙醇清洗三遍后，用乙醇清洗一遍。室温干燥 Fe-Co-NiO _X@C@SiO ₂-NH ₂。 In step S130, the stirred product is magnetically separated and washed with isopropanol and ethanol to obtain iron-cobalt-nickel-amino-iron alloy Fe-Co-NiO _X @C@SiO ₂ -NH ₂ magnetic nanoparticles; magnetic separation product Fe-Co-NiO _X @C@SiO ₂ -NH ₂ , after cleaning three times with isopropanol, clean it with ethanol once. Dry Fe-Co-NiO _X @C@SiO ₂ -NH ₂ at room temperature.

步骤S140，将所述氨基铁钴镍合金Fe-Co-NiO _X@C@SiO ₂-NH ₂磁性纳米颗粒超声分散在2-吗啉缓冲液中，并加入乙基-(3-二甲基氨基丙基)碳酰二亚胺EDC和羧基聚乙二醇COOH-PEG，在机械搅拌条件下进行室温反应；取上述干燥产物，超声均匀分散在2-吗啉缓冲液中(MES，pH＝4.5-6.5)，并加入20毫克1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(EDC)和100毫克羧基聚乙二醇(COOH-PEG)。在300转/分机械搅拌条件下，室温反应2小时； Step S140, ultrasonically disperse the amino-iron-cobalt-nickel alloy Fe-Co-NiO _X @C@SiO ₂ -NH ₂ magnetic nanoparticles in 2-morpholine buffer solution, and add ethyl-(3-dimethyl Aminopropyl) carbodiimide EDC and carboxyl polyethylene glycol COOH-PEG are reacted at room temperature under mechanical stirring conditions; take the above-mentioned dried product and uniformly disperse it in 2-morpholine buffer (MES, pH= 4.5-6.5), and add 20 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 100 mg of carboxypolyethylene glycol (COOH-PEG). React at room temperature for 2 hours under the condition of 300 rpm mechanical stirring;

步骤S150，磁性分离机械搅拌后的产物，用超纯水和乙醇依次清洗得到聚乙二醇铁钴镍合金Fe-Co-NiO _X@C@SiO ₂-PEG磁性纳米颗粒。磁性分离生成产物，用超纯水和乙醇依次清洗产物，用于洗去多余未反应碳酰二亚胺EDC、羧基聚乙二醇COOH-PEG和2-吗啉缓冲液MES。60℃真空干燥，得到最终产物Fe-Co-NiO _X@C@SiO ₂-PEG。 In step S150, the mechanically stirred product is magnetically separated, and washed with ultrapure water and ethanol in sequence to obtain polyethylene glycol Fe-Co-NiO _X @C@SiO ₂ -PEG magnetic nanoparticles. Magnetic separation produces the product, and the product is washed sequentially with ultrapure water and ethanol to wash away excess unreacted carbodiimide EDC, carboxyl polyethylene glycol COOH-PEG and 2-morpholine buffer MES. Vacuum drying at 60°C to obtain the final product Fe-Co-NiO _X @C@SiO ₂ -PEG.

本申请提供一种纳米磁珠，其磁核为铁钴镍合金Fe-Co-NiO _X，所述磁核采用前述方法制得，其表面由内到外依次至少包覆有碳层和无机二氧化硅层，且最外层共价接枝有聚乙二醇。本申请实施例中，纳米磁珠表层包覆碳层、无机二氧化硅层、共价接枝聚乙二醇的方法可参照前述图1所对应实施例。本申请纳米磁珠由于采用铁钴镍合金作为磁核，因此具有强磁响应能力，能在外界磁场条件下从溶液环境中有效快速分离。另外，磁珠粒径小且均一，粒径分布在500-600nm之间，在溶液环境中具备良好的悬浮性，有利于磁珠和溶液中DNA的相互接触，促进分离富集过程。 The present application provides a nano magnetic bead, the magnetic core of which is an iron-cobalt-nickel alloy Fe-Co-NiO _{X. The} magnetic core is prepared by the aforementioned method, and its surface is sequentially covered with at least a carbon layer and an inorganic layer from the inside to the outside. Silicon oxide layer, and the outermost layer is covalently grafted with polyethylene glycol. In the embodiments of the present application, the method for covering the carbon layer, the inorganic silica layer, and the covalent grafting of polyethylene glycol on the surface of the nano magnetic beads can refer to the embodiment corresponding to FIG. 1. Because the nano magnetic beads of the present application adopt iron-cobalt-nickel alloy as the magnetic core, they have strong magnetic response capability and can be effectively and quickly separated from the solution environment under the external magnetic field condition. In addition, the particle size of the magnetic beads is small and uniform, with a particle size distribution between 500-600nm, and has good suspension in the solution environment, which is beneficial to the mutual contact of the magnetic beads and the DNA in the solution, and promotes the separation and enrichment process.

本申请提出一种游离DNA提取试剂盒。该游离DNA提取试剂盒包括样本裂解液、结合液、一次洗涤液、二次洗涤液、洗脱液和前述铁钴镍合金纳米磁珠。其中，样本裂解液成份包括无核酸酶水、试剂A和试剂B，试剂A为异硫氰酸胍、盐酸胍、碘化钠、乙酸锂、高氯酸锂和高氯酸钠中至少一种，试剂B为十二烷基苯磺酸钠、脂肪酸甘油酯、Tween20、Triton-X100、SDS、NP40和聚氧乙烯型非离子表面活性剂中至少一种；结合液中除样本裂解液的成份外还包括试剂C，试剂C为异丙醇、丁醇、甲醇和无水乙醇中至少一种。本申请实施例中，试剂A为强离子性离液盐，可使细胞裂解，蛋白质和多糖沉淀；试剂B为表面活性剂，可破坏裂解核酸/蛋白复合物，使DNA释放出；试剂C为DNA沉淀剂。由于采用前述高磁性微粒径纳米磁珠，可最大程度提高分离提取效率，提高分离提取后游离DNA纯度，可直接用于下游检测。This application proposes a free DNA extraction kit. The free DNA extraction kit includes a sample lysis solution, a binding solution, a first washing solution, a second washing solution, an eluent and the aforementioned iron-cobalt-nickel alloy nano magnetic beads. Among them, the components of the sample lysis solution include nuclease-free water, reagent A and reagent B. Reagent A is at least one of guanidine isothiocyanate, guanidine hydrochloride, sodium iodide, lithium acetate, lithium perchlorate, and sodium perchlorate , Reagent B is at least one of sodium dodecylbenzene sulfonate, fatty acid glyceride, Tween20, Triton-X100, SDS, NP40 and polyoxyethylene type non-ionic surfactant; the binding solution except for the components of the sample lysate It also includes reagent C, which is at least one of isopropanol, butanol, methanol and absolute ethanol. In the examples of this application, reagent A is a strong ionic chaotropic salt, which can lyse cells and precipitate proteins and polysaccharides; reagent B is a surfactant, which can destroy the lysed nucleic acid/protein complex and release DNA; reagent C is DNA precipitation agent. Due to the use of the aforementioned high-magnetic micro-particle size nano magnetic beads, the separation and extraction efficiency can be maximized, and the purity of the free DNA after separation and extraction can be improved, which can be directly used for downstream detection.

