JP7138677B2 - Nucleic acid pretreatment kit and nucleotide sequence analysis method - Google Patents

Description

The present invention relates to a nucleic acid pretreatment kit for isolating and purifying nucleic acids for base sequence analysis from biological samples, and a method for base sequence analysis of nucleic acids isolated using the kit.

Base sequence analysis of nucleic acids contained in biological samples such as blood, serum, cells, urine, and feces isolated from animals and plants is useful for medical examinations, food safety and hygiene management, and the like. In addition, the relationship between various diseases and genomic DNA sequences has been clarified, and along with this, the analysis of nucleotide sequences such as polymorphisms and mutations has attracted increasing attention.

In order to eliminate the adverse effects caused by various contaminants such as proteins, sugars, and lipids, it is necessary to pretreat the biological sample and separate and purify the nucleic acid when analyzing the base sequence of nucleic acids in the biological sample. . After separation and purification, the nucleic acid is amplified by PCR or the like, if necessary, and then analyzed by a base sequence analyzer.

Multi-capillary base sequence analyzers that can analyze multiple samples simultaneously are widespread, and it is common to create a library consisting of multiple specimen samples prepared in advance and analyze the base sequences of each sample at the same time. is done in Moreover, in recent years, next-generation sequencing techniques employing various principles have been developed, and the ability to analyze base sequences has improved dramatically.

Since the analysis by the base sequence analyzer is automated, at the stage of base sequence analysis, mix-up of specimen samples and contamination between samples due to human error hardly occur. On the other hand, in pretreatments such as isolation and purification of nucleic acids from biological samples for base sequence analysis, operations such as pipetting and transfer of samples to another container are performed in an open system. Therefore, contamination between samples may occur in the pretreatment operation for nucleotide sequence analysis, and there is a risk of lowering the reliability of test results.

In Patent Documents 1 to 3, the inside of the container is separated into a plurality of spaces by a water-insoluble or sparingly soluble aqueous phase separation medium such as an oil phase or a gel medium, and cell lysis is performed in the spaces partitioned by the separation medium. Pretreatment devices loaded with aqueous liquids such as liquids, washing liquids, and nucleic acid elution liquids have been proposed. Specifically, Patent Document 1 discloses a device in which a plurality of droplets and magnetic silica beads capable of selectively adsorbing nucleic acids are present in an encapsulating medium such as oil charged in a container. there is In this device, nucleic acid purification and amplification reactions can be carried out by sequentially moving magnetic silica beads present in a droplet into other droplets by manipulating a magnetic field.

In Patent Document 2, aqueous liquid layers such as cell lysate, washing liquid, nucleic acid eluate, etc., and gel layers that are sparingly soluble or insoluble in water are alternately layered in a tubular container having an open end that can be closed on one side. A device is disclosed. Patent Document 3 discloses a chip device in which aqueous liquid layers and gel layers are alternately arranged in grooves formed on a substrate surface. In these devices, multiple aqueous liquids are partitioned by gel, and magnetic silica beads are sequentially moved along the length of the tube to the cell lysate, washing solution, and nucleic acid elution solution, thereby purifying nucleic acids. can be implemented.

The pretreatment devices disclosed in Patent Documents 1 to 3 can perform sample pretreatment in a closed system, thereby suppressing contamination between specimen samples and improving the reliability of base sequence analysis. Moreover, these closed-system pretreatment devices can be applied to nucleic acid amplification operations such as PCR, in addition to separation and purification of nucleic acids from biological samples. If separation and purification of nucleic acids from biological samples and nucleic acid amplification are carried out in the same closed-system device, it is possible to analyze the samples obtained by the pretreatment device as they are with the base sequence analysis equipment, thereby preventing contamination between samples. Nation can be minimized. Furthermore, since multiple types of liquids are loaded in one closed container, the number of operations to transfer the sample to another container is reduced, and the risk of sample mix-up due to human error can be reduced.

Japanese Patent Application Laid-Open No. 2008-012490 WO2012/086243 international pamphlet WO2013/094322 international pamphlet

Even when using the closed-system pretreatment devices disclosed in Patent Documents 1 to 3, etc., it is necessary to transfer the container when preparing an analysis library or injecting a sample into a base sequence analyzer. Therefore, there is a risk of sample mix-up due to human error. In order to prevent erroneous detection due to such mix-ups, each container for loading specimen samples is attached with identification information that can be recognized from the outside, such as a barcode. The method of consistently managing the code for the process up to analysis is widespread.

However, in the method of inheriting and managing the identification information attached to the container on a process-by-process basis, it is difficult to completely eliminate human errors such as code reading mistakes and code confusion. The closed-system pretreatment device as described above does not require large-scale equipment, so it is suitable for use in clinical settings. Most of them are carried out in remote locations. Therefore, it is difficult for a single entity to consistently manage everything from nucleic acid pretreatment to base sequence analysis, and the reliability of testing decreases due to code reading mistakes and code confusion. contains risks.

In view of such problems, the present invention aims to provide a method for improving the reliability of testing by consistently retaining information identifying specimen samples in the process from nucleic acid pretreatment to base sequence analysis. .

In the pretreatment stage of separating and purifying nucleic acid from a biological sample, an individual identification nucleic acid having a different base sequence for each sample is added to the sample. to analyze the base sequence of the individual identification nucleic acid. This makes it possible to consistently retain information identifying specimen samples. The present invention relates to a nucleotide sequence analysis method applying this method and a nucleic acid pretreatment kit used for the pretreatment.

A pretreatment kit is used to separate nucleic acids from a biological sample containing nucleic acids and contaminants. The pretreatment kit includes a particle manipulation vessel, nucleic acid-capturing particles capable of selectively binding to nucleic acids, an aqueous phase separation medium that is insoluble or sparingly soluble in water, multiple types of aqueous liquids, and nucleic acids for individual identification.

Two of the multiple types of aqueous liquids are a nucleic acid capture liquid for binding nucleic acids contained in the biological sample to the nucleic acid capture particles, and a nucleic acid recovery liquid for recovering the nucleic acids bound to the surfaces of the nucleic acid capture particles. be. A cell lysate or the like is used as the nucleic acid capture solution. As the nucleic acid recovery liquid, a nucleic acid elution liquid or the like for eluting the nucleic acid bound to the surface of the nucleic acid-capturing particles from the particle surface is used.

