JPH0662897A - Method for measuring specimen of nucleic acid and measuring device therefor - Google Patents

Abstract

PURPOSE:To enable use of a reagent common in items in detecting oligonucleotide sequence of an analytical target of a nucleic acid specimen. CONSTITUTION:A nucleic acid specimen is blended with a labeled common polynucleotide reagent and a nonbinding polynucleotide reagent specific to measurement target having a sequence complementary to an oligonucleotide sequence of the detection target and a sequence complementary to the labeled polynucleotide and the labeled amount is measured.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring a nucleic acid sample, a measuring device and a reagent kit used therefor. In particular, the present invention is effective for detecting the presence or absence of a particular oligonucleotide sequence that is an analyte of interest present in a nucleic acid sample.

[0002]

2. Description of the Related Art Detection or identification of pathogenic microorganisms is essential for diagnosing infectious diseases. Therefore, the antibody titer of the patient serum against a specific antigen of a specific pathogenic microorganism is measured, but such measurement merely sees the shadow of infectious disease and does not confirm its true form. It takes time to detect and identify microorganisms that cause infectious diseases by culturing, and there is a drawback that practical diagnosis and treatment of diseases cannot be made in time.

By examining the presence or absence of a specific base sequence in a nucleic acid sample, it becomes possible to identify the microorganisms that cause the infectious disease, and it becomes possible to make a diagnosis before the onset of the infectious disease. Examples of this type of prior art include “Analytical Biochemistry, v
138, pp. 267-284 (1984) "is known. This method uses single-stranded DNA having a sequence complementary to the base sequence of the nucleic acid to be detected (this is called a DNA probe). Is used as a reagent that reacts specifically.
The presence or absence of the target pathogen can be determined by detecting the presence or absence of the base sequence complementary to the base sequence of the DNA probe in the sample.

As a method using a DNA probe, a method called dot hybridization is known. This method is a single-stranded DNA obtained by denaturing a sample.
(SS-DNA) is bound to a solid phase, radioisotope-labeled SS-DNA is allowed to act on this solid phase to form hybrid with SS-DNA in the solid phase, and then unreacted labeled SS-DNA is removed. Then, the solid-phase radiation is measured.

As a modification of this method, there is sandwich hybridization. This method can reduce the background due to adsorption and is particularly effective when an impure sample is used. This method uses at least two DNA fragments derived from the target nucleic acid to be identified. One DNA fragment is bound to a solid phase and used as a capture reagent. The other fragment is labeled as a detection reagent and added together with the sample solubilized in the hybridization solution. If there is a chlorine sequence homologous to both reagents in the sample, that sequence should hybridize to both the capture and detection reagents. Whether or not they have hybridized can be known through the labeling on the solid phase. Japanese Examined Patent Publication No. 3-78120 proposes a method of using a restriction enzyme to enable measurement of a nucleic acid sample in a short time. In this method, a single-stranded polynucleotide to be measured, a solid phase and a solution in which a label is bound and a single-stranded polynucleotide capable of forming a single-stranded polynucleotide and a double-stranded polynucleotide to be measured are bound. Double-stranded polynucleotide is formed by contacting in the medium, a restriction enzyme is allowed to act on the formed double-stranded polynucleotide, the double-stranded polynucleotide is cleaved, and a solution or solid-phase labeled substance is measured. To do.

[0006]

In the measurement of nucleic acid using a DNA probe, a reagent containing a different DNA probe for each measurement target (oligonucleotide sequence of interest for detection of nucleic acid analyte present in nucleic acid sample) Need to prepare. To develop a DNA probe,
Due to the large amount of labor required, the development of measuring reagents tends to be delayed, and infectious bacteria can be detected by detecting the presence or absence of an oligonucleotide (specific base) sequence that is an analyte of interest present in a nucleic acid sample. It has been a factor to delay the spread of the method of detecting.

Further, in order to reduce the measurement error due to non-specific adsorption or the like, a method of recognizing the double strand formed by binding and selectively cleaving the double strand is effective. However, when the analysis is performed using different DNA probes for each measurement target, the double strands must be recognized by different restriction enzymes for each measurement target, and this must be selectively cleaved. Therefore, in developing a measurement reagent, it was necessary to prepare different restriction enzymes for each measurement target to form a reagent kit. The development of measurement reagents tends to be delayed because a great deal of labor is required to develop a restriction enzyme that recognizes a special oligonucleotide (specific chlorine) sequence and selectively cleaves it. The restriction enzymes developed became very expensive. This is also another factor that delays the spread of the method for detecting infectious bacteria by detecting the presence or absence of an oligonucleotide as an analyte of interest in a nucleic acid sample.

An object of the present invention is to make it possible to use a common labeling reagent even when the oligonucleotides to be measured are different.

Another object of the present invention is to make it possible to use a common solid-phase reagent even when the oligonucleotides to be measured are different.

Another object of the present invention is to make it possible to commonly use a restriction enzyme for cleaving a double-stranded polynucleotide even when the oligonucleotide to be measured is different.

