JP3996416B2 - B / F separation method in nucleic acid hybridization - Google Patents

B / F separation method in nucleic acid hybridization Download PDF

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JP3996416B2
JP3996416B2 JP2002085233A JP2002085233A JP3996416B2 JP 3996416 B2 JP3996416 B2 JP 3996416B2 JP 2002085233 A JP2002085233 A JP 2002085233A JP 2002085233 A JP2002085233 A JP 2002085233A JP 3996416 B2 JP3996416 B2 JP 3996416B2
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magnetic
reaction vessel
nucleic acid
magnetic force
reaction
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JP2003164279A (en
Inventor
浩平 丸山
雄司 宇田川
是 松永
越男 根本
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Juki株式会社
是 松永
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Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an efficient B / F separation method after completion of a hybridization reaction.
[0002]
[Prior art]
The nucleic acid hybridization method is widely used as a means for specifically detecting a specific gene. Among them, the nucleic acid probe-immobilized particles are used as probes, the target nucleic acid in the liquid sample and the nucleic acid probe-immobilized particles are hybridized in the reaction container, and then solid / liquid separation (B / F separation) is performed. The hybridization method for measuring the amount of the target nucleic acid hybridized above is particularly noted because it is a simple and rapid hybridization method.
[0003]
Recently, magnetic particles have attracted attention as particles for immobilizing nucleic acid probes from the viewpoint of simplicity of B / F separation and the like. As the B / F separation means using the magnetic particles, (1) a method in which a magnetic body is arranged on the outer side surface of the reaction vessel and the magnetic particles are adsorbed and held on the inner side surface of the reaction vessel (patent No. 3115501), (2) a method of carrying out by arranging a rotating magnetic body on the outer side surface of the reaction vessel and adsorbing and desorbing magnetic particles on the inner side surface of the reaction vessel (Japanese Patent Laid-Open No. 11-156231), (3 ) A method of using a magnetic device in a sleeve separated from a solution and rotating and / or raising and lowering the magnetic device to adsorb magnetic particles on the outer wall of the sleeve, thereby collecting and resuspending the magnetic particles (JP-A-8-29425). No.) is known.
[0004]
[Problems to be solved by the invention]
However, when B / F separation is carried out by the methods (1) to (3) above, the reaction vessel and sleeve are usually made of plastic, and depending on the type of magnetic particles, they are easily adsorbed on the plastic. Therefore, even if the magnetic force is turned off, it remains adsorbed on the inner wall and sleeve of the reaction vessel, and the magnetic particles to be measured are not recovered well, and the number of particles is reduced. It turned out that. Further, it is necessary to move to another measuring container at the time of measurement.
Accordingly, an object of the present invention is to provide a highly sensitive and rapid B / F separation means that can collect magnetic particles without fail.
[0005]
[Means for Solving the Problems]
The present inventor examined B / F separation after the completion of the nucleic acid hybridization reaction. As a result, if the probe-immobilized magnetic particles were immobilized only at the bottom of the reaction vessel by controlling the magnetic force, the supernatant was aspirated. The liquid part can be easily removed, and the magnetic particles can be collected without fail after injecting the cleaning liquid after injecting the cleaning liquid in the immobilized state or by moving the magnetic particles by controlling the magnetic force. Since magnetic particles move when -OFF is repeated, washing can be performed efficiently and easily, allowing high-sensitivity measurement, and as it is for measurement after B / F separation (without transferring the reaction vessel to another vessel) The present invention has been completed by finding that it can be transferred.
[0006]
That is, the present invention is a B / F separation method after the completion of a nucleic acid hybridization reaction using nucleic acid probe-immobilized magnetic particles as a probe, and the following B / F separation steps (1), (2) and (3) And a magnetic force control is performed so that the magnetic particles are immobilized only at the bottom of the reaction vessel.