Among the components of these kits, at least the aqueous phase separation medium and the nucleic acid recovery solution are loaded in the particle manipulation container. The particle manipulation container may be configured to be separable in the vicinity of the location where the nucleic acid recovery solution is loaded.

In the first form of the kit of the present invention, the aqueous phase separation medium and the aqueous liquid are all loaded into the particle manipulation vessel. The second form of the kit of the present invention includes a nucleic acid capture operation container in addition to the particle manipulation container, and the nucleic acid capture solution is loaded in the nucleic acid capture operation container. The nucleic acid-capturing particles may be contained within the container, or may be provided separately from the container. When the kit is provided with the nucleic acid-capturing particles loaded in the container, the nucleic acid-capturing particles are preferably contained in the nucleic acid-capturing liquid.

The identifying nucleic acid is included in the kit either contained in at least one of the aqueous liquids or attached to the surface of the nucleic acid-capturing particles. When the nucleic acid for individual identification is contained in the nucleic acid recovery solution, the recovery rate of the nucleic acid for individual identification is increased. When the nucleic acid for individual identification is contained in the nucleic acid-capturing solution or bound to the surface of the nucleic acid-capturing particles, the nucleic acid in the biological sample and the nucleic acid for individual identification coexist from the time the biological sample is added to the kit. Since the operation is performed in the state, the reliability of the inspection is enhanced. In particular, as in the second mode, the nucleic acid capturing solution is loaded in the nucleic acid capturing operation container and the sample is transferred from the nucleic acid capturing operation container to the particle manipulation container.

The base sequence of the individual identification nucleic acid includes an identification sequence consisting of a non-complementary base sequence to the nucleic acid contained in the biological sample. When analyzing the base sequence of the nucleic acid in the biological sample collected using the kit of the present invention, the base sequence of the individual identification nucleic acid is also analyzed. The reliability of the test is increased by checking whether the base sequence of the identification sequence portion obtained by base sequence analysis matches the base sequence of the identification sequence of the individual identification nucleic acid included in the kit. be done.

The individual identification nucleic acid may contain other sequences on the 3' and 5' sides of the identification sequence. When the nucleic acid for individual identification contains a complementary base sequence to the nucleic acid contained in the biological sample on the 3′ side and / or 5′ side of the identification sequence, during amplification of the nucleic acid contained in the biological sample by PCR etc. , the nucleic acid of the identification sequence of the identification nucleic acid can also be amplified.

Identification information that can be recognized from the outside of the container is attached to the particle manipulation container, and the identification information attached to the particle manipulation container is preferably associated with the base sequence of the identification sequence. By associating the identification information attached to the operation container with the identification sequence, traceability can be ensured and collation during base sequence analysis can be easily performed. The identification information is preferably attached in a form recognizable by an optical method or an electromagnetic method.

When the nucleic acid capture solution is loaded in the nucleic acid capture operation container and the nucleic acid for individual identification is contained in the nucleic acid capture solution or bound to the surface of the nucleic acid capture particles, the identification information is stored in the nucleic acid capture operation container. is attached, and it is preferable that the identification information attached to the nucleic acid capturing operation container is associated with the base sequence of the identification sequence. Furthermore, it is preferable that the identification information attached to the nucleic acid capturing operation container and the identification information attached to the particle operation container can be associated with each other.

Since the kit of the present invention can separate and purify nucleic acids from a biological sample in a closed container, contamination between samples can be suppressed even when a large number of test samples are handled at the same time. In addition, by using the kit of the present invention, the state in which the nucleic acid of the biological sample and the nucleic acid for individual identification coexist is consistently maintained from the pretreatment stage of separating and purifying the nucleic acid from the biological sample to base sequence analysis. be.

Even in the unlikely event that contamination or a mix-up of samples occurs, cross-containment of containers or contamination can be detected by matching the base sequence of the identification sequence portion of the individual identification nucleic acid. That is, by using the kit of the present invention, the risk of contamination and mix-up of specimens can be reduced, and the occurrence thereof can be detected, so that the reliability of genetic testing and the like based on nucleic acid base sequence analysis can be enhanced.

1 is a schematic cross-sectional view showing a configuration example of a pretreatment kit of a first embodiment; FIG. FIG. 4 is a schematic cross-sectional view showing a configuration example of a pretreatment kit of a second form;

The nucleic acid pretreatment kit of the present invention is used for pretreatment of nucleic acids for base sequence analysis. Specifically, the kit of the present invention is used for separation of nucleic acids from biological samples containing nucleic acids and contaminants. The isolated nucleic acid is subjected to base sequence analysis after being amplified as necessary.

Nucleic acids contained in biological samples include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). An individual identifying nucleic acid is DNA, RNA, or peptide nucleic acid (PNA). Nucleic acids may be single-stranded or double-stranded.

Examples of biological samples containing nucleic acids include biological samples such as animal and plant tissues, body fluids and excretions, nucleic acid-containing bodies such as cells, protozoa, fungi, bacteria and viruses. Body fluids include blood, cerebrospinal fluid, saliva, milk and the like, and excretions include feces, urine, sweat and the like. Combinations of these may also be used. Cells include leukocytes in blood, platelets, exfoliated cells of mucosal cells such as buccal cells, leukocytes in saliva, etc., and combinations thereof can also be used. A biological sample containing nucleic acids may be prepared in the form of, for example, a cell suspension, a homogenate, or a mixture with a cell lysate. These biological samples contain a wide variety of contaminants in addition to the target nucleic acid. For example, blood contains contaminants such as proteins, sugars, and lipids eluted from cells, in addition to nucleic acids.

The pretreatment kit includes nucleic acid-capturing particles, an aqueous phase separation medium, an aqueous liquid, and a particle manipulation container for containing these, and further includes an identification nucleic acid.