[0011]

According to the present invention, in detecting at least one kind of oligonucleotide sequence present in a nucleic acid sample, the nucleic acid sample and a reagent containing a labeled polynucleotide for other analytical items are used. A common labeled polynucleotide reagent that is also used as, and a base complementary to at least a part of the labeled polynucleotide having a base sequence complementary to at least a part of the base sequence of the oligonucleotide to be measured. A polynucleotide probe for binding a label having a sequence was mixed.

In this mixing step, a common solid-phase nucleotide reagent containing a polynucleotide bound to a solid phase and also used as a reagent for other analytical items, and complementary to at least a part of the above-mentioned oligonucleotide sequence. Can be mixed with a solid-phase binding polynucleotide probe having a sequence complementary to at least a part of the sequence of the polynucleotide bound to the solid phase.

As the restriction enzyme, by using a restriction enzyme that recognizes and cleaves a double-stranded site formed by the labeled polynucleotide and the polynucleotide of the polynucleotide probe for label binding, a reagent for a plurality of analytical items is obtained. Can be shared as. A signal derived from the label cleaved and released by the action of the restriction enzyme is detected. Another embodiment of the restriction enzyme is a restriction enzyme that recognizes and cleaves a double-stranded site formed by a polynucleotide bound to a solid phase and a nucleotide of a polynucleotide probe for solid phase binding.

[0014]

[Function] The labeled polynucleotide reagent and / or the solid-phase nucleotide reagent are each bound with a common polynucleotide that does not depend on the analyte (oligonucleotide sequence of nucleic acid). A DNA probe containing both a base sequence and a base sequence that recognizes the base sequence of the nucleic acid to be analyzed is used. During the analysis reaction, the above-mentioned DNA probe binds to the solid-phase nucleotide or the labeled nucleotide via the common polynucleotide, respectively, so that substantially the same reaction result as in the conventional direct binding can be obtained. By this method, a great deal of commonization of reagents can be achieved, and there is no need to prepare different reagents for each measurement target as in the conventional case.

1 (A) to 1 (D) conceptually show the binding mode between nucleotides according to the present invention. In the embodiment of FIG. 1 (A), a sample (oligonucleotide having a specific base sequence) S has a sequence common to labeled common polynucleotide 1 and a sequence complementary to at least a part of the oligonucleotide base sequence of the sample. This is an example of binding with a polynucleotide 2 as a probe having a sequence complementary to at least a part of the base sequence of the common polynucleotide 1. In this case, probe 2 is an unlabeled polynucleotide. In the embodiment of FIG. 1 (B), the sample S is bound to the labeled polynucleotide 1 by the polynucleotide 2 as a probe, and the sample S is bound to the common polynucleotide 3 bound to the solid phase M. , An example of binding with a polynucleotide 4 as a probe having a sequence complementary to at least a part of the oligonucleotide base sequence of the sample and having a sequence complementary to at least a part of the base sequence of the common polynucleotide 3. .

FIG. 1C and FIG. 1D show the case where there are plural kinds of analytes. When the oligonucleotide base sequences as the analytes are different, the nucleic acid samples corresponding to them are generally processed in separate reaction vessels. As shown in FIG. 1 (C), the first oligonucleotide sequence S1 is bound by the probe 2 to the labeled common polynucleotide 1 as in the case of FIG. 1 (A). On the other hand, the second oligonucleotide sequence S2 is the same as the labeled common polynucleotide sequence 1 with the probe 5
Combined by This probe 5 is an unlabeled polynucleotide having a sequence complementary to at least a part of the base sequence of the common polynucleotide 1 and having a sequence complementary to at least a part of the base sequence of the second oligonucleotide. is there.

The embodiment of FIG. 1 (D) is an example in which the plural kinds of oligonucleotide sequences S1 and S2 of FIG. 1 (C) are individually bound to a common polynucleotide 3 bound to a solid phase M. Is. In this case, S1 in FIG. 1D and S in FIG. 1B are the same type. The probe 6 has a sequence complementary to at least a part of the second oligonucleotide base sequence S2 and a sequence complementary to at least a part of the base sequence of the common polynucleotide 3 bound to the solid phase M. It has a polynucleotide.

In these FIGS. 1 (A) to 1 (D), the same reference numerals represent the same base sequences. As can be understood from these figures, according to the present invention, the labeling reagent and the solid-phase reagent can be shared even when there are plural kinds of oligonucleotide base sequences of the nucleic acid sample as the analysis target. To support multiple types of measurements,
The polynucleotide probe reagent may be changed.

In order to reduce an error due to non-specific adsorption or the like, a method of recognizing a double strand formed by a bond between single-stranded nucleotides with a restriction enzyme and selectively cutting it is effective. By binding to a solid phase or label before cleavage with a DNA probe consisting of a common polynucleotide that does not depend on the measurement target, the measurement item (the oligonucleotide sequence of interest for the detection of the nucleic acid analyte present in the nucleic acid sample) It becomes possible to apply common restriction enzymes that do not depend on them. Therefore, for each analysis, a common reagent and an unbound polynucleotide having a base sequence specific to the measurement target may be prepared.

