JP3215752B2 - Analysis method and analyzer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to relates <br/> the analytical method and analytical equipment, in particular, measurement after labeling the measurement object with particles, the concentration of the measurement object is measured direct the particles into the flow cell It relates to analytical methods and analytical equipment to.

[0002]

2. Description of the Related Art Detection and identification of pathogenic microorganisms is essential for diagnosis of infectious diseases. However, it takes time to detect and identify the causative microorganism of an infectious disease by culture, and it often falls short of diagnosis and treatment of the disease.

[0003] In recent years, it has become possible to identify pathogens of infectious diseases and diagnose infectious diseases before the onset of disease by examining the presence or absence of a specific base sequence in a sample using the technique of nucleic acid hybridization.

That is, a method using a DNA probe. This utilizes a single-stranded DNA having a sequence complementary to the base sequence of a nucleic acid to be detected (this is called a DNA probe) as a specific reaction reagent. The presence or absence of the target pathogen can be determined by detecting the presence or absence of a base sequence complementary to the base sequence of the DNA probe in the sample.

Various specific methods have been proposed. For example, Japanese clinical study, 47, 737-754 (198)
9) etc. As an example, there is a method called a dot hybridization method. In this method, a single-stranded DNA (ss-DNA) obtained by denaturing a sample is bound to a solid phase, and ss-DNA labeled with a radioisotope is allowed to act on the solid phase to form ss-DNA on the solid phase. In this method, unreacted labeled ss-DNA is removed after a hybrid is formed, and the radioactivity of the solid phase is measured.

As a modification of this method, there is a sandwich hybridization method. This method can reduce the background due to adsorption, and is particularly effective when using an impure sample. In this method, at least two DNA fragments derived from the labeled nucleic acid to be identified are used. One DNA fragment is bound to a solid phase and used as a capture reagent. The other DNA fragment is labeled as a detection reagent and added to the hybridization solution together with the sample sample solubilized. If a base sample homologous to both reagents is present in the sample, that sequence should hybridize to both the capture reagent and the detection reagent. Whether or not hybridization has occurred can be known through labeling on the solid phase.

Also, Japanese Patent Publication No. 3-78120 proposes a method using a restriction enzyme. In this method, a single-stranded polynucleotide to be measured is fixed to a single-stranded polynucleotide to which a labeled substance is bound and which can form a double-stranded polynucleotide by reacting with the single-stranded polynucleotide to be measured. The phase and the solution are contacted in solution to form a double-stranded polynucleotide, a restriction enzyme is applied to the formed double-stranded polynucleotide to cleave the double-stranded polynucleotide, and a labeled solution or solid phase is formed. Measure.

[0008] Similarly, a method using a restriction enzyme or a reagent into which a selectable cleavage site has been introduced is disclosed in JP-A-2-92300.

[0009] On the other hand, although there is no association with the nucleic acid analysis, a method using a reaction vessel provided with a discharge hole below a filter in an immunoassay is disclosed in JP-A-2-228562, JP-A-2-228563 and others. Has been proposed. This prior art is used as a method for washing reagents on a solid phase,
/ F separation (bound part -bound- and free part -fr
As a simple method of performing the operation of (separation from ee-), pressure is applied from above the filter of the reaction vessel, and the liquid is discharged through a discharge hole provided below the filter.

Although there is no association with nucleic acid analysis, Japanese Patent Application Laid-Open No. Hei 4-273065 proposes a highly sensitive immunoassay method using microparticles as a label. In this conventional technique, microparticles are used as a label, and microparticles in proportion to the amount of an object to be measured are captured in a reaction solid phase by a specific reaction such as an antigen-antibody reaction, and then released, and the number of microparticles in the released liquid is counted. By doing so, the amount of the object to be measured is obtained. A liquid to be measured containing a free substance is introduced into a flow cell, and pulse-like fluorescence generated when the fine particles pass through a laser beam irradiated from a direction orthogonal to the flow is detected, and the number of pulses is counted. Is counted. In this conventional technique, the measurement is performed after releasing the label, ie, the fine particles, once captured on the reaction solid phase, so that the influence of the label that non-specifically binds to the solid phase can be avoided. By using microparticles as labels and counting them, highly linear detection is realized even at low concentrations.

[0011]

SUMMARY OF THE INVENTION As described above, in the conventional nucleic acid detection method, a DNA probe labeled with a radioactive isotope is widely used. The use of isotopes adds to the cost and requires consideration of handling safety and disposal. In addition, the conventional techniques of dot hybridization and sandwich hybridization have a problem when the amount of the nucleic acid to be detected is small. It is hard to say that it is suitable.