(1) A step of sucking and removing the supernatant by controlling the magnetic force so that the magnetic particles are immobilized only at the bottom of the reaction vessel, (2) the magnetic particles in a state where the magnetic particles are immobilized or by controlling the magnetic force A step of injecting a cleaning liquid into the reaction vessel after mobilizing the particles, (3) Insert a permanent magnet into the non-penetrating hole of the magnet holding rod to make it permanent Hold the permanent magnet so that the end face of a part of the magnet protrudes, Magnet holding rod Does not exert magnetic force on the outside A magnetic rod, which is disposed at the bottom of the reaction vessel Rotate Change the direction of the magnetic flux The process of moving the magnetic particles in the cleaning liquid by repeatedly turning on and off the magnetic force.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
In the B / F separation method of the present invention, the magnetic force may be controlled so that the nucleic acid probe-immobilized magnetic particles are immobilized only at the bottom of the reaction vessel. Specifically, the magnetic force may be controlled by disposing a magnetic force generation source at the bottom of the reaction vessel and turning the magnetic force at the bottom of the reaction vessel on and off. More specifically, (1) means for turning on and off the magnetic force by shifting the permanent magnet arranged at the lower part of the reaction vessel in the vertical direction (FIG. 1), (2) the permanent magnet in the horizontal direction on the lower surface of the reaction vessel. For example, a means for turning the magnetic force on and off by shifting the pitch so that it is completely removed by half pitch or (3) means for controlling the rotation of the ferromagnetic material provided with the permanent magnet, for example, a permanent magnet arranged at the bottom of the reaction vessel Means (Fig. 2) for turning on and off the magnetic force by rotating the embedded magnetic rod such as iron and changing the direction of the magnetic flux, and the end face of a part of the magnet arranged at the bottom of the reaction vessel protrudes Means for rotating the magnetic rod holding the magnet to turn the magnetic force on and off (FIG. 3), (4) means for horizontally turning the shield material of the permanent magnet to turn the magnet on and off, (5 ) Round electromagnet Means for the ON-OFF of more force, (6) means force the horseshoe electromagnet to the ON-OFF, include means such that the ON-OFF of the magnetic force using electromagnets (7) without core.
Of these magnetic ON / OFF means, the means (1), (2) and (3) are preferred because they are simple. For example, when the means shown in FIG. 3 is employed, the magnetic flux extends in the end face extending direction without being attracted to the inside of the magnet holding rod, so that the magnetic force of the magnet can be effectively directed to the reaction vessel. The hole for inserting the magnet does not penetrate and has a sufficient thickness so that the magnetic force does not leak. As a result, the magnetic flux on the holding side end surface passes through the inside of the magnet holding rod and does not exert a magnetic force on the outside of the holding rod, so that the magnetic force to the reaction vessel can be completely cut off when the magnetic force is cut off.
[0008]
The B / F separation step of the present invention is performed by repeating the following steps (1) and (2) a predetermined number of times after completion of the hybridization reaction (means a).
(1) A step of sucking and removing the supernatant by controlling the magnetic force so that the magnetic particles are immobilized only on the bottom of the reaction vessel,
(2) A step of injecting a cleaning liquid into the reaction vessel after mobilizing the magnetic particles in a state where the magnetic particles are immobilized or by controlling the magnetic force.
[0009]
In the B / F separation step of the present invention, the following steps (1), (2) and (3) may be performed after the hybridization reaction (means b).
(1) A step of sucking and removing the supernatant by controlling the magnetic force so that the magnetic particles are immobilized only on the bottom of the reaction vessel,
(2) A step of injecting a cleaning liquid into the reaction vessel after mobilizing the magnetic particles by immobilizing the magnetic particles or controlling the magnetic force,
(3) A step of moving the magnetic particles in the cleaning liquid by repeatedly turning on and off the magnetic force.
[0010]
Here, in the step (1), the state in which the magnetic particles are immobilized only at the bottom of the reaction vessel by turning on the magnetic force at the bottom of the reaction vessel is shown on the right side of FIGS. . Usually, a reaction vessel for hybridization, such as a 24-well to 96-well microplate, has a small bottom surface, so if the magnetic particles are immobilized only at the bottom, a plastic such as magnetic beads produced by magnetic bacteria as magnetic particles. Even when particles that are easily adsorbed are used, there is almost no adsorption.
[0011]
In step (2), even if the magnetic force at the lower part of the reaction vessel is turned off, the magnetic particles remain collected at the bottom of the vessel, but diffuse to some extent by injecting the cleaning liquid. After that, the magnetic force is turned on again, and the cleaning liquid may be sucked and discarded in a state where the magnetic particles are immobilized on the bottom. However, it is also possible to perform cleaning with the magnetic force turned on from the beginning. Thereby, further cleaning is performed quickly and efficiently.
[0012]
In the means a, the step (1) and the step (2) may be repeated once or more, for example, 1 to 3 times.