研究表明，在强离子性离液盐的作用下，可通过作用于蛋白质、多糖和磷脂等，破坏细胞完整结构；同时，该类盐可以使磁珠及其硅层吸附DNA的能力可以大大增强。其机理可能为：①高浓度离液盐可以降低磁珠材料表面电荷，从而减少磁珠表面和带负电荷的DNA间的静电斥力；②高浓度盐可通过形成水合离子降低水的活性，使DNA和磁珠表面脱水，促使DNA聚合到磁珠表面；③高浓度离液盐可破坏氢键，使双链DNA转变为单链DNA，单链DNA暴露出来的自由碱基与磁珠表面硅层形成氢键，促进DNA的进一步吸附。作为强离子性离液盐，盐酸胍、碘化钠、高氯酸钠等盐在较高浓度时，可使变性的蛋白质和变性DNA表现出比其正确折叠或天然构象时更高的热动力学稳定性，继而可促进DNA与磁珠的可逆性结合。同时，异硫氰酸胍除据上述功能，还可作为高效的促溶剂，具有较强蛋白变性和降解、抑制核酸酶从而减少提取过程中核酸降解的作用。Studies have shown that under the action of strong ionic chaotropic salts, it can destroy the complete structure of cells by acting on proteins, polysaccharides and phospholipids; at the same time, this type of salt can greatly enhance the ability of magnetic beads and their silicon layers to adsorb DNA . The mechanism may be: ①High concentration of chaotropic salt can reduce the surface charge of magnetic bead material, thereby reducing the electrostatic repulsion between the surface of magnetic bead and negatively charged DNA; ②High concentration of salt can reduce water activity by forming hydrated ions, so that The surface of DNA and magnetic beads is dehydrated, which promotes DNA polymerization to the surface of magnetic beads; ③High concentration of chaotropic salt can break the hydrogen bond and convert double-stranded DNA into single-stranded DNA. Free bases exposed by single-stranded DNA and silicon on the surface of magnetic beads The layer forms hydrogen bonds to promote further adsorption of DNA. As a strong ionic chaotropic salt, guanidine hydrochloride, sodium iodide, sodium perchlorate and other salts at higher concentrations can make denatured proteins and denatured DNA exhibit higher thermodynamics than their correct folding or natural conformation. The chemical stability can then promote the reversible combination of DNA and magnetic beads. At the same time, in addition to the above-mentioned functions, guanidine isothiocyanate can also be used as a highly effective solvent-promoting agent, with strong protein denaturation and degradation, inhibiting nucleases and reducing nucleic acid degradation during extraction.

本申请实施例中，一次洗涤液的成份可与结合液相同。本申请实施例中一次洗涤液也包括无核酸酶水、试剂A、试剂B和试剂C，只是比例不同。具体的，在一较佳实施例中一次洗涤液的pH值为4-6；由无核酸酶水、摩尔浓度为0.5-4M的试剂A与质量浓度为0.5％-10％的试剂B，在避光条件下搅拌至溶解、然后用Tris缓冲液或Tris-EDTA溶液调节并与15％-55％体积的试剂C混合制成。In the embodiment of the present application, the composition of the primary washing liquid may be the same as that of the binding liquid. The one-time washing solution in the embodiment of this application also includes nuclease-free water, reagent A, reagent B and reagent C, but the ratio is different. Specifically, in a preferred embodiment, the pH value of a washing solution is 4-6; from nuclease-free water, reagent A with a molar concentration of 0.5-4M and reagent B with a mass concentration of 0.5%-10%, Stir to dissolve under dark conditions, then adjust with Tris buffer or Tris-EDTA solution and mix with 15%-55% of reagent C in volume.

本申请实施例中，所述二次洗涤液为无核酸酶水、摩尔浓度为0.1-1M氯化钠溶液、Tris缓冲液或者TE缓冲液中的一种；洗脱液为无核酸酶水，Tris缓冲液或者TE缓冲液中的一种。In the embodiment of this application, the secondary washing solution is one of nuclease-free water, a molar concentration of 0.1-1M sodium chloride solution, Tris buffer or TE buffer; the eluate is nuclease-free water, One of Tris buffer or TE buffer.

本申请实施例中，样本裂解液pH值为4-6；由无核酸酶水、摩尔浓度为2-6M的试剂A与质量浓度为5％-20％的试剂B，在避光条件下搅拌至溶解、然后用Tris缓冲液或Tris-EDTA溶液调节制成。In the example of this application, the pH value of the sample lysate is 4-6; it is composed of nuclease-free water, reagent A with a molar concentration of 2-6M and reagent B with a mass concentration of 5%-20%, stirred under dark conditions To dissolve, then adjust it with Tris buffer or Tris-EDTA solution.

本申请实施例中，结合液的pH值为4-6；由无核酸酶水、摩尔浓度为1-6M的试剂A与质量浓度为5％-20％的试剂B，在避光条件下搅拌至溶解、然后用Tris缓冲液或Tris-EDTA调节并与15％-55％体积的试剂C混合制成。In the example of this application, the pH value of the binding solution is 4-6; it is composed of nuclease-free water, reagent A with a molar concentration of 1-6M and reagent B with a mass concentration of 5%-20%, stirred under dark conditions To dissolve, then adjust with Tris buffer or Tris-EDTA and mix with 15%-55% of the volume of reagent C.

本申请实施例中，可采用磁核为铁钴镍合金Fe-Co-NiO _X的纳米磁珠，还可在磁核的表层由内到外依次至少包覆有碳层和无机二氧化硅层，且在最外层共价接枝有聚乙二醇，粒径分布在500-600nm之间。 In the embodiments of the present application, the magnetic core is Fe-Co-NiO _X nano magnetic beads, and the surface of the magnetic core can be coated with at least a carbon layer and an inorganic silica layer in sequence from the inside to the outside. , And the outermost layer is covalently grafted with polyethylene glycol, the particle size distribution is between 500-600nm.

本申请实施例中，游离DNA提取试剂盒溶液的制备可包括样本裂解液配制、裂解/结合液配制、一次洗涤液配制、二次洗涤液配制、洗涤液配制等步骤，具体步骤流程如下：In the embodiment of the application, the preparation of the free DNA extraction kit solution may include sample lysis solution preparation, lysis/binding solution preparation, primary washing solution preparation, secondary washing solution preparation, and washing solution preparation steps. The specific steps are as follows:

1、样本裂解液的配制。裂解液配置方法为：取一定量无核酸酶水，加入2-6M试剂A与5％-20％质量浓度试剂B，避光条件下不断搅拌至溶解，用Tris 缓冲液或Tris-EDTA溶液调节pH值为4-6。1. Preparation of sample lysate. The lysis solution configuration method is: take a certain amount of nuclease-free water, add 2-6M reagent A and 5%-20% mass concentration reagent B, keep stirring until it dissolves in the dark, adjust with Tris buffer or Tris-EDTA solution The pH is 4-6.

2、结合液的配制。裂解结合液配置方法为：取一定量无核酸酶水，加入1-6M试剂A，0.5％-20％质量浓度试剂B，在避光条件下不断搅拌至溶解后用Tris缓冲液或Tris-EDTA溶液调节pH值为4-6，并与15％-55％体积的试剂C混合均匀。2. Preparation of binding solution. The lysis and binding solution configuration method is: take a certain amount of nuclease-free water, add 1-6M reagent A, 0.5%-20% mass concentration reagent B, and keep stirring under dark conditions until it dissolves, then use Tris buffer or Tris-EDTA Adjust the pH of the solution to 4-6, and mix with 15%-55% of reagent C by volume.

3、一次洗涤液的配制。洗涤液配置方法为：取一定量无核酸酶水，加入0.5-4M试剂A，0.5％-10％(w/v)试剂B，在避光条件下不断搅拌至溶解后用Tris缓冲液或Tris-EDTA溶液调节pH值为4-6，并与15％-55％体积的试剂C混合均匀。3. The preparation of a washing liquid. The washing solution configuration method is: take a certain amount of nuclease-free water, add 0.5-4M reagent A, 0.5%-10% (w/v) reagent B, keep stirring under dark conditions until dissolved, then use Tris buffer or Tris -EDTA solution adjusts the pH to 4-6, and mixes with 15%-55% of reagent C by volume.

4、二次洗涤液的配制。二次洗涤液为无核酸酶水、0.1-1M氯化钠溶液，Tris缓冲液或者TE缓冲液中的一种。4. Preparation of secondary washing liquid. The secondary washing solution is one of nuclease-free water, 0.1-1M sodium chloride solution, Tris buffer or TE buffer.

5、洗脱液的配制。洗脱液为无核酸酶水、Tris缓冲液或者TE缓冲液中的一种。5. Preparation of eluent. The eluent is one of nuclease-free water, Tris buffer or TE buffer.

