Patent Application
20200032241
The present application is being filed with a Sequence Listing. The Sequence Listing is submitted electronically in in ASCII format via EFS-Web in the form of a text file. Said ASCII copy, created on Mar. 26, 2019, is named FP17-0614-00US-XX_SEQ-PW Ref-Soei-0501091.txt and is 3.35 KB in size, the contents of which are incorporated herein by reference in their entirety.
The present invention relates to a nucleic acid extraction method and a kit used therefor.
The BOOM method is one of the methods for extracting nucleic acid from biological samples (for example, see Patent Literature 1). In this method, nucleic acid is adsorbed on silica particles and is isolated from the biological sample, in the presence of a chaotropic compound, which is a substance that robs hydration water from biopolymers such as nucleic acid, and destabilize the higher-order structure of the biopolymers.
An example of method using the BOOM method is a method comprising lysing the biological sample and fixing silica and the like and the nucleic acid, in the presence of a chaotropic compound and/or an alkanol, wherein the fixing of the nucleic acid is performed within a temperature range of 36 to 75° C. (for example, see Patent Literature 2).
Patent Literature 1: Japanese Unexamined Patent Publication No. H2-289596
Patent Literature 2: Japanese Unexamined Patent Publication No. 2008-529509
In order to extract nucleic acid, a lysing step is necessary to break the membrane structure of the cell and release the contents outside the cell. As a lysing method, an enzymatic lysis method using proteolytic enzymes is widely used in commercial nucleic acid extraction kits. Moreover, when extracting nucleic acid from cells having special cell walls such as gram-positive bacteria or fungi among microorganisms, a pre-pretreatment with a cell wall degrading enzyme such as lysozyme and zymolyase is separately required. Therefore, depending on the type of biological material, the corresponding kit or protocol is generally different.
Moreover, many of the kits using the enzymatic lysis method take most of the time on the lysing step, relative to the time for all of the steps for nucleic acid extraction. Moreover, a centrifugal separation step is also often needed. Therefore, it is not suitable for applications where promptness is required.
The present invention was made in consideration of these issues, therefore, the aim of the present invention is to provide a method for extracting nucleic acid and a kit used therefor, that can reduce the amount of time necessary to extract nucleic acid from a biological sample.
The present invention provides a method for extracting nucleic acid from biological samples, comprising steps of: (i) mixing a biological sample, an anionic surfactant and a chaotropic compound to obtain a lysate; (ii) mixing the lysate, an alkanol having a boiling point higher than 75° C., a chaotropic compound and silica particles to adsorb the nucleic acid on the silica particles; (iii) washing the silica particles having the nucleic acid adsorbed thereon with a washing liquid; and (iv) eluting the nucleic acid adsorbed on the silica particles with an eluent, wherein at least steps (i) and (ii) are performed at a temperature higher than 75° C.
In the above method, step (iv) is preferably performed at a temperature higher than 75° C.
The alkanol having a boiling point higher than 75° C. may be 1-propanol.
The anionic surfactant is preferably lithium dodecyl sulfate.
The chaotropic compound may be a guanidinium salt.
The present invention also provides a nucleic acid extraction kit comprising a first lysis solution comprising an anionic surfactant, a second lysis solution comprising a chaotropic compound and an adsorbing liquid comprising an alkanol having a boiling point higher than 75° C., a chaotropic compound and silica particles.
In the kit, the alkanol having a boiling point higher than 75° C. may be 1-propanol.
In the kit, the anionic surfactant is preferably lithium dodecyl sulfate.
In the kit, the chaotropic compound may be a guanidinium salt.
According to the present invention, a method for extracting nucleic acid and a kit used therefor, that can reduce the amount of time necessary to extract nucleic acid from a biological sample, can be provided.
The following describes in detail the embodiments of the present invention. However, the present invention is not limited to the following embodiments.
The method for extracting nucleic acid from a biological sample, according to the present embodiment, comprises (i) mixing a biological sample, an anionic surfactant and a chaotropic compound to obtain a lysate.