[Nucleic acid-capturing particles]
Nucleic acid-capturing particles are particles capable of selectively binding nucleic acids. Silica particles or particles whose surfaces are coated with silica are preferably used as particles capable of selectively binding nucleic acids. When nucleic acid-capturing particles are present in a liquid containing nucleic acids and contaminants, nucleic acids selectively bind to the particle surfaces. Nucleic acid can be recovered in the nucleic acid recovery solution by moving the nucleic acid-bound particles to a washing solution to remove contaminants adhering to the surface, and then moving the particles to the nucleic acid recovery solution.

From the viewpoint of facilitating particle manipulation in a liquid or gel medium, the particle diameter of the nucleic acid-capturing particles is preferably 1 mm or less, more preferably 0.1 μm to 500 μm. The shape of the particles is desirably spherical with a uniform particle size, but may be irregular in shape and have a certain degree of particle size distribution as long as the particles can be manipulated.

The action of magnetic fields, electric fields, gravitational fields, ultrasonic fields, etc. can perform operations such as agglomeration, dispersion, movement, etc. of particles. Among them, magnetic field operation is preferable because aggregation, dispersion and movement of particles can be easily and accurately performed. Magnetic particles are preferably used as the nucleic acid-capturing particles when the particles are manipulated by a magnetic field. Examples of magnetic substances that constitute magnetic particles include iron, cobalt, nickel, and compounds, oxides, and alloys thereof.

Commercially available magnetic particles may be used as nucleic acid-capturing particles. Commercially available magnetic particles for capturing nucleic acids whose surfaces are coated with silica include Dynabeads (registered trademark) sold by Life Technologies and MagExtractor (registered trademark) sold by Toyobo.

[Water-based liquid]
The aqueous liquid provides a site for chemical manipulations such as immobilization of nucleic acids on the surfaces of nucleic acid-capturing particles and separation. In separation and purification of nucleic acids in a biological sample by particle manipulation, a nucleic acid capture solution for binding nucleic acids contained in the biological sample to the nucleic acid capture particles, and a nucleic acid recovery solution for recovering the nucleic acids bound to the surfaces of the nucleic acid capture particles. is used. As the aqueous liquid, it is preferable to use a washing liquid in addition to the nucleic acid capture liquid and the nucleic acid recovery liquid. By exposing the particles having nucleic acids bound to their surfaces to the washing liquid, contaminants and the like adhering to the particle surfaces can be removed, and the purity of the nucleic acids can be improved.

<Nucleic acid capture solution>
The nucleic acid-capturing liquid provides a field for binding the nucleic acid released in the liquid to the surface of the nucleic acid-capturing particles. The nucleic acid-trapping solution is preferably one that also has the action of destroying cells and releasing nucleic acids into the solution, that is, a cell lysate. Examples of cell lysates that can be used as nucleic acid-trapping solutions include buffer solutions containing chaotropic substances, chelating agents such as EDTA, Tris-HCl, and the like. Cell lysates can also contain detergents such as Triton X-100. Chaotropic substances include guanidine hydrochloride, guanidine isothiocyanate, potassium iodide, urea, and the like. In addition to the above, the cell lysate may contain proteolytic enzymes such as protease K, various buffers, salts, other various adjuvants, and organic solvents such as alcohol. In the presence of these substances, nucleic acids bind specifically to surfaces such as silica particles. Therefore, when a biological sample containing nucleic acids and nucleic acid-capturing particles are added to a cell lysate, nucleic acids selectively bind to the surfaces of the nucleic acid-capturing particles.

<Washing liquid>
The cleaning liquid is used to remove contaminants adhering to the particle surfaces and the like. The washing solution should be capable of liberating components other than nucleic acids (proteins, sugars, lipids, etc.) and reagents used for lysing cells into the washing solution while maintaining the nucleic acid immobilized on the particle surface. I wish I had. Examples of the cleaning liquid include aqueous solutions of high salt concentration such as sodium chloride, potassium chloride and ammonium sulfate, and aqueous alcohol solutions such as ethanol and isopropanol.

<Nucleic acid recovery solution>
The nucleic acid recovery liquid is used to recover nucleic acids bound to the surface of the nucleic acid-capturing particles. Methods for recovering the nucleic acid bound to the particle surface into a liquid include a method in which the particles are recovered while the nucleic acid remains bound to the particle surface, and a method in which the nucleic acid bound to the particle surface is eluted into the liquid and recovered as a solution. method. When nucleic acids are recovered while bound to the particle surface, an aqueous solution having the same composition as the washing solution is preferably used as the nucleic acid recovery solution. When nucleic acids are eluted into a liquid, a nucleic acid elution liquid is used as the nucleic acid recovery liquid for eluting the nucleic acids bound to the surfaces of the nucleic acid-capturing particles from the particle surfaces.

As a nucleic acid elution solution, water or a buffer solution containing a low concentration of salt can be used. Specifically, Tris buffer, phosphate buffer, distilled water, and the like can be used. Among them, it is common to use 5-20 mM Tris buffer adjusted to pH 7-9. By moving particles with nucleic acids bound to their surfaces into a nucleic acid elution solution, the nucleic acids can be eluted from the particle surfaces and recovered in the nucleic acid elution solution.

[Aqueous phase separation medium]
The aqueous phase separation medium is arranged in the container so as to be in contact with two or more aqueous liquids, and separates the inside of the container into a plurality of spaces. Each of the spaces partitioned by the separation medium is filled with an aqueous liquid such as a cell lysate, a washing liquid, or a nucleic acid recovery liquid.

In order to prevent contamination among a plurality of aqueous media, a substance insoluble or sparingly soluble in water is used as the medium for aqueous phase separation. The aqueous phase separation medium may be in a liquid state or a (semi)solid state as long as it is insoluble or sparingly soluble in water and is immiscible with an aqueous liquid.

Liquid separation media include hydrocarbons such as alkanes, perfluoroalkanes, mineral oils, silicone oils, fatty acids, fatty acid esters, fatty acid amides, fatty acid ketones, and fatty acid amines. The (semi-)solid separation medium is preferably one that allows the nucleic acid-capturing particles to penetrate and is less likely to be mixed with an aqueous liquid through the through-holes of the particles. From this point of view, a gel-like substance is preferably used. When particles enter the gel and move in the gel, the gel is perforated, but the self-repairing action of the gel's restoring force immediately closes the holes formed in the gel. Therefore, almost no water-based liquid flows through the through-holes due to the particles.