[0020]

[Example] Detection of a specific base sequence in a nucleic acid sample
It is carried out by applying the principle of hybridization.

In the present invention, the nucleic acid to be measured is preferably in a free state in the solution, but it may be bound to the support. Alternatively, the base sequence of the nucleic acid to be detected may be bound to the solid phase via the common solid-phased polynucleotide reagent and the polynucleotide reagent.

According to one method according to the present invention, a nucleic acid sample, a labeled first polynucleotide and a detector are used to detect the presence of the analyte oligonucleotide sequence present in the nucleic acid sample. A second polynucleotide having a sequence complementary to the target oligonucleotide sequence and having a sequence complementary to the labeled polynucleotide is mixed.

Furthermore, a third polynucleotide reagent bound to the solid phase and a fourth polynucleotide reagent having a sequence complementary to the oligonucleotide sequence to be detected and having a sequence complementary to the polynucleotide bound to the solid phase. A step of mixing with a polynucleotide reagent may be provided. The reaction in this case will be described below step by step as an example.
The order of the reactions in the present invention is not limited to this.

(1) The third polynucleotide reagent bound to the solid phase and the fourth polynucleotide are mixed.

(2) The fourth polynucleotide reagent released in the solution is removed by washing.

(3) Reaction with a nucleic acid sample containing an oligonucleotide which is a single-stranded analyte.

(4) Mix with the second polynucleotide reagent.

(5) The second polynucleotide reagent released in the solution is removed by washing.

(6) React with the labeled first polynucleotide reagent.

(7) The labeled first polynucleotide reagent released in the solution is removed by washing. (8) A restriction enzyme that recognizes the double-stranded portion and selectively cuts it is made to act.

(9) Detect the signal derived from the label released in the liquid.

That is, as the labeled first polynucleotide, the label is bound to a common polynucleotide that does not depend on each detection target. A DNA probe containing both a base sequence complementary to this common polynucleotide and a base sequence that recognizes the base sequence of the nucleic acid to be detected is defined as the second polynucleotide. During the analysis reaction, the DNA probe (second polynucleotide) binds to the label via the common polynucleotide (first polynucleotide), so that the oligonucleotide to be detected is substantially the same as the conventional one. The reaction result is similar to that obtained by directly binding to.

Similarly, since the DNA probe (fourth polynucleotide) binds to the solid phase via the common solid-phased polynucleotide (third polynucleotide), it is practically used as a detection target as in the conventional case. The same reaction result can be obtained as when the oligonucleotide is directly bound to the immobilized nucleotide. In addition, for the label or solid phase, DN
Since the A probe (second and fourth polynucleotides) are connected by a common polynucleotide that does not depend on the measurement target, a common restriction enzyme acts on the double-stranded site to dissociate or cleave the double-stranded site. Then, the label bound to the probe can be released in the liquid.

The label applicable to the present invention is selected from fluorescent labels, luminescent labels, enzymes, microparticles, etc. depending on the type of detector used. Generally, these labels are double-stranded portions formed by a DNA probe containing a polynucleotide and a base sequence that recognizes the base sequence complementary to the common polynucleotide and the base sequence of the oligonucleotide to be analyzed. It is released into the solution by cutting or dissociating. Therefore, the amount of signal derived from the label released in the liquid is proportional to the amount of the base sequence of the analyte.
The signal derived from the label can be detected by a conventionally known method. When particles are used as the label, a commercially available flow cytometer can be used.

When particles are used as the labeling substance, the average particle size of the particles is preferably in the range of 0.01 to 10.0 um, particularly 0.01 to 1.0 um.
As the particles to be used, it is preferable to use fluorescent fine particles and magnetic particles. The fluorescent fine particles may be latex particles or inorganic particles having a fluorescent material layer formed on the surface thereof, or may be particles of a mixture of a particle forming composition and a fluorescent material. In addition to this, gel such as Sephadex or ion exchange resin can be used.

When using a flow cytometer,
Fluorescent microparticles can be preferably used. Fluorescence is detected by a flow cytometer and the number of particles passing through the flow cell is counted. The fluorescent substances used for the fluorescent particles are known substances such as fluorescein, coumarin derivatives, rhodamine derivatives and umbelliferone.

It is also possible to use particles which do not have a label such as fluorescence. In this case, the particles can be optically measured by measuring the scattering intensity or the turbidity of dust originating from the particles.

The size of the polynucleotide used as the DNA probe is about 15 bases or more, preferably 50 bases or more.
Base or higher. Usually, there will be a region in the polynucleotide reagent component that has homology to a sequence in the nucleic acid sample, and usually the sequence of interest will have at least 6 bases, usually at least 12 bases. The region for hybridization is 16 bases or more and usually does not exceed about 1 kbp. Although complete homology is not required, at least about 50% homology is preferred and at least 80% homology is more preferred.

