WO2020237781A1 - 一种提高无细胞体液中微量核酸捕获率的方法 - Google Patents

一种提高无细胞体液中微量核酸捕获率的方法 Download PDF

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WO2020237781A1
WO2020237781A1 PCT/CN2019/094963 CN2019094963W WO2020237781A1 WO 2020237781 A1 WO2020237781 A1 WO 2020237781A1 CN 2019094963 W CN2019094963 W CN 2019094963W WO 2020237781 A1 WO2020237781 A1 WO 2020237781A1
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liquid
nucleic acid
centrifuge tube
cell
capture rate
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French (fr)
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黄彦钦
宋永茂
袁瑛
郑树
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浙江大学
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

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  • the invention belongs to the field of biotechnology, and relates to a method for improving the capture rate of trace nucleic acids in cell-free body fluids, which is used to increase the nucleic acid capture rate when enriching and purifying nucleic acids from cell-free body fluids, and is particularly suitable for enriching from serum or plasma.
  • a method for improving the capture rate of trace nucleic acids in cell-free body fluids which is used to increase the nucleic acid capture rate when enriching and purifying nucleic acids from cell-free body fluids, and is particularly suitable for enriching from serum or plasma.
  • Liquid biopsy is a hot research topic in the current biomedical field.
  • Liquid biopsy mainly refers to the extraction of trace nucleic acid substances from cell-free body fluids such as serum, plasma, and cerebrospinal fluid of living organisms, and further testing is used to indicate disease.
  • Trace nucleic acid substances mainly refer to DNA, but also include various types of RNA.
  • the nucleic acid substances extracted from cell-free serum and plasma are mainly released by dead cells and circulate in the blood system of the body. They can be called free nucleic acids, and their main components are called free DNA.
  • Free DNA has the characteristics of DNA released by apoptotic cells, which is manifested in that the extracted and purified DNA has product bands at about 170 bp and its integer multiples of length during electrophoresis, and there are fewer large fragments above 1000 bp. Therefore, DNA extracted from cell-free serum or plasma is significantly different from genomic DNA extracted from cells.
  • liquid biopsy is to detect nucleic acid substances extracted and purified from body fluids with cell-free serum and plasma as the main target, so as to achieve the purpose of assisting diagnosis of diseases, and may be used as a diagnostic standard for certain diseases in the future.
  • Nucleic acid substances in cell-free serum or plasma are very small. Serum or plasma as the object of nucleic acid extraction and purification, its initial processing volume is much larger than that of tissue extraction. At the same time, there may be unclear components in serum and plasma that affect nucleic acid adsorption and capture, which greatly affects nucleic acid extraction. effectiveness.
  • the kits generally used for nucleic acid extraction from tissue cells can hardly obtain effective extraction. At present, there are a few specialized commercial kits that can provide more efficient extraction. Among them, the QIAamp Circulating Nucleic Acid series of the German QIAGEN company has the best effect.
  • QIAGEN’s kit is mainly column purification type.
  • the kit uses a large volume of column binding solution to dilute the effect of large volume of serum or plasma on the extraction efficiency. It can successfully capture trace nucleic acids, but the cost is relatively high (currently priced at 50 (Approximately 10,000 yuan is required for this withdrawal).
  • the other magnetic bead adsorption method has many companies providing products, but the magnetic bead method has an unsatisfactory adsorption and capture rate of trace nucleic acids in a large volume of liquid, especially when the amount of free nucleic acid is small. Compared with the column method, the magnetic bead method has a low nucleic acid capture rate, and nucleic acid materials will inevitably be lost when the magnetic beads are washed.
  • the magnetic bead method requires high magnetic bead particle diameter, suspension dispersion, uniformity, etc., and the cost of the kit is still relatively high.
  • the magnetic bead extraction method also has higher requirements for the operator's operation technique, and the extraction effect is not stable enough.
  • the purpose of the present invention is to provide a method for improving the capture rate of trace nucleic acids in cell-free body fluids, including protein lysis, column binding, washing and elution, etc.
  • the key is to add the following steps between protein lysis and column binding :
  • Cell-free body fluids include human and animal body fluids, including plasma, serum, amniotic fluid, cerebrospinal fluid, pleural fluid, ascites, pericardial effusion, sinus effusion, synovial effusion, mammary duct fluid, and aqueous humor.