本申请的游离核酸提取试剂盒包括样本裂解液、结合液、一次洗涤液、二次洗涤液、洗脱液和纳米磁珠。纳米磁珠的保存条件是2-8℃冷藏，其它溶液室温，避光保存。使用前，将二次洗涤液按照1:4的比例与无水乙醇混合。纳米磁珠溶液为棕色溶液，使用纳米磁珠溶液之前，请先平衡至室温，然后涡旋震荡使纳米磁珠重悬混匀。其他溶液在室温状态下(15-30℃)应为无色澄清液体。如观察到沉淀物，应将试剂置于37℃加热直至沉淀溶解。The free nucleic acid extraction kit of the present application includes a sample lysis solution, a binding solution, a primary washing solution, a secondary washing solution, an eluent and nano magnetic beads. The storage conditions of nano magnetic beads are refrigerated at 2-8℃, and other solutions are stored at room temperature and protected from light. Before use, mix the secondary washing liquid with absolute ethanol in a ratio of 1:4. The nano magnetic bead solution is a brown solution. Before using the nano magnetic bead solution, please equilibrate to room temperature, and then vortex to resuspend and mix the nano magnetic beads. Other solutions should be colorless and clear liquids at room temperature (15-30°C). If a precipitate is observed, the reagent should be heated at 37°C until the precipitate is dissolved.

本申请还提供一种游离DNA的提取方法，由其前述的游离DNA提取试剂盒完成，所述方法包括以下步骤：This application also provides a method for extracting free DNA, which is completed by the aforementioned free DNA extraction kit, and the method includes the following steps:

步骤S11，按照先添加蛋白酶K，然后添加样本，最后加入裂解液的顺序，在离心管中依次添加各个组分，震荡混匀各组分并孵育，以进行样品裂解；Step S11, in the order of adding proteinase K first, then adding the sample, and finally adding the lysis solution, add each component in the centrifuge tube, shake and mix each component and incubate to perform sample lysis;

步骤S12，将纳米磁珠和结合液按比例配制成纳米磁珠结合液；Step S12, preparing the nano magnetic beads and the binding liquid into a nano magnetic bead binding liquid in proportion;

步骤S13，将制备好的纳米磁珠结合溶液加入样本中，震荡混匀后静置，并弃掉上清；Step S13, adding the prepared nano-magnetic bead binding solution to the sample, shaking and mixing, and then standing, and discarding the supernatant;

步骤S14，使用一次洗涤液清洗游离DNA；Step S14, use a washing solution to clean the free DNA;

步骤S15，使用二次洗涤工作液清洗游离DNA；所述二次洗涤工作液由二次洗涤液用无水乙醇稀释得到；Step S15, using a secondary washing working solution to clean the free DNA; the secondary washing working solution is obtained by diluting the secondary washing solution with absolute ethanol;

步骤S16，加入洗脱液，震荡以使纳米磁珠重悬，并进一步震荡以使纳米磁珠上的游离DNA洗脱。Step S16, adding the eluent, shaking to resuspend the nano magnetic beads, and further shaking to elute the free DNA on the nano magnetic beads.

本申请实施例中，利用前述游离DNA提取试剂盒提取DNA的使用方法包括以下样本裂解、使用纳米磁珠吸附游离DNA、使用洗涤液清洗游离DNA、二次洗涤游离DNA及从纳米磁珠上洗脱游离DNA几个步骤，具体的，各步骤详细操作流程如下：In the examples of this application, the method of using the aforementioned free DNA extraction kit to extract DNA includes the following sample lysis, using nano magnetic beads to adsorb free DNA, using washing solution to wash free DNA, secondary washing of free DNA and washing from nano magnetic beads There are several steps to remove free DNA. Specifically, the detailed operation process of each step is as follows:

A.样本裂解A. Sample lysis

1.依照试剂盒操作说明所示，于15毫升的大离心管中依次添加各个组分：蛋白酶(20毫克/毫升)80微升；血浆样本4毫升；样本裂解液400微升。注意：避免将蛋白酶K直接与样本裂解液混合。1. According to the instructions of the kit, add each component in a large 15 ml centrifuge tube: 80 μl protease (20 mg/ml); 4 ml plasma sample; 400 μl sample lysate. Note: Avoid mixing proteinase K directly with the sample lysate.

2.涡旋震荡，以混匀上述大离心管中各组分，并于60℃孵育20分钟。2. Vortex to mix the components in the large centrifuge tube, and incubate at 60°C for 20 minutes.

3.孵育结束后，将该含有血浆的大离心管平衡至室温。3. After the incubation, equilibrate the large centrifuge tube containing plasma to room temperature.

B.使用纳米磁珠吸附游离DNAB. Use nano magnetic beads to adsorb free DNA

4.依照试剂盒操作说明所示制备纳米磁珠结合溶液并混匀(注意：使用磁珠试剂前，请先平衡至室温，然后涡旋震荡该试剂使之重悬混匀)：游离DNA裂解/结合液5毫升；纳米磁珠60微升。4. Prepare the nano-magnetic bead binding solution and mix well according to the instructions of the kit (Note: Before using the magnetic bead reagent, please equilibrate to room temperature, then vortex the reagent to resuspend and mix): Free DNA lysis / Binding solution 5 ml; Nano magnetic beads 60 μl.

5.将制备好的纳米磁珠结合溶液加入大离心管的血浆样本中，涡旋震荡或颠倒10次以上混匀(注意：避免过度震荡，防止产生大量泡沫)。5. Add the prepared nanomagnetic bead binding solution to the plasma sample in the large centrifuge tube, vortex or invert more than 10 times to mix (note: avoid excessive shaking to prevent a lot of foam).

6.中高速摇晃离心管10分钟，以促进游离DNA与纳米磁珠的结合。6. Shake the centrifuge tube at medium and high speed for 10 minutes to promote the combination of free DNA and nanomagnetic beads.

7.将上述大离心管置于磁力架上静置5分钟，或待管内溶液澄清且磁珠聚于管壁。7. Place the above-mentioned large centrifuge tube on a magnetic stand for 5 minutes, or wait until the solution in the tube is clear and the magnetic beads are collected on the tube wall.

8.小心弃掉上清(可使用移液枪移走上清，或是在磁力架上小心倾倒掉上清)。8. Carefully discard the supernatant (you can use a pipette to remove the supernatant, or carefully dump the supernatant on a magnetic stand).

C.使用洗涤液清洗游离DNAC. Use washing solution to wash free DNA

9.将上述大离心管从磁力架上取下，加入1毫升洗涤液，并涡旋震荡以重悬纳米磁珠。9. Remove the above large centrifuge tube from the magnetic stand, add 1 ml of washing solution, and vortex to resuspend the nano magnetic beads.

10.将已重悬的纳米磁珠溶液转移入一个新1.5毫升离心管，并暂时保留上一步所用的大离心管(该步骤考虑管中可能有残余磁珠，以待清洗)。10. Transfer the resuspended nanomagnetic bead solution into a new 1.5ml centrifuge tube, and temporarily keep the large centrifuge tube used in the previous step (this step considers that there may be residual magnetic beads in the tube for cleaning).

11.将该1.5毫升离心管置于磁力架上静置1分钟，待管内磁珠聚集于管壁。11. Place the 1.5 ml centrifuge tube on the magnetic stand and let it stand for 1 minute until the magnetic beads in the tube gather on the tube wall.

12.利用1.5毫升离心管内的上清液来润洗上一步所用的大离心管的管壁和盖子，将残余磁珠的润洗液转移回该1.5毫升离心管，继而弃掉上一步所用大离心管。12. Use the supernatant in the 1.5 ml centrifuge tube to rinse the wall and lid of the large centrifuge tube used in the previous step, transfer the rinse solution of residual magnetic beads back to the 1.5 ml centrifuge tube, and then discard the large centrifuge tube used in the previous step. Centrifuge tube.

13.将上述1.5毫升离心管置于磁力架上静置2分钟，待管内溶液澄清且磁珠聚于管壁。13. Put the above 1.5 ml centrifuge tube on the magnetic stand and let it stand for 2 minutes, and wait until the solution in the tube is clear and the magnetic beads gather on the tube wall.

14.用移液枪小心弃掉上清。14. Use a pipette to carefully discard the supernatant.