Next, an embodiment of the present invention will be shown.

A sample containing 1 mg / ml double-stranded DNA was dissolved in 0.14 M NaCl, boiled for 10 minutes, and then rapidly cooled with ice to obtain single-stranded DNA. The obtained single-stranded DNA solution was diluted with a hybridization buffer to prepare a sample for preparing a calibration curve. As the hybridization buffer, 5 × SSC, 0.55 BSA,
0.5% PVA and 1% SDS were used.

Phosphate buffer (PBS: 0.14M NaC
20 dissolved in 0.01% phosphate buffer (pH 7.2)
0 μg / ml of the first single-stranded polynucleotide was added to a 0.1% polystyrene latex particle A containing a fluorescent dye.
The mixture was added to the solution (particle size: 0.2 μm) and kept at 37 ° C. for 2 hours. The single-stranded polynucleotide labeled with the latex particles A having a small particle size is used as a labeling reagent 1.

Single-stranded DNA to be detected in nucleic acid sample
A second polynucleotide reagent having a sequence complementary to the oligonucleotide sequence and having a sequence complementary to the fluorescence-labeled polynucleotide (first single-stranded polynucleotide) is prepared, and a probe reagent 2 and To do.

Next, the third single-stranded polynucleotide is
A microtiter plate was added and incubated at 37 ° C for 2 hours. This is the polynucleotide reagent 3 bound to the solid phase. In this case, the wall of the reaction container also serves as the reagent 3.

A fourth polynucleotide reagent having a sequence complementary to the oligonucleotide sequence of the single-stranded DNA to be detected and also having a sequence complementary to the third polynucleotide 3 bound to the solid phase is used. Prepare and use it as the probe reagent 4.

50 μl of the probe reagent 4 was added to the microtiter plate (reagent 3) on which the third single-stranded polynucleotide was immobilized, and the mixture was kept at 65 ° C. for 3 hours. Excess reagent 4 was then washed with PBS and removed from the plate. Add 10 to this microtiter plate.
A nucleic acid standard sample for preparing a ul calibration curve was added, and the mixture was further incubated at a temperature of 65 ° C. for 3 hours. After removing the interfering components contained in the sample from the plate by washing with PBS, 50 ul of the probe reagent 2 was added and the temperature was further kept at 65 ° C. for 2 hours. Excess reagent 2 was removed by washing with PBS. Next, 10 ul of the labeling reagent 1 was added. 65
After incubating at the temperature for 3 hours, excess reagent 1 was washed with PBS and removed from the plate.

Finally, a restriction enzyme that recognizes and cleaves the double-stranded portion where the single-stranded nucleotides are bound to the latex particle A as a label that remains after binding to the microtiter plate as a result of the reaction. And released into the liquid in the plate. The restriction enzyme used does not recognize the base sequence specific to the measurement sample and cut the double strand, but recognizes the common base sequence site that does not depend on the measurement target and cuts the double strand. did.

The latex particles A containing the released fluorescent dye were counted using a commercially available flow cytometer device. The results are shown in Table 1.

[0048]

[Table 1]

An example of a nucleic acid sample automatic analyzer to which the present invention is applied will be described with reference to FIG.

FIG. 2 is a schematic diagram showing the overall structure of an automatic analyzer suitable for nucleic acid analysis. The sample table 11 includes a plurality of sample containers 33 containing samples such as blood.
Are rotatably arranged. Also, the reaction table 12
In the above, a plurality of reaction vessels 34 are rotatably arranged in a constant temperature bath. The reaction container 34 is composed of a reaction chamber and a small chamber having a filter at the top and small holes in the wall. A suction device is arranged so that the excess labeled particles are discharged to the outside of the reaction container through a small hole provided in the wall of the small chamber of the reaction container 34. Each of the tables 11 and 12 is configured to be rotatable so that the desired sample container 33 or reaction container 34 is stopped at the suction position by the pipetting nozzle 43 by the pulse motors 91 and 92 whose operations are controlled by the controller 46. There is. The pipetting nozzle 43 transfers the sample in the sample container 33. A fixed amount of the sample containing nucleic acid is sucked by the pipetting nozzle 43 and discharged into the reaction container 34 transferred to the sample discharging position.