  • the centrifuge tubes used are made of polymer compounds, including plastic centrifuge tubes mainly made of polypropylene, polyethylene, polyvinyl chloride, polystyrene, and polycarbonate. Preferably it is made of polypropylene. Contains cylindrical plastic centrifuge tubes with sharp, round and square bottoms.
  • the protein lysate formula of the present invention must contain two key components: proteinase K and protein denaturant. This is a well-known and commonly used formula.
  • the pH value of the solution is between 7.5-8, but the pH value is between 7-9. Can also play a proteolytic role.
  • the protein lysate of the present invention can also contain Tris, EDTA and other conventional components with metal ion chelation and pH buffering, which are mainly used to adjust the pH and chelate metal ions, all of which are conventional methods.
  • the amount of proteinase K in the protein lysate may vary according to the amount of serum or plasma to be processed and its protein content.
  • the amount of proteinase K added can be excessive, but not insufficient.
  • the proteinase K content is 2ug/ ⁇ l, it can meet the general protein content of plasma or serum protein lysis requirements, which is sufficient for most plasma or serum.
  • Protein denaturants include sodium lauryl sulfate 0.2-20% by mass, acetic acid 0.5-8mol/L, guanidine hydrochloride 0.9-8mmol/L, guanidine thiocyanate 0.9-8mmol/L, 4-8mmol /L of urea, and the above-mentioned protein denaturant does not precipitate a mixed solution.
  • the above protein denaturants are all known and commonly used formulations.
  • the temperature and time of the protein lysis incubation in the present invention can be different according to the amount of sample processed.
  • Proteinase K has a certain activity between 30°C and 80°C, but the activity is relatively high between 35°C and 60°C, preferably incubation at 55°C.
  • the amount of sample processed is small (such as ⁇ 100 microliters)
  • incubation at 35°C or 80°C can achieve the effect of protein lysis, and the incubation time is 10 minutes.
  • the incubation time should be greater than 1 hour, preferably 3 hours, overnight incubation is also acceptable, but should not exceed 16 hour.
  • Low dielectric constant solvent refers to a solvent with a dielectric constant less than pure water at 20°C, and includes ethanol, isopropanol, acetic acid, acetone, n-hexanol, and carbon tetrachloride. Preferred is ethanol or isopropanol.
  • a solvent with a low dielectric constant is added to the lysis solution.
  • the low dielectric constant solvent used for addition should be pure, that is, the content should be >99%.
  • the amount to be added varies according to the relative difference between the dielectric constant of the solvent and the dielectric constant of water.
  • the dielectric constant of water is 78 at 20°C and that of ethanol is 24.
  • the mass content of ethanol in the final centrifugal solution should be greater than 60%, the addition amount is generally 2 times the volume of the original solution.
  • the dielectric constant of isopropanol is 18 at 20°C, the mass content of isopropanol in the final centrifugal solution should be greater than 33%, and the amount of isopropanol added is generally 1 time the volume of the original solution.
  • the dielectric constants of acetone, n-hexanol, acetic acid and carbon tetrachloride are respectively 21, 13, 6 and 2 at 20°C, and their addition amount is generally not less than 1.5 times, 0.75 times, 0.5 times and 0.5 times of the original solution volume. Can play a role.
  • the pyrolysis system liquid of the present invention is centrifuged at room temperature after adding a low-dielectric constant solvent.
  • the environment temperature of the centrifuge tube during centrifugation is room temperature, and the temperature is between 4°C and 35°C, preferably 4°C or 20°C. °C.
  • Centrifuge at medium or high speed means that the centrifugal force is between 1,000 grams and 30,000 grams, both of which can make nucleic acids adhere to the wall.
  • the centrifugal force is 8000 grams, and the centrifugal force is centrifuged for 40 minutes.
  • the 2ml centrifuge tube is tilted at 30 degrees, it can be firmly adhered to the wall when it is centrifuged for 2 hours at 1000 grams of centrifugal force, and it can be firmly attached to the wall when it is centrifuged at 4000 grams for 50 minutes.
  • the centrifugal force is 30,000 grams, it only needs to centrifuge for 10 minutes to firmly adhere to the wall.
  • the position of the centrifuge tube during centrifugation should be such that the long diameter direction of the centrifuge tube and the direction of the centrifugal axis form a centrifugal inclination angle, the centrifugal inclination angle can be between 20-70 degrees, preferably 30-40 degrees, the purpose is to reduce the attachment of nucleic acids in the solution
  • the distance required to travel on the wall of the centrifuge tube allows the nucleic acid to adhere to the wall of the centrifuge tube faster.