15.将1.5毫升离心管从磁力架上取下，加入1毫升洗涤液并涡旋震荡 30秒。15. Remove the 1.5 ml centrifuge tube from the magnetic stand, add 1 ml washing solution and vortex for 30 seconds.

16.将1.5毫升离心管于台式离心机中快速离心，将粘壁的残液甩至管底，将该离心管置于磁力架上静置2分钟，待管内溶液澄清且磁珠聚于管壁。16. Quickly centrifuge the 1.5 ml centrifuge tube in a bench top centrifuge, shake the remaining liquid on the wall to the bottom of the tube, place the centrifuge tube on the magnetic stand for 2 minutes, wait until the solution in the tube is clear and the magnetic beads are collected in the tube wall.

17.用移液枪小心弃掉上清。17. Use a pipette to carefully discard the supernatant.

D.二次洗涤游离DNAD. Second washing of free DNA

18.将二次洗涤液预先以1:4的体积比用无水乙醇稀释。处理每个样品需要至少2毫升制备好的二次洗涤工作液(20％二次洗涤液)。18. Dilute the second washing solution with absolute ethanol in a volume ratio of 1:4 in advance. To process each sample, at least 2 ml of the prepared secondary washing working solution (20% secondary washing solution) is required.

19.将1.5毫升离心管从磁力架上取下，加入1毫升已经制备好的二次洗涤工作液，并涡旋震荡30秒。19. Remove the 1.5 ml centrifuge tube from the magnetic stand, add 1 ml of the prepared secondary washing working solution, and vortex for 30 seconds.

20.将1.5毫升离心管于台式离心机中快速离心，将粘壁的残液甩至管底，并将该离心管置于磁力架上静置2分钟，待管内溶液澄清且磁珠聚于管壁。20. Quickly centrifuge the 1.5 ml centrifuge tube in a bench top centrifuge, shake the remaining liquid on the wall to the bottom of the tube, and place the centrifuge tube on a magnetic stand for 2 minutes. Wait until the solution in the tube is clear and the magnetic beads are collected. Pipe wall.

21.用移液枪小心弃掉上清。21. Use a pipette to carefully discard the supernatant.

22.重复19-21步操作，对磁珠进行二次洗涤。22. Repeat steps 19-21 to wash the magnetic beads twice.

23.将1.5毫升离心管从磁力架上取下,将1.5毫升离心管于台式离心机中快速离心，将粘壁的残液甩至管底。之后将1.5毫升离心管置于磁力架上，待管内溶液澄清且磁珠聚于管壁。23. Remove the 1.5 ml centrifuge tube from the magnetic stand, quickly centrifuge the 1.5 ml centrifuge tube in a bench top centrifuge, and shake the remaining liquid on the wall to the bottom of the tube. Then place the 1.5 ml centrifuge tube on the magnetic stand, and wait until the solution in the tube is clear and the magnetic beads are collected on the tube wall.

24.用移液枪小心弃掉1.5毫升管底部残存液体。24. Use a pipette to carefully discard the remaining liquid at the bottom of the 1.5 ml tube.

25.将1.5毫升离心管置于磁力架上，开盖3分钟室温晾干(注意：若环境湿度较大，则可适当延长干燥时间，确保乙醇完全挥发，但无需过度干燥磁珠)。25. Place the 1.5ml centrifuge tube on the magnetic stand, open the lid for 3 minutes to dry at room temperature (Note: If the ambient humidity is high, the drying time can be appropriately extended to ensure that the ethanol is completely volatilized, but there is no need to over-dry the magnetic beads).

E.从纳米磁珠上洗脱游离DNAE. Elute free DNA from nanomagnetic beads

26.将1.5毫升离心管从磁力架上取下，依照试剂盒操作说明所示加入80微升游离DNA洗脱液。26. Remove the 1.5 ml centrifuge tube from the magnetic stand, and add 80 microliters of free DNA eluate according to the kit operating instructions.

27.在涡旋上充分震荡1.5毫升离心管以使纳米磁珠重悬，并进一步震荡5分钟以使纳米磁珠上的游离DNA洗脱下来。27. Fully vortex the 1.5 ml centrifuge tube to resuspend the nanomagnetic beads, and further vortex for 5 minutes to elute the free DNA on the nanomagnetic beads.

28.将1.5毫升离心管于台式离心机中快速离心，将粘壁残液甩至管底。并将该离心管置于磁力架上，待管内溶液澄清且磁珠聚于管壁。28. Centrifuge the 1.5 ml centrifuge tube quickly in a bench top centrifuge, and shake the sticky wall residue to the bottom of the tube. Place the centrifuge tube on the magnetic stand, and wait until the solution in the tube is clear and the magnetic beads are gathered on the tube wall.

29.使用不沾粘、不含有DNase和RNase的常规离心管收集含有游离DNA的上清洗脱液。29. Use a non-sticky conventional centrifuge tube that does not contain DNase and RNase to collect the upper washing and dewatering solution containing free DNA.

30.收获的游离DNA产物样品短期可置于4℃冷藏，长期保存需置于-20℃冷冻。30. The harvested free DNA product samples can be stored at 4°C for short-term storage, and stored at -20°C for long-term storage.

31.如果需要对游离DNA样品做定性定量分析,推荐使用Agilent所属设备及试剂(Bioanalyzer 2100以及配套试剂High Sensitivity DNA Analysis Kit)，以确保对样本足够低的检测限(5pg/μL)。31. If you need to do qualitative and quantitative analysis of free DNA samples, it is recommended to use Agilent's equipment and reagents (Bioanalyzer 2100 and supporting reagents High Sensitivity DNA Analysis Kit) to ensure a sufficiently low detection limit (5pg/μL) for the sample.

本申请的游离DNA提取试剂盒对人类血浆或尿液来源的自然条件下的游离DNA的卓越的抽提能力，以下是验证方式及验证结论：The cell-free DNA extraction kit of the present application has excellent extraction capabilities for cell-free DNA under natural conditions derived from human plasma or urine. The following are the verification methods and verification conclusions:

1、通过2000g、4℃离心10分钟将无细胞血浆从人全血样本中分离出来，并进一步通过16000g、4℃离心10分钟去除细胞碎片等杂质得清洁血浆。取4毫升该血浆用本申请的游离DNA提取试剂盒抽提游离DNA，同时用其他同类主流抽提试剂抽提游离DNA。使用安捷伦2100生物分析仪分析抽提产物。1. Separate the cell-free plasma from a human whole blood sample by centrifugation at 2000g for 10 minutes at 4°C, and further centrifuge at 16000g for 10 minutes at 4°C to remove cell debris and other impurities to obtain clean plasma. Take 4 ml of this plasma to extract free DNA with the free DNA extraction kit of this application, and use other similar mainstream extraction reagents to extract free DNA. The extracted products were analyzed using Agilent 2100 Bioanalyzer.

图3A展示了使用本申请的游离DNA提取试剂盒抽提血浆中cfDNA(曲线①)和其他同类主流抽提试剂抽提游离DNA(曲线②)的结果。本申请试剂所抽提血浆中cfDNA(曲线①)160bp-180bp片段主峰位(左边箭头指示)平均浓度为285pg/μL，300bp-600bp片段伴峰位(右边箭头指示)平均浓度为66pg/μL；右边箭头显示其他同类主流抽提试剂所抽提血浆中cfDNA(曲线②)160bp-180bp片段主峰位(左边箭头指示)平均浓度为192pg/μL，300bp-600bp片段主峰位(右边箭头指示)平均浓度为19pg/μL。Figure 3A shows the results of using the free DNA extraction kit of the present application to extract cfDNA from plasma (curve ①) and other similar mainstream extraction reagents to extract free DNA (curve ②). The average concentration of the main peak of the 160bp-180bp fragment (indicated by the arrow on the left) in the plasma extracted by the reagents of this application is 285pg/μL, and the average concentration of the peak position of the 300bp-600bp fragment (indicated by the arrow on the right) is 66pg/μL; The arrow on the right shows the main peak of cfDNA (curve ②) 160bp-180bp fragment in plasma extracted by other similar mainstream extraction reagents (the arrow on the left) average concentration is 192pg/μL, the average concentration of the main peak of 300bp-600bp fragment (the arrow on the right) It is 19pg/μL.