[0013]
Step (3) of the means b is a means that enables sufficient washing and removes nonspecifically adsorbed substances with respect to magnetic particles even though the washing liquid is injected only once into the reaction vessel. According to the means b, it is not necessary to repeat the step (1) and the step (2) twice, so that the step of removing the supernatant after the washing liquid is injected can be omitted. Therefore, it is possible to reduce the risk of erroneously sucking magnetic particles by the supernatant suction operation.
[0014]
The principle of moving the magnetic particles in the cleaning liquid by repeating ON / OFF of the magnetic force in the lower part of the reaction vessel is as follows. That is, the magnetic particles having spontaneous magnetization lie so that the N pole-S pole of each bead contacts when the magnetic field applied from below is zero. When a large N-pole magnetic field is applied from the bottom, the magnetic particles exceed the magnetic force interaction between the magnetic particles, and the S-pole side of the magnetic particles faces downward so that the magnetic particles rotate just 90 degrees on the spot. In the magnetic force interaction between the magnetic particles, the poles repelling each other face each other. When the forcible magnetic field is gradually reduced from this state, the magnetic particles cause an inversion phenomenon called “rising” when the magnetic interaction between the magnetic particles exceeds. In this way, if the magnetic field is forcibly controlled and the cycle of strong → weak → cutting is repeated, the magnetic particles move around in the cleaning solution, and it becomes possible to release the nonspecifically adsorbed substances on the surface of the magnetic particles. .
[0015]
If the magnetic force is turned off after completion of the step (2) or step (3), the B / F separation-completed solid phase remains in the reaction vessel. Further, by transferring the reaction vessel subjected to the B / F separation to a measurement unit (not shown) as it is, it becomes possible to measure the light emission amount or the fluorescence amount of the magnetic particles collected on the bottom.
[0016]
The magnetic particles used in the present invention are not particularly limited as long as they are insoluble in an aqueous solution and exhibit magnetism. For example, FeO, γ-FeO, Co-γ-FeO, (NiCuZn) O · FeO, (CuZn) O · FeO, (MnZn) O · FeO, (NiZn) O · FeO, SrO · 6FeO, BaO · 6FeO, SiO 2 Coated with FeO (particle size of about 200A) [see Enzyme Microb. Tecnol., Vol2, p.2-10 (1980)], various polymer materials (nylon, polyacrylamide, polystyrene, etc.) and composite fine particles And magnetic bacteria particles synthesized by the bacteria in the cells. The magnetic bacterial particles are particularly preferred because the magnetic domains are in a single domain region in a certain direction within the particles, so that magnets can be collected in a narrow region at the bottom of the reaction vessel by placing them at the bottom of the reaction vessel.
[0017]
Magnetic bacteria were discovered in the United States in the 1970s, and magnetite (Fe) having a particle size of about 50 to 100 nm was found in the cells. Three O Four ) Holds chain-like particles called magnetosomes, each consisting of about 10 to 20 single crystal particles. Magnetic bacteria can detect geomagnetism by holding this magnetosome and recognize the direction of magnetic field lines. Magnetic bacteria are microaerobic bacteria that can swim along the magnetic field lines from the aerobic water surface to the microaerobic sediment surface by sensing geomagnetism.
As shown in ANALYTICAL CHEMISTRY, VOL. 63, No. 3, FEBRUARY 1,1991 P268-P272, such a magnetic bacterium can be isolated and cultivated in large quantities.
The magnetic particles in this magnetic bacterium are hexagonal cylinders with a very uniform particle size and shape, high purity, and about 50 emu / g when the magnetization of the microbial cells containing the particles is converted per fine particle.
Further, the coercive force is 230 Oe, and it has been confirmed that it has a single domain structure. Further, since the particle surface is covered with an organic thin film, the magnetic particles have characteristics that they are present stably with little metal elution and excellent in dispersibility in an aqueous solution.
[0018]
Known methods for extracting magnetic bacterial particles from magnetic bacteria include physical pressure crushing using a French press, alkaline boiling, enzyme treatment, ultrasonic crushing treatment, etc. Crushing with a French press is suitable. After extraction, magnetic bacterial particles may be separated with a magnet or the like.
[0019]
Nucleic acid probe-immobilized magnetic particles are not limited as long as DNA, RNA, or PNA that serves as a probe for a target gene is immobilized on the magnetic particles. In addition, substances other than these can also be used as long as they have specific binding ability to DNA sequences. As the DNA that can serve as a probe for the target gene, a DNA known per se can be used. For immobilization of the probe to the magnetic particles, it is preferable to immobilize the probe by a covalent bond using an ordinary cross-linking agent.