In the prior art described in Japanese Patent Publication No. 3-78120, measurement is performed by releasing a labeled substance attached to a single-stranded polynucleotide by a reaction. However, since the measurement of the released label is performed in a manner depending on the concentration of the measurement target in the entire reaction solution, the detection sensitivity is reduced when the measurement target in the sample is extremely small. Also, the shape of the reaction vessel is not mentioned, and if a normal bottomed reaction vessel is used, the washing step and the B / F separation work become complicated, which is also suitable from the viewpoint of automation of measurement. It is hard to say that.

The prior art described in Japanese Patent Application Laid-Open No. 2-92300 has the same problem as Japanese Patent Publication No. 3-78120.

JP-A-2-228562 and JP-A-2-2-2
In the prior art described in No. 28563, the washing step and the B / F separation operation are performed by using a reaction vessel provided with a discharge hole below the filter to pressurize the filter from above, and discharge the liquid through the discharge hole. Easy going. However, the measurement of the released label is a method in which the reaction solution remaining on the filter is taken together with the reaction container to the detector. This method requires a special detector capable of accommodating the reaction vessel, and also has a problem in sensitivity because the measurement is performed through the wall of the vessel.

In the prior art described in Japanese Patent Application Laid-Open No. 4-2730065, in order to measure the released labeled fine particles, a liquid to be measured containing the fine particles is introduced into a flow cell, and the number of the fine particles is measured optically. Therefore, high-sensitivity measurement is possible, but this conventional technique also uses a bottomed reaction vessel, which complicates the washing process and B / F separation work, which is also called automation of measurement. It is hard to say that it is suitable from the point of view.

An object of the present invention, the analysis methods and analysis equipment can measure nucleic acids in the sample automatically and sensitive
It is to provide.

[0017]

Means for Solving the Problems In order to achieve the above object, the analysis method of the present invention comprises (a) two kinds of fine particles having different particle diameters, one of which is a small particle having a label. Particles, and the other fine particles are prepared as large particles having a larger particle diameter than the small particles;
(C) using a filter having a pore size larger than the particle size of the small particles and smaller than the particle size of the large particles to bind the unbound fine particles through the measurement object in the sample. Removing small particles, (d) specifically cleaving small particles and large particles bound via the object to be measured, (e) separating only small particles using the filter, (F) measuring the concentration of the measurement target in the sample by measuring the label of the small particles.

In order to achieve the above object, the analyzer of the present invention comprises two kinds of fine particles having different particle sizes,
An analyzer for measuring the concentration of an object to be measured in a sample using one of the small particles as a small particle with a label, and the other fine particle as a large particle having a larger particle size than the small particle. (A) at least one reaction chamber, a filter arranged at the bottom of the reaction chamber and having a pore size larger than the small particles and smaller than the large particles, further below the filter; A liquid flow resistance member provided, a reaction container having a small hole for discharging the liquid that has passed through the filter and the liquid flow resistance member from the reaction chamber, and (b) a turntable in which a plurality of the reaction containers are arranged; (C) dispensing and supplying means for injecting the sample, the reagent containing the two kinds of microparticles, the washing liquid, and the reagent for specifically cutting the binding site into the reaction chamber of the reaction vessel;
A pressure difference generating means for generating a pressure difference between the reaction chamber and the small hole and discharging the liquid in the reaction chamber from the small hole; and (e) the pressure difference generating means included in the liquid discharged from the small hole. And a measuring means for measuring the label of the small particles and measuring the concentration of the measurement target in the sample.

In the above-mentioned analyzer, the measuring means preferably has a flow cell into which a liquid is introduced through a small hole of the reaction vessel, and measures optical characteristics based on a label of a small particle passing through the flow cell. To measure the concentration of the object to be measured in the sample. Preferably, the small hole of the reaction container is formed in a discharge nozzle provided at a lower end of the reaction container, and the discharge nozzle is directly inserted into an inlet of the injection chamber of the flow cell.

[0020]

[0021]

[0022]

[0023]

According to the analysis method of the present invention, two particles having different particle sizes are used.
Using two types of microparticles and a filter with a pore size between those particle sizes, unbound small particles after the two types of microparticles are bound via the measurement target in the sample are discharged by passing through the filter (B / F separation), and the small particles after cutting the combined substance of the small particles and the large particles are also separated by passing through a filter (B / F separation). When the cleaning liquid is injected, the cleaning liquid is also discharged through the filter, and the reaction chamber and the filter are cleaned. As described above, the B / F separation and the washing process are performed using two kinds of microparticles and a filter having a pore diameter intermediate between the two kinds of microparticles. Therefore, the reaction solution or the washing solution is inclined by tilting the container or using a pipetting nozzle. An operation such as removal is not required, and the B / F separation and the washing process are extremely simplified, and the automation of the measurement is facilitated. Also,
The separated fluorescent microparticles can be directly introduced into the flow cell, and the operation time can be reduced. Further, since the label can be measured by the flow cell, highly sensitive measurement is possible.

