WO2007058077A1

WO2007058077A1 - Gene test method, microreactor for gene test and gene test system

Info

Publication number: WO2007058077A1
Application number: PCT/JP2006/321964
Authority: WO
Inventors: Akihisa Nakajima; Yasuhiro Sando; Kusunoki Higashino; Youichi Aoki
Original assignee: Konica Minolta Medical & Graphic, Inc.
Priority date: 2005-11-18
Filing date: 2006-11-02
Publication date: 2007-05-24
Also published as: JPWO2007058077A1

Abstract

It is intended to provide a microreactor for gene test and a gene test system which enable highly reliable and highly sensitive analysis. Namely, a gene test system comprising chip components for individual specimens, each carrying a reagent and a liquid-feeding element, and controlling and detection components which are separately provided as the main body of the apparatus. These microreactor chips, by which a positive control, a negative control and specimens are simultaneously analyzed in the course of nucleic acid amplification, have a channel structure free from internal control. Since the internal control is omitted from the channel structure of the microreactor as described above, simplified channels and elements can be provided. Furthermore, the accuracy and reliability of the data can be examined thereby, which ensures the acquisition of accurate analytical data.

Description

Specification

Genetic testing method, genetic testing microreactor, and genetic testing system

Technical field

The present invention relates to a genetic testing method, a genetic testing microreactor, and a genetic testing system.

Background art

[0002] In recent years, by making full use of micromachine technology and ultrafine processing technology, devices and means (for example, pumps, valves, flow paths, sensors, etc.) for performing conventional sample preparation, chemical analysis, chemical synthesis, etc. have been developed. A system that has been miniaturized and integrated on a single chip has been developed. This is also called TAS (Micro total Analysis System), bioreactor, lab 'on-chips', biochip, and is used in medical examination' diagnostic field, environmental measurement field, agricultural production field. Its application is expected. When complex processes, skilled procedures, and instrumentation are required, automated, accelerated and simplified microanalysis systems are not only cost, required sample volume, and time, but also time Analysis is possible anywhere.

[0003] In the field where various tests such as clinical tests are performed, results are obtained quickly regardless of the location. Even in the measurement using these analysis chips, the quantitativeness, accuracy of analysis, etc. are important. Since the analysis chip has severe restrictions on its size and shape, establishing a highly reliable liquid delivery system with a simple configuration is an issue. Therefore, there is a need for a microfluidic control element that has high accuracy and excellent reliability. The present inventors have already proposed a microphone pump system suitable for this (Patent Documents 1 and 2).

[0004] Specific problems and demands that should be solved as actual problems have been raised for microreactors that provide simple and rapid inspection means, and their solutions are desired. For example, in order to perform an inspection on a chip, it is the greatest challenge required for a microreactor to minimize the amount of sample and reduce the amount of reagent required. However, depending on the sample, the concentration of the gene or nucleic acid to be detected may be very dilute. Chi Since the amount of sample that can be introduced into the cell is limited, such sample amount will not fall within the measurable range unless it is concentrated. For detection of genes and nucleic acids, it is advantageous to use an amplification reaction by PCR (polymerase chain reaction). In that case, it is customary to analyze the controls in parallel to avoid contamination, failure of replication, nonspecific amplification, failure such as amplification failure due to reaction inhibition, and pitfalls. The present inventors have already proposed a microreactor having a channel configuration for that purpose (Patent Document 3). When control for accurate analysis is performed on the chip, complicated flow paths and fluid control elements must be configured and arranged, and fluids must be mixed and divided, which often involves various technical difficulties. .

Patent Document 1: Japanese Patent Laid-Open No. 2001-322099

Patent Document 2: JP 2004-108285 A

Patent Document 3: Japanese Patent Application No. 2004-138959

Disclosure of the invention

Problems to be solved by the invention

[0005] In the present invention made in view of the above situation, positive control and negative control analysis are performed in parallel with the analysis of a sample at the time of nucleic acid amplification, but the internal control can be omitted. We propose a genetic testing microreactor, a genetic testing method using a genetic testing microreactor, and a genetic testing system including a genetic testing microreactor.

Means for solving the problem

[0006] The genetic testing method of the present invention comprises:

At least in one chip

A sample container into which a sample or DNA extracted from the sample is injected;

A reagent container for storing a reagent used in the nucleic acid amplification reaction;

A positive control accommodating part for accommodating a positive control;

A negative control accommodating portion for accommodating a negative control;

An amplification section for performing a nucleic acid amplification reaction;

A detection part in the downstream flow path and A flow path communicating with each of these accommodating portions,

A gene testing method using a genetic testing microreactor provided with a constant amount of a reagent contained in the reagent containing part is fed to a flow path, and then divided into three parts, and the divided reagents are respectively separated. In the flow path

(0 Mixed liquid (1) generated by mixing with the sample sent from the sample container,

(ii) Force of positive control housing Mixture formed by mixing with sent positive control (2), and

(iii) Negative control storage capacity Mixture produced by mixing with the sent negative control (3)

Then, the mixture (1) and the mixture (2) were further divided into two parts,

Amplification reaction is performed on the fluid mixture (1) only, the mixture of the mixture (1) and the mixture (2), each fluid of the mixture (2) only, and only the mixture (3) in the amplification section. Is detected by each detector.