The lysate is obtained, for example, by adding and mixing the biological sample, a first lysis solution comprising the anionic surfactant and a second lysis solution comprising the chaotropic compound, in a microtube placed on a heat block, then heating it.
The biological sample, the first lysis solution comprising the anionic surfactant and the second lysis solution comprising the chaotropic compound may be mixed at the same time, or either the first lysis solution comprising the anionic surfactant or the second lysis solution comprising the chaotropic compound may be mixed with the biological sample to obtain a mixed liquid, then the mixed liquid may be mixed with the other solution.
In this description, “biological sample” refers to a specimen that are subjected to the extraction of nucleic acid, and, specifically, is a part or the entirety of the cell, tissue, or organ of viruses, phages, bacteria, fungi and living organisms, and includes specimens collected directly from living body as well as those obtained from the environment such as water, soil or air. Examples of biological sample include blood, throat swabs, nasopharyngeal swabs, sputum, spinal fluid, urine, feces and saliva.
The nucleic acid may be a naturally occurring nucleic acid such as DNA or RNA.
Examples of anionic surfactants include lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate. The anionic surfactant is preferably lithium dodecyl sulfate (LDS), since it hardly precipitates at low temperatures.
An example of chaotropic compounds is a guanidinium salt. The guanidinium salt may be, for example, guanidinium (iso)thiocyanate (GuSCN) or guanidinium hydrochloride.
After the step (i), (ii) the lysate, an alkanol having a boiling point higher than 75° C., a chaotropic compound and silica particles are mixed to adsorb the nucleic acid on the silica particles.
To adsorb the nucleic acid on the silica particles, for example, the adsorbing liquid comprising the alkanol having a boiling point higher than 75° C., the chaotropic compound and the silica particles is added to the microtube containing the above lysate and placed on a heat block, and is mixed, then heated.
From a viewpoint of reducing the amount of time necessary from the lysis to the adsorption, the temperature at which step (i) and step (ii) are performed is a temperature higher than 75° C., and is preferably 80° C. or more and more preferably 85° C. or more. The temperature at which step (i) and step (ii) are performed is preferably 100° C. or less, more preferably 98° C. or less, and further preferably 95° C. or less. The temperature at which step (i) and step (ii) are performed is, for example, 75 to 100° C., 75 to 98° C., 75 to 95° C., 80 to 100° C., 80 to 98° C., 80 to 95° C., 85 to 100° C., 85 to 98° C., or 85 to 95° C.
Setting the temperature at which step (i) and step (ii) are performed both to a temperature higher than 75° C. allows to reduce the amount of time necessary for the temperature change of the liquid temperature. Therefore, it allows to reduce the amount of time necessary from the lysis of the biological sample to the adsorption of the nucleic acid. As a result, the amount of time necessary for the extraction of nucleic acid (the amount of time necessary from the lysis of the biological sample to the elution of the nucleic acid) can also be reduced.
The boiling point of the alkanol used in step (ii) is a temperature higher than 75° C., and is preferably 80° C. or more and more preferably 85° C. or more. The boiling point of the alkanol used in step (ii) is preferably 120° C. or less, more preferably 110° C. or less, and further preferably 100° C. or less. The boiling point of the alkanol used in step (ii) is, for example, 75 to 120° C., 75 to 110° C., 75 to 100° C., 80 to 120° C., 80 to 110° C., 80 to 100° C., 85 to 120° C., 85 to 110° C., or 85 to 100° C.
The alkanol having a boiling point higher than 75° C. is, for example, 1-propanol, ethanol, isopropanol and the like.
A specific example of the chaotropic compound is the same as the specific example of the chaotropic compound in step (i).
Examples of silica particles include particles constituted of alkyl silica, aluminum silicate (also referred to as “zeolite”), silica having an amino group, and the like. From a viewpoint of easier collection, the silica particles are preferably magnetic silica particles.
After step (ii), (iii) the silica particles having the nucleic acid adsorbed thereon are washed with a washing liquid.