On the reagent table 35, various reagent liquid containers necessary for the analysis operation are placed. That is, a container 91 containing a fluorescent particle-labeled polynucleotide reagent 1 which is a first reagent commonly used for a plurality of analysis items A and B whose target is a nucleic acid, and a container 91 common to the analysis items A and B A container 30 containing a polynucleotide reagent 3 bound to a particle solid phase, which is a third reagent used as
Second polynucleotide reagent 2, which corresponds to B, respectively
Container 90a and 90b containing 5 and containers 36a and 36b containing fourth polynucleotide reagents 4 and 6 corresponding to analysis items A and B, respectively, and a restriction enzyme solution containing container 38.
A container for a cleaning solution and other necessary buffer solution is held on the reagent table 35. The pulse motor 93 whose operation is controlled by the controller 46 rotates the reagent table 35 by 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. That is, the first reagent is injected by the nozzle 43 into the reaction container 34 at the first reagent dispensing position. Further, the second reagent 90a or 90b is selectively injected into the reaction container 34 by the nozzle 43 at the second reagent dispensing position according to the type of the oligonucleotide to be analyzed.
Similarly, the third and fourth reagents are also injected into the reaction container 34 at the reagent dispensing position by the nozzle 43.

The automatic pipette mechanism 39 includes a movable arm 42.
Pipetting nozzle 43 and arm 4 attached to
A rotation drive unit 45 for horizontally rotating 2 and an arm 4
It is provided with an up-and-down drive unit for moving up and down 2, a syringe pump 40 connected to a nozzle 43 via a tube 41, and a cleaning liquid tank 37 also serving as an extruding liquid. With the movement of the arm 42, the pipetting nozzle 43 rotates around the sample suction position on the sample table 11, the reagent dispensing position on the reaction table 12, the injection chamber inlet 48 of the flow cell 47, and the nozzle cleaning tank 44. Can rotate and can descend and rise in each position.

A membrane filter is installed above the reaction vessel 34. Of the sample reacted with the solid phase in the reaction chamber of the reaction container 34, the first polynucleotide reagent labeled with fluorescent particles, and the second and fourth polynucleotide reagents The hybridization reaction liquid is injected into the membrane filter portion of the reaction container 34 by the pipetting nozzle 43 at the reaction liquid dispensing position. At the filter washing position, the suction device operates to discharge the unreacted fluorescent particle-labeled polynucleotide reagent smaller than the pore diameter of the filter to the outside of the reaction container through the small hole provided in the wall of the small chamber of the reaction container 34. . The cleaning liquid is supplied to the reaction container 34 by the nozzle 43.
The filter is thoroughly washed by repeatedly injecting it into the inner filter and operating the suction device. At the restriction enzyme liquid dispensing position, the filter portion of the reaction container 34 that has been washed is discharged by the nozzle 43 into the restriction enzyme liquid storage container 3
The restriction enzyme solution is dispensed from 8. After a lapse of a predetermined time, the suction device is operated at the measurement liquid take-out position to take out the measurement liquid and introduce it into the sheath flow cell 47 by the nozzle 43.

The internal structure of the sheath flow cell 47 is the same as that used in a known flow cytometer, but a reagent injection chamber equivalent to that shown in JP-A No. 2-80937 is provided above. It is open. Therefore, the nozzle 43 can enter the injection chamber inlet 48 and discharge the liquid to be measured into the sheath flow cell 47. Liquid delivery pump 9
The sheath liquid in the sheath liquid tank 16 is fed at a constant flow rate by the sheath liquid tank 16, flows along the inner wall of the flow cell 47, and the waste liquid reservoir 95
Is discharged to. The liquid to be measured introduced into the flow cell is
It flows through the center of the sheath fluid flow.

The laser light source 49 has an oscillation wavelength of 488 nm.
Argon laser can be emitted, and the beam width of this laser beam is expanded by the beam expander 50, and then the laser beam is narrowed down by the lens 51 and applied to the sheath flow cell 47 so as to be focused on the flow of the liquid to be measured. . An objective lens 5 for a microscope is used to collect the fluorescence from the flow cell 47.
2 is used. A spatial filter 54 and a wavelength selection filter 55 are provided in front of the photomultiplier 53 as a photoelectric detector to remove scattered light and Raman light. The output of the photomultiplier 53 is amplified by the preamplifier 18 and then by the linear amplifier 19, and the lower limit wave height discriminator 20 is amplified.
The nozzle is removed by a and the upper limit wave height discriminator 20b. After that, the counter 56 detects the pulse between the two thresholds.
Accumulate with.

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

At the time when the reaction container 34 holding the sample reaches the second reagent dispensing time, the nozzle 43 sucks a predetermined amount of the second reagent 90a or 90b and transfers it to the dispensing position. Dispense into When this operation is completed, the reaction table 12 rotates counterclockwise by 360 ° + one pitch (1 cycle) of the reaction container and stops. Now
Allow 20 hours between rotating and stopping the reaction table
Letting 20 seconds be one cycle of 20 seconds, the above operation is repeated. In other words, the position of the specific reaction container 34 into which the second reagent has been dispensed advances counterclockwise by one pitch of the reaction container as the cycle progresses while the turntable is stopped. When looking at a specific reaction container into which the second reagent has been dispensed, the first reagent is dispensed at a position where the turntable is stopped by one pitch of the reaction container. As a result, the sample, the first reagent and the second reagent are dispensed into the reaction container 34, and the reaction proceeds. Further, when the reaction container 34 reaches the dispensing time of the fourth reagent, the nozzle 43 sucks a predetermined amount of the fourth reagent 36a or 36b and dispenses it into the reaction container 34 which is transferred to the dispensing position. When the reaction container 34 comes to the dispensing position of the third reagent, a certain amount of the third reagent is sucked by the nozzle 43 and dispensed into the reaction container 34 being transferred to the dispensing position. This reaction is recorded for 20 seconds as a cycle for a certain period of time until the liquid is drained by the drainage device attached to the table 12 and washed by the washing device.