Moreover, the genetic testing method of the present invention is the genetic testing method described above,

Furthermore, the probe accommodating portion is arranged in communication with the flow path for accommodating the probe to be hybridized with the gene to be detected amplified by the nucleic acid amplification reaction,

The amplification product generated in the amplification part is mixed with the probe accommodated in the probe accommodating part in the flow path to bind or hybridize with the probe, and the target gene is detected based on the reaction product,

Similarly, the positive control and the negative control are configured such that their amplification products are combined or hybridized in the flow path with the probe accommodated in the probe accommodating portion, and detection is performed based on the reaction product. It is characterized by that. The microreactor for genetic testing of the present invention is

At least in one chip

A positive control accommodating part for accommodating a positive control;

A negative control accommodating portion for accommodating a negative control; An amplification section for performing a nucleic acid amplification reaction;

A detection part in the downstream flow path and

A flow path communicating with each of these accommodating portions,

A microreactor for genetic testing provided with

A first liquid dividing means for dividing a predetermined amount of the reagent accommodated in the reagent accommodating portion fed into the flow path into three parts, a first reagent, a second reagent and a third reagent;

(0) A mixed liquid (1) generating unit that generates a mixed liquid (1) by mixing the first reagent divided by the first liquid dividing unit and the sample sent from the sample storage unit;

GO A mixed liquid (2) generating unit that mixes the second reagent divided by the first liquid feeding dividing unit and the positive control sent from the positive control storage unit to generate a mixed liquid (2); ,

(iii) Mixed liquid (3) generated by mixing the third reagent divided by the first liquid dividing means and the negative control sent from the negative control storage unit to produce a mixed liquid (3) And comprising

Further, the liquid mixture (1) generated in the generating section is divided into two liquid feeding and dividing means (1), and the liquid mixture (2) generated in the generating section (2) is mixed. A third liquid feeding and dividing means for dividing into two,

The amplification unit includes an amplification unit that amplifies only the mixture (1), an amplification unit that amplifies the mixture of the mixture (1) and the mixture (2), and the mixture (2). The amplifying unit that amplifies only the mixed solution (3) and the amplifying unit that amplifies only the mixed solution (1) are amplified. A detection unit for detecting the amplification product obtained, and a detection unit for detecting the amplification product obtained by the amplification reaction in the amplification unit that amplifies the mixture of the mixture (1) and the mixture (2). An amplification reaction is carried out in a detection section for detecting an amplification product obtained by amplification reaction in an amplification section that amplifies only the mixture (2), and an amplification section in which amplification reaction is performed only in the mixture (3). It is also characterized by four forces with the detection unit that detects the resulting amplification product.

The gene testing microreactor of the present invention is the gene testing microreactor. In

And a probe storage unit that is disposed in communication with each of the detection units and stores a probe that is hybridized to the amplification product.

Each of the detection units detects a reaction product in which the amplification product and a probe delivered from the probe storage unit are combined or hybridized.

[0009] Further, the gene testing microreactor of the present invention is the gene testing microreactor,

The first, second and third liquid feeding and dividing means are:

Branched microchannels,

A liquid feed control unit that blocks passage of liquid at a pressure lower than a preset pressure, and allows passage of liquid at a pressure higher than a preset pressure;

A backflow prevention unit for preventing backflow of liquid in the flow path;

Consisting of

It is characterized by controlling the liquid feeding and the amount of liquid feeding in the branched flow path

[0010] The genetic test system of the present invention comprises:

A device main body in which a micropump and a temperature control device are integrated together with a detection device that optically detects nucleic acid, and the above-described genetic testing microphone reactor that can be attached to the device main body. Nucleic acid measurement is performed by attaching a microreactor.

[0011] Further, the genetic test system of the present invention, in the genetic test system,

The micropump is

A first channel in which the channel resistance changes according to the differential pressure;

A second flow path whose rate of change in flow path resistance with respect to a change in differential pressure is smaller than that of the first flow path; a pressure chamber connected to the first flow path and the second flow path;

An actuator for changing the pressure inside the pressurizing chamber;

It is provided with.

The invention's effect [0012] The genetic test system of the present invention has a system configuration in which reagents' chips that are microreactors for each specimen, each equipped with an element for a liquid delivery system ', and a control' detection component that is a device main body are separated. It is. Therefore, the problems of false or insufficient amplification, cross-contamination and carry-over contamination for microanalysis and amplification reactions are avoided.

[0013] The microreactor for gene testing of the present invention has a flow channel configuration that can simultaneously analyze positive control and negative control in order to eliminate the influence of reaction inhibition, contamination, and increase in nottag round. Therefore, since the accuracy and reliability of the results can always be verified, the accuracy of the analysis results finally obtained in the form of numerical data can be ensured. Therefore, the accuracy of the analysis result finally obtained in the form of numerical data can be secured in the same manner as described above for the genetic testing method using such a genetic testing mic reactor.

[0014] In order to employ a flow path configuration that can omit internal control, a simplified flow path configuration and arrangement of elements are possible. Brief Description of Drawings

[0015] FIG. 1 is a schematic diagram of a genetic test system comprising a microreactor and an apparatus main body.

FIG. 2 is a schematic view of a microreactor (chip) for genetic testing. Note that the micropump belongs to a separate device from the microreactor.