The material, composition, etc. of the gel that can be used as the separation medium are not particularly limited, and may be a physical gel or a chemical gel. For example, as described in WO2012/086243, a water-insoluble or sparingly soluble liquid substance is heated, a gelling agent is added to the heated liquid substance, and then cooled below the sol-gel transition temperature. By doing so, a physical gel is formed. As described in WO2015/001629, chemical gels such as silicone gels are easy to load into a container, exhibit little change in properties due to usage environment (temperature, etc.), and generate gel-derived contaminants. It has the advantage that it is difficult to generate

[Container for Particle Manipulation]
The particle manipulation vessel accommodates the above aqueous liquid and aqueous phase separation medium. Nucleic acids are separated and purified by particle manipulation in the particle manipulation container. The particle manipulation container has an opening for loading a biological sample or nucleic acid-capturing particles into the container. The opening of the particle manipulation vessel may be closable. After loading the biological sample and the nucleic acid-capturing particles into the container, the opening is closed to make the inside of the container a closed system, thereby preventing contamination from the outside. Closing of the opening can be performed by an appropriate method such as sealing with a sealing member such as a lid or plug, heat sealing of the opening portion, or the like.

The material and shape of the particle manipulation container are not particularly limited as long as they can hold the aqueous liquid and the aqueous phase separation medium. As for the shape of the container, a tubular body with an inner diameter of about 1 to 2 mm and a length of about 50 mm to 200 mm, and a linear groove with a width of about 1 to 2 mm, a depth of about 0.5 to 1 mm, and a length of about 50 mm to 200 mm are formed. Another example is a structure in which another flat plate member is attached to the upper surface of a flat plate member that has been laminated.

When the container is a tubular body, the cross-sectional shape of the tube is not particularly limited, and may be a circular, elliptical, polygonal, or other appropriate shape. From the viewpoint of operability when loading a biological sample or particles into a tubular body, as shown in FIG. 1, the tubular container may be formed so that the open end has a wider inner diameter. The shape of the particle manipulation container is not limited to a tubular shape or a planar shape, and the moving path of the particles may have a branched structure such as a cross or a T shape. Alternatively, a cone-shaped container such as an Eppendorf tube may be used.

Materials for particle manipulation containers include polyolefins such as polypropylene and polyethylene, fluorine resins such as tetrafluoroethylene, organic materials such as polyvinyl chloride, polystyrene, polycarbonate, and cyclic polyolefins; inorganic materials such as ceramics, glass, silicone, and metals. materials. When magnetic particles are used as the nucleic acid-capturing particles, the particles are manipulated by magnetic field manipulation from outside the container (for example, movement of a magnet along the outer wall surface of the container). In this case, the material of the container is preferably permeable to the magnetic field. When optical measurement is performed during or after particle manipulation, or when light irradiation is performed, a material having light transmittance is preferably used. Further, if the container is light-transmissive, it is preferable because the state of particle manipulation in the container can be visually confirmed.

The particle manipulation container may be integrally molded, or may be configured by combining a plurality of members. When the container is configured by combining a plurality of members, the container may be separable at the combined portion. Moreover, even when the container is integrally molded, the container may be configured so as to be separable by a method such as providing a portion (separation line) where the thickness of the wall surface of the container is locally small. When the container for particle manipulation is configured to be separable, after the pretreatment by particle manipulation is completed, by separating the portion containing the liquid after recovery of the nucleic acid (nucleic acid recovery solution) from the other portions, It is possible to reduce the size of the container containing the nucleic acid to be tested and improve the storage efficiency.

[Nucleic acid for individual identification]
The kit of the present invention includes an individual identification nucleic acid. The individual identification nucleic acid contains an identification sequence consisting of a non-complementary base sequence to the nucleic acid contained in the biological sample. The identification sequences of the individual identification nucleic acids contained in each of the plurality of kits are different from each other. The nucleic acid recovered in the nucleic acid recovery solution using the kit contains the nucleic acid derived from the biological sample and the nucleic acid for individual identification.

In general, kits used for the isolation and purification of nucleic acids and the like are assigned a code such as a number for each individual, and samples are identified by matching the code attached to the kit with the sample. . Nucleic acids isolated using the kit are transferred from the particle manipulation container included in the kit to another container for amplification and base sequence analysis. Therefore, if the container is mixed up or contamination occurs, the result is that the base sequence of a sample different from that of the target sample is analyzed.

When the kit of the present invention is used, the nucleic acid in the nucleic acid recovery solution contains the sample-derived nucleic acid and the individual identification nucleic acid, and even when the nucleic acid is transferred to another container in the subsequent processes, Coexistence is maintained. At the time of base sequence analysis, in addition to the base sequence of the sample-derived nucleic acid (sequencing target), the base sequence of the identification sequence portion of the individual identification nucleic acid is analyzed, and the base sequence of the identification sequence portion obtained by analysis is , to check whether or not it matches the base sequence of the identification sequence of the individual identification nucleic acid contained in the kit. If the base sequences of the identification sequence portion match, it can be guaranteed that the target sample is correctly analyzed.

Although the number of bases in the identification sequence is not particularly limited, it is preferably 5 bases or more, preferably 7 bases or more, in order to identify a sufficient number of individuals. In the case of 5 bases, 4 ⁵ =1024 identification sequences can be generated. However, when the identification sequence portion specifically hybridizes with the nucleic acid contained in the biological sample that is the specimen, it adversely affects the subsequent amplification reaction and sequence analysis, causing erroneous detection. Therefore, the identification sequence is selected from base sequences that are non-complementary to nucleic acids contained in the biological sample.

A nucleic acid for individual identification having a desired nucleotide sequence can be synthesized by known methods such as solid-phase synthesis and liquid-phase synthesis. When PNA is used as an individual identification nucleic acid, an individual identification nucleic acid having a desired sequence may be synthesized using a peptide nucleic acid monomer by a peptide synthesis method such as the Fmoc method or the tBoc method. The individual identification nucleic acid may be labeled with a fluorescent label, radioisotope label, electrochemical label, affinity label, epitope label, or the like.