2、通过16000g、4℃离心10分钟获得无细胞尿液20mL，用本申请的游离DNA提取试剂盒抽提游离DNA，同时用其他同类主流抽提试剂抽提游离DNA。使用安捷伦2100生物分析仪分析抽提产物。2. Obtain 20 mL of cell-free urine by centrifugation at 16000 g for 10 minutes at 4°C. Use the free DNA extraction kit of this application to extract free DNA, and use other similar mainstream extraction reagents to extract free DNA. The extracted products were analyzed using Agilent 2100 Bioanalyzer.

图3B展示了使用本申请的游离DNA提取试剂盒抽提尿液中cfDNA(曲线①)和其他同类主流抽提试剂抽提游离DNA(曲线②)的结果。本申请试剂所抽提血浆中cfDNA(曲线①)160bp-180bp片段主峰位(左边箭头指示)平均浓度为341pg/uL，300bp-600bp片段伴峰位(右边箭头指示)平均浓度为59pg/μL；右边箭头显示其他同类主流抽提试剂所抽提血浆中cfDNA(曲线②)160bp-180bp片段主峰位(左边箭头指示)平均浓度为185pg/μL，300bp-600bp片段主峰位(右边箭头指示)平均浓度为23pg/μL。Figure 3B shows the results of using the free DNA extraction kit of the application to extract cfDNA in urine (curve ①) and other similar mainstream extraction reagents to extract free DNA (curve ②). The average concentration of the main peak of cfDNA (curve ①) 160bp-180bp fragment (indicated by the left arrow) in the plasma extracted by the reagent of this application is 341pg/uL, and the average concentration of the peak position of the 300bp-600bp fragment (indicated by the right arrow) is 59pg/μL; The arrow on the right shows the main peak of cfDNA (curve ②) 160bp-180bp fragments in plasma extracted by other mainstream extraction reagents of the same kind (the arrow on the left) has an average concentration of 185pg/μL, and the average concentration of the main peak of 300bp-600bp fragments (indicated by the arrow on the right) It is 23pg/μL.

从图3A和图3B可看出，本申请游离DNA提取试剂盒在血浆和尿液两种生物样本中都展示出卓越的抽提能力。本申请试剂所抽提血浆中cfDNA与其他同类主流抽提试剂所抽提血浆中cfDNA比较(图3A)，160bp-180bp片段主峰位平均浓度高0.48倍，300bp-600bp片段伴峰位(右边箭头指示)平均浓度高2.48倍；本申请试剂所抽提尿液中cfDNA与其他同类主流抽提试剂所抽提尿液中cfDNA比较(图3B)，160bp-180bp片段主峰位平均浓度高0.85倍，300bp-600bp片段伴峰位(右边箭头指示)平均浓度高1.57倍。It can be seen from FIG. 3A and FIG. 3B that the free DNA extraction kit of the present application exhibits excellent extraction capabilities in both biological samples, plasma and urine. The cfDNA in the plasma extracted by the reagent of this application is compared with the cfDNA in the plasma extracted by other similar mainstream extraction reagents (Figure 3A). The average concentration of the main peak of 160bp-180bp fragment is 0.48 times higher, and the peak position of 300bp-600bp fragment is associated with the peak (right arrow Indication) The average concentration is 2.48 times higher; the cfDNA in the urine extracted by the reagent of this application is compared with the cfDNA in the urine extracted by other similar mainstream extraction reagents (Figure 3B). The average concentration of the 160bp-180bp fragment at the main peak position is 0.85 times higher. The 300bp-600bp fragment with peak position (indicated by the arrow on the right) has an average concentration 1.57 times higher.

游离DNA提取试剂盒的高抽提效率，以最大程度保证下游操作的质量，分离提取的游离DNA可直接应用于下游的各类分子生物学检测，如测序以及各种PCR。图4A和4B展示了使用游离DNA提取试剂盒抽提血浆中的cfDNA，通过荧光定量PCR检测β-球蛋白的水平，得到的扩增曲线和C _T值的结果。图4A展示了将血液样本使用游离DNA提取试剂盒抽提，抽提产物设置3复孔，经过荧光定量PCR检测β-球蛋白的水平，扩增曲线重合；图4B展示了上述荧光定量PCR 3复孔扩增曲线对应的C _T值，理论标准差σ值＜0.05，具有统计学意义。 The high extraction efficiency of the free DNA extraction kit guarantees the quality of downstream operations to the greatest extent. The separated free DNA can be directly applied to various downstream molecular biology tests, such as sequencing and various PCR. Figures 4A and 4B show the results of the amplification curve and _CT value obtained by using a free DNA extraction kit to extract cfDNA from plasma and detecting the level of β-globulin by fluorescent quantitative PCR. Figure 4A shows the blood sample is extracted using the free DNA extraction kit, the extraction product is set in 3 multiple wells, and the level of β-globulin is detected by fluorescence quantitative PCR, and the amplification curves overlap; Figure 4B shows the above fluorescence quantitative PCR 3 The C _T value corresponding to the multiple-well amplification curve, the theoretical standard deviation σ value is less than 0.05, which is statistically significant.

二)游离DNA提取试剂盒对50-3000bp范围的DNA片段的抽提效率。2) The extraction efficiency of free DNA extraction kit for DNA fragments in the range of 50-3000bp.

为了验证50-3000bp范围的DNA片段的抽提得率，将梯状条带DNA ladder加入血清中，并用本申请的游离DNA提取试剂盒抽提DNA。使用安捷伦2100生物分析仪分析抽提产物并与DNA ladder原液对比。In order to verify the extraction yield of DNA fragments in the range of 50-3000 bp, the ladder-shaped DNA ladder was added to the serum, and the DNA was extracted with the free DNA extraction kit of the present application. Use Agilent 2100 bioanalyzer to analyze the extracted products and compare with the original DNA ladder solution.

验证血清中DNA抽提得率的步骤：Steps to verify the extraction rate of DNA in serum:

预备4毫升牛血清(FBS，赛默飞公司)，按照提取富集操作步骤进行蛋白酶K处理。Prepare 4 ml of bovine serum (FBS, Thermo Fisher Scientific), and perform proteinase K treatment according to the extraction and enrichment procedure.

将20微升梯状条带DNA ladder(50-3000bp，Sigma Aldrich公司)添加至80微升TE缓冲液进行稀释，涡旋混合均匀。Add 20 microliters of ladder-shaped DNA ladder (50-3000bp, Sigma Aldrich) to 80 microliters of TE buffer for dilution, and vortex to mix evenly.

蛋白酶K处理样品后，将50μL稀释后的DNA ladder添加至血清中；剩余50微升稀释后的DNA ladder用作参考原液(建议在添加DNA ladder之前先对血清进行蛋白酶K处理。因为外源性的DNA ladder在血清中会被降解，若不进行蛋白酶K处理会导致DNA ladder抽提得率降低)。After proteinase K processes the sample, add 50μL of the diluted DNA ladder to the serum; the remaining 50μL of the diluted DNA ladder is used as the reference stock solution (it is recommended to treat the serum with proteinase K before adding the DNA ladder. Because of exogenousity The DNA ladder will be degraded in the serum. Without proteinase K treatment, the extraction rate of DNA ladder will decrease).

按照游离DNA提取试剂盒说明书剩余步骤进行抽提。在洗脱步骤，用50微升洗脱液进行洗脱，得到50μL产物。Follow the remaining steps of the free DNA extraction kit instructions for extraction. In the elution step, elution was performed with 50 μl of eluent to obtain 50 μL of product.

使用安捷伦2100生物分析仪和高灵敏DNA分析试剂盒计算不同片段大小DNA的得率，分析和比较血清DNA抽提产物和参考原液的结果。计算DNA产量时，使用安捷伦2100生物分析仪检测抽提的DNA浓度，由于洗脱液和稀释原液都是50微升，两者浓度的比较实际也反应了DNA总量的比较。The Agilent 2100 Bioanalyzer and High Sensitive DNA Analysis Kit were used to calculate the yields of different fragment sizes of DNA, and to analyze and compare the results of serum DNA extraction products and reference stock solutions. When calculating the DNA yield, use the Agilent 2100 Bioanalyzer to detect the concentration of the extracted DNA. Since the eluate and the diluted stock solution are both 50 microliters, the comparison of the two concentrations actually reflects the comparison of the total amount of DNA.