[FIG. 3] FIG. 3 shows a conceptual diagram of a sample configuration of a sample and a control.

[FIG. 4] FIG. 4 shows an embodiment of the microreactor for gene testing of the present invention, and shows an embodiment corresponding to FIG. 3 (c).

[FIG. 5] FIG. 5 shows a configuration around the pump connection portion of the microreactor when the micropump 11 is separated from the microreactor. (a) is a diagram showing a configuration of a pump unit for feeding a driving liquid, and (b) is a diagram showing a configuration of a pump unit for feeding a reagent.

FIG. 6 shows a piezo pump, (a) is a sectional view showing an example of the pump, and (b) is a top view thereof. (C) is a sectional view showing another example of a piezo pump.

FIG. 7 shows an example of a flow path configuration for dividing a fluid into three on the flow path. FIGS. 8 (a) and 8 (b) are cross-sectional views along the flow path axis direction of the liquid feeding control unit.

[Fig. 9] Fig. 9 is a view for explaining a fixed-quantity liquid feeding mechanism using a check valve.

FIG. 10 is a system schematic diagram of a micro analysis system including a microreactor and an apparatus main body.

Explanation of symbols

1 Main unit

2 Microreactor (inspection chip)

3 Peltier element

4 Heater

5 photodiode

6 LED

7 Reagent reservoir

11 Micro pump (piezo pump)

12 Pump connection

13 Liquid feed controller

15 flow path

15A reagent filling channel

15B branch flow path

16 Check valve

17 Reservoir

18 Reagent storage

19 specimens

20 Sample storage

21a Stop liquid reservoir

21b Denaturing solution container

21c Hybridization buffer housing

21d Cleaning liquid container

21e Gold colloid container If probe DNA compartment

1g Probe for positive control DNA housing 1h Positive control housing

11 Negative control housing

1j Dilution buffer

1k Positive control probe DNA compartment 11 Probe DNA compartment

2 Detection unit (Streptazibine adsorption unit)

3 Waste liquid reservoir

Drive fluid storage

5 Sealing liquid container

6 Air vent flow path

7 liquid

1 Reagent

1 Upper board

2 Board

3 Diaphragm

Piezoelectric element

5 Pressurization chamber

6 First flow path

7 Second channel

8 1st chamber

9 Second liquid chamber

1 Restriction flow path

0 Reagent solution

0 Drive fluid

1 Silicon substrate

2 ports 73 ports

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, a genetic testing microreactor according to the present invention and a genetic testing system including the microreactor, a micropump, various control devices, and a detection device will be described. In the present specification, the “specimen” is a fluid containing the nucleic acid to be measured. A “gene” refers to DNA or RNA that carries genetic information that expresses a certain function, but may also be referred to simply as DNA or RNA that is a chemical entity. These and the polynucleotide are collectively referred to as “nucleic acid”. “Element” refers to a functional component installed in the microreactor. The “fine channel” is a channel formed in the microreactor of the present invention, and is also simply referred to as “channel”. The fluid flowing in the fine channel is usually a liquid.

[0018] Control in gene analysis

Nucleic acid amplification methods such as the PCR method and the ICAN method are widely used because even a very small amount of DNA can be amplified hundreds of thousands to several million times. Due to the extremely high amplification factor, the effects of contamination such as cross-contamination and carry-over contamination are extremely serious. Furthermore, it is said that false amplification or non-specific amplification is not uncommon. For this reason, always performing positive control and internal control simultaneously in parallel with sample analysis is essential for checking that PCR reactions are occurring correctly, especially in gene amplification by PCR. For example, if any problem arises, it is effective to verify whether it originates from the set conditions, reagents, operation, analysis system, or sample. In genetic testing using a microreactor, specimen processing, amplification, and detection are performed almost automatically. For this reason, it is extremely important to ensure that the accuracy and reliability of final analysis results can always be confirmed. The setting and validation of these controls, which are useful for determining false positives and false negatives in amplification reactions on microreactors, follow conventional analytical techniques.

[0019] The internal control is used as an internal standard for target nucleic acid (DNA, RNA), monitoring of amplification, or quantification. It is particularly useful for quantitative analysis. The internal control sequence is placed on both sides of the sequence different from the sample. Since the same primer as the ima has a sequence that can be hybridized, it can be amplified in the same manner as the sample. Nucleic acids (DNA, RNA) used for internal control may be those described in publicly known technical literature. If internal control cannot be successfully amplified, it may be possible that a sample-derived interfering factor was present.

[0020] The sequence of the positive control is a specific sequence for detecting the sample, and the portion where the primer hybridizes and the sequence between them are the same as the sample. This is useful for detecting interfering factors in added reagents, verifying the appropriateness of set conditions, and nonspecific interactions. If amplification of the positive control is unsatisfactory, there may be operational problems such as reagent defects, reagent delivery, and mixing failure.

[0021] Negative control is used for background correction, such as checking for the presence or absence of contamination that can be used as ultrapure water instead of nucleic acid (DNA, RNA), correction of fluorescence, and color development of substances mixed in reagents, etc. Used for. If an amplified gene is detected in the negative control, it is possible that a gene other than the sample is mixed.

[0022] When all positive control, negative control, and internal control are performed in the same flow path under the same reagent and the same conditions in a separate analysis channel from the sample, it is shown in 03 (a). In addition, it is necessary to install six detectors.