  • the nucleic acid is attached to one side of the centrifuge tube wall, which also facilitates subsequent pouring or suction of liquid on the other side.
  • the liquid in the centrifuge tube is removed by direct pouring, or suction, or by pouring and suction, and the total amount of residual liquid cannot exceed 50 microliters.
  • the nucleic acid in the liquid is adsorbed on the siliconized adsorption membrane of the purification column, after adding the cleaning solution and centrifugation, the nucleic acid is eluted with the conventional eluent to obtain the purified cell-free protein-containing liquid Nucleic acid substance.
  • the post-column binding solution is a high-salt low-pH binding solution applied to conventional column-type nucleic acid purification siliconized adsorption membranes.
  • the well-known common formula contains sodium chloride, lithium chloride, sodium molybdate, guanidine hydrochloride, guanidine sulfur (isothio)cyanate, sodium salicylate, ammonium sulfate, sodium acetate with a molar concentration between 2-8 moles.
  • the percentage is a mixture of polyethylene glycol 8000 and the above compounds between 5-15%. It is also possible to use a column binding solution with an undisclosed formula in the nucleic acid extraction kit on the commercial market.
  • the principle that the method of the present invention can produce the corresponding effect may be that after the protein-containing body fluids such as plasma and serum are lysed, the centrifugal force is not less than 1000 grams at room temperature in the presence of a relatively high concentration of low dielectric constant solvent for a long time. , Can make the nucleic acid scattered in the liquid adhere to the wall of the plastic centrifuge tube. When a small amount of nucleic acid is in close contact with the wall of the plastic centrifuge tube, it can be firmly adsorbed on the wall of the centrifuge tube without loosening and falling off like a large amount of nucleic acid adheres to the wall. This is an unknown discovery.
  • Another possible principle is that after protein-containing body fluids are lysed, there are still some non-protein substances such as lipids. These substances may be attached to the plastic tube wall before the trace nucleic acid to form a film. When the trace nucleic acid falls on it At this time, it can firmly adsorb trace nucleic acid. Through the firm adsorption of nucleic acid, the liquid part can be removed as much as possible, which removes the factors that affect the adsorption of nucleic acid through the column in the original serum and plasma, so that the column binding liquid for reconstituted trace nucleic acid can perform adsorption normally.
  • the beneficial effect of the present invention is that the method can use conventional protein lysis formulas and conventional nucleic acid column combined purification methods, only need to add key centrifugal liquid removal steps, do not need to use a large volume of binding liquid, do not need to use magnetic beads, simple operation, No additional skills are required, the reagent cost is low, and compared with the general tissue cell nucleic acid extraction kit, the nucleic acid capture rate can be increased to 3-8 times.
  • the nucleic acid extraction is similar to the special plasma serum free nucleic acid extraction kit, which is an economical and effective method.
  • liquid biopsy can be applied on a large scale in the future, a more economical and effective method is needed to extract trace nucleic acid from plasma or serum.
  • Our invention provides a very economical method that can significantly increase the nucleic acid capture rate when common nucleic acid extraction reagents are used to extract trace nucleic acids from body fluids such as plasma and serum through simple operations.
  • the invention can significantly increase the nucleic acid capture rate when conventional reagents are used to extract trace nucleic acids in cell-free body fluids, solve the problem of the need for economical, effective, and stable methods for extracting trace nucleic acids from cell-free body fluids, and is beneficial to the promotion of liquid biopsy application.
  • Figure 1 is a 50ml polypropylene plastic centrifuge tube used to purify 6ml plasma.
  • FIG. 2 shows that the centrifuge tube forms a fixed centrifugal tilt angle with the centrifuge shaft when it is placed in the centrifuge.
  • Figure 3 is a centrifuge tube with lacing up to pour liquid.
  • Figure 4 shows the vortexing after adding the column binding solution of reconstituted adherent nucleic acid.
  • Example 1 Improve the nucleic acid capture rate of purified free DNA from a larger amount of 6 ml plasma
  • 6 ml of plasma is contained in a round-bottomed 50 ml polypropylene centrifuge tube.
  • the centrifuge tube is connected with a tie 1 and a cap 2, and contains 6 ml of plasma 3.
  • After the lysis is complete add another volume (8 ml) of isopropanol to form 16 ml of liquid. Put it into the centrifuge hole of the centrifuge as shown in Figure 2.