[0020]
The B / F separation means of the present invention is preferably used for hybridization using an automated hybridization apparatus. As the hybridization apparatus, as shown in FIG. 4, at least (a) a denaturer unit 5 including a reaction container holder and a heating / cooling apparatus, and (b) a reaction container holder and a heating / cooling apparatus. (C) A B / F separation unit comprising a reaction vessel holder and a magnetic force control device, and capable of controlling the magnetic force so that the nucleic acid probe-immobilized magnetic particles are immobilized only on the bottom of the reaction vessel 7, (d) a chip rack storage section 8 including a chip rack 15, (e) a cleaning liquid section 10 including a cleaning liquid storage container, (f) a waste liquid section 9 including a waste liquid storage container, and (g) a plurality of Equipped with a chip nozzle 14, a mechanism for attaching / detaching a chip to / from the chip nozzle, a mechanism for the attached chip to suck / inject a processing solution, and a reaction vessel to be freely held and detached. It has a robot hand mechanism 13 capable, head portion 12 becomes provided with a freely movable arm unit to X-Z-axis direction, and a more composed automated nucleic acid hybridization device.
[0021]
If this hybridization apparatus is used, the following steps (1) to (10) are automatically performed, and then the amount of labeled nucleic acid in the reaction vessel is measured, whereby the nucleic acid in the sample can be detected.
(1) A reaction vessel in which nucleic acid probe-immobilized magnetic particles and labeled sample nucleic acid are injected and mixed is installed in the deinator unit 5, and the temperature in the reaction vessel is set to the nucleic acid deaner temperature by a heating / cooling device. The temperature is set, and the temperature is maintained for a predetermined time so that the sample nucleic acid is single-stranded. At this time, the magnetic particles in the reaction vessel are moved by magnetic force control.
(2) The arm unit is operated to transfer the reaction vessel of the detainer unit 5 to the annealing unit 6.
(3) The temperature in the reaction vessel is set to the annealing temperature of the nucleic acid by the heating / cooling device of the annealing unit 6, and annealing is performed while maintaining the temperature for a predetermined time.
(4) The arm unit is moved to the annealing unit 6 and the reaction vessel is transferred to the B / F separation unit 7.
(5) Operate the magnetic force control device to immobilize the sample nucleic acid bound to the magnetic particles at the bottom of the reaction vessel.
(6) The arm unit is moved to the chip rack storage 8 and the chip 16 is mounted on the chip nozzle 14.
(7) The arm unit is moved to the B / F separation unit 7 and the supernatant in the reaction vessel is sucked by the tip nozzle 14.
(8) The arm unit is moved to the waste liquid section 9 and the suction supernatant in the tip nozzle is discharged into the waste container of the waste liquid section 9.
(9) Move the arm unit to the cleaning liquid unit 10, suck the cleaning liquid from the cleaning liquid container, and inject the cleaning liquid into the reaction container of the B / F separation unit 7.
(10) The washing operation of (7) to (9) is repeated a predetermined number of times, and finally the washing solution is injected into the reaction vessel.
[0022]
Furthermore, a hybridization apparatus in which all reactions in hybridization, ie, denature, annealing, and B / F separation are all performed in one reaction vessel is more preferable. Examples of such an apparatus include a hybridization apparatus having a reaction section equipped with a reaction container holder, a heating / cooling apparatus, and a magnetic force control apparatus. More specifically, at least (A) a reaction unit 17 including a reaction vessel holder, a heating / cooling device, and a magnetic force control device, and (B) a chip rack / waste solution unit including a chip rack and a waste liquid storage container. 18, (C) a cleaning liquid container 19 including a cleaning liquid storage container and a heating / cooling device, and (D) a plurality of tip nozzles, and a mechanism for attaching / detaching the tips to / from the tip nozzles. An automatic nucleic acid hybridization apparatus comprising a head unit 12 having an arm unit that has a mechanism for sucking and injecting a processing solution and moving freely in the XZ axis direction (FIG. 5). It is. Here, the magnetic force control apparatus in the reaction part (A) can control the magnetic force so that the nucleic acid probe-immobilized magnetic particles are immobilized only on the bottom part of the reaction vessel.