As described above, the internal control is included in the amplification reagent, and is amplified and reacted together in each of the three analyzes of the specimen, the positive control, and the negative control, and the detection is performed in a separate detection unit. However, from the significance of internal control analysis, it is preferable to combine positive control with internal control. Although the internal control will surely hybridize with the primer, the sequence to be amplified is different from the sample, so during amplification, only the sample or only the internal control may be amplified, making the control meaningless. There may not be. In consideration of this point, the present inventor devises the division of the fluid and the configuration of the flow path in the microreactor for genetic testing of the present invention, as described in “Control measurement and flow path configuration”. This eliminates internal control analysis and By using the sub-control, the number of detectors could be reduced to four. According to the configuration of the flow path, it is more simplified than the conventional method, and since the restrictions are large in terms of size, a complicated flow path is preferable for a microreactor that should be avoided. Furthermore, sufficient mixing is realized in the flow path of the nucleic acid amplification section. Differences such as amplification due to insufficient diffusion of nucleic acids and reagents are unlikely to occur between samples and controls.

[0023] Overview of microreactor and genetic testing system for genetic testing

Fig. 1 is a schematic diagram of a genetic testing system comprising a genetic testing microreactor (hereinafter sometimes simply referred to as a microreactor) that can be attached to and detached from the device main body, and Fig. 2 is a schematic diagram of the genetic testing microreactor. It is. The present invention can be arbitrarily modified and changed in various embodiments in accordance with the spirit of the present invention, and these are included in the present invention. That is, the structure, configuration, arrangement, shape, dimensions, material, method, method, etc. of the whole or a part of the gene testing microreactor (test chip) and test apparatus of the present invention are consistent with the gist of the present invention. As long as it can be various.

[0024] The test chip 2 shown in FIG. 1 and FIG. 2, that is, the microreactor for gene testing, is a single chip manufactured by appropriately combining one or more members such as plastic resin, glass, silicon, ceramics, etc. It is. The vertical and horizontal sizes are usually several tens of mm and the height is several mm. Preferably, the micro flow path and the housing of the microreactor are formed of plastic resin that is easy to be molded by calorie and inexpensive, and easy to dispose of by incineration. Among these, polystyrene resin is preferable because it has excellent moldability and can easily form a detection part on a fine channel having a strong tendency to adsorb streptavidin and the like as will be described later. The microchannel is formed by a microfabrication technique with a width and height force of about m to several hundreds / zm, for example, a width of about 100 m and a depth of about 100 m. In addition, in such a microreactor, in order to optically detect a fluorescent substance or a product of a color reaction, the detection part covering at least the detection part of the microchannel on the surface of the microreactor is preferably a transparent member, preferably Must be transparent plastic. (B) and (c) of FIG. 3 show an example of a typical flow path configuration of the microreactor for gene testing of the present invention.

[0025] FIG. 10 is a system overview of a micro analysis system including a microreactor and an apparatus main body. FIG. The genetic test system of the present invention includes a micropump 11, a control device (not shown) for controlling the micropump 11, a temperature control device (not shown) for controlling the heater 4 and the Peltier element 3, and a detection device (LED6 And a photodiode 5) are integrated into a device main body 1 and a genetic testing microreactor 2 that can be attached to the device main body 1. When the sample liquid is injected into the sample storage part of the microreactor 2 in which the reagent is sealed in advance and the microphone reactor is attached to the main body 1 of the genetic test system, the mechanical connection for operating the liquid feed pump, If necessary, control electrical connections are also made. Therefore, when the apparatus main body 1 and the microreactor 2 are joined, the flow path of the microreactor is also activated. Preferably, once the analysis is started, a series of consecutive steps are taken, including sample and reagent delivery, nucleic acid amplification based on mixing, nucleic acid-probe binding reactions, reactant detection, and optical measurements. The measurement data is stored in a file together with the necessary conditions and recorded items, and nucleic acid measurement is performed automatically.

[0026] The detection device irradiates the detection unit on the flow path with measurement light such as an LED, and detects transmitted light or reflected light by optical detection means such as a photodiode or a photomultiplier tube. Device. There are various optical devices with different principles as means for optical detection, but an ultraviolet-visible spectrophotometer is desirable. Preferably, the genetic testing system of the present invention is incorporated in the apparatus main body 1 together with the liquid feeding means and the temperature control apparatus including the above-described micropump, which detects the nucleic acid contained in the sample optically. It has become the composition.

[0027] A control system related to each control of liquid feeding, temperature, and reaction, a unit responsible for optical detection, data collection and processing, together with the micropump and the optical device, the main body 1 of the gene detection system of the present invention. Constitute. The apparatus main body 1 is commonly used for samples by mounting the microreactor 2 on the apparatus main body 1. The reaction and detection such as amplification described above are performed under conditions set in advance with respect to the order of delivery, volume, timing, etc., and a gene inspection system as a program along with control of the mic pump and temperature, and data processing for optical detection. It is built into the software installed on the PC. In the conventional analysis chip, when performing different analysis or synthesis, it is necessary to configure a microphone port fluid device corresponding to the changed contents each time. Unlike this, in the present invention, desorption Replace only the above microreactor 2 possible. If it is necessary to change the control of each element, change the control program stored in the main unit 1 as appropriate.