  • the centrifugal inclination angle 6 formed by the long diameter 4 of the 50 ml centrifuge tube and the centrifuge shaft 5 of the centrifuge is 30 degrees. Centrifuge in this centrifuge with a centrifugal force of 1000 g for 2 hours at 4 degrees Celsius. After the centrifugation is completed, immediately take out the centrifuge tube and open the lid. Pour and turn over the centrifuge tube as shown in Figure 3, and pour all the liquid 8 in the centrifuge tube through the opening 7 on the opposite side of the centrifuge tube tie to pour all the liquid in the tube. Place the centrifuge tube from which the liquid has been poured into the original centrifuge again. Note that the direction of the centrifuge tube tie must be the same as the previous centrifuge.
  • sucking off the liquid add 1500 microliters of conventional nucleic acid-passing column binding solution 9 into the centrifuge tube, and shake and mix on a vortex shaker. When vortexing, the liquid in the centrifuge tube rotates quickly and forms a vortex. As shown in Figure 4, the surrounding liquid 10 rises against the wall of the centrifuge tube.
  • Example 2 Improve the nucleic acid capture rate of purified free DNA from a relatively small amount of 450 microliters of serum
  • the container for containing serum is a commonly used 2 ml polypropylene cone-bottom centrifuge tube.
  • the operation diagram please refer to the attached drawings in Example 1.
  • After adding 450 microliters of serum to the centrifuge tube add 150 microliters of commonly used proteinase K and protein denaturant, and incubate in a constant temperature metal bath at 55 degrees for 3 hours to fully lyse the protein in the serum.
  • the liquid in the centrifuge tube is 600 microliters. Rise.

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Abstract