[0023]
If this hybridization apparatus is used to automatically perform the following steps (1) to (8) and then measure the amount of labeled nucleic acid in the reaction vessel, the nucleic acid in the sample can be detected.
(1) Install a reaction vessel in which the nucleic acid probe-immobilized magnetic particles and labeled sample nucleic acid are injected and mixed in the reaction section, and set the temperature in the reaction vessel to the nucleic acid denaturer temperature using a heating / cooling device. The temperature is maintained for a predetermined time to make the sample nucleic acid single-stranded. At this time, the magnetic particles in the reaction vessel are moved by magnetic force control.
(2) The temperature in the reaction vessel is changed to the annealing temperature of the nucleic acid, and annealing is performed while maintaining the temperature for a predetermined time.
(3) Operate the magnetic force control device to immobilize the sample nucleic acid bound to the magnetic particles at the bottom of the container.
(4) Operate the arm unit to move to the chip rack / waste liquid section and attach the chip to the chip nozzle.
(5) The arm unit is moved to the reaction part, and the supernatant in the reaction container is sucked with a tip nozzle.
(6) The arm unit is moved to the tip rack / waste liquid section, and the suction supernatant in the tip nozzle is discharged to the waste container.
(7) The arm unit is moved to the cleaning liquid section, the cleaning liquid whose temperature is adjusted to the annealing temperature in advance by the heating / cooling device is sucked from the cleaning liquid storage container, and the cleaning liquid is injected into the reaction container of the reaction section.
(8) The cleaning operation of (5) to (7) is repeated a predetermined number of times, and finally the cleaning liquid is injected into the reaction vessel.
[0024]
Hereinafter, specific examples of the hybridization apparatus used in the B / F separation method of the present invention will be described with reference to the drawings.
FIG. 5 is a conceptual diagram showing the internal configuration of the automatic nucleic acid hybridization apparatus.
The reaction unit 17 in FIG. 5 converts the sample nucleic acid into a single strand (denature), then performs hybridization (annealing) between the single-stranded sample nucleic acid and a specific nucleic acid probe, and then performs B / F separation. It is a reaction part to be performed, and includes a holder 20 for storing and holding the reaction vessel, and a heating / cooling device and a magnetic force control device 25 for adjusting the temperatures of the deenergizer and the annealing (FIG. 6).
[0025]
As shown in FIG. 6, the heating / cooling device includes a heater 21 that heats the reaction vessel holder 20, a chiller 22 that cools the heater 21, a sensor 23 that controls the temperature, and a temperature control device that controls the temperature. 24.
The temperature control device 24 is a temperature at which the deenergization proceeds in the reaction vessel in the deinator (generally around 95 ° C., but may be lower if the length of the sample nucleic acid is short). In annealing, it is fixed so as to be controlled at an annealing temperature (usually 40 to 70 ° C.). The reaction time is also controlled by the timer function of the temperature controller.
[0026]
As described above, the magnetic force control device 25 may be any device that can control the magnetic force so that the nucleic acid probe-immobilized magnetic particles are immobilized only on the bottom of the reaction vessel. FIG. 6 shows a magnetic force control device by means (FIG. 1) for turning on and off the magnetic force by shifting a permanent magnet arranged in the lower part of the reaction vessel in the vertical direction. As the magnetic force control device, a magnetic force control device for controlling the rotation of a ferromagnetic material provided with a permanent magnet, for example, rotating a magnetic rod embedded with a permanent magnet arranged at the bottom of the reaction vessel as shown in FIG. 2 or FIG. It is preferable to use a magnetic force control device using means for turning the magnetic force on and off.
[0027]
At the time of the deenergization and annealing reaction, the magnetic force at the lower part of the reaction vessel is turned off as shown on the left side of FIGS. After completion of the annealing reaction, washing and B / F separation are performed by means a or b.
[0028]
Reference numeral 18 in FIG. 5 denotes a chip rack / waste liquid part which discharges waste liquid sucked up from the reaction part at the time of B / F separation / washing, a chip rack holding a disposable chip for dispensing cleaning liquid and reagents, A waste liquid storage container is provided at the lower part for retaining. In this manner, space can be saved by arranging the chip rack and the waste liquid storage container perpendicular to the moving plane of the head portion.
In the chip rack, a hole is formed to support the tapered portion of the disposable chip (hereinafter, disposable chip), and the disposable chip is inserted before the measurement is started.