[0028] In the genetic test system of the present invention, all components are miniaturized and are in a form that is convenient to carry. Therefore, the genetic test system is not restricted by the place and time of use, and has good workability and operability. Since a large number of micropump units used for liquid feeding are incorporated in the main body of the apparatus, the microreactor chip can be used as a disposable type.

[0029] The microreactor and gene testing system for gene testing of the present invention can be suitably used particularly for testing genes or nucleic acids. In that case, a mechanism for nucleic acid amplification is mounted on the microreactor. However, it can be said that the basic structure of nucleic acids other than genes is almost the same. For example, a reverse transcriptase storage unit may be installed. Normally, it is sufficient to change the specimen pretreatment unit, reagents, probes, etc. In that case, the arrangement and number of liquid supply elements will change.

[0030] Microreactor for genetic testing

The microreactor for gene testing of the present invention has at least a sample storage unit, a reagent storage unit, a liquid feeding / dividing unit, a micropump connection unit, and a fine channel in one chip, and the respective parts are connected with each other through the fine channel. . A pump connection is provided upstream of each container, and a micro pump separate from the microreactor is connected to these pump connections, and the driving liquid is supplied from each micro pump to thereby supply the sample in each container. The liquid (the sample liquid containing the nucleic acid to be measured) and the reagent are extruded and merged. The nucleic acid is measured by flowing the combined solution to the flow path constituting the nucleic acid amplification section provided downstream thereof, and then to the flow path constituting the detection section. Furthermore, in addition to each storage unit, flow path, and pump connection unit, each element such as a liquid feed control unit, backflow prevention unit, reagent quantification unit, and mixing unit is installed at a functionally appropriate position by microfabrication technology. ing.

[0031] Reagent storage unit

In the microreactor for gene testing of the present invention, necessary reagents 31 (including a cleaning solution) are preliminarily encapsulated in a predetermined amount in a reagent storage unit 18 in the microreactor (FIG. 2). A plurality of reagent containers 18 are provided corresponding to the plurality of reagents 31. Therefore, this departure The bright microreactor is ready for immediate use without having to fill the required amount of reagent each time it is used, so it can be tested quickly regardless of location or time. When analyzing nucleic acids in a specimen, reagents necessary for the measurement are generally known. Examples of reagents for genetic testing include various reagents used for nucleic acid amplification, probes used for detection, and coloring reagents.

[0032] Reagents and the like built in the microreactor are subjected to sealing treatment on the surface of the reagent part in order to prevent evaporation, leakage, mixing of bubbles, contamination, modification, etc., and a sealing material (sealing liquid) It is enclosed by. These sealing materials (contained in the sealing liquid container 25 in FIG. 5) are solid or gelled under refrigerated conditions in which the microreactor is stored before use. If it becomes, it will melt | dissolve and will be in a fluid state. As such a sealing material, a water-insoluble plastic material can be used. Preferably, it has a solubility power in water of S 1% or less and a melting point of 8 ° C. to room temperature (25 ° C. ) And an aqueous solution of gelatin.

[0033] Each part of the reagent storage unit and the mixing unit for mixing the reagents (and the sample storage unit if necessary) is cooled by the Peltier element 3. The Peltier element 3 may be provided on the upper surface of the chip, which is a microreactor of each part, or on both upper and lower surfaces of the chip. By cooling the reagent container or the like, it is possible to prevent the reagent from being altered by heating. Usually, if the temperature is kept below 4 ° C, the reagent can be sufficiently prevented from being altered.

[0034] · Control housing

The positive control accommodating portion accommodates a positive control that is a specific sequence for detecting the sample and in which the primer hybridizes and the sequence between them is the same as that of the sample. The negative control container contains sterilized ultrapure water instead of the specimen as a negative control.

[0035] Control measurement and flow path configuration

Conceptual diagram 3 shows the flow path configuration for sample and control measurements in the genetic testing microreactor of the present invention.

[0036] The flow path configuration of Fig. 3 (a) includes an internal control in the amplification reagent divided into three, and the specimen, the positive control, and the negative control are independent of each other. It is a conventional method in which it is amplified by flowing in a separate flow path. In the arrangement, various flow from the reagent storage section at the uppermost stream part and the flow path to the reagent division to basically three flow paths (flow paths that branch into three flow paths and reach each waste liquid storage part). The reagent is configured to flow. The analysis channel on the left is a channel for analyzing the sample, the analysis channel in the center is a channel for positive control, and the analysis channel on the right is a channel for negative control.

[0037] In the present invention, the flow path configuration of FIGS. 3 (b) and 3 (c) is employed. In the case of the flow path configuration shown in Fig. 3 (b), a 4-split technique, a 3-liquid simultaneous mixing technique, etc. are further required. In the present invention, FIG. 4 shows a specific flow path configuration in which the flow path configuration of FIG. 3 (c) is particularly preferred. In this system, a micropump is connected to the genetic test microreactor of the present invention via a pump connection part, and a fixed amount of the amplification reagent stored in the reagent storage part is sent to the flow path, and the liquid splitting means ( The first solution dividing means) divides a fixed amount of the reagent into three parts, the first reagent, the second reagent, and the third reagent, and the divided reagents (first reagent, second reagent, and third reagent). In each flow path

The liquid mixture (1) and the liquid mixture (2) are further divided into liquid feed dividing means (in this case, for convenience, the second liquid feed dividing means for dividing the liquid mixture (1) into two, the liquid mixture (2) is divided into 2 Divided into two by the third liquid feeding and dividing means), then mixed liquid (1) only, mixed liquid (1) and mixed liquid (2), mixed liquid (2) only, mixed liquid (3) In this method, each fluid is subjected to an amplification reaction in the amplification section, and the amplification product is detected in each detection section.