The individual identification nucleic acid may contain other nucleotide sequences on the 3' side and/or 5' side of the identification sequence. For example, when the individual identification nucleic acid contains a common nucleotide sequence on the 3' and/or 5' side of the identification sequence, the common nucleotide sequence can be recognized as the start point or end point of the identification sequence. In addition, if an adapter sequence capable of binding to a carrier such as a flow cell for base sequence analysis or particles is included on the 5' side of the identification sequence, the base sequence of the identification sequence can be easily analyzed.

Either the 3' side or the 5' side of the identification sequence may contain a complementary base sequence to the nucleic acid (target) to be analyzed for base sequence contained in the biological sample. In this case, PCR can be performed using the individual identification nucleic acid as either the FW primer or the RW primer. Therefore, the complementary sequence portion of the individual identification nucleic acid is annealed with the target nucleic acid for base sequence analysis, and a fragment obtained by linking the target nucleic acid and the antisense sequence of the identification sequence can be amplified. By analyzing the base sequence of this fragment, the base sequence of the target nucleic acid for base sequence analysis and the base sequence of the identification sequence of the individual identification nucleic acid can be analyzed simultaneously.

Both the 3' side and the 5' side of the identification sequence may contain a base sequence complementary to the nucleic acid to be analyzed for base sequence contained in the biological sample. For example, if an individual identification nucleic acid having the same sense sequence as the FW primer on the 3' side of the identification sequence and the antisense sequence of the RW primer on the 5' side of the identification sequence is used, amplification of the nucleic acid to be analyzed by PCR can be performed in parallel. can be used to amplify sense and antisense fragments of the identifying nucleic acid.

If the 3' side of the identification sequence contains a complementary base sequence (complementary sequence) to the nucleic acid to be analyzed in the biological sample, and the 5' side of the identification sequence contains an adapter sequence, i.e., individual identification When the nucleic acid for identification contains an adapter sequence, an identification sequence and a complementary sequence from the 5' side, the base sequence of the nucleic acid to be analyzed by PCR, the base sequence of the identification sequence of the individual identification nucleic acid, and the adapter sequence are obtained by PCR. Fragments can be amplified and analyzed simultaneously with the base sequence of the identification sequence of the individual identification nucleic acid.

As described above, the base sequence other than the identification sequence contained in the individual identification nucleic acid may be appropriately designed according to the base sequence analysis method and the like.

[Kit composition and pretreatment operation]
<First form>
In the first form of the nucleic acid pretreatment kit of the present invention, all of the aqueous phase separation medium and the aqueous liquid constituting the kit are loaded in the particle manipulation vessel. In the pretreatment operation using this kit, a biological sample such as blood is added to the particle manipulation container, and the nucleic acid is allowed to bind to the nucleic acid capturing particles in the particle manipulation container.

FIG. 1 is a schematic cross-sectional view showing a configuration example of the pretreatment kit of the first embodiment. The pretreatment kit 1 contains a nucleic acid recovery solution 38, a third aqueous phase separation medium 23, a second washing solution 32, and a second aqueous phase separation medium in a tubular particle manipulation container 10 having an opening at the top, from the bottom of the container. A medium 22, a first washing liquid 31, a first aqueous phase separation medium 21, and a nucleic acid capturing liquid 35 are loaded. The aqueous phase separation media 21, 22, 23 are gel layers, and aqueous liquids 31, 32, 33 are filled in spaces partitioned by the inner walls of the containers and the gel layers.

A lid 13 is attached to the opening of the container 10 so that it can be opened and closed. By closing the lid after adding the biological sample into the container 10 with the lid 13 open, the opening of the container can be closed and the interior of the container can be made into a closed system.

In the form shown in FIG. 1, magnetic particles 70 as nucleic acid-trapping particles are contained in the cell lysate 35 as the nucleic acid-trapping liquid. The nucleic acid-trapping particles may be contained in the cell lysate in advance, or the nucleic acid-trapping particles may be added to the cell lysate immediately before adding the biological sample to the cell lysate. Also, the nucleic acid-capturing particles may be added to the cell lysate at the same time as or after the addition of the biological sample to the cell lysate. That is, the nucleic acid-capturing particles may be provided in advance in the kit 1, or may be provided separately from the kit 1 as one component of the pretreatment kit.

The nucleic acid in the biological sample binds to the surfaces of the nucleic acid-capturing particles by stirring the cell lysate containing the biological sample and the nucleic acid-capturing particles to disperse the nucleic acid-capturing particles in the liquid. The stirring method is not particularly limited, and includes a method of vibrating the container 10 with a vortex mixer or the like, a method of generating a liquid flow by pipetting or the like, a method of moving particles in the cell lysate and stirring, and the like. When the nucleic acid-capturing particles are magnetic particles, the particles can be moved and dispersed in the cell lysate by changing the strength and direction of the magnetic field applied from the outside of the container.

After the nucleic acid is bound to the particle surface, the particle 70 is moved in the longitudinal direction of the container 10 by magnetic field manipulation. Washing is performed by passing the particles through the aqueous phase separation medium 21 and moving the particles into the first washing liquid 31 to disperse the particles in the washing liquid. After passing through the aqueous phase separation medium 22 and washing in the second washing liquid 32 , the particles 70 are passed through the aqueous phase separation medium 23 and transferred to the nucleic acid recovery liquid 38 . When the nucleic acid recovery solution 38 is a nucleic acid elution solution, the nucleic acids bound to the particle surfaces are eluted by dispersing the nucleic acid capturing particles 70 in the nucleic acid elution solution, and the nucleic acids can be recovered in the nucleic acid recovery solution 38.