The step (iii) may comprise, for example, a step of adding a first washing liquid comprising an alcohol and an anionic surfactant to the silica particles having the nucleic acid adsorbed thereon, followed by stirring, and then separating the silica particles having the nucleic acid adsorbed thereon and the first washing liquid, and a step of adding a second washing liquid comprising polyethylene glycol to the silica particles after the separation, followed by stirring, and then separating the silica particles having the nucleic acid adsorbed thereon and the second washing liquid.
The alcohol may be 2-propanol, 1-propanol or ethanol. The alcohol may also be a mixture of these.
The anionic surfactant may be, for example, sodium dodecyl sulfate (SDS), or may be lithium dodecyl sulfate. The anionic surfactant may also be a mixture of these.
The average molecular weight of polyethylene glycol may be 200 to 10000. An example of such polyethylene glycol includes polyethylene glycol 4000 (PEG4000). In the present description, the average molecular weight of polyethylene glycol means the value measured in accordance with the average molecular weight test of each macrogol (polyethylene glycol) described in the Japanese Pharmacopoeia, Sixteenth Edition.
After step (iii), (iv) the nucleic acid adsorbed on the silica particles is eluted with an eluent.
The nucleic acid can be eluted, for example, by the following method. Namely, the silica particles on which the nucleic acid is adsorbed to and the eluent are first mixed and stirred, then heated. After stirring again, the nucleic acid can be eluted by separating the silica particles and the solution.
From a viewpoint of eluting the adsorbed nucleic acid efficiently and in a short time, the temperature at which step (iv) is performed is a temperature higher than 75° C., and is preferably 80° C. or more and more preferably 85° C. or more. The temperature at which step (iv) is performed is preferably 100° C. or less, more preferably 98° C. or less, and further preferably 95° C. or less. The temperature at which step (iv) is performed is, for example, 75 to 100° C., 75 to 98° C., 75 to 95° C., 80 to 100° C., 80 to 98° C., 80 to 95° C., 85 to 100° C., 85 to 98° C., or 85 to 95° C.
Setting the temperature at which step (iv) is performed to a temperature higher than 75° C. similarly as the temperature at which step (ii) is performed, allows to perform the extraction of nucleic acid (from the lysis of the biological sample to the elution of the nucleic acid) without changing the heating conditions of the heater such as a heat block. Therefore, the amount of time necessary for changing the temperature inside the heater and of the solution can be reduced, and the amount of time necessary for the extraction of the nucleic acid (the amount of time necessary from the lysis of the biological sample to the elution of the nucleic acid) can further be reduced.
Setting all the temperatures at which step (i), step (ii) and step (iv) are performed to a temperature higher than 75° C. allows to obtain a high-yield nucleic acid, in a short time, from a biological sample. Moreover, if the temperatures at which step (i), step (ii) and step (iv) are performed are all temperatures higher than 75° C., there may be a single heater and a plurality of heaters set at different temperatures is not required, since it is possible to perform step (i), step (ii) and step (iv) under the same range of temperature conditions.
Examples of eluents include sterile water and buffer solutions with a low salt concentration. A buffer solution with a low salt concentration is, for example, a buffer solution comprising 10 mM of tris-hydrochloric acid (Tris-HCl).
The above step (i) to step (iv) may be performed manually, or may be performed fully automatically using a nucleic acid extractor.
Each solution necessary to the method according to the present embodiment can be packaged in advance, and utilized as a nucleic acid extraction kit. Namely, the nucleic acid extraction kit comprises a first lysis solution comprising an anionic surfactant. Specific examples of anionic surfactants are the same as the specific examples of anionic surfactants in the step (i) of the method for extracting nucleic acid from a biological sample.
As the concentration in the mixed liquid of the biological sample and the first lysis solution, the concentration of the anionic surfactant is preferably 0.01 to 10 mass %, more preferably 0.1 to 5 mass %, and further preferably 0.5 to 3 mass %.
The first lysis solution may further comprise tris-hydrochloric acid (Tris-HCl) and ethylenediaminetetraacetic acid (EDTA).