The above-described analyzer of FIG. 2 is a device 11, 3 for supplying a plurality of nucleic acid samples for measuring different oligonucleotides corresponding to different analysis items to the reaction container 34.
9 and a common labeling reagent 91 containing a labeled polynucleotide are dispensed into different reaction containers for different oligonucleotides to be measured, and are unlabeled for specifically binding the oligonucleotides to be labeled and the labeled polynucleotides. The probe reagents 90a and 90b are selectively dispensed into reaction containers corresponding to different oligonucleotides,
And a reagent dispensing device 35, 39 that dispenses a restriction enzyme reagent 38 that recognizes a double-stranded portion formed by a paired nucleotide sequence, and a detection signal derived from a label released by the action of the restriction enzyme. Detecting device 47,5 for obtaining
3 and so on. Further, the reagent dispensing apparatus dispenses the common solid-phase reagent 30 containing the polynucleotide bound to the solid phase into any reaction container for different oligonucleotides to be measured, and binds to the oligonucleotide and the solid phase. The probe reagents 36a and 36b that specifically bind to the polynucleotide are selectively dispensed according to the corresponding reaction vessels.

In the automatic analyzer shown in FIG. 2, the same number of label binding polynucleotide probe reagents and solid phase binding polynucleotide probe reagents are used according to the type of oligonucleotide of the nucleic acid derived from the microorganism to be measured. By preparing on the reagent table 35 and preparing three common reagents on the reagent table 35, hepatitis B virus (HBV), hepatitis C virus (HCV), human papilloma virus (HPV), adult T-cell leukocytes Infectious microorganisms such as virus (HTLV-I) can be detected one after another.

Since the reaction processes for each measurement target item of the nucleic acid analysis are similar, here, HBV (B
Taking the case of hepatitis B virus) as an example, the analytical operation process in the reaction container 34 will be described. The analysis item is specified by the type of particle probe added after the start of the operation.

In the particle solid phase polynucleotide solution container 30, a latex particle reagent (solid phase diameter 0.9 μm) on which single-stranded HBV-DNA probe type 3 is immobilized is prepared. On the other hand, fluorescent particle-labeled polynucleotide liquid container 91
First, a fluorescent labeled latex particle reagent in which single-stranded HBV-DNA probe type 1 is bound to fluorescent latex particles (label diameter 0.1 μm) containing a coumarin derivative as a fluorescent substance is prepared. Also, the container 90a
Single-stranded HBV-DNA probe type 2, container 36a
First, the single-stranded HBV-DNA probe 4 is prepared. Single-stranded HBV-DNA probe type 1 and single-stranded HBV-DN
A probe type 2, single-stranded HBV-DNA probe type 3 and single-stranded HBV-DNA probe type 4 had complementary nucleotide sequences. Also single chain HB
As V-DNA probe type 2 and single-stranded HBV-DNA probe type 4, those having a nucleotide sequence complementary to the nucleic acid component to be measured were used.

As the restriction enzyme solution in the container 38, two strands formed by hybridizing a single-stranded HBV-DNA probe type 1 and a single-stranded HBV-DNA probe type 2 bound to fluorescent-labeled latex particles were used. HaeIII, for example, is prepared as a restriction enzyme for cutting the strand DNA.

When the analysis operation is started, the single-stranded HBV-DNA probe type 2 from the container 90a and the single-stranded HBV-DNA probe 4 from the container 36a are collected by the nozzle 43, and the corresponding reaction container 34 is stored. It is specified that the reaction container is for HBV analysis by discharging the reaction container into the reaction chamber.

The sample dispensed from the sample container 33 by the nozzle 43 is added to the reaction chamber of the reaction container 34 thus prepared. HBV-in the sample
DNA reacts with single-stranded HBV-DNA probe type 2 and single-stranded HBV-DNA probe type 4.
The reaction was allowed to run for 15 minutes.

Next, the latex particle reagent is sucked into the reaction chamber of the corresponding reaction container 34 by sucking a fixed amount of the latex particle reagent from the container 30 on the reagent table 35 with the nozzle 43.