By including the individual identification nucleic acid in advance in the nucleic acid recovery solution 38, a mixture of the biological sample (specimen)-derived nucleic acid and the individual identification nucleic acid can be obtained. The individual identification nucleic acid can be included in a kit other than the nucleic acid recovery solution 38 . For example, if the nucleic acid-capturing solution (cell lysate) contains the individual-identifying nucleic acid, by dispersing the nucleic acid-capturing particles in the nucleic acid-capturing solution, the individual-identifying nucleic acid can be obtained in addition to the nucleic acid derived from the biological sample. , bound to the particle surface. Further, if the individual identification nucleic acid is contained in another aqueous liquid such as a washing liquid, the individual identification nucleic acid binds to the particle surface when the nucleic acid-capturing particles move into the aqueous liquid. By moving the nucleic acid-capturing particles to which the biological sample-derived nucleic acid and the individual identification nucleic acid are bound into the nucleic acid recovery liquid 38 by magnetic field operation or the like, the biological sample-derived nucleic acid and the individual identification nucleic acid are mixed in the nucleic acid recovery liquid 38. can be recovered.

Nucleic acid-capturing particles having individual identification nucleic acids bound to their surfaces in advance may also be used. The nucleic acid-capturing particles to which the individual identification nucleic acid is bound may be contained in the nucleic acid-capturing solution in advance, and may be added to the nucleic acid-capturing solution immediately before, simultaneously with, or after the addition of the biological sample to the nucleic acid-capturing solution. Nucleic acid-capturing particles may be added. When the kit is provided with the nucleic acid-capturing particles and the individual-identifying nucleic acid contained in the nucleic acid-capturing solution, the individual-identifying nucleic acid is bound to the surfaces of the nucleic acid-capturing particles.

The nucleic acid collected in the particle manipulation container is amplified by PCR or the like as necessary, and then sequenced by a base sequence analyzer. If post-recovery nucleic acid amplification, nucleotide sequence analysis, or other operations are performed in a separate location from particle manipulation, the nucleic acid sample should be placed in the particle manipulation container in order to prevent contamination, etc. caused by opening the container. It is preferable to move (delivery) to another place while being accommodated.

When the particle manipulation container is configured to be separable, the part containing the nucleic acid recovery solution may be separated from the other parts, and the separated container may be used for delivery. From the viewpoint of increasing the storage efficiency in a container storage rack or the like used for delivery, the particle manipulation container is preferably separated in the vicinity of the location where the nucleic acid recovery solution is loaded. For example, in the form shown in FIG. Preferably, the containers are separated at a point. When the container is separated at the location where the aqueous phase separation medium 23 is loaded, a closed system in which the nucleic acid recovery solution 38 is sealed in the space formed by the inner wall surface of the container after separation and the aqueous phase separation medium 23 is formed. maintained, reducing the risk of contamination.

As shown in FIG. 1, the container 10 preferably has identification information 14 that can be identified from the outside. Examples of identification information include information recognizable by optical techniques such as character strings, bar codes, and two-dimensional codes, and information recognizable by electromagnetic techniques such as IC chips and IC tags. This identification information is associated with the base sequence of the individual identification nucleic acid contained in the kit.

Any method can be used to associate the identification information attached to the container with the identification sequence of the individual identification nucleic acid. When the identification information is a character string, a method of describing the base sequence of the identification sequence as a character string as it is, a method of coding the base sequence, and the like can be used. One example of encoding the base sequence of DNA is a method of replacing four kinds of bases (A, G, C, T) with 2-bit information (00, 01, 10, 11) and quantifying them. . Since one base has 2-bit information, 10-bit identification information can be generated if the identification sequence has 5 bases, and 20-bit identification information can be generated if the identification sequence has 10 bases. The coded identification information may be encrypted. The identification information may be attached to the container as numbers or characters, or may be attached to the container as mechanically readable identification information such as a bar code, two-dimensional code, or IC.

The identification information attached to the container and the identification sequence of the individual identification nucleic acid may be associated via a database. For example, a coded number is attached to the container as identification information, and the serial number of the container and the identification sequence of the nucleic acid for individual identification contained in the container are put into a database, so that the container identification information and can be associated with an identification sequence.

The position where the identification information 14 is attached to the container 10 is not particularly limited as long as the identification information can be read after the nucleic acid is recovered in the nucleic acid recovery solution. When the container for particle manipulation is configured to be separable, it is preferable that the identification information is attached at a position where the identity of the nucleic acid recovery solution and the identification information of the container can be maintained. For example, when the particle manipulation container is configured to be separable in the vicinity of the location where the nucleic acid recovery solution is loaded, it is preferable that the identification information of the container is attached to the location where the nucleic acid recovery solution is loaded.

<Second form>
FIG. 2 is a schematic cross-sectional view showing a configuration example of the pretreatment kit of the second form. The second form of pretreatment kit includes a nucleic acid capture operation container 15 in addition to the particle operation container 10 . In the first mode, all the aqueous liquids including the nucleic acid capturing solution and the nucleic acid recovery solution are loaded in the particle manipulation container 10, whereas in the second mode, the nucleic acid capturing solution 35 is used for the nucleic acid capturing operation. A container 15 is loaded. That is, the second form of the pretreatment kit includes a first kit 101 in which the aqueous liquids 31, 32, 38 and the aqueous phase separation media 21, 22, 23 are filled in the particle manipulation container 10, and It is configured by combining with a second kit 102 in which a nucleic acid capturing solution 35 is loaded in a container 15 . In the pretreatment operation using this kit, a biological sample such as blood is added to the nucleic acid capturing operation container 15, and the nucleic acid is bound to the nucleic acid capturing particles.

When nucleic acid-capturing particles such as magnetic silica particles are stored in a liquid for a long period of time, aggregation of the particles may occur. In addition, contaminants contained in a biological sample, especially denatured proteins produced by lysis of cells, etc., mask the surfaces of particles, and have the effect of causing the particles to adhere to each other and aggregate. Aggregation of the nucleic acid-capturing particles reduces the chance of contact between the particle surface and the nucleic acid, which tends to inhibit the binding of the nucleic acid to the particle surface. Therefore, in order to increase the recovery efficiency of nucleic acids, strong vibration should be applied to the container and agitation should be performed so as to crush the aggregates of the particles and eliminate the aggregation when the nucleic acids are bound to the nucleic acid-capturing particles. is preferred.