The nucleic acid extraction kit further comprises a second lysis solution comprising a chaotropic compound.
Specific examples of the chaotropic compound are the same as the specific examples of the chaotropic compound in step (i) of the method for extracting nucleic acid from a biological sample.
As the concentration in the mixed liquid of the biological sample, the first lysis solution and the second lysis solution, the concentration of the chaotropic compound contained in the second lysis solution is preferably 0.01 to 5 M, more preferably 0.1 to 4.5 M, and further preferably 1 to 4 M.
The second lysis solution may further comprise tris-hydrochloric acid (Tris-HCl).
The nucleic acid extraction kit further comprises an adsorbing liquid comprising an alkanol having a boiling point higher than 75° C., a chaotropic compound and silica particles.
Specific examples and preferable aspects of the alkanol having a boiling point higher than 75° C., the chaotropic compound and the silica particles are the same as the specific examples and preferable aspects of the alkanol having a boiling point higher than 75° C., the chaotropic compound and the silica particles in the method for extracting nucleic acid from a biological sample.
As the concentration in the mixed liquid of the above lysate and adsorbing liquid, the concentration of the alkanol having a boiling point higher than 75° C. is preferably 1 to 99 mass %, more preferably 10 to 90 mass %, and further preferably 20 to 70 mass %.
As the concentration in the mixed liquid of the above lysate and adsorbing liquid, the concentration of the chaotropic compound contained in the adsorbing liquid is preferably 0.01 to 5 M, more preferably 0.1 to 4 M, and further preferably 1 to 4.5 M.
The nucleic acid extraction kit may further comprise a first washing liquid, a second washing liquid and an eluent in the method for extracting nucleic acid from a biological sample.
The nucleic acid extraction kid according to the present embodiment is applicable to a wide range of applications. With the nucleic acid extraction kit according to the present embodiment, the extraction of nucleic acid from a wide range of biological materials such as bacteria, fungi and viruses contained in clinical samples such as blood, throat swabs, nasopharyngeal swabs, sputum, spinal fluid, urine, feces and saliva, is possible with the same kit. Moreover, it is possible to provide a method for extracting nucleic acid and a kit used therefor, that can reduce the amount of time necessary for the extraction compared to a general method.
The present invention will now be described in more detail based on Examples, but the present invention is not intended to be limited to Examples.
The bacteria and viruses shown in the following Table 1 were used. S. pneumoniae (also referred to as “S.P”) were cultivated from a culture plate, suspended in a normal saline solution and adjusted to a bacteria liquid concentration of McF#1 by measuring the turbidity, to be used as specimen (1). B. pertussis (also referred to as “B.P”) were cultivated from a culture plate, suspended in a normal saline solution and adjusted to a bacteria liquid concentration of McF#1 by measuring the turbidity, to be used as specimen (2). S. cerevisiae (also referred to as “S.C”) were cultivated from a culture plate, suspended in a normal saline solution and adjusted to a bacteria liquid concentration corresponding to OD=6.0 by measuring the turbidity at a wavelength of 600 nm, to be used as specimen (3). Human adenovirus 2 (also referred to as “ADV”) was infected to A549 cells and cultured, and what was recovered from the culture supernatant was used as specimen (4). Influenza A virus (subtype H3N2, also referred to as “FluA”) was infected to MDCK cells and cultured, and its culture supernatant was used as specimen (5).
S. pneumoniae, S.P
B. pertussis, B.P
S. cerevisiae, S.C
As the nucleic acid extraction reagents, the solutions with the compositions shown below were prepared.