Further, the fluorescent labeled latex particle reagent is
A fixed amount is sucked from the container 91 on the reagent table 35 by the nozzle 43 and discharged into the reaction chamber of the corresponding reaction container 34. The reaction container 34 is kept at a predetermined temperature (37 ° C.) for a certain time (1
Maintain reaction on turntable for 5 minutes. As a result, the single-stranded HBV-
DNA probe type 1 and further single-stranded HBV-DNA
Probe type 3 reacts. At the reaction liquid dispensing position on the turntable 2, a suction pump is arranged below the reaction container 34. When the reaction container 34 is transferred to the position, the reaction liquid is injected into the filter portion of the reaction container 34 by the pipetting nozzle 43. Filter has a pore size of 0.6
A um, membrane filter with a diameter of 10 mm was used.
At the filter cleaning position on the reaction table 12, the cleaning liquid was injected into the filter section in the reaction container 34 by the nozzle 43 and the suction device was operated repeatedly. Then, by rotating the reagent table 35, the restriction enzyme liquid container 38 is positioned at the suction position, and the liquid containing the restriction enzyme HaeIII 25 in the container 38 is pipetted into the pipetting nozzle 43.
Is inhaled by a predetermined amount and is discharged to the filter portion of the corresponding reaction container 34 on the turntable 2. In the filter section, single-stranded HBV bound to fluorescent labeled latex particles
-Double-stranded DN formed by hybridizing DNA probe type 1 and single-stranded HBV-DNA probe type 2
A is cut at a predetermined cutting point. As a result, the fluorescent labeled latex particles float in the liquid in the reaction container 34.
The liquid to be measured containing these educts is introduced into the sheath flow cell 47 at the position for taking out the liquid to be measured, the suction device is operated to take out the liquid to be measured, and is sucked by the pipetting nozzle 43.

As the polynucleotide reagent, a synthetic product was used.

Applied Biosystems Model 3
Using the 81A DNA synthesizer,
An oligonucleotide having a length of 30 bases was synthesized by the amidite method and used.

The second polynucleotide reagent, which is a reagent corresponding to each of the analysis items, has a sequence complementary to both the first polynucleotide commonly used for the item and the sample. Therefore, by preparing a polynucleotide having a sequence complementary to the first common polynucleotide, separately synthesizing a polynucleotide having a sequence complementary to the sample corresponding to each analysis item, and connecting the two. Obtainable. Similarly, the fourth polynucleotide reagent, which is a reagent corresponding to each of the analysis items, has a sequence complementary to both the third polynucleotide commonly used for the item and the sample. Therefore, by preparing a polynucleotide having a sequence complementary to the third common polynucleotide, separately synthesizing a polynucleotide having a sequence complementary to the sample corresponding to each analysis item, and connecting the two. Obtainable.

[0070]

EFFECTS OF THE INVENTION According to the present invention, since a reagent common to a plurality of kinds of oligonucleotides to be measured in a nucleic acid sample can be used, preparation of reagents required for measurement becomes easy.

[Brief description of drawings]

FIG. 1 is a diagram for explaining an aspect of hybridization used in the present invention.

FIG. 2 is a diagram showing a schematic configuration of an automatic nucleic acid sample analyzer according to an embodiment of the present invention.

Description of symbols S, S1, S2 ... Oligonucleotide, 1 ... Labeled polynucleotide, 2, 5 ... Unlabeled polynucleotide, 3 ... Solid-phase polynucleotide, 4, 6 ... Binding polynucleotide, 30 ... Solid-phase reagent Container, 33 ... Sample container, 34 ...
Reaction container, 36a, 36b, 90a, 90b ... Individual reagent container, 38 ... Restriction enzyme solution container, 43 ... Pipetting nozzle, 47 ... Sheath flow cell.

Claims (14)