In the analyzer according to the present invention, the sample and the reagent containing the above two kinds of fine particles are injected into the reaction chamber of the reaction vessel by using a dispensing supply means, and the two kinds of fine particles are mixed in the reaction chamber. The sample is bound via the measurement object in the sample, and then a pressure difference is generated between the reaction chamber and the small hole using the pressure difference generating means, so that the unbound small particles resist the resistance of the liquid flow resistance member. And remove through a filter. At this time, a washing solution is injected as needed using the dispensing and supplying means to wash the reaction chamber and the filter. Next, a reagent that specifically cuts the bonding site is injected into the reaction chamber using the dispensing supply means, the bonded substance of the small particles and the large particles is cut, and the cut small particles are again cut using the pressure difference generating means. Is filtered through a filter against the resistance of the liquid flow resistance member. Also at this time, the cleaning liquid is injected using the dispensing supply means as needed. The small particles, which are microparticles with a label separated in this way, are measured by a measuring means,
It is preferably introduced into a flow cell, and the label of the small particles is measured, and the concentration of the measurement target in the sample is measured. Thus, the analysis method of the present invention can be performed.

The small hole of the reaction vessel is formed in a discharge nozzle provided at the lower end of the reaction vessel, and this discharge nozzle is directly inserted into the inlet of the flow chamber, so that the non-measurement liquid can be easily transferred to the flow cell. Measurement time is reduced.

[0026]

[0027]

First, an embodiment of the analysis method of the present invention will be described with reference to FIG. First, a polynucleotide (DNA probe) 402 bonded to a microparticle 401 with a label
And a microparticle 403 having no label
(DNA probe) 40 bound to DNA
4. Prepare a second reagent containing Here, the polynucleotide (DNA probe) 40 of the first and second reagents is used.
2,404 has a nucleotide sequence complementary to the sample DNA as the measurement object in the sample,
Those having no complementary nucleotide sequence are used. Further, the fine particles 401 of the first reagent are small particles, and the fine particles 403 of the second reagent are large particles having a larger particle diameter than the fine particles 401 of the first reagent. It is preferable to use fluorescent microparticles as the microparticles 401 with a label.

After the first and second reagents are prepared in this manner, the sample and the first reagent are injected into the reaction chamber of the reaction vessel (FIG. 1), and the sample DNA 405 in the sample and the first reagent The inside is hybridized with the fluorescent particle-labeled DNA probe 402 to form a double strand (FIG. 1). Then
The second reagent is injected into the reaction chamber of the reaction vessel (FIG. 1), and the sample DNA 405 in the sample and the non-fluorescent particle-bound DNA probe 404 in the second reagent are further hybridized to form a double strand. (FIG. 1).

In this way, two kinds of fine particles 401,
After binding 403 to the sample DNA 405, the reaction is performed using a filter 406 having a pore size larger than the particle size of the microparticles 401 in the first reagent and smaller than the particle size of the microparticles 403 in the second reagent. The solution is discharged through a filter 406 to remove unbound excess fluorescent particle-labeled DNA probe 402, and a first B / F separation is performed (FIG. 1).
). At this time, a cleaning liquid is injected as necessary to wash the reaction container and the filter 406. These operations are
The reaction vessel used so far may be provided with a mechanism capable of controlling the discharge of the reaction solution together with the filter 406,
This may be performed using a separate reaction chamber having a filter 406 and a discharge opening.

Next, an appropriate restriction enzyme solution is injected into the reaction chamber to cause the restriction enzyme 407 to act, thereby cutting the double-stranded site formed by the hybridization (FIG. 1).
As a result, the fluorescent fine particles 401 trapped in the filter 406 are released, and the released fluorescent fine particles 401 are separated together with the reaction solution through the filter 406 in the same manner as described above (FIG. 1). Thus, the second B
/ F separation is performed. Also at this time, a cleaning liquid is added as needed.

The effluent containing the fluorescent fine particles 401 thus separated is introduced into the flow cell, and the fluorescence based on the fine particles passing through the flow cell is detected, and the number of particles is counted. Based on the counted number of particles, the concentration of the sample DNA as the measurement object in the sample can be calculated.

According to the above-described analysis method, B / F separation and washing steps are performed using two types of microparticles 401 and 403 having different particle diameters and a filter 406 having a pore diameter intermediate between those particle diameters. Therefore, it is not necessary to perform an operation such as tilting the container or taking out the reaction liquid or the cleaning liquid using a pipetting nozzle, and the B / F separation and the cleaning step are extremely simplified. Therefore, automation of the measurement is facilitated. In addition, the separated fluorescent microparticles 401 can be directly introduced into the flow cell, and the operation time can be reduced. In addition, since the label is counted using the flow cell, highly sensitive measurement is possible.