[0029]
Reference numeral 19 in FIG. 5 denotes a cleaning liquid unit, which includes a cleaning liquid container and a heating / cooling device. The washing liquid is controlled to the same temperature as the annealing temperature by a heating / cooling device so that the nucleic acid in the reaction vessel does not undergo non-specific adsorption or dissociation due to temperature changes during washing.
A plurality of cleaning liquid portions can be provided as necessary. For example, when an immune reaction is performed in order to detect hybridized nucleic acid, two washing liquid parts are required.
[0030]
Reference numeral 12 in FIG. 5 denotes a head unit having an arm unit movable in the XZ axis direction. The arm unit moves the tip nozzle, a mechanism for moving the head unit, and the head unit. Thus, a mechanism for attaching / detaching a chip to / from the chip nozzle and a mechanism for sucking / injecting a processing liquid (waste liquid or cleaning liquid) from the mounted chip are constituted.
[0031]
This hybridization apparatus can be further provided with a reagent part (E) comprising a reagent container as required. For example, when a label for detecting the hybridized nucleic acid is necessary, the labeling reagent (for example, alkaline phosphatase-labeled anti-DIG Fab ′ fragment (anti-DIG-AP) or the like) or an enzyme coloring substrate (for example, alkaline phosphatase) It is necessary to provide a reagent part for storing a phosphatase luminescent substrate or the like.
On the other hand, when the sample nucleic acid labeled in advance is used, it is not necessary to provide this reagent part.
[0032]
The nucleic acid hybridization method used here is a two-step method (sandwich method) even if the hybridization method is a one-step method as long as it utilizes hybridization of nucleic acids in a sample and magnetic particles immobilized with a nucleic acid probe. It may be. In addition, the nucleic acid probe to be used may be any of single-stranded DNA, RNA, or PNA.
[0033]
【Example】
A specific example of a nucleic acid detection method using the above hybridization apparatus (see FIG. 5) is shown below.
1. Preparation process
(1) Production of magnetic bacteria particles
For the production of magnetic bacterial particles, isolated bacterial bacterium Magnetopsirillum sp. AMB-1 (Matsunaga et al. 1991) was used for about 7 days at room temperature using MSGM medium (Blakemore et al. 1979) (100 L). Cultured under anaerobic conditions. After 3 days of culture, 4 mL of the iron quinate solution was added to 1 L of the culture solution. The cultured cells were collected at 10,000 g at 4 ° C. using a continuous centrifuge. It was suspended using 10 mM phosphate buffered saline (PBS, pH 7.4). This microbial cell is 1500 kg / cm 2 Under the above conditions, the powder was crushed using a French press (Otake Seisakusho, 5501M), and magnetic bacteria particles were magnetically recovered from the crushed cells using a neodymium-boron (Nd-B) magnet. The obtained magnetic bacterial particles were washed three or more times in PBS using an ultrasonic cleaner (Kaijo Denki Co. Ltd., CA4481), then suspended in PBS and stored at 4 ° C.
[0034]
(2) Synthesis of detection probe
A genus-specific region is searched in the 16S rDNA region of cyanobacteria, and a region where a difference of 2 to 3 or more bases is observed between each genus within 15 to 20 bases is found. DNA was designed. For the designed probe DNA, a synthetic oligo DNA labeled with biotin at the 5 ′ end was prepared.
[0035]
(3) Preparation of DNA-immobilized magnetic bacterial particles
Modification to magnetic bacterial particles utilized amino groups that are thought to be present on magnetic bacterial membranes. First, 1 mg of magnetic bacterial particles (BMPs) extracted and purified from the magnetic bacterial strain AMB-1 were suspended in 1 mL of PBS containing 2.5% glutaraldehyde, and reacted at room temperature for 30 minutes, so that the magnetic bacterial particle film An aldehyde group was introduced into the amino group. After the reaction, it was magnetically recovered and washed 3 times with PBS.
After washing, 100 μg of streptavidin (New England Bio Labs.) Was suspended in 1 mL of PBS with respect to 1 mg of modified BMPs and reacted at room temperature for 2 hours to crosslink BMPs and streptavidin. After cross-linking, after magnetic recovery and washing with PBS three times, NaBH is used to suppress nonspecific adsorption of DNA. Four The unreacted aldehyde group was reduced to give streptavidin-immobilized BMPs (SA-BMPs). 300 μg of the prepared SA-BMPs was subjected to avidin-biotin reaction with 300 μl of oligo DNA labeled with biotin at the 5 ′ end in 300 μl of PBS to prepare oligo DNA-immobilized magnetic bacterial particles (DNA-BMPs).