[Claims] 1. A measuring method for detecting a sequence of a specific oligonucleotide as an analysis item present in a nucleic acid as a sample to be measured, which contains a labeled nucleotide and is also used as a reagent for other analysis items. A common labeled nucleotide and the specific oligonucleotide of the sample have a sequence complementary to at least a part of the specific oligonucleotide sequence and a sequence complementary to at least a part of the labeled nucleotide. Binding with a polynucleotide probe for label binding having a common solid-phase nucleotide that contains nucleotides bound to a solid phase and is also used as a reagent for other analytical items, and the specific oligonucleotide of the sample With a sequence complementary to at least a portion of the above specific oligonucleotide sequence. And binding by a solid-phase binding polynucleotide probe having a sequence complementary to at least a portion of the nucleotides bound to the solid phase, and the solid phase was bound to the specific oligonucleotide and the label A method for measuring a nucleic acid sample, which comprises cleaving the conjugate with a restriction enzyme to separate the solid phase from the label. 2. A method for detecting at least one kind of oligonucleotide sequence present in a nucleic acid sample, which is commonly used as a reagent for other analysis items, containing the above-mentioned nucleic acid sample and a labeled polynucleotide. A labeled polynucleotide reagent, and a polynucleotide probe for label binding having a sequence complementary to at least a part of the labeled polynucleotide and having a sequence complementary to at least a part of the oligonucleotide sequence, A method for measuring a nucleic acid sample, which comprises a step of mixing. 3. A common solid-phase nucleotide reagent containing a polynucleotide bound to a solid phase, which is also used as a reagent for other analytical items, in the measuring method according to claim 2, A polynucleotide probe for solid phase binding, which has a sequence complementary to at least a part and has a sequence complementary to at least a part of the sequence of the polynucleotide bound to the solid phase, is further mixed in the mixing step. A method for measuring a nucleic acid sample, comprising: 4. The method according to claim 2 or 3, wherein a restriction enzyme that recognizes and cleaves a double-stranded portion formed by the labeled polynucleotide and the nucleotide of the label-binding polynucleotide probe is used. A method for measuring a nucleic acid sample, which comprises a releasing step of causing the reaction and a step of detecting a signal derived from a label released by this cleavage. 5. The measuring method according to claim 3, wherein a restriction enzyme that recognizes and cleaves a double-stranded portion formed by the polynucleotide bound to the solid phase and the nucleotide of the polynucleotide probe for solid phase binding is used. A method for measuring a nucleic acid sample, which comprises a releasing step of causing the reaction and a step of detecting a signal derived from a label released by this cleavage. 6. The method for measuring a nucleic acid sample according to claim 2, wherein the common labeled polynucleotide reagent contains a polynucleotide labeled with particles. 7. A method for measuring a first nucleic acid sample for detecting a first oligonucleotide sequence as an analyte and a second nucleic acid sample for detecting a second oligonucleotide sequence as an analyte. , The first nucleic acid sample, a common labeled polynucleotide reagent containing a labeled polynucleotide, and the labeled polynucleotide having a sequence complementary to at least a part of the first oligonucleotide sequence. A step of mixing a first label-binding polynucleotide probe having a sequence complementary to at least a part of nucleotides, the second nucleic acid sample, the common label polynucleotide reagent, and the second And a sequence complementary to at least a part of the labeled polynucleotide, which has a sequence complementary to at least a part of the oligonucleotide sequence Method of measuring the nucleic acid sample which comprises the steps of a second label attachment for polynucleotide probes are mixed with. 8. The measuring method according to claim 7, wherein a restriction enzyme that recognizes a double-stranded portion formed by the labeled polynucleotide and the nucleotide of the first polynucleotide probe for binding and binding is allowed to act on A method for measuring a nucleic acid sample, which comprises a step of cleaving a double-stranded portion. 9. The method according to claim 8, wherein the double-stranded portion formed by the labeled polynucleotide and the nucleotide of the second polynucleotide probe for label binding is cleaved by the action of the restriction enzyme. A method for measuring a nucleic acid sample, which comprises a step. 10. The measuring method according to claim 7, wherein for the first nucleic acid sample, a common solid-phase nucleotide reagent containing a polynucleotide bound to a solid phase and at least the first oligonucleotide sequence are included. The second nucleic acid sample is mixed with a first solid-phase binding polynucleotide probe having a sequence complementary to a part thereof and a sequence complementary to at least a part of the sequence of the polynucleotide bound to the solid phase. For the common solid-phase nucleotide reagent above,
A second having a sequence complementary to at least a part of the second oligonucleotide sequence and a sequence complementary to at least a part of the sequence of the polynucleotide bound to the solid phase
A method for measuring a nucleic acid sample, which comprises mixing the solid-phase binding polynucleotide probe described in 1. 11. The method according to claim 10, wherein the polynucleotide bound to the solid phase and the first or second polynucleotide are combined.
A method for measuring a nucleic acid sample, which comprises a step of causing a restriction enzyme that recognizes a double-stranded portion formed by nucleotides of the solid-phase binding polynucleotide probe to act to cleave the double-stranded portion. 12. A reagent kit for use in detecting an oligonucleotide to be measured present in a nucleic acid sample, comprising a reagent containing a labeled polynucleotide and at least the sequence of the oligonucleotide to be measured. An unlabeled probe reagent comprising a polynucleotide having a sequence complementary to a part thereof and a sequence having a sequence complementary to at least a part of the sequence of the labeled polynucleotide. 13. An apparatus for supplying a plurality of nucleic acid samples for measuring different oligonucleotides corresponding to different analysis items, and a common labeling reagent containing a labeled polynucleotide are dispensed together for different oligonucleotides to be measured. Then, an unlabeled probe reagent that specifically binds the above-mentioned oligonucleotide and the above-mentioned labeled polynucleotide is selectively dispensed to each of the above-mentioned different oligonucleotides, and a dinucleotide formed by a pair of nucleotide sequences is formed. A reagent dispensing device that dispenses a restriction enzyme reagent that recognizes a double-stranded portion, and a nucleic acid sample characterized by comprising a detection device that obtains a detection signal derived from a label released by the action of the restriction enzyme measuring device. 14. The measuring device according to claim 13, wherein the reagent dispensing device dispenses together a common solid-phase reagent containing a polynucleotide bound to a solid phase for different oligonucleotides to be measured, A device for measuring a nucleic acid sample, which selectively dispenses a probe reagent that specifically binds a nucleotide and the polynucleotide bound to the solid phase.