[0038] When the amplification reaction is performed, it is necessary to heat the heater 4 to approximately 50 ° C or higher.

The heater ₄ can be provided on the upper surface of the chip, which is a microreactor, or on both upper and lower surfaces of the chip.

[0039] In this embodiment, only four amplifying units and four detecting units are required. In that case, sample amplification Product (amplification of mixture (1) only), mixture of sample and positive control amplification product (amplification of mixture of mixture (1) and mixture (2)), positive control amplification product (mixture ( (A) amplification only) and negative control amplification products (amplification of mixture (3) only) will be detected by each probe DNA. The role for internal control is the detection of the second amplification product. That is, since the sequences obtained by the amplification reaction of the sample and the positive control and the mixture of the sample and the positive control are all the same, detection is performed using the same probe.

[0040] The detection result may be judged only when the negative control is negative, the positive control is positive, and the mixture of the sample and the positive control is positive. In other cases, there will be some error and a retest will be required. Use a positive control probe DNA to detect a reliable mixture of sample and positive control amplification products (amplification of mixture (1) and mixture (2)). If the amplification is unsatisfactory and there is no problem with amplification of the mixture (2) alone, it is possible that the amplification is disturbed due to the sample.

[0041] On the other hand, the flow path configuration shown in FIG.

A micropump is connected to the microreactor for genetic testing of the present invention via a pump connection part, and a certain amount of the sample accommodated in the sample accommodating part and a certain amount of the positive control accommodated in the positive control accommodating part, respectively. Liquid is sent to the flow path and divided into two parts by the liquid feed dividing means, and a predetermined amount of the amplification reagent contained in the reagent container is sent to the flow path, and divided into four parts by the liquid feed dividing means. Each reagent in the flow channel (0 mixed with one of the two divided specimens to produce a mixed liquid (1),

GO Mixture prepared by mixing the other of the two divided samples and one of the two divided positive controls (2)

(iii) Mixture formed by mixing with the other of the two divided positive controls (3), and

(iv) Mixture formed by mixing with the negative control sent from the negative control container (4)

Amplification reaction was performed on the mixture (1) to (4) in the amplification section, and the amplification products were This is a method of detecting by the detector.

[0042] In this embodiment, four amplifying units and four detecting units are required. That is, the amplification product of the sample (amplification of the mixture (1) only), the mixture of the sample and the positive control amplification product (amplification of the mixture (2)), the amplification product of the positive control (mixture (3)) Amplification) and negative control amplification products (amplification of the mixture (4) only) can be detected. In this case as well, the role for internal control is played by the detection of the second amplification product. In this case, the force that requires four-part fluid technology and three-liquid simultaneous mixing technology is not technically easy.

[0043] In the flow path configuration of Figs. 3 (b) and (c), there are two, three or four divisions of the fluid, and also a combination of fluids that mix and mix two or three liquids. The split and merge are not the same in the sample, positive control and negative control (in Fig. 3 (a), split and merge are the same in all cases). For this reason, for example, it is necessary to adjust the flow rate by dividing the fluid into a ratio of 2: 1, rather than 1: 1.

[0044] As described above, the mixing of the reagent and the reagent and the mixing of the specimen and the reagent may be performed at a desired ratio in a single mixing unit, or may be performed by dividing either force or both into a plurality. A merging portion may be provided and mixed so that the desired mixing ratio is finally obtained. However, it is necessary to arrange and control a micropump, a liquid feeding element, and a flow path that can accurately and exactly realize a predetermined amount of fluid to be quantified and fed, a fluid division, and a merging.

[0045] Micropump and pump connection

In the microreactor for gene testing of the present invention, a micropump 11 for feeding a predetermined amount of the contents of the sample storage unit 20, the reagent storage unit 18 and the control storage unit is provided separately from the microreactor. The micropump 11 is connected to the upstream side of the reagent storage unit 18 and supplies the driving liquid to the reagent storage unit side by the micropump 11 to push the reagent and the like into the flow path and send the liquid. The micropump unit is incorporated in the main body of the apparatus separate from the microreactor, and the microreactor is connected to the microreactor from the pump connection portion 12 by attaching the microreactor to the main body of the microreactor (see FIG. Four).

In this embodiment, a piezo pump is used as a micro pump! That is, the first flow path in which the flow path resistance changes according to the differential pressure, A second flow path whose rate of change in flow path resistance with respect to a change in differential pressure is smaller than that of the first flow path; a pressure chamber connected to the first flow path and the second flow path;

An actuator for changing the pressure inside the pressurizing chamber;

This is a piezo pump equipped with (Fig. 6). The details are described in Patent Documents 1 and 2 above.