图5A展示了使用本申请游离DNA提取试剂盒抽提DNA的验证结果：将梯状条带DNA ladder加入血清中，并用本申请的游离DNA提取试剂盒抽提DNA。使用安捷伦2100生物分析仪分析抽提产物(曲线①)并与梯状条带DNA ladder原液对比(曲线②)。安捷伦2100生物分析仪分析抽提产物(曲线①)曲线50bp–3000bp范围的DNA片段平均浓度为1854pg/μL，梯状条带DNA ladder原液(曲线②)曲线50bp-3000bp范围的DNA片段平均浓度为1893pg/μL，比较得本申请游离DNA提取试剂盒呈现超过95％的抽提效率。Figure 5A shows the verification result of DNA extraction using the free DNA extraction kit of the present application: adding a ladder-shaped DNA ladder to the serum, and extracting DNA using the free DNA extraction kit of the present application. Use Agilent 2100 bioanalyzer to analyze the extracted products (curve ①) and compare with the stock solution of ladder-shaped DNA ladder (curve ②). The average concentration of DNA fragments in the 50bp-3000bp range of the Agilent 2100 bioanalyzer (curve ①) curve is 1854pg/μL, and the average concentration of DNA fragments in the 50bp-3000bp range of the ladder-shaped DNA ladder stock solution (curve ②) curve is Compared with 1893pg/μL, the free DNA extraction kit of this application shows an extraction efficiency of more than 95%.

三)本申请游离DNA提取试剂盒呈现高度可重复的DNA抽提效果。3. The free DNA extraction kit of this application presents a highly repeatable DNA extraction effect.

将梯状条带DNA ladder稀释于血清中，并用本申请的游离DNA提取试剂盒抽提。使用安捷伦2100生物分析仪分析不同实验批次间的抽提产物，体现出游离DNA提取试剂盒在不同实验间高度的一致性和可重复性。The ladder-shaped DNA ladder is diluted in serum and extracted with the free DNA extraction kit of the present application. The Agilent 2100 bioanalyzer was used to analyze the extraction products between different experimental batches, which demonstrated the high consistency and repeatability of the free DNA extraction kit between different experiments.

图5B展示了使用安捷伦2100生物分析仪分析不同实验批次间的抽提产物的结果。本申请游离DNA提取试剂盒呈现高度可重复的DNA抽提效果。具体的，安捷伦2100生物分析仪分析抽提产物与梯状条带DNA ladder原液曲线基本重合，分析抽提产物曲线50bp–3000bp范围的DNA片段平均浓度为1867pg/μL，梯状条带DNA ladder原液曲线50bp–3000bp范围的DNA片段平均浓度为1865pg/μL，不同批次间试剂盒抽提得率理论标准差σ值＜0.001，统计学无差异。Figure 5B shows the results of using the Agilent 2100 Bioanalyzer to analyze the extracted products between different experimental batches. The free DNA extraction kit of this application presents a highly repeatable DNA extraction effect. Specifically, the Agilent 2100 Bioanalyzer analyzes the extraction product and the ladder-shaped DNA ladder stock solution curve basically coincides, analyzes the extraction product curve 50bp-3000bp range of DNA fragments with an average concentration of 1867pg/μL, ladder-shaped DNA ladder stock solution The average concentration of DNA fragments in the 50bp-3000bp range of the curve is 1865pg/μL, and the theoretical standard deviation σ value of the kit extraction rate between different batches is less than 0.001, and there is no statistical difference.

四)本申请游离DNA提取试剂盒对突变DNA片段卓越的富集分离能力(图5C和5D)。将人工合成的约170bp并含有c.2573T>G(L858R)突变的EGFR片段作为标准品，用本申请的游离DNA提取试剂盒提取，并最终洗脱入与原片段标准品同样体积的洗脱液。同时对样品以及标准品进行荧光定量PCR检测。图5C展示了血清中人工合成并含有突变的EGFR片段的标准品经由游离DNA提取试剂盒抽提，抽提产物设置3复孔，经过荧光定量PCR探针法检测c.2573T>G(L858R)突变水平，扩增曲线重合；图5D展示了上述荧光定量PCR 3复孔扩增曲线对应的C _T值，理论标准差σ值＜0.05，具有统计学意义。 Four) The free DNA extraction kit of the application has excellent enrichment and separation capabilities for mutant DNA fragments (Figures 5C and 5D). The artificially synthesized EGFR fragment of about 170 bp and containing c.2573T>G (L858R) mutation was used as a standard, extracted with the free DNA extraction kit of this application, and finally eluted into the same volume as the original fragment standard. liquid. At the same time, fluorescence quantitative PCR detection is performed on samples and standards. Figure 5C shows the artificially synthesized and mutated EGFR fragment standard in the serum was extracted by the free DNA extraction kit, and the extraction product was set in 3 multiple wells, and the fluorescent quantitative PCR probe method was used to detect c.2573T>G (L858R) The mutation level and the amplification curve overlap; Figure 5D shows the _CT value corresponding to the above-mentioned fluorescence quantitative PCR 3 multiple-well amplification curve, and the theoretical standard deviation σ value is less than 0.05, which is statistically significant.

以上的仅为本申请的部分或优选实施例，无论是文字还是附图都不能因此限制本申请保护的范围，凡是在与本申请一个整体的构思下，利用本申请说明书及附图内容所作的等效结构变换，或直接/间接运用在其他相关的技术领域均包括在本申请保护的范围内。The above are only part or preferred embodiments of this application. Neither the text nor the drawings can limit the scope of protection of this application. Anything made with the content of the specification and drawings of this application is based on the concept of the whole application. The equivalent structure transformation, or direct/indirect application in other related technical fields are all included in the protection scope of this application.

Claims

一种纳米磁珠的制备方法，所述纳米磁珠用于提取游离DNA，其特征在于，所述方法包括以下步骤：A method for preparing nano magnetic beads, which is used for extracting free DNA, characterized in that the method includes the following steps:

将氯化铁、氯化钴和氯化镍等比混合，得到金属盐混合物；Mix ferric chloride, cobalt chloride and nickel chloride in equal ratios to obtain a metal salt mixture;

将乙二醇与所述金属盐混合物在机械搅拌条件下充分混合，得到乙二醇与金属盐混合物；Fully mixing ethylene glycol and the metal salt mixture under mechanical stirring conditions to obtain a ethylene glycol and metal salt mixture;

在乙二醇与金属盐混合物中加入乙酸钠，并继续进行搅拌，得到混合溶液；Add sodium acetate to the mixture of ethylene glycol and metal salt, and continue to stir to obtain a mixed solution;

将混合溶液倒入非金属材质反应釜中；Pour the mixed solution into the non-metallic reactor;

将反应釜放入控温烘箱中，控制烘箱按预设程序工作，使混合溶液热反应，形成铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒； Put the reaction kettle into a temperature-controlled oven, and control the oven to work according to a preset program to thermally react the mixed solution to form Fe-Co-NiO _X magnetic nanoparticles;

磁性分离所述铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒，并对其进行清洗和干燥。 Magnetically separate the Fe-Co-NiO _X magnetic nanoparticles of Fe-Co-Ni alloy, and clean and dry them.
根据权利要求1所述的纳米磁珠的制备方法，其特征在于，在执行完所有步骤后还包括：The method for preparing magnetic nanobeads according to claim 1, characterized in that, after performing all the steps, it further comprises:

称量1份干燥后的铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒，以重量计按1:18的比例配比葡萄糖，加入质量为200倍磁性纳米颗粒的超纯水并在机械搅拌条件下混合均匀； Weigh 1 part of dried Fe-Co-Ni alloy Fe-Co-NiO _X magnetic nanoparticles, mix with glucose in a ratio of 1:18 by weight, add ultra-pure water with a mass of 200 times the magnetic nanoparticles and stir mechanically Mix well under conditions;