In the above embodiment, the fluorescent particle-labeled DN
A First reagent containing probe 402 and non-fluorescent particle-bound D
The injection order of the second reagent including the NA probe 404 may be reversed or simultaneous.

Next, an embodiment of an apparatus for carrying out the above-described analysis method and a reaction vessel used in the analysis method is shown in FIG.
This will be described with reference to FIG.

FIG. 2 is a schematic diagram showing the entire configuration of an automatic analyzer for carrying out the analysis method of the present invention, and FIGS. 3 to 5 are diagrams showing the configuration of a reaction vessel used in the analysis method. .

In FIG. 1, the automatic analyzer of this embodiment has four turntables 1, 2, 3, and 35.
On the turntable 1, a plurality of sample containers 33 containing samples such as blood are arranged in a circle. The turntable 2 has a plurality of reaction vessels 34a arranged in a circle, and the turntable 3 has a plurality of reaction vessels 34b arranged in a circle. Further, the turntable 35 is a reagent table in which various reagent liquid containers required for the analysis operation are arranged in a circle. That is, the containers 90a, 90b, and 90c containing the fluorescent particle-labeled probe liquid as the first reagent corresponding to the analysis items A, B, and C, respectively, in which the nucleic acid is to be measured, and the particle binding as the second reagent. A container 36a containing the probe liquid,
36b, 36c, a restriction enzyme solution container 38, a container for a washing solution and other necessary buffers, and the like are held on a reagent table 35. The particles contained in the particle-bound probe solution as the second reagent have a larger particle size than the fluorescent particles contained in the fluorescent particle-labeled probe solution as the first reagent.

The turntables 1, 2, 35 are provided with pulse motors 91, controlled by a controller (not shown).
By 92 and 93, a desired sample container 33, reaction container 34a or reagent liquid container is rotatable so as to be stopped at a suction position or a discharge position by the pipetting nozzle 43. The turntable 3 is also rotatable by a pulse motor 94 controlled by a controller (not shown) so that the desired reaction vessel 34b is stopped at a position matching the reaction vessel 34a on the turntable 2. Also, turntable 2
Is provided with a driving device 95 for moving the turntable 2 up and down.

As shown in FIG. 3, the reaction vessel 34a has a plurality of reaction chambers 341 and 342 and a plurality of sample chambers 343 and 34.
5, a reaction chamber 341 which is one of the reaction chambers has an open bottom, and a filter 346 is disposed at the opened bottom. The reaction vessel 34b is, as shown in FIG.
It has a cap 347 at the upper part and a discharge nozzle 348a having a small hole 348 at the lower part. The cap 347 can be connected to the lower opening of the filter 346 of the reaction vessel 34a as shown in FIG. 5, and if there is no pressure difference between the reaction chamber 341 and the small hole 348 when it is connected to the lower opening. It has a structure as a liquid flow-down resistance member that holds the liquid in the reaction chamber 341. Specifically, the cap 347 is made of an elastic material, and a cross-shaped cut is made in the material as shown in FIG. 4 (B). It is a structure that closes when there is no difference. In this embodiment, the pressure difference generated between the reaction chamber 341 and the small hole 348 is controlled by the pressurizing device 101 connected to the pressurizing source 102 as shown in FIG.
It is generated by pressing from the 41 side.

The filter 346 is larger than the particle diameter of the fluorescent particles contained in the fluorescent particle-labeled probe liquid as the first reagent and smaller than the particle diameter of the particles contained in the particle-bound probe liquid as the second reagent. It has a pore size.

Reference numeral 39 denotes an automatic pipette mechanism, which is provided with the pipetting nozzle 4 attached to the movable arm 42.
3, a rotation drive unit 45 for horizontally rotating the movable arm 42, and a vertical drive unit 4 for vertically moving the movable arm 42
4, a syringe pump 40 connected to the nozzle 43 via the tube 41, a cleaning liquid tank 37 also serving as an extruding liquid, and a nozzle cleaning tank 96. With the operation of the movable arm 42, the pipetting nozzle 43 moves the sample suction position on the turntable 1, the reagent suction position on the turntable 35,
The sample / reagent dispensing position on the turntable 2 and the nozzle cleaning tank 96 can be rotated as a radius of rotation.
Can be lowered and raised at each position.

The turntables 1, 2, 3, 35 and the automatic pipette mechanism are arranged in a reaction thermostat 98.

The operation of the above apparatus is as follows. First,
The transfer of the sample in the sample container 33 is performed by the pipetting nozzle 43. That is, a certain amount of the sample is sucked by the pipetting nozzle 43, and the sample chamber 343 in the reaction vessel 34a transferred to the sample discharge position.
The liquid is discharged into the reaction chamber 342, and after being diluted if necessary, discharged into the reaction chamber 342 by the pipetting nozzle 43.