First lysis solution: 233.3 mM Tris-HCl (pH 7.5), 23.3 mM EDTA, 4.6 (w/w) % LDS
Second lysis solution: 100 mM Tris-HCl (pH 7.5), 4.25 M GuSCN
Adsorbing liquid: 47.3 (w/w) % 1-propanol, 2.5 M GuSCN, magnetic silica particles
First washing liquid: 41.0 (w/w) % 2-propanol, 1 M NaCl, 1.1 (w/w) %
Second washing liquid: 9.5 (w/w) % PEG 4000, 1 M NaCl
Nucleic acid was extracted from S.P, B.P, S.C, ADV and FluA fully automatically using a nucleic acid extractor, by the following procedure. Namely, 3 μL of the S.P specimen (1) was added to 200 μL of a normal saline solution, to obtain biological sample A. Similarly, 2 μL of the B.P specimen (2), 3 μL of the S.C specimen (3), 3 μL of the ADV specimen (4) and 1 μL of the FluA specimen (5) were each added to 200 μL of a normal saline solution, to obtain biological samples B to E. The biological samples A to E and 150 μL of the first lysis solution were mixed in microtubes, then stirred for 7 s. Next, they were heated on a heat block at 110° C. for 1 min. While continuing to heat on the heat block, 350 μL of the second lysis solution was further added and stirred for 7 s, then heated on the heat block at 110° C. for 1 min. While continuing to heat on the heat block, 800 μL of the adsorbing liquid was further added, then stirred for 7 s. Subsequently, after heating on the heat block at 110° C. for 1 min, they were stirred for 7 s and further heated on the heat block at 110° C. for 1 min. After separating the magnetic silica particles having the nucleic acid adsorbed thereon and the solutions, 900 μL of the first washing liquid was added and stirred for 7 s. After separating again the magnetic silica particles having the nucleic acid adsorbed thereon and the solutions, 900 μL of the second washing liquid was added and stirred for 7 s. After collecting the magnetic silica particles and discarding the supernatant, 250 μL of the eluent was added and stirred for 7 s. After letting it rest on the heat block at 110° C. for 2 min, they were stirred for 7 s and the magnetic silica particles having the nucleic acid adsorbed thereon and the solutions were separated, to obtain nucleic acid extract liquids I to V. The liquid temperature was monitored and it was confirmed that the liquid temperature higher than 75° C. during the lysis step, the adsorption step and the elution step.
The nucleic acids were quantified by conducting a PCR reaction using the nucleic acids in the obtained nucleic acid extract liquids I to V as templates. Namely, the PCR was first performed with the reagent compositions and under the reaction conditions shown below, using the primer sets and probes shown in the following Table 2. Regarding Example 1, the same extraction and quantification were performed three times. The results of the average value and the standard error are shown in
S.pneumoniae
B.pertussis
S.cerevisiae
Composition of the PCR reaction solution and conditions of the PCR reaction
Nucleic Acid Extract Liquid I or IV
Nucleic Acid Extract Liquid II
Nucleic Acid Extract Liquid III
Nucleic Acid Extract Liquid V
Nucleic acid was extracted by spiking the specimen (1) in the sample from a healthy subject. First, 3 μL of specimen (1) was added to 200 μL of throat swab suspension, to obtain biological sample F. Similarly, 3 μL of specimen (1) was added to 200 μL of nasopharyngeal swab suspension, 3 μL of specimen (1) was added to 200 μL of blood serum, 3 μL of specimen (1) was added to 200 μL of blood (containing the anticoagulant EDTA-2K), 3 μL of specimen (1) was added to 200 μL of blood (containing the anticoagulant sodium citrate) and 3 μL of specimen (1) was added to 200 μL of blood (containing the anticoagulant heparin), to obtain biological samples G to K respectively. The nucleic acid extract liquids VI to X were obtained in the same way as Example 1.
In regard to the obtained nucleic acid extract liquids VI to X, nucleic acid was quantified by conducting PCR reactions the same way as Example 1.