提供了一种提高无细胞体液中微量核酸捕获率的方法，其中在蛋白裂解和过柱结合之间加入了使用低介电常数溶液离心的步骤。

Description

一种提高无细胞体液中微量核酸捕获率的方法 技术领域
本发明属于生物技术领域，涉及一种提高无细胞体液中微量核酸捕获率的方法，用于提高从无细胞体液中富集和纯化核酸时的核酸捕获率，特别适用于从血清或血浆中富集和纯化微量DNA的场合。
背景技术
液体活检是当前生物医学领域研究的热点。液体活检主要是指从活体的血清、血浆、脑脊液等无细胞体液中提取微量核酸物质，进一步检测用于提示疾病。微量核酸物质主要指DNA，但也包括各类RNA。从无细胞血清和血浆所提取的核酸物质主要为死亡细胞所释放，在体内血液系统中循环，可称为游离核酸，其主要成份被称为游离DNA。游离DNA具有凋亡细胞所释放DNA的特征，表现为所提取纯化的DNA电泳时在约170bp及其整数倍长度的位置有产物条带，而较少1000bp以上的大片段。因此，无细胞血清或血浆中提取DNA相比于细胞提取的基因组DNA有显著的不同。
当身体存在疾病，疾病导致的细胞死亡可释放核酸物质，此时游离核酸携带了疾病的信息。液体活检的含义正是通过对以无细胞血清和血浆为主要目标的体液中提取纯化的核酸物质进行检测，从而达到辅助诊断疾病的目的，并有可能将来在某些疾病中作为诊断标准。
无细胞血清或血浆中的核酸物质是极微量的。血清或血浆作为核酸提取纯化对象，其初始所需处理的体积远较提取组织时为大，同时血清血浆中可能还存在未明确的影响核酸吸附捕获的成份，这极大的影响了核酸的提取效率。一般用于组织细胞核酸提取的试剂盒几乎无法获得有效提取。目前有少数专用商业化试剂盒可提供较为高效的提取，其中以德国QIAGEN公司的QIAamp Circulating Nucleic Acid系列试剂盒效果为优。QIAGEN公司试剂盒主要为过柱纯化型，该试剂盒采用大体积过柱结合液去稀释大体积血清或血浆对提取效率的影响，可成功捕获到微量核酸，但成本较高(目前售价50次提取需近1万元人民币)。另一种磁珠吸附法有较多公司提供产品，但磁珠法在大体积液体中对微量核酸的吸附捕获率无法令人满意，特别在游离核酸量较少时。与过柱法比较，磁珠法核酸捕获率偏低，在清洗磁珠时不可避免的会损失核酸物质。此外，磁珠法对磁珠颗粒直径、悬浮分散度、均一性等要求高，试剂盒成本仍然较高。磁珠法提取对操作者的操作手法要求也较高，提取效果也不够稳定。
发明内容
本发明的目的是提供一种提高无细胞体液中微量核酸捕获率的方法，包括蛋白裂解，过柱结合，清洗和洗脱等步骤，其中关键是在蛋白裂解和过柱结合之间加入以下步骤：
(1)向盛有已裂解蛋白体液的离心管中加入0.5-3倍体积的低介电常数溶剂，混匀；
(2)在常温下中高速离心；
(3)用直接倾倒液体，或者吸除液体，或者倾倒后再加以离心再吸除的方法去除液体成份；
(4)用核酸过柱结合液复溶贴壁核酸。
具体通过以下步骤实现：
(1)将无细胞体液加入高分子化合物材质的塑料离心管中，再加入常用的蛋白酶K和蛋白变性剂为主要成份的蛋白裂解液，在蛋白酶K适宜工作温度下孵育，使液体中蛋白大分子裂解，目的使后续高速离心时不出现大量蛋白沉淀，而液体中的微量核酸在蛋白酶K去除核酸酶功能的保护下得以完整保存。
无细胞体液，包含人体和动物的体液，包含血浆、血清、羊水、脑脊液、胸水、腹水、心包积液、窦腔积液、滑囊积液、乳腺管液体和眼房水。
所使用的离心管为高分子化合物材质，包含以聚丙烯、聚乙烯、聚氯乙烯、聚苯乙烯、聚碳酸酯为主要材质的塑料离心管。优选为聚丙烯材质。包含尖底、圆底、方底的圆柱形塑料离心管。
本发明所述蛋白裂解液配方必须包含蛋白酶K和蛋白变性剂两种关键成份，此为公知常用配方，其溶液PH值在7.5-8之间为最佳，但PH值在7-9之间也可发挥蛋白裂解作用。本发明所述蛋白裂解液还可包含Tris，EDTA等其他具有金属离子螯合作用、pH值缓冲作用的常规成份，主要用于调节PH值及螯合金属离子，均为常规方法。
所述蛋白裂解液中蛋白酶K用量根据所需处理的血清或血浆量和其蛋白含量不同可有不同，蛋白酶K加入量可以过量，但不可以不足。在血清(血浆)与蛋白裂解液组成的混合液中，蛋白酶K含量为2ug/微升时，可满足一般蛋白含量的血浆或血清蛋白裂解要求，对于绝大部分血浆或血清已足够。