[0047] Solution dividing means

In the present invention, when analyzing a positive control and a negative control at the same time for one sample, the reagents and the sample are divided into two or more and sent to the respective analysis channels. There is a need. The liquid feed dividing means is provided for that purpose. As shown in FIG. 4, the liquid feed dividing means includes a branched fine channel, a liquid feed control unit 13 and a backflow prevention unit (check valve 16).

[0048] The liquid feeding control unit 13 blocks the passage of the liquid until the liquid feeding pressure in the forward direction (usually the direction in which the liquid is pushed out by the pump, that is, the downstream direction) reaches a predetermined pressure, Allow fluid to pass by applying hydraulic pressure. The liquid feeding control unit 13 is composed of a portion with a narrowed channel diameter, thereby restricting the liquid that has reached the throttle channel (fine channel) 51 from one end side from passing to the other end side. The specific flow channel mode is a trickle having a cross-sectional area smaller than the cross-sectional area of these adjacent flow channels formed between these flow channels so as to connect the flow channels adjacent to both sides in series. This is realized by using a road. For example, the throttle channel 51 is formed to have a length and width of about 30 m × 30 m with respect to a channel of 150 m × 150 m connected in series on both sides.

[0049] In order to extrude the liquid from the end 51a of the narrow diameter flow path 51 to the large diameter flow path 15, a predetermined liquid feeding pressure is required for surface tension. Therefore, since the stop and passage of the liquid can be controlled by the pump pressure from the micro pump, for example, the movement of the liquid is temporarily stopped at a predetermined position of the flow path, and the force at this position is also increased at a desired timing. The pumping can be resumed.

[0050] In addition, the backflow prevention unit for preventing the backflow of the liquid in the flow path is a check valve 16 (see FIGS. 4 and 9) in which the valve body closes the flow path opening due to the backflow pressure, or the valve body is deformed. An active valve force that presses the valve body to the flow path opening by means to close the opening. [0051] As a check valve used in the flow path of the gene testing microreactor of the present invention, a microsphere is used as a valve body, and an opening formed in the substrate is opened and closed by the movement of the microsphere, thereby allowing liquid to pass therethrough. And a check valve that shuts off is preferred. Alternatively, a check valve of a type in which the opening is opened and closed by moving up and down the upper side of the opening by a flexible substrate force hydraulic pressure stacked on the substrate and extending at the end of the opening. Also good.

[0052] In the fine flow path of the microreactor for genetic testing of the present invention, the micropump, the liquid feed control unit 13 that can control the passage of the liquid by the pump pressure, and the backflow prevention that prevents the backflow of the liquid in the flow path. The division (check valve) 16 controls the division of each liquid in the branched flow path and delivers a fixed amount of liquid. Reagents and specimens are divided at an appropriate ratio by the action of the powerful liquid dividing means and the micropump 11.

FIG. 7 shows a system in which a nucleic acid amplification reagent is mixed, divided into equal parts, and allowed to flow downstream. Reagents such as a pyotin-modified chimera primer that specifically hybridizes to the gene to be detected, a DNA polymerase having strand displacement activity, and an endonuclease are housed in the reagent housings 18a, 18b, and 18c. A piezo pump 11, which is a micropump built in the main body of the apparatus separate from the microreactor, is connected to the upstream side of the storage unit by a pump connection unit 12, and is passed from each reagent storage unit to the downstream flow path 15 a. The reagent is fed by the pump.

[0054] The flow path 15a, the flow path to the next process branched from the flow path 15a, and the liquid feeding control units 13a and 13b constitute a merging portion of the flow path, and the reagent fed from each reagent storage unit Cut off the tip of the mixed solution, and after the mixing state is stabilized, divide the reagent mixed solution into three equal parts and send it to the next step. Each reagent storage unit contains more than three times the amount of reagent that flows downstream after division, and a predetermined amount of reagent mixture is sent to the three branched channels 15b, 15c, and 15d. .

[0055] Quantitative liquid feeding mechanism

A check valve is provided at an appropriate position in the flow path between the reagent storage unit, the nucleic acid amplification unit, and the detection unit. In addition to the above, it is preferable to provide a check valve at an appropriate position for preventing contamination as well as cross contamination as described above.

[0056] It is desirable to provide a check valve in order to prevent liquid backflow and accurately perform a predetermined liquid feeding. However, as a suitable mechanism using a check valve, the quantitative liquid feeding mechanism shown in FIG. 9 can be exemplified. In this mechanism, a predetermined amount of reagent is filled in the flow path (reagent filling flow path 15A) between the check valve 16 and the hydrophobic valve 13a. A branch channel 15B that branches from the reagent-filled channel 15A and communicates with the micropump 11 that feeds the driving liquid is provided.

[0057] The reagent is quantitatively fed as follows. In FIG. 9, the reagent solution 60 is first introduced into the reagent-filling flow path 15A at a solution feeding pressure (a pressure lower than a preset pressure) at which the reagent solution 60 does not pass from the check valve 16 side to the hydrophobic valve 13a first. Fill reagent solution 60 by feeding. Next, at the liquid feeding pressure that allows the reagent solution 60 to pass through the hydrophobic valve 13a first (pressure equal to or higher than a preset pressure), the micropump 11 applies the branch flow path 15B to the reagent filling flow path 15A. By feeding the driving liquid 70 in the force direction, the reagent liquid 60 filled in the reagent filling flow path 15A is pushed out from the liquid feeding control unit 15A first, and thereby the reagent liquid 60 is quantitatively fed. . It should be noted that by providing a large-volume storage section 7 in the reagent filling channel 15A, the quantitative variation is reduced. This quantitative feeding mechanism is also used for quantitative mixing of reagents and quantitative feeding of specimens.