[0036]
(4) Preparation of sample DNA
Extraction of genomic DNA from cyanobacteria was performed by an improved method of MagExtractor-genome- extraction. Prokaryotic microorganisms were amplified by PCR using 16S rDNA amplification primers RSF-1 and RSR-2 (E. coli 1523-1542nt antisense) (Kawaguchi et al. 1992). went.
At the time of gene amplification, a labeled sample nucleic acid is prepared by performing PCR using a dUTP labeled with a marker such as a fluorescent dye, alkaline phosphatase, ferrocene or the like that can be detected by fluorescence, luminescence, or electrochemical signal. be able to. For example, when synthesizing FITC-labeled 16S rDNA, PCR may be performed using FITC-labeled dUTP which is a fluorescent substance.
[0037]
(5) Installation of reagents in the equipment
{Circle around (1)} 100 μg of probe DNA-immobilized magnetic particles and 100 μl of sample DNA are mixed in a reaction vessel such as a cucumber plate, and after sufficient stirring, placed in the reaction section of this apparatus.
(2) After sterilizing the tip rack holding the tip nozzle, place it in the tip rack / waste liquid section.
(3) A cleaning liquid container for storing the cleaning liquid is installed in the cleaning section.
[0038]
2. De nature process
The start switch of the apparatus is turned on, the temperature control function of the heating / cooling apparatus of the reaction section 1 is operated, and the reaction vessel is warmed to 95 ° C. By holding for 5 minutes, the sample nucleic acid in the reaction vessel is made into a single strand.
[0039]
3. Annealing process
By cooling the temperature to 60 ° C. and holding for 10 minutes as it is, annealing of the nucleic acid probe-immobilized magnetic bacterial particles and the sample nucleic acid in the container is performed.
[0040]
4). B / F separation
After the annealing is completed, a magnetic control device installed immediately below the container is operated to generate a magnetic attractive force, and the magnetic particles are collected on the bottom surface of the container. Wait about 3 minutes after operating the magnetic controller. At this time, the reaction vessel is maintained at 60 ° C.
[0041]
5). Cleaning process
The chip unit held in the chip rack / waste liquid part is mounted by moving the arm unit moved onto the chip rack / waste liquid part downward and fitting the chip nozzle and the disposable chip. The arm unit is moved to the reaction part, lowered as it is, stopped at an appropriate position, and the supernatant in the reaction vessel is sucked by the tip nozzle, moved to the waste liquid part and discharged.
Thereafter, the arm unit is moved to the cleaning liquid section, the cleaning liquid maintained at 60 ° C. is sucked from the cleaning liquid storage container, the arm unit is moved again to the reaction section, and this is injected into the reaction container. After waiting for about 3 minutes, the supernatant in the reaction vessel is again sucked with the tip nozzle and discharged to the tip rack / waste liquid part. This cleaning operation is repeated three times, and finally the cleaning liquid is injected to complete the operation.
[0042]
Even after the operation is completed, the magnetic attractive force of the reaction part is maintained, and the temperature is controlled to be maintained at 0 to 15 ° C., preferably 4 ° C. Thereby, the magnetic substance in the reaction solution can be kept in a state of being aggregated at the bottom of the container, and the properties of the solution can be prevented from being altered. The temperature control of the cleaning liquid part is cut off. In addition, by attaching a cover for blocking light from outside the apparatus to the reaction container, it is possible to prevent deterioration of the fluorescent substance incorporated in the nucleic acid.
[0043]
6). detection
Remove the reaction vessel from the apparatus and transfer the fluorescent plate reader (FLUOstar) without changing the vessel. TM ) To measure the change in light generated in the reaction vessel.
As a result, the strongest luminescence was observed from the PCR product sample of Microcystis aeruginosa NIES-98, and it was revealed that the genus Microcystis can be specifically detected.
[0044]
【The invention's effect】
According to the B / F separation method of the present invention, magnetic particles do not remain adsorbed in the reaction vessel, and high sensitivity can be measured by collecting all the magnetic particles. Further, for the measurement after B / F separation. The transfer can be carried out as it is (without transferring the reaction vessel to another vessel), and hybridization using an apparatus with automated hybridization can be carried out quickly and easily.