In the second form of the kit, a nucleic acid capture operation container 15 is prepared separately from the particle operation container 10 filled with aqueous phase separation media 21, 22, 23 and aqueous liquids 31, 32, 38, and the like. With the nucleic acid-trapping liquid 35 and the magnetic particles 70 loaded in the nucleic acid-trapping operation container 15, binding of nucleic acids to the surfaces of the nucleic acid-trapping particles is carried out. Even if strong vibration is applied to the nucleic acid capture operation container 15 using a vortex mixer or the like, the aqueous phase separation medium and aqueous liquid loaded in the particle operation container 10 are not affected at all, and the inner wall surface of the container and the water are not affected. It is possible to maintain the state in which the aqueous liquid is charged in the space partitioned by the phase separation medium. Therefore, it is possible to apply a stronger external force for stirring than in the case where nucleic acids are bound to the surfaces of the nucleic acid-capturing particles in the particle manipulation container.

In addition, since the nucleic acid capture operation container does not need to provide a space for storing the aqueous phase separation medium, the nucleic acid recovery solution, etc., it is more compact than the particle operation container and has a high degree of freedom in the shape of the container. Therefore, a shape suitable for dispersing the nucleic acid-capturing particles in a liquid by stirring can be adopted. Therefore, binding of nucleic acids to the surface of the nucleic acid-capturing particles in the nucleic acid-capturing operation container improves the efficiency of dispersing the nucleic acid-capturing particles in the liquid, even when the nucleic acid-capturing particles are agglomerated. recovery efficiency can be improved.

The nucleic acid-capturing operation container 15 has an opening for adding a biological sample or nucleic acid-capturing particles into the container and removing them from the container. The opening of the nucleic acid capturing operation container may be closable. It is preferable to use an openable and closable sealing member such as a lid or a plug to close the opening.

The material and shape of the nucleic acid capturing operation container are not particularly limited as long as they can hold a nucleic acid capturing solution such as a cell lysate and nucleic acid capturing particles. The shape of the container is preferably designed so that the particles can be efficiently dispersed in the liquid. As the material of the container, those mentioned above as the material constituting the particle manipulation container can be used.

In the form shown in FIG. 2, the cell lysate 35 loaded in the nucleic acid trapping operation container 15 contains magnetic particles 70 as nucleic acid trapping particles. The nucleic acid-trapping particles may be contained in advance in the cell lysate, or may be added to the cell lysate immediately before use. Also, the nucleic acid-capturing particles may be added to the cell lysate at the same time as or after the addition of the biological sample to the cell lysate. That is, the nucleic acid-capturing particles may be provided in a state of being included in the second kit 102 in advance, or may be provided separately from the second kit 102 as one component of the pretreatment kit.

Binding of nucleic acids in a biological sample to nucleic acid-capturing particles can be performed by the same method as in the first mode. As described above, in this embodiment, the nucleic acid is bound to the surface of the nucleic acid-capturing particles in the nucleic acid-capturing operation container, so that the efficiency of dispersing the particles in the liquid can be improved. Accompanying this, the amount of nucleic acid bound to the particle surface can be increased, and the recovery efficiency of nucleic acid can be enhanced.

After binding the nucleic acid, the nucleic acid-capturing particles are moved into the particle manipulation container 10 , and then the particles 70 are moved in the container 10 by magnetic field manipulation as in the first embodiment, and the nucleic acid is recovered in the nucleic acid recovery solution 38 . When transferring the nucleic acid-trapping particles from the nucleic acid-trapping operation container 15 to the particle manipulation container 10, the particles may be transferred together with the nucleic acid-trapping liquid, or the particles alone may be transferred. When only the particles are transferred to the particle manipulation container 10, there is no problem even if the nucleic acid capturing liquid adheres to the surface of the particles.

FIG. 2 shows a mode in which the aqueous phase separation medium 21 is arranged as the uppermost layer on the open end side of the particle manipulation container 10, but the uppermost layer may be an aqueous liquid such as a washing liquid. In particular, when transferring only the nucleic acid-bound particles from the nucleic acid capture operation container to the particle manipulation container, from the viewpoint of simplifying the particle manipulation process in the particle manipulation container, Particles are preferably added to the deposited aqueous liquid.

Also in the second mode, a mixture of the biological sample-derived nucleic acid and the individual identification nucleic acid is obtained in the nucleic acid recovery liquid 38 by including the individual identification nucleic acid in the kit in advance. The individual identification nucleic acid may be contained in either the particle manipulation container 10 of the first kit 101 or the nucleic acid capture manipulation container 15 of the second kit 102 . In addition, the nucleic acid for individual identification can also be included in the container for nucleic acid capturing operation 15 by adding particles to the surface of which the nucleic acid for individual identification is bound in advance to the container for nucleic acid capturing operation. Also in the second mode, a mixture of the biological sample-derived nucleic acid and the individual identification nucleic acid can be recovered in the nucleic acid solution.

Both the particle manipulation container and the nucleic acid capture manipulation container may contain an individual identification nucleic acid. When the individual identifying nucleic acids are contained in both the particle manipulation container and the nucleic acid capturing manipulation container, the identification sequences of the respective individual identifying nucleic acids may be the same or different. When the identification sequence of the individual-identifying nucleic acid contained in the particle manipulation container and the identification sequence of the individual-identifying nucleic acid contained in the nucleic acid capture operation container are the same, one type of identification sequence is used during base sequence analysis. If it is confirmed that only one sample is detected, it can be confirmed that there is no sample mix-up or contamination.

If the identification sequence of the individual identification nucleic acid contained in the particle manipulation container and the identification sequence of the individual identification nucleic acid contained in the nucleic acid capture operation container are different, the base sequences of both identification sequences during base sequence analysis By analyzing , it is possible to confirm that there is no sample mix-up or contamination. The individual identification nucleic acid contained in the particle manipulation container and the individual identification nucleic acid contained in the nucleic acid capture operation container may have different functions. For example, one individual identification nucleic acid has a base sequence corresponding to the FW primer in addition to the identification sequence, and the other individual identification nucleic acid has a base sequence corresponding to the RW primer in addition to the identification sequence. By designing identification nucleic acids, PCR can be performed using these two types of individual identification nucleic acids as a pair of primers.