The pulse motor 93 rotates the reagent table 35 at a desired angle, and positions the designated reagent container at a required timing so as to stop at the suction position by the nozzle 43. The first reagent is injected into the reaction chamber 342 of the reaction container 34a at the first reagent dispensing position by the nozzle 43 (see FIG. 1). Further, the second reagent is supplied to the reaction chamber 3a of the reaction vessel 34a at the second reagent dispensing position by the nozzle 43.
42 (see FIG. 1).

Now, when the reaction container 34a comes to the dispensing position of the first reagent, a certain amount of the first reagent is aspirated by the nozzle 43 and is transferred to the reaction chamber 342 in the reaction container 34a being transferred to the dispensing position. Dispense. When this operation is completed, the turntable 2 is stopped by rotating counterclockwise by 360 ° + one pitch (one cycle) of the reaction vessel. Now, assuming that the time during which the turntable rotates and stops is 20 seconds, the above operation is repeated with 20 seconds as one cycle.
That is, the specific reaction container 34 into which the first reagent has been dispensed.
In a, as the cycle proceeds, the position in the state where the turntable 2 is stopped advances in the counterclockwise direction by one pitch of the reaction vessel. When looking at a specific reaction container into which the first reagent has been dispensed, the second reagent is dispensed at a position where the turntable 2 is stopped, for example, by one pitch of the reaction container. Thus, the sample, the first reagent, and the second reagent are dispensed into the reaction chamber 342 in the reaction container 34a, and the reaction proceeds.

The hybridization reaction solution of the sample, the particle-labeled probe and the fluorescent particle-labeled probe reacted in the reaction chamber 342 of the reaction vessel 34a is supplied to the reaction vessel 3 by the pipetting nozzle 43 at the reaction solution dispensing position.
4a is injected into the reaction chamber 341. In the filter cleaning position, the pressurizing device 101 is operated, and the filter 346 is activated.
The unreacted fluorescent particle probe in the unreacted first reagent having a pore diameter smaller than the pore size is discharged out of the reaction vessel through the filter 346 (see FIG. 1). By repeatedly injecting the cleaning liquid into the reaction chamber 341 by the nozzle 43 and operating the pressurizing device 101, the filter 346 is thoroughly cleaned.

When the cleaning of the reaction chamber 341 is completed, as shown in FIG. 6, the turntable 2 is lowered by the operation of the driving device 95 at the container connecting position, and the container 34b on the turntable 3 is connected to the reaction container 34a. After that, the driving device 9
5 operates in the reverse direction to raise the turntable 2.
Thereafter, the restriction enzyme solution is dispensed from the restriction enzyme solution container 38 by the nozzle 43 at the restriction enzyme solution dispensing position (see FIG. 1). After a lapse of a predetermined time, the drive device 95 is operated at the measurement liquid take-out position to process the turntable 2, and the discharge nozzle 348a of the container 34b is set to the inlet 48 of the sheath flow cell 47 as shown in FIG.
And the pressurizing device 101 is operated, and the measuring solution is
48 and is introduced into the sheath flow cell 47 (FIG. 1).
reference).

The internal structure of the sheath flow cell 47 is the same as that used in a known flow cytometer, but above it is a reagent injection chamber equivalent to that shown in JP-A-2-80937. Is open. Therefore, the liquid to be measured can be discharged from the injection chamber inlet 48 into the sheath flow cell 47 through the small hole 348. Then, the sheath liquid in the sheath liquid tank 6 is sent at a constant flow rate by the liquid sending pump 9, flows along the inner wall of the flow cell 47, and is discharged to the waste liquid reservoir 95. The liquid to be measured introduced into the flow cell 47 flows through the center of the flow of the sheath liquid.

The laser light source 49 has an oscillation wavelength of 488 nm.
The laser beam is broadened by a beam expander 50 and then squeezed by a lens 51 to irradiate a sheath flow cell 47 so as to focus on the flow of the liquid to be measured. . To collect the fluorescence from the flow cell 47, a microscope objective lens 5 is used.
2 is used. A spatial filter 54 and a wavelength selection filter 55 are provided before a photomultiplier 53 as a photoelectric detector to remove scattered light and Raman light. After the output of the photomultiplier 53 is amplified by the preamplifier 18 and then amplified by the linear amplifier 19, the lower limit wave height discriminator 20a
And noise is removed by the upper limit wave height discriminator 20b. Thereafter, pulses between the two thresholds are integrated by the counter 56.

A high voltage is applied to the photomultiplier 53 via the transformer 96 and the high-voltage power supply 97. The sample number, counting result, calibration curve, histogram of fluorescence measurement, and the like are output to the display 57, the printer 22, and the floppy disk 58. In addition, it can communicate with a personal computer via the interface 59.