[Brief description of the drawings]
FIG. 1 is a diagram showing an example of magnetic force control for turning on and off a magnetic force by shifting a permanent magnet arranged at a lower part of a reaction vessel in a vertical direction.
FIG. 2 is a diagram showing an example of magnetic force control for turning on and off a magnetic force by rotating a magnetic rod embedded with a permanent magnet disposed under a magnetic particle and changing the direction of the magnetic flux.
FIG. 3 is a diagram showing an example of magnetic force control in which a magnetic rod that holds a magnet is rotated so that a part of the end surface of the magnet protrudes, and the magnetic force is turned on and off.
FIG. 4 is a diagram showing an internal configuration of an example of a hybridization apparatus.
FIG. 5 is a diagram showing an internal configuration of an example of a hybridization apparatus.
6 is a diagram showing an internal configuration of a reaction unit of the hybridization apparatus of FIG.
[Explanation of symbols]
1: Reaction vessel
2: Solution
3: Magnetic particles
4: Magnet
5: De-Nature part
6: Annealing club
7: B / F separation part
8: Chip rack storage
9: Waste liquid part
10: Cleaning liquid part
11: Reagent part
12: Head
13: Robot hand mechanism
14: Nozzle
15: Chip rack
16: Chip
17: Reaction part
18: Chip rack and waste liquid section
19: Cleaning liquid part
20: Reaction vessel holder
21: Heater
22: Chiller
23: Temperature sensor
24: Temperature control device
25: Magnetic control device

Claims (3)

  1. B / F separation method after completion of nucleic acid hybridization reaction using nucleic acid probe-immobilized magnetic particles as probes, wherein the following B / F separation steps (1), (2) and (3) are performed, and the magnetic particles The B / F separation method is characterized in that the magnetic force is controlled so as to be immobilized only at the bottom of the reaction vessel.
    (1) A step of sucking and removing the supernatant by controlling the magnetic force so that the magnetic particles are immobilized only at the bottom of the reaction vessel, (2) the magnetic particles in a state where the magnetic particles are immobilized or by controlling the magnetic force A step of injecting a cleaning liquid into the reaction vessel after mobilizing the particles, (3) inserting the permanent magnet into a non-penetrating hole of the magnet holding rod and holding the permanent magnet so that a part of the end face of the permanent magnet protrudes; the outside of the magnet retaining bars a magnetic rod which does not adversely force by Rukoto the magnetic substance rods arranged in the lower portion of the reaction vessel is rotated changing the direction of the magnetic flux, by repeating the oN-OFF of the magnetic force Moving the magnetic particles in the cleaning liquid.
  2.   The B / F separation method according to claim 1, wherein the hybridization reaction is carried out in a reaction section comprising a reaction vessel holder, a heating / cooling device, and a magnetic force control device.
  3.   The hybridization reaction comprises at least (A) a reaction container holding tool, a heating / cooling device and a magnetic force control device, C) A cleaning liquid part including a cleaning liquid container and a heating / cooling device, and (D) a mechanism for mounting / removing a chip to / from the chip nozzle, and a process for processing the mounted chip. And a mechanism for sucking and injecting a solution, and an automatic nucleic acid hybridization device comprising a head unit including an arm unit that can move freely in the XZ axis direction. Item 3. The B / F separation method according to Item 1 or 2.
JP2002085233A 2001-09-21 2002-03-26 B / F separation method in nucleic acid hybridization Active JP3996416B2 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
JP2001-289940 2001-09-21
JP2001289940 2001-09-21
JP2002085233A JP3996416B2 (en) 2001-09-21 2002-03-26 B / F separation method in nucleic acid hybridization

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Application Number Priority Date Filing Date Title
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US8852862B2 (en) 2004-05-03 2014-10-07 Handylab, Inc. Method for processing polynucleotide-containing samples
JP4595063B2 (en) * 2005-03-18 2010-12-08 農工大ティー・エル・オー株式会社 Reactor
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US9186677B2 (en) 2007-07-13 2015-11-17 Handylab, Inc. Integrated apparatus for performing nucleic acid extraction and diagnostic testing on multiple biological samples
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USD787087S1 (en) 2008-07-14 2017-05-16 Handylab, Inc. Housing
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WO2012142516A1 (en) 2011-04-15 2012-10-18 Becton, Dickinson And Company Scanning real-time microfluidic thermo-cycler and methods for synchronized thermocycling and scanning optical detection
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