蛋白变性剂包括质量百分比为0.2-20％的十二烷基硫酸钠，0.5-8mol/L的乙酸，0.9-8mmol/L的盐酸胍，0.9-8mmol/L的硫氰酸胍，4-8mmol/L的尿素，以及上述蛋白变性剂不析出沉淀的混合液。以上蛋白变性剂均为公知常用配方。
本发明所述蛋白裂解孵育的温度和时间根据所处理样品量的多少可有不同。蛋白酶K在30℃和80℃之间均有一定的活性，但以35℃-60℃之间活性相对较高，优选的以55℃孵育为佳。当所处理样品量较少(如<100微升)时，35℃或者80℃孵育均可达到裂解蛋白的作 用，且孵育时间10分钟即可。当处理样品量较大时(如>100微升)时，优选的以50-60℃之间孵育，孵育时间应大于1小时，优选的为3小时，过夜孵育亦可，但不应超过16小时。
(2)过柱结合：蛋白裂解完成后，向离心管中加入原有液体体积0.5倍至3倍体积的低介电常数溶剂，在离心管与离心轴形成倾角的状态下，于不低于4℃且不大于35℃的环境温度下，中速或高速离心使液体中微量核酸牢固贴附于塑料离心管管壁。离心后直接开盖倒除液体，留有残液者再次离心并用移液器吸除液体的方法将离心管中液体成份去除，目的为使离心管中液体尽量去除。
低介电常数溶剂是指在20℃时介电常数小于纯水的溶剂，包含乙醇、异丙醇、乙酸、丙酮、正己醇和四氯化碳。优选的为乙醇或异丙醇。本发明所述在蛋白裂解完成后，向裂解液中加入低介电常数的溶剂。所述用于添加的低介电常数溶剂应为纯品，即含量应>99％。所添加量根据溶剂的介电常数与水的介电常数的相对大小差异而不同，水在20℃时介电常数为78，乙醇为24，此时乙醇在最终离心溶液中的质量含量应大于60％，加入量一般为原溶液体积的2倍。异丙醇在20℃时介电常数为18，异丙醇在最终离心溶液中的质量含量应大于33％，异丙醇加入量一般为原溶液体积的1倍。丙酮、正己醇、乙酸和四氯化碳在20℃时介电常数分别为21，13，6和2，其加入量为一般不小于原溶液体积的1.5倍，0.75倍，0.5倍和0.5倍即可发挥作用。
本发明所述的裂解体系液体中加入低介电常数溶剂后在常温下离心，包含离心时离心管的环境温度为常温，温度介于4℃和35℃之间，优选的为4℃或20℃。中速或高速离心。中速或高速离心是指离心力于1000克-30000克，均有使核酸贴壁粘附的作用。优选离心力为8000克，离心力离心40分钟。
离心力越小，使核酸牢固贴附所需的时间越长，对于2ml离心管30度倾斜时，当1000克离心力时离心2小时方可牢固贴壁，当4000克离心力离心时50分钟可牢固贴壁，当30000克离心力时只需离心10分钟即可牢固贴壁。离心时离心管的位置应使离心管长径方向与离心轴方向形成离心倾角，离心倾角可在20-70度之间，优选的为30-40度角状态，目的为减少溶液中核酸贴附于离心管壁所需行进的距离，使核酸更快贴附于离心管壁。同时，核酸贴附于离心管壁的一侧，也有利于后续在另一侧倾倒或吸除液体。
通过直接倾倒，或吸除，或倾倒加吸除的方法将离心管内液体去除，残留液体总量不能超过50微升。
(3)向离心管中加入常规过柱结合液，涡旋振荡充分混匀。涡旋振荡时，离心管内液体快速旋转，其贴壁液面升高，可使用离心管正立和倒置交叉涡旋振荡的方法使旋转液体触及离心管管壁有可能吸附核酸的所有位置。涡旋振荡使贴壁核酸重新溶解于过柱结合液中。 后续按常规方法过核酸纯化柱，将液体中核酸吸附于纯化柱的硅化吸附膜，加入清洗液离心后，再用常规洗脱液将核酸洗脱，即可获得纯化的无细胞含蛋白液体中核酸物质。
过柱结合液为应用于常规柱式核酸纯化硅化吸附膜的高盐低PH值的结合液。公知常见配方包含摩尔浓度为2-8摩尔之间的氯化钠、氯化锂、钼酸钠、盐酸胍、硫(异硫)氰酸胍、水杨酸钠、硫酸铵、乙酸钠，质量百分比为5-15％之间的聚乙二醇8000以及上述化合物的混合液。也可使用商品市场上核酸提取试剂盒中未公开配方的过柱结合液。