Next, a specific example will be described taking nucleic acid analysis as an example. Since the reaction process for each measurement target item in nucleic acid analysis is similar, the analysis operation process in the reaction container will be described here taking the case of HBV (hepatitis B virus) as an example. The analysis item is specified by the type of the particle probe added after the start of the operation.

In the fluorescent particle-labeled probe liquid container 90a, single-stranded HBV-DNA probe type 1 was bound to fluorescent-labeled latex particles (0.1 μm in diameter) containing a coumarin derivative as a fluorescent substance. Prepare a fluorescent labeled latex particle reagent. On the other hand, a latex particle reagent (having a diameter of 0.1 μm) having single-stranded HBV-DNA probe type 2 immobilized thereon is placed in the particle-bound probe solution container 36a.
9um) is prepared. Each single-chain HBV-D
NA probe (single-stranded HBV-DNA probe type 1
And the single-stranded HBV-DNA probe type 2) had a nucleotide sequence complementary to the nucleic acid component to be measured, but did not have a nucleotide sequence complementary to each other.

The restriction enzyme solution is a restriction enzyme that cuts a double-stranded DNA formed by hybridizing a single-stranded HBV-DNA probe type 1 bound to fluorescent labeled latex particles with a nucleic acid component to be measured. For example, Hae
Prepare III.

When the analysis operation is started, a predetermined amount of the fluorescent labeled latex particle reagent is sucked from the container 60a on the reagent table 35 by the nozzle 43 by the nozzle 43, and is discharged into the corresponding reaction chamber 342 in the corresponding reaction container 34a. Specifies that the reaction vessel is for HBV analysis.

The reaction vessel 34a thus prepared
Next, the sample separated from the sample container 33 by the nozzle 43 is added to the reaction chamber 342. The HBV-DNA in the sample reacts with the fluorescently labeled latex particles to which the single-stranded HBV-DNA probe type 1 is bound.
The reaction was allowed to run for 15 minutes.

Next, a certain amount of latex particle reagent is sucked from the latex particle reagent liquid container 34a by the nozzle 43 and discharged into the corresponding reaction chamber 342 of the reaction container 34a. The reaction vessel 34a is kept at a predetermined temperature (37 ° C.) for a certain time (1
Maintain the reaction on the turntable for 5 minutes). As a result, the single-stranded HBV-
DNA probe type 2 reacts.

At the dispensing position of the reaction solution on the turntable 2, the pressurizing device 1 is placed above the reaction chamber 341 of the reaction vessel 34a.
01 is arranged. When the reaction container 34a is transferred to the position, the reaction solution is injected into the reaction chamber 341 of the reaction container 34a by the pipetting nozzle 43. As the filter 346, a membrane filter having a pore diameter of 0.6 μm and a diameter of 10 mm was used. At the filter cleaning position, the cleaning liquid is supplied by the nozzle 43 to the reaction chamber 34 in the reaction vessel 34a.
1 and the operation of the pressurizing device 101 was repeated. Next, the rotation of the turntable 3 causes the reaction vessel 43b to move to the corresponding reaction vessel 34a on the turntable 2.
After the reaction with the reaction chamber 341, the restriction enzyme solution container 38 is positioned at the suction position by rotation of the reagent table 35,
A certain amount of the liquid containing the restriction enzyme HaeIII in the container 38 is sucked by the pipetting nozzle 43 and discharged into the corresponding reaction chamber 341 of the reaction container 34 a on the turntable 2. In the reaction chamber 341, the double-stranded DNA formed by hybridizing the single-stranded HBV-DNA probe type 1 bound to the fluorescent-labeled latex particles and the nucleic acid component to be measured is cut at a predetermined cleavage site. Thereby, the fluorescent-labeled latex particles float in the liquid in the reaction chamber 341. The liquid to be measured containing these loose substances is discharged from the sheath flow cell 47 by operating the pressurizing device 101 at the position where the liquid to be measured is taken out.

FIG. 8 shows another embodiment of the reaction vessel used in the analysis method of the present invention. In this embodiment, the reaction vessel 6
Numeral 0 has an integral structure different from those shown in FIGS. That is, the reaction vessel 60 has a vessel section provided with a filter 346 and a vessel section provided with a discharge nozzle 607a having a cap 355 and a small hole 607 as a liquid flow resistance member. In this embodiment, since the two container portions are integrated as described above, the turntable 3 becomes unnecessary.