Also in the second embodiment, the particle manipulation container 10 may be configured to be separable in the vicinity of the location where the nucleic acid recovery solution is loaded. Further, it is preferable that identification information 14 associated with the base sequence of the individual identification nucleic acid is attached to the particle manipulation container 10 . When an individual identification nucleic acid is contained in the particle manipulation container 10, the association between the identification information 14 attached to the nucleic acid capture manipulation container and the base sequence of the identification sequence can be performed in the same manner as in the first embodiment.

When an individual identification nucleic acid is contained in the nucleic acid capturing operation container 15 (including a case where nucleic acid capturing particles bound with an individual identifying nucleic acid are added to the nucleic acid capturing operation container), the nucleic acid capturing operation container 15 A mixture of the nucleic acid derived from the biological sample (specimen) and the nucleic acid for individual identification is obtained inside. Therefore, even if a container mix-up occurs when the sample is transferred from the nucleic acid capturing operation container 15 to the particle manipulation container 10, the base sequence of the identification sequence of the individual identification nucleic acid can be analyzed during base sequence analysis. , can detect false positives caused by mix-ups.

When nucleic acid for individual identification is contained in the nucleic acid trapping operation container 15, it is preferable that the nucleic acid trapping operation container 15 is provided with identification information 19 that can be recognized from the outside of the container. The identification information 19 attached to the nucleic acid capturing operation container 15 is preferably associated with the identification sequence of the individual identification nucleic acid.

In order to ensure traceability even after the sample in the nucleic acid capture operation container 15 is transferred to the particle operation container 10, the identification information 14, 19 is attached to the particle operation container 10 and the nucleic acid capture operation container 15, respectively. and these identification information are preferably relatable. For example, the identification information 19 and the identification information 14 can be associated by previously associating the nucleic acid capturing operation container 15 and the particle operation container 10 with the same identification information.

In order to reduce erroneous detection due to misreading of the identification information or human error such as confusion, it is necessary to associate the identification information 19 of the nucleic acid capture operation container 15 with the identification information 14 of the particle operation container 10 when using the kit. preferably. For example, by reading both the identification information 19 and the identification information 14 when moving the sample in the nucleic acid capture operation container 15 to the particle operation container 10, the two can be associated. In addition, the identification information 19 is detachably attached to the nucleic acid-trapping container 15 using a seal or the like, and when the sample in the nucleic acid-trapping container 15 is transferred to the particle manipulation container 10, the identification information 19 can be removed. may be separated from the nucleic acid capturing operation container 15 and attached to the particle operation container 10 to associate the identification information of both.

[Nucleotide sequence analysis]
In the base sequence analysis method of the present invention, the base sequence of the nucleic acid recovered with the pretreatment kit is analyzed. The method for analyzing the base sequence is not particularly limited. Nucleic acids recovered from the pretreatment kit may undergo further treatments such as fragmentation, ligation, amplification, etc. before being subjected to nucleotide sequence analysis. These treatments can be appropriately performed according to the method of analyzing the base sequence. Even when the nucleic acid recovered from the kit is transferred to another container for use in these treatments, the coexistence of the sample-derived nucleic acid and the individual identification nucleic acid is maintained.

In the base sequence analysis, the base sequence of the identification sequence of the individual identification nucleic acid is analyzed in addition to the base sequence of the nucleic acid in the biological sample. Verification is performed as to whether or not the base sequence of the identification sequence portion obtained by the base sequence analysis matches the base sequence of the identification sequence of the individual identification nucleic acid contained in the kit. If the base sequences of the identification sequences do not match, it is considered that the samples have been mixed up. If a plurality of identification sequences are detected even though the kit contains only one type of individual identification nucleic acid, contamination is considered to have occurred. In this way, by analyzing the base sequence of the identification sequence portion, erroneous detection due to sample mix-up or contamination can be detected.

As described above, in the nucleic acid separation and purification operation using the kit of the present invention, nucleic acid separation and purification from a biological sample can be performed in a sealed container. It is possible to reduce the risk of defects such as contamination of Also, even if contamination or mix-up of specimens should occur, erroneous detection based on these can be detected by collating the base sequences of the identification sequence portions of the individual identification nucleic acids. Therefore, by separating and purifying the nucleic acid using the kit of the present invention, the reliability of genetic testing and the like based on nucleic acid base sequence analysis can be enhanced.

10 Vessel for particle operation 15 Vessel for nucleic acid capture operation 21, 22, 23 Aqueous phase separation medium 31, 32 Washing liquid 35 Nucleic acid capture liquid 38 Nucleic acid recovery liquid 14, 19 Identification information 70 Nucleic acid capture particles

Claims (1)

A base sequence analysis method using a nucleic acid pretreatment kit for separating nucleic acids from a biological sample containing nucleic acids and contaminants,
The nucleic acid pretreatment kit comprises
A particle manipulation container; a nucleic acid-capturing particle group capable of selectively binding to nucleic acids; an aqueous phase separation medium that is insoluble or poorly soluble in water; a plurality of types of aqueous liquids;
The plurality of types of aqueous liquids include a nucleic acid capturing liquid for binding the nucleic acid contained in the biological sample to the nucleic acid capturing particle group, a washing liquid for removing contaminants adhering to the surface of the nucleic acid capturing particle group, and including a nucleic acid recovery solution for recovering the nucleic acid bound to the surface of the nucleic acid-capturing particle group;
At least the aqueous phase separation medium and the nucleic acid recovery solution are loaded in the particle manipulation container,
The base sequence of the individual identification nucleic acid includes an identification sequence consisting of a base sequence non-complementary to the nucleic acid contained in the biological sample,
The base sequence analysis method includes
a step of allowing the nucleic acid in the biological sample and the individual identification nucleic acid to coexist in the particle manipulation container in a state of being bound to the nucleic acid-capturing particle group;
By moving the nucleic acid-capturing particle group to which the nucleic acid in the biological sample and the nucleic acid for individual identification are bound from the nucleic acid-capturing solution to the nucleic acid recovery solution via the washing solution, recovering the nucleic acid in the biological sample and the nucleic acid for individual identification ;
analyzing the base sequence of the recovered nucleic acid ; and
A base sequence analysis method, comprising the step of verifying whether the base sequence of the identification sequence portion obtained by the analysis matches the base sequence of the identification sequence of the individual identification nucleic acid .

Images