本发明所述方法能产生相应效果的原理可能在于血浆、血清等含蛋白体液在裂解蛋白后，在较高浓度的低介电常数溶剂存在下，在常温下不小于1000克离心力较长时间离心，即可使液体中散在的核酸贴附聚集于塑料离心管管壁。而微量核酸与塑料离心管壁紧密接触时，可牢固吸附于离心管管壁，而不会像大量核酸贴壁出现松解掉落，这是尚未公知的发现。另一可能的原理是含蛋白体液在裂解蛋白后，仍存在一些脂类等非蛋白物质，这些物质可能先于微量核酸贴附于塑料管壁，形成一层膜，当微量核酸落于其上时，可将微量核酸牢固吸附。通过核酸牢固吸附，可将液体部分尽量去除，这便去除了原血清血浆中影响核酸过柱吸附的因素，使复溶微量核酸的过柱结合液能正常发挥吸附作用。
本发明的有益效果在于该方法可使用常规的蛋白裂解配方和常规的核酸过柱结合纯化方法，只需加入关键的离心去液步骤，无需使用大体积结合液，无需使用磁珠，操作简单，不需要额外的技巧，试剂成本低，与一般组织细胞核酸提取试剂盒相比，可将核酸捕获率提高至3-8倍。核酸提取所得与专用血浆血清游离核酸提取试剂盒相似，是一种经济又有效的方法。
液体活检如在将来可大规模应用，血浆或血清中微量核酸提取方法还需要更经济且有效的方法。我们的发明便提供了一种十分经济的方法，可通过简单的操作将普通核酸提取试剂用于从血浆、血清等体液中提取微量核酸时的核酸捕获率显著提高。
针对现有从无细胞血清、血浆、脑脊液等体液中提取纯化微量核酸时，核酸捕获率不高，或者成本较高，或者效果不稳定的不足，采用了将无细胞体液中蛋白经常规方法裂解后，加入低介电常数溶剂，在常温下离心，使核酸贴壁牢固粘附于塑料离心管管壁，离心完成后立即通过直接倾倒液体或加以吸除的方法，尽量去除管内液体部分，用核酸过柱结合液复溶管中贴壁的微量核酸，再用常规方法过柱提取的技术方案。该发明可将常规试剂用于提取无细胞体液中微量核酸时的核酸捕获率显著提高，解决了从无细胞体液中提取微量核酸需要经济、有效、稳定的方法的问题，有利于液体活检的推广应用。
附图说明
图1是纯化6ml血浆时所用的50ml聚丙烯塑料材质离心管。
图2是离心管置于离心机内时与离心机轴形成固定的离心倾角。
图3是离心管系带朝上倾倒液体。
图4是加入复溶贴壁核酸的过柱结合液后涡旋振荡。
具体实施方式
下面给出本发明的具体实施方式并结合附图加以说明。
实施例1：提高从较大量的6毫升血浆中纯化游离DNA的核酸捕获率
如图1中所示，本实例中6毫升血浆盛装在一个圆底50毫升聚丙烯材质的离心管中，该离心管有系带1与按盖2相连，内装有6毫升血浆3。向离心管内加入蛋白酶K和蛋白变性剂共2毫升，合计为8毫升。在55度水浴中孵育3小时，使蛋白充分裂解。裂解完成后，再加入一倍体积(8毫升)异丙醇，形成16毫升液体。将其置入离心机的离心孔中如图2，此时50毫升离心管的长径4与离心机的离心轴5形成的离心倾角6为30度。在该离心机中4摄氏度下，1000克离心力离心2小时。离心完成后，立即取出离心管，打开盖子。如图3倾倒并翻转离心管，将离心管内全部液体8通过离心管系带对侧的开口处7将管内液体全部倒除。再次将已倒除液体的离心管置入原离心机，注意离心管系带朝向须与前次离心时保持一致。在4摄氏度，1000克离心力下离心5分钟。第二次离心完成后，立即取出离心管，用200微升尖头移液器吸头将管内残余液体吸除。注意吸除液体时，保持离心管系带朝上，此时吸除液体不会影响贴壁核酸。吸除液体后，向离心管内加入常规核酸过柱结合液9共1500微升，在涡旋振荡器上振荡混匀。涡旋振荡时，离心管内液体快速旋转并形成涡旋，如图4周边液体10贴着离心管壁上升，可使用离心管正立和倒置交叉涡旋振荡的方法使液面旋转到有可能存在贴壁核酸的所有离心管壁。涡旋振荡混匀5分钟后，按常规过柱结合、清洗和洗脱，即可获得血浆中提取的游离核酸物质。与不增加本发明所述步骤的一般核酸提取方法相比，增加本发明所述方法后，获得游离核酸的量可增加至3-8倍。
实施例2：提高从较小量的450微升血清中纯化游离DNA的核酸捕获率
本实例盛装血清的容器为常用的2毫升聚丙烯材质的锥底离心管，操作图示可参考实施例1所标注附图。离心管加入450微升血清后，再加入常用的蛋白酶K和蛋白变性剂共150微升，在55度恒温金属浴内孵育3小时，使血清中蛋白充分裂解，此时离心管内液体共600微升。裂解完成后，再加入1200微升的无水乙醇，上下颠倒15次充分混匀，此时管内共1800微升液体。将离心管置入离心倾角为40度的离心机中，在室温下，离心力为8000克下，离心40分钟。注意离心时，离心管管盖系带须部分朝上，以此作为离心管位置朝向的标记。离心完成后，立即取出离心管，打开盖子，缓慢翻转离心管，自离心管系带的对侧直接倾倒出液体，并用无尘纸吸除管口液体，使液体去除。再加入500微升常用核酸过柱结合液，涡旋 振荡5分钟使贴壁核酸充分溶解。常规过柱吸附、清洗和洗脱，即可获得游离DNA。与不增加本发明所述提高核酸捕获率方法的一般方法相比，增加本发明所述步骤后，所获得的游离DNA量增加至3-8倍。