FIG. 9 shows another embodiment of the reaction vessel.
Alternatively, reaction vessels 34a and 34c as shown in FIG. 10 may be used. In this embodiment, the reaction vessel 34c has a discharge nozzle 351a having a small hole 351 formed in the wall of the vessel. In this case, the automatic analyzer shown in FIG. 2 requires a suction device in addition to the pressure device 101. This suction device is connected to a discharge nozzle 351a having a small hole 351 of the container 34c when the two containers 34a and 34c are connected as shown in FIG. A pressure difference is generated before and after the cap 350, which is a member, to move the reaction liquid in the reaction chamber 341 into the container 34c.

The operation of the automatic analyzer shown in FIG. 2 using the reaction vessels 34a and 34c of the present embodiment is as follows.

First, the transfer of the sample in the sample container 33 is performed by the pipetting nozzle 43.
A certain amount of the sample is aspirated by the pipetting nozzle 43 and is transferred to the sample discharge position.
After being discharged into the sample chamber 343 in the inside and diluted as necessary, it is discharged into the reaction chamber 342 by the pipetting nozzle 43.

The pulse motor 93 rotates the reagent table 35 at a desired angle, and positions the designated reagent container at the required timing so as to stop at the suction position by the nozzle 43. That is, the first reagent is injected into the reaction chamber 342 of the reaction container 34a at the first reagent dispensing position by the nozzle 43. Further, the second reagent is injected into the reaction chamber 342 of the reaction container 34a at the second reagent dispensing position by the nozzle 43.

The hybridization reaction solution of the sample, the particle-labeled probe and the fluorescent particle-labeled probe reacted in the reaction chamber 342 of the reaction vessel 34a is supplied to the reaction vessel 3 by the pipetting nozzle 43 at the reaction solution dispensing position.
4a is injected into the reaction chamber 341. In the filter cleaning position, the pressurizing device 101 is operated, and the filter 346 is activated.
The unreacted fluorescent particle probe in the unreacted first reagent smaller than the pore size of is passed through the filter 346 and discharged out of the reaction vessel. By repeatedly injecting the cleaning liquid into the reaction chamber 341 by the nozzle 43 and operating the pressurizing device 101, the filter 346 is thoroughly cleaned.

When the cleaning of the reaction chamber 341 is completed, as shown in FIG. 6, the turntable 2 is lowered by the operation of the driving device 95 at the container connecting position, and the container 34c on the turntable 3 is connected to the reaction container 34a. After that, the driving device 9
5 operates in the reverse direction to raise the turntable 2.
Thereafter, at the restriction enzyme solution dispensing position, the restriction enzyme solution is dispensed from the restriction enzyme solution container 38 by the nozzle 43. After a lapse of a predetermined time, a suction device (not shown) is connected to the discharge nozzle 351a, and the reaction solution in the reaction chamber 341 is sucked through the small hole 351 so that the reaction liquid in the reaction chamber 341 is transferred to the container 43c.
Move toward. Thereafter, the container 34a and the container 34c are separated, and the container 34c moves to the position for taking out the measurement liquid.
At the measurement liquid take-out position, the measurement liquid is taken out of the container 34 c by the nozzle 43 and introduced into the sheath flow cell 47.

As still another embodiment of the reaction vessel, FIG.
And a reaction vessel 34a and a reaction vessel 34 as shown in FIG.
It may be a combination with f. In this case, the reaction vessel 34f
Is different from a nozzle 353a having a suction hole 353 and a discharge nozzle 354a having a small hole 354 for discharge. It is taken out from 354 and introduced into the sheath flow cell 47.

The reaction vessel shown in FIG. 11 and FIG. As in the case shown in FIG. 8, the reaction vessel 60 shown in FIG.
And a cap 356 and small holes 608 and 60 as a liquid flow resistance member.
9 may be integrated with the container portion provided with the nozzles 608a and 609a, respectively. In this case, the turntable 3 becomes unnecessary.

Further, a fluorescent particle-labeled probe solution or a particle-bound probe solution can be put in one of the sample chambers of the reaction vessel, for example, the sample chamber 345 of the reaction vessel 34a, together with the storage solution. In this case, the container for storing the first reagent or the second reagent does not enter, and the measurement time can be further reduced.

[0067]

According to the present invention, the B / F separation and the washing process are performed using two kinds of fine particles having different particle diameters and a filter having a pore diameter intermediate between the particle diameters. There is no need to perform an operation such as taking out a reaction solution or a washing solution using a pipetting nozzle, so that B / F separation and a washing process become extremely simple, and automation of measurement becomes easy. Further, the separated fluorescent microparticles can be directly introduced into the flow cell, and the operation time can be reduced. Further, since the label can be measured by the flow cell, highly sensitive measurement is possible.

According to the present invention, the small hole of the reaction vessel is formed in the discharge nozzle provided at the lower end of the reaction vessel, and the discharge nozzle is directly inserted into the inlet of the flow cell for measurement. You can save time.