For the preparation of the biological sample H, powdered serum from healthy subject was restored with purified water and used as a sample from healthy subject, and extraction and quantification were performed three times to obtain the results of N=3. For the preparation of the biological samples F, G, I, J and K, healthy subject samples from three donors each were used, and extraction and quantification were performed once and the results were considered as the results of N=3. The results of N=3 are shown in
Except that specimen (4) was used instead of specimen (1), the nucleic acid extract liquid was obtained and quantified by conducting a PCR reaction the same way as Example 2. The results are shown in
Except performing in an environment where the elution temperature was set at room temperature (25° C.), 50° C., 80° C. or 110° C., the extract liquid was obtained from the biological sample A of specimen (1), a PCR reaction was performed and the nucleic acid was quantified the same way as Example 1. The results are shown in
Except performing in an environment where the elution temperature was set at room temperature (25° C.), 50° C., 80° C. or 110° C., the extract liquid was obtained from the biological sample A of specimen (1), a PCR reaction was performed and the nucleic acid was quantified the same way as Example 1. The results are shown in
Using the QIAAMP™ DNA mini kit (by QIAGEN), nucleic acid was extracted from a biological sample containing S.P. Namely, except using specimen (1) as a sample instead of centrifuged sediment and using 10 mM Tris-HCl (pH 7.5) instead of Buffer AE or purified water, nucleic acid was extracted in accordance with the recommended protocols of the kit. The recommended protocols of the kit are Appendix D: Protocol for Bacteria, Isolation of genomic DNA from Gram-positive bacteria and Protocol: DNA purification from Tissues described in QIAAMP™ DNA Mini and Blood Mini Handbook (Fifth edition). Regarding the nucleic acid extracted using the QIAAMP™ DNA mini kit (by QIAGEN), nucleic acid was quantified by conducting a PCR reaction the same way as Example 1. The result is shown in
Using the QIAAMP™ DNA mini kit (by QIAGEN), nucleic acid was extracted from a biological sample containing B.P. Namely, except using specimen (2) as a sample instead of centrifuged sediment, performing the protease K treatment for 10 min and using 10 mM Tris-HCl (pH 7.5) instead of Buffer AE or purified water, nucleic acid was extracted in accordance with the recommended protocols of the kit. The recommended protocols of the kit are Appendix D: Protocol for Bacteria, Isolation of genomic DNA from bacterial plate cultures and Protocol: DNA purification from Tissues described in QIAAMP™ DNA Mini and Blood Mini Handbook (Fifth edition). In regard to the extracted nucleic acid, the nucleic acid was quantified the same way as in Reference Example 1. The result is shown in
Using the QIAAMP™ DNA mini kit (by QIAGEN), nucleic acid was extracted from a biological sample containing S.C. Namely, except using specimen (3) as a sample instead of centrifuged sediment for each, performing the protease K treatment for 10 min and using 10 mM Tris-HCl (pH 7.5) instead of Buffer AE or purified water, nucleic acid was extracted in accordance with the recommended protocols of the kit. The recommended protocols of the kit are Appendix E: Protocol for Yeast and Protocol: DNA purification from Tissues described in QIAamp DNA Mini and Blood Mini Handbook (Fifth edition). In regard to the extracted nucleic acid, the nucleic acid was quantified the same way as Reference Example 1. The result is shown in
Using the QIAAMP™ MinElute Virus Spin kit (by QIAGEN), nucleic acid was extracted from a biological sample containing ADV. Namely, except using 3 μL of specimen (4) in 200 μL of normal saline solution instead of blood plasma or blood serum described in the recommended protocol of the kit, nucleic acid was extracted in accordance with the recommended protocol of the kit. The recommended protocol of the kit is Protocol: Purification of Viral Nucleic Acids from Plasma or Serum described in QIAAMP™ MinElute Virus Spin Handbook (2010 edition).
Regarding the nucleic acid extracted using the QIAAMP™ MinElute Virus Spin kit (by QIAGEN), it was quantified by conducting a PCR reaction the same way as Example 1. The result is shown in
In regard to Examples 1 to 3 and Reference Examples 1 to 4, the amount of time it took for the extraction are shown below in Table 3.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2016-193987 | Sep 2016 | JP | national |
This patent application is the National Phase of International Application No. PCT/JP2017/033741, filed Sep. 19, 2017, which claims the benefit of priority to JP Patent Application No. 2016-193987, filed Sep. 30, 2016. The entire contents of the foregoing applications are is incorporated herein by reference, including all text, tables, sequence listings and drawings.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2017/033741 | 9/19/2017 | WO | 00 |