Claims (10)

  1. 一种提高无细胞体液中微量核酸捕获率的方法，包括蛋白裂解，过柱结合，清洗和洗脱等步骤，其特征在于，在蛋白裂解和过柱结合之间加入以下步骤：
    (1)向盛有已裂解蛋白体液的离心管中加入0.5-3倍体积的低介电常数溶剂，混匀；
    (2)在常温下中高速离心；
    (3)用直接倾倒液体，或者吸除液体，或者倾倒后再加以离心再吸除的方法去除液体成份；
    (4)用核酸过柱结合液复溶贴壁核酸。
  2. 根据权利要求1所述的一种提高无细胞体液中微量核酸捕获率的方法，其特征在于，通过以下步骤实现：
    (1)蛋白裂解：将无细胞体液加入离心管中，再加入蛋白裂解液孵育；
    (2)蛋白裂解完成后，向离心管中加入原有液体体积0.5倍至3倍体积的低介电常数溶剂，在离心管与离心轴形成倾角的状态下，于不低于4℃且不大于35℃的环境温度下，中高速离心使液体中微量核酸牢固贴附于塑料离心管管壁，离心后直接开盖倒除或吸除液体，留有残液者再次离心并用移液器吸除液体的方法将离心管中液体成份去除，目的为使离心管中液体尽量去除；
    (3)向离心管中加入常规过柱结合液，涡旋振荡充分混匀，涡旋振荡时，离心管内液体快速旋转，其贴壁液面升高，使离心管正立和倒置交叉涡旋振荡的方法使旋转液体触及离心管管壁有可能吸附核酸的所有位置，涡旋振荡使贴壁核酸重新溶解于过柱结合液中，后续按常规方法过核酸纯化柱，将液体中核酸吸附于纯化柱的硅化吸附膜，加入清洗液离心后，再用常规洗脱液将核酸洗脱，即可获得纯化的无细胞含蛋白液体中核酸物质。
  3. 根据权利要求2所述的一种提高无细胞体液中微量核酸捕获率的方法，其特征在于，步骤(1)所述蛋白裂解液的PH值在7.5-8之间。
  4. 根据权利要求2所述的一种提高无细胞体液中微量核酸捕获率的方法，其特征在于，步骤(1)所述蛋白裂解孵育的温度在30℃和80℃之间，但以35℃-60℃之间活性相对较高，优选的以55℃孵育为佳。当所处理样品量较少(如<100微升)时，35℃或者80℃孵育均可达到裂解蛋白的作用，且孵育时间10分钟即可。当处理样品量较大时(如>100微升)时，优选的以50-60℃之间孵育，孵育时间应大于1小时，优选的为3小时，过夜孵育亦可，但不应超过16小时。
  5. 根据权利要求2所述的一种提高无细胞体液中微量核酸捕获率的方法，其特征在于，步骤(2)低介电常数溶剂是指在20℃时介电常数小于纯水的溶剂，包含乙醇、异丙醇、乙酸、丙酮、正己醇和四氯化碳；所述用于添加的低介电常数溶剂应为纯品，即含量应>99％。
  6. 根据权利要求2所述的一种提高无细胞体液中微量核酸捕获率的方法，其特征在于，步骤(2)所述的裂解体系液体中加入低介电常数溶剂后在常温下离心，包含离心时离心管的环境温度为常温，温度介于4℃或20℃；高速离心时间在10-120分钟之间，离心力在1000克-30000克之间。
  7. 根据权利要求2所述的一种提高无细胞体液中微量核酸捕获率的方法，其特征在于，步骤(2)通过直接倾倒，或吸除，或倾倒加吸除的方法将离心管内液体去除。
  8. 根据权利要求2所述的一种提高无细胞体液中微量核酸捕获率的方法，其特征在于，步骤(3)过柱结合液为用常规柱式核酸纯化硅化吸附膜的高盐低PH值的结合液。
  9. 根据权利要求1或2所述的一种提高无细胞体液中微量核酸捕获率的方法，其特征在于，离心管长径方向与离心力方向应形成离心倾角，角度为20-70度之间；离心管包含尖底、圆底、方底的圆柱形塑料离心管，离心管材质主要成份包含聚丙烯、聚乙烯、聚氯乙烯、聚苯乙烯、聚碳酸酯。
  10. 根据权利要求6所述的一种提高无细胞体液中微量核酸捕获率的方法，其特征在于，离心力为8000克，离心力离心40分钟。
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