将混和均匀的溶液倒入非金属材质反应釜中；Pour the evenly mixed solution into the non-metallic reactor;

将非金属材质反应釜放入控温烘箱中，控制烘箱按预设程序工作，开始溶剂热反应，得到表层包覆有碳层的铁钴镍合金Fe-Co-NiO _X@C磁性纳米颗粒。 Put the non-metallic material reaction kettle into a temperature-controlled oven, control the oven to work according to a preset program, start the solvothermal reaction, and obtain Fe-Co-NiO _X @C magnetic nanoparticles with an iron-cobalt-nickel alloy coated with a carbon layer on the surface.
根据权利要求2所述的纳米磁珠的制备方法，其特征在于，在执行完所有步骤后还包括：The method for preparing nano magnetic beads according to claim 2, characterized in that, after all the steps are performed, it further comprises:

用乙醇和氨水进行超声分散表层包覆有碳层的铁钴镍合金Fe-Co-NiO _X@C磁性纳米颗粒，对超声分散后形成的溶液进行机械搅拌，并在机械搅拌过程中加入正硅酸乙酯； Use ethanol and ammonia to ultrasonically disperse the Fe-Co-NiO _X @C magnetic nanoparticles of iron-cobalt-nickel alloy coated with a carbon layer, mechanically stir the solution formed after ultrasonic dispersion, and add ortho silicon during the mechanical stirring process Ethyl ester

磁性分离所生成的产物，得到表层包覆有无机二氧化硅的铁钴镍合金Fe-Co-NiO _X@C@SiO ₂磁性纳米颗粒； Magnetically separated products to obtain Fe-Co-NiO _X @C@SiO ₂ magnetic nanoparticles coated with inorganic silica on the surface;

在表层包覆有无机二氧化硅的铁钴镍合金Fe-Co-NiO _X@C@SiO ₂磁性纳米颗粒表层共价接枝聚乙二醇。 The surface layer of Fe-Co-NiO _X @C@SiO ₂ magnetic nanoparticles coated with inorganic silicon dioxide is covalently grafted with polyethylene glycol.
根据权利要求3所述的纳米磁珠的制备方法，其特征在于，所述在表层包覆有无机二氧化硅的铁钴镍合金Fe-Co-NiO _X@C@SiO ₂磁性纳米颗粒表层共价接枝聚乙二醇的步骤包括： The method for preparing nano magnetic beads according to claim 3, wherein the iron-cobalt-nickel alloy Fe-Co-NiO _X @C@SiO ₂ magnetic nanoparticles coated with inorganic silica on the surface layer has The steps of valence grafting polyethylene glycol include:

将所述铁钴镍合金Fe-Co-NiO _X@C@SiO ₂磁性纳米颗粒用异丙醇进行超声分散； Ultrasonically dispersing the iron-cobalt-nickel alloy Fe-Co-NiO _X @C@SiO ₂ magnetic nanoparticles with isopropanol;

在氮气保护下，对超声分散后的产物进行机械搅拌，并在搅拌过程中加入氨丙基三乙氧基硅烷；Under the protection of nitrogen, mechanically stir the product after ultrasonic dispersion, and add aminopropyltriethoxysilane during the stirring process;

磁性分离搅拌后的产物并用异丙醇和乙醇进行清洗，得到氨基铁钴镍合金Fe-Co-NiO _X@C@SiO ₂-NH ₂磁性纳米颗粒； Magnetically separate the stirred product and clean it with isopropanol and ethanol to obtain amino-iron-cobalt-nickel alloy Fe-Co-NiO _X @C@SiO ₂ -NH ₂ magnetic nanoparticles;

将所述氨基铁钴镍合金Fe-Co-NiO _X@C@SiO ₂-NH ₂磁性纳米颗粒超声分散在2-吗啉缓冲液中，并加入乙基-(3-二甲基氨基丙基)碳酰二亚胺EDC和羧基聚乙二醇COOH-PEG，在机械搅拌条件下进行室温反应； The amino-iron-cobalt-nickel alloy Fe-Co-NiO _X @C@SiO ₂ -NH ₂ magnetic nanoparticles were ultrasonically dispersed in 2-morpholine buffer solution, and ethyl-(3-dimethylaminopropyl) was added. ) Carbodiimide EDC and carboxyl polyethylene glycol COOH-PEG are reacted at room temperature under mechanical stirring conditions;

磁性分离机械搅拌后的产物，用超纯水和乙醇依次清洗得到聚乙二醇铁钴镍合金Fe-Co-NiO _X@C@SiO ₂-PEG磁性纳米颗粒。 The product after magnetic separation and mechanical stirring is washed with ultrapure water and ethanol in turn to obtain polyethylene glycol Fe-Co-NiO _X @C@SiO ₂ -PEG magnetic nanoparticles.
根据权利要求1所述的纳米磁珠的制备方法，其特征在于，所述将反应釜放入控温烘箱中，控制烘箱按预设程序工作，使混合溶液热反应，形成铁钴镍合金Fe-Co-NiO _X磁性纳米颗粒的步骤中包括： The method for preparing nano magnetic beads according to claim 1, wherein the reaction kettle is placed in a temperature-controlled oven, and the oven is controlled to work according to a preset program to cause the mixed solution to react thermally to form an iron-cobalt-nickel alloy Fe -The steps of Co-NiO _X magnetic nanoparticles include:

在2小时内将温度从30℃提升至150℃，并维持在150℃进行6小时反应，最后在2小时内将温度降至室温。The temperature was increased from 30°C to 150°C within 2 hours, and the reaction was maintained at 150°C for 6 hours, and finally the temperature was reduced to room temperature within 2 hours.
一种游离DNA提取试剂盒，其特征在于，包括样本裂解液、结合液、一次洗涤液、二次洗涤液、洗脱液和纳米磁珠，所述样本裂解液成份包括无核酸酶水、试剂A和试剂B，所述试剂A为异硫氰酸胍、盐酸胍、碘化钠、乙酸锂、高氯酸锂和高氯酸钠中至少一种，所述试剂B为十二烷基苯磺酸钠、脂肪酸甘油酯、Tween20、Triton-X100、SDS、NP40和聚氧乙烯型非离子表面活性剂中至少一种；所述结合液中除样本裂解液的成份外还包括试剂C，所述试剂C为异丙醇、丁醇、甲醇和无水乙醇中至少一种；所述一次洗涤液的成份与结合液相同；所述二次洗涤液为无核酸酶水、摩尔浓度为0.1-1M氯化钠溶液、Tris缓冲液或者TE缓冲液中的一种；所述洗脱液为无核酸酶水，Tris缓冲液或者TE缓冲液中的一种，所述纳米磁珠其磁核为铁钴镍合金 Fe-Co-NiO _X，粒径分布在300-400nm之间，表层由内到外依次至少包覆有碳层和无机二氧化硅层，且在最外层共价接枝有聚乙二醇。 A free DNA extraction kit, which is characterized in that it includes a sample lysis solution, a binding solution, a primary washing solution, a secondary washing solution, an eluent, and nano magnetic beads. The sample lysis solution includes nuclease-free water, reagents A and reagent B, the reagent A is at least one of guanidine isothiocyanate, guanidine hydrochloride, sodium iodide, lithium acetate, lithium perchlorate and sodium perchlorate, and the reagent B is dodecylbenzene At least one of sodium sulfonate, fatty acid glyceride, Tween20, Triton-X100, SDS, NP40 and polyoxyethylene type nonionic surfactant; the binding solution includes reagent C in addition to the components of the sample lysate, so The reagent C is at least one of isopropanol, butanol, methanol and absolute ethanol; the composition of the primary washing liquid is the same as the binding liquid; the secondary washing liquid is nuclease-free water with a molar concentration of 0.1- One of 1M sodium chloride solution, Tris buffer or TE buffer; the eluent is one of nuclease-free water, Tris buffer or TE buffer, and the magnetic core of the nano magnetic beads is The iron-cobalt-nickel alloy Fe-Co-NiO _{X has a} particle size distribution between 300-400nm. The surface layer is covered with at least a carbon layer and an inorganic silica layer from the inside to the outside, and the outermost layer is covalently grafted with Polyethylene glycol.
根据权利要求6所述的游离DNA提取试剂盒，其特征在于，所述样本裂解液的pH值为4-6；由无核酸酶水、摩尔浓度为2-6M的试剂A与质量浓度为5％-20％的试剂B，在避光条件下搅拌至溶解、然后用Tris缓冲液或Tris-EDTA溶液调节制成。The cell-free DNA extraction kit of claim 6, wherein the pH of the sample lysate is 4-6; it is composed of nuclease-free water, reagent A with a molar concentration of 2-6M and a mass concentration of 5 %-20% Reagent B, stirred to dissolve under dark conditions, and then adjusted with Tris buffer or Tris-EDTA solution.
根据权利要求6所述的游离DNA提取试剂盒，其特征在于，所述结合液的pH值为4-6；由无核酸酶水、摩尔浓度为1-6M的试剂A与质量浓度为5％-20％的试剂B，在避光条件下搅拌至溶解、然后用Tris缓冲液或Tris-EDTA调节并与15％-55％体积的试剂C混合制成。The free DNA extraction kit according to claim 6, wherein the pH of the binding solution is 4-6; it is composed of nuclease-free water, reagent A with a molar concentration of 1-6M and a mass concentration of 5% -20% Reagent B, stir to dissolve under dark conditions, then adjust with Tris buffer or Tris-EDTA and mix with 15%-55% Reagent C by volume.
根据权利要求6所述的游离DNA提取试剂盒，其特征在于，所述一次洗涤液的pH值为4-6；由无核酸酶水、摩尔浓度为0.5-4M的试剂A与质量浓度为0.5％-10％的试剂B，在避光条件下搅拌至溶解、然后用Tris缓冲液或Tris-EDTA溶液调节并与15％-55％体积的试剂C混合制成。The free DNA extraction kit according to claim 6, wherein the pH value of the washing solution is 4-6; it is composed of nuclease-free water, reagent A with a molar concentration of 0.5-4M and a mass concentration of 0.5 %-10% Reagent B, stirred to dissolve under dark conditions, then adjusted with Tris buffer or Tris-EDTA solution and mixed with 15%-55% Reagent C by volume.
一种游离DNA的提取方法，其特征在于，利用权利要求6所述的游离DNA提取试剂盒完成，所述方法包括以下步骤：A method for extracting free DNA, characterized in that it is completed by using the free DNA extraction kit of claim 6, and the method comprises the following steps:

按照先添加蛋白酶K，然后添加样本，最后加入裂解液的顺序，在离心管中依次添加各个组分，震荡混匀各组分并孵育，以进行样品裂解；In the order of adding proteinase K first, then adding the sample, and finally adding the lysis solution, add each component in the centrifuge tube, shake and mix each component and incubate to perform sample lysis;

将纳米磁珠和结合液按比例配制成纳米磁珠结合液；Prepare the nano magnetic beads and the binding liquid in proportion to make the nano magnetic bead binding liquid;

将制备好的纳米磁珠结合溶液加入样本中，震荡混匀后静置，并弃掉上清；Add the prepared nanomagnetic bead binding solution to the sample, shake and mix, and then stand still, and discard the supernatant;

使用一次洗涤液清洗游离DNA；Use a washing solution to clean free DNA;

使用二次洗涤工作液清洗游离DNA；所述二次洗涤工作液由二次洗涤液用无水乙醇稀释得到；Use a secondary washing working solution to clean the free DNA; the secondary washing working solution is obtained by diluting the secondary washing solution with absolute ethanol;

加入洗脱液，震荡以使纳米磁珠重悬，并进一步震荡以使纳米磁珠上的游离DNA洗脱。Add the eluent, shake to resuspend the nano magnetic beads, and further shake to elute the free DNA on the nano magnetic beads.
根据权利要求10所述的方法，其特征在于，所述按照先添加蛋白酶K，然后添加样本，最后加入裂解液的顺序，依次添加各个组分，震荡混匀各组分，并孵育进行样品裂解的步骤中包括：The method according to claim 10, characterized in that, in the order of adding proteinase K first, then adding the sample, and finally adding the lysis solution, each component is added in sequence, the components are shaken and mixed, and the sample is incubated for lysis The steps include:

在15毫升的大离心管中依次添加质量浓度为20毫克/毫升的蛋白酶80微升、血浆样本4毫升和样本裂解液400微升；Add 80 μl of protease with a mass concentration of 20 mg/ml, 4 ml of plasma sample, and 400 μl of sample lysate into a large 15 ml centrifuge tube;

震荡大离心管以混匀其中各组分，并将大离心管置于60℃环境中孵育20分钟；Shake the large centrifuge tube to mix the components, and incubate the large centrifuge tube at 60°C for 20 minutes;

孵育结束后，将大离心管平衡至室温。After the incubation, equilibrate the large centrifuge tube to room temperature.
根据权利要求10所述的方法，其特征在于，所述将制备好的纳米磁珠结合溶液加入样本中，震荡混匀后静置，并弃掉上清的步骤中包括：The method according to claim 10, wherein the step of adding the prepared nanomagnetic bead binding solution to the sample, shaking and mixing, and then standing, and discarding the supernatant comprises:

将制备好的纳米磁珠结合溶液加入大离心管的血浆样本中并震荡混匀；Add the prepared nanomagnetic bead binding solution to the plasma sample in the large centrifuge tube and shake and mix;

摇晃离心管以促进游离DNA与纳米磁珠的结合；Shake the centrifuge tube to promote the combination of free DNA and nanomagnetic beads;

将上述大离心管置于磁力架上静置至管内溶液澄清且磁珠聚于管壁。Place the above-mentioned large centrifuge tube on a magnetic stand and let it stand until the solution in the tube is clear and the magnetic beads gather on the tube wall.
根据权利要求10所述的方法，其特征在于，所述使用一次洗涤液清洗游离DNA的步骤中包括：The method according to claim 10, wherein the step of using a washing solution to clean free DNA comprises:

将大离心管从所述磁力架上取下，加入1毫升洗涤液，并涡旋震荡以重悬纳米磁珠；Remove the large centrifuge tube from the magnetic stand, add 1 ml of washing solution, and vortex to resuspend the nano magnetic beads;

将已重悬的纳米磁珠溶液转移入一个1.5毫升离心管；Transfer the resuspended nanomagnetic bead solution into a 1.5ml centrifuge tube;

将所述1.5毫升离心管置于磁力架上至管内磁珠聚集于管壁；Placing the 1.5 ml centrifuge tube on a magnetic stand until the magnetic beads in the tube gather on the tube wall;

利用1.5毫升离心管内的上清液来润洗所述大离心管的管壁和管盖，将残余磁珠的润洗液转移回所述1.5毫升离心管；Use the supernatant in the 1.5 ml centrifuge tube to rinse the tube wall and cap of the large centrifuge tube, and transfer the rinse solution of residual magnetic beads back to the 1.5 ml centrifuge tube;

将所述1.5毫升离心管置于磁力架上静置至管内溶液澄清且磁珠聚于管壁；Place the 1.5 ml centrifuge tube on a magnetic stand and let it stand until the solution in the tube is clear and the magnetic beads gather on the tube wall;

弃掉上清；Discard the supernatant;

将1.5毫升离心管从磁力架上取下，加入1毫升洗涤液并涡旋震荡该1.5毫升离心管30秒；Remove the 1.5 ml centrifuge tube from the magnetic stand, add 1 ml of washing solution and vortex the 1.5 ml centrifuge tube for 30 seconds;

将1.5毫升离心管于台式离心机中快速离心，将粘壁的残液甩至管底，并将该离心管置于磁力架上静置至管内溶液澄清且磁珠聚于管壁。Centrifuge the 1.5 ml centrifuge tube quickly in a bench top centrifuge, shake the remaining liquid on the wall to the bottom of the tube, and place the centrifuge tube on a magnetic stand until the solution in the tube is clear and the magnetic beads gather on the tube wall.