[0069]

[Brief description of the drawings]

FIG. 1 is a diagram illustrating an operation procedure of an analysis method according to an embodiment of the present invention.

FIG. 2 is a schematic diagram showing an overall configuration of an analyzer according to one embodiment of the present invention.

3A and 3B are views showing one embodiment of a reaction vessel used in the apparatus shown in FIG. 2, wherein FIG. 3A is a longitudinal sectional view and FIG. 3B is a top view.

FIG. 4 is a view showing one embodiment of a reaction vessel used in the apparatus shown in FIG. 2, (A) is a longitudinal sectional view, and (B) is a top view.

5 is a longitudinal sectional view showing a state where the container shown in FIG. 3 and the container shown in FIG. 4 are combined.

6A and 6B are diagrams showing an operation procedure for connecting the container shown in FIG. 3 and the container shown in FIG. 4, wherein FIG. 6A shows a state before connection, FIG. 6B shows a state at the time of connection, and FIG. Indicates the status.

FIG. 7 is a longitudinal sectional view showing a state in which a discharge nozzle of the reaction container shown in FIGS. 3 and 4 is inserted into a flow cell.

FIG. 8 is a longitudinal sectional view of a reaction vessel according to another embodiment of the present invention.

FIG. 9 is a longitudinal sectional view of a reaction vessel according to still another embodiment of the present invention.

10 is a sectional view of a lower portion of the reaction vessel shown in FIG.

FIG. 11 is a longitudinal sectional view of a reaction vessel according to still another embodiment of the present invention.

FIG. 12 is a sectional view of a lower portion of the reaction vessel shown in FIG.

FIG. 13 is a longitudinal sectional view of a reaction vessel according to still another embodiment of the present invention.

[Explanation of symbols]

1, 2, 3 Turntable 33 Sample container 34a, 34b Reaction container 35 Turntable (reagent table) 36a, 36b, 36c Fluorescent particle labeled probe liquid container 38 Restriction enzyme liquid container 42 Movable arm 43 Pipetting nozzle 46 Controller 47 Sheath flow cell 48 Injection chamber inlet 56 Counter 90a, 90b, 90c Particle labeled probe liquid container 94 Nozzle washing tank 102 Compressor 401 Sample DNA 402 Fluorescent particle labeled DNA probe 403 Non-fluorescent particle labeled DNA probe 404 Filter 405 Restriction enzyme

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-6-317594 (JP, A) JP-A-6-113896 (JP, A) JP-A-62-298257 (JP, A) JP-A-4- 16764 (JP, A) JP-A-4-273065 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/68 G01N 33/543

Claims (4)

(57) [Claims] (A) two types of microparticles having different particle sizes, one of which is a small particle with a label, and the other of which has a larger particle size than the small particle. Preparing large particles, (b) binding the two types of microparticles via an object to be measured in a sample,
(C) removing unbound small particles by using a filter having a pore size larger than the particle size of the small particles and smaller than the particle size of the large particles, and (d) binding through the object to be measured. Specifically cleaving the combination of small and large particles,
(E) separating only the small particles by using the filter; and (f) measuring the concentration of the measurement target in the sample by measuring the label of the small particles. 2. Two kinds of fine particles having different particle diameters, one of which is a small particle with a label, and the other is a large particle having a larger particle diameter than the small particle. (A) at least one reaction chamber, which is disposed at a bottom of the reaction chamber, and is larger than a particle size of the small particles; A reaction vessel having a filter having a pore size smaller than the particle diameter of the particles, a liquid flow-down resistance member provided further below the filter, and a small hole for discharging the liquid that has passed through the filter and the liquid flow-down resistance member from the reaction chamber. (B) a turntable on which a plurality of the reaction vessels are arranged; and (c) a specimen, a reagent containing the two kinds of microparticles, a washing solution, and a binding site specifically cut in a reaction chamber of the reaction vessel. Try to do (D) a pressure difference generating means for generating a pressure difference between the reaction chamber and the small hole, and discharging the liquid in the reaction chamber from the small hole; A) a nucleic acid analyzer, comprising: a measuring unit that measures a label of the small particles contained in the liquid discharged from the small hole and measures the concentration of the measurement target in the sample. 3. The analyzer according to claim 2, wherein the measuring means has a flow cell into which a liquid is introduced through a small hole of the reaction vessel, and the optical means based on the labeling of the small particles passing through the flow cell. An analyzer characterized by measuring characteristics and measuring the concentration of an object to be measured in a sample. 4. The analyzer according to claim 3, wherein the small hole of the reaction vessel is formed in a discharge nozzle provided at a lower end of the reaction vessel, and the discharge nozzle is directly inserted into an inlet of the injection chamber of the flow cell. An analyzer characterized by the above-mentioned.