[0058] Specimen container

The specimen container 20 into which the specimen or DNA extracted from the specimen is injected has the following configuration (see Fig. 2). The sample storage unit 20 communicates with the sample injection unit to temporarily store a sample (containing nucleic acid) and supply the sample to the mixing unit. The sample injected into the sample storage unit 20 is connected to the micropump 11 (not shown) via the pump connection unit 12, and is fed to the mixing unit immediately downstream of the amplification unit by these actions.

[0059] In addition, the portion where the sample is injected on the upper surface of the sample container (sample injection portion) is made of an elastic material such as a rubber-like material in order to prevent external leakage, infection and contamination, and to ensure sealing. It is desirable that the stopper is formed or covered with a resin or reinforcing film such as polydimethylsiloxane (PDMS).

[0060] · Sample

The sample to be measured in the present invention is not particularly limited to a biological sample itself! However, for example, most of biological samples such as whole blood, plasma, serum, buffy coat, urine, feces, saliva, sputum, etc. Most samples fall under this category. In the case of genetic testing, it remains as a nucleic acid that serves as a template for amplification reactions. Genes, DNA or RNA can be analyzed. The specimen may also have been prepared or isolated from sample forces that may contain such nucleic acids. Therefore, in addition to the above samples, cell cultures; nucleic acid-containing samples such as viruses, bacteria, molds, yeasts, plants and animals; samples that may contain or contain microorganisms, and other nucleic acids. Therefore, any sample that has the possibility of being covered is considered.

The microreactor for genetic testing of the present invention requires a very small amount of specimen as compared with the case of manual work performed using a conventional apparatus. For example, a blood sample of about 2 to 3 L is simply injected into a chip with a length and width of several centimeters. For example, in the case of a gene, the DNA is 0.001 to 100 ng.

[0062] Nucleic acid amplification unit

The form of the amplification part in which the nucleic acid amplification reaction is performed is not particularly limited, and various forms and modes are conceivable. The reaction part of the nucleic acid amplification part is basically desirably a wide liquid reservoir-like flow path, but may be a fine flow path sufficiently long for the reaction. In this case, it is desirable to provide a control means for switching the liquid feeding direction of the combined liquid of the liquids fed to the reaction section and repeatedly moving the combined liquid back and forth in the reaction section. This causes a flow velocity gradient due to viscosity from the center of the microchannel to the inner wall of the channel. Under these conditions, the diffusion of the reagent due to the switching liquid supply in the longitudinal direction of the channel becomes two-dimensional, and the gene and the reagent The probability of encountering is increased. Alternatively, a nucleic acid non-adsorbing bead is further held inside the flow channel of the amplification section, and when the nucleic acid is amplified, the bead is moved inside the flow channel to increase the amplification efficiency.

[0063], nucleic acid amplification method

In gene testing using the microreactor for gene testing of the present invention, the nucleic acid amplification method is not limited. For example, the DNA amplification technique can use the PCR amplification method which is widely used in various fields. Various conditions for implementing the amplification technique have been studied in detail, and are described in various documents including improvements. In PCR amplification, a force that requires temperature control to be raised or lowered between three temperatures has already been proposed by the present inventors, which enables temperature control suitable for a microchip (Japanese Patent Application Laid-Open No. 2004-4). —See publication 108285). This device system is used as a microarray for genetic testing according to the present invention. What is necessary is just to apply to the amplification channel of an actor. As a result, the heat cycle can be switched at high speed, and the micro flow path is a micro reaction cell with a small heat capacity, so that nucleic acid amplification can be performed in a much shorter time than the conventional method that is performed manually.

[0064] The ICAN (Isothermal chimera primer initiated nucleic acid amplification) method, which was recently developed as an improvement of PCR, has the feature that nucleic acid amplification can be performed in a short time under any constant temperature at 50 to 65 ° C. (See Japanese Patent No. 3433929). Therefore, the I CAN (registered trademark) method is a suitable amplification technique because simple temperature control is sufficient in the microreactor for genetic testing of the present invention.

[0065] The nucleic acid amplification reaction may be another PCR modification method, and the microreactor for genetic testing of the present invention is flexible enough to cope with any change in the design of the flow path. When using any nucleic acid amplification reaction, details of the technology are disclosed and can be easily introduced by those skilled in the art.

[0066] Detection unit

In the microreactor for gene testing of the present invention, a detection unit for detecting the amplified nucleic acid is provided in the flow path downstream of the nucleic acid amplification unit. Piotin-affinity protein (avidin, streptavidin) adsorbed on the detection section on the microchannel is combined with either the biotin labeled with the probe substance or the 5 'end of the primer used in the nucleic acid amplification reaction. Bind specifically. As a result, the probe labeled with piotin or the amplified nucleic acid is trapped by this detection unit.

[0067] A method for detecting the separated amplified nucleic acid is not particularly limited, but as a preferred embodiment, it is basically carried out in the following steps. That is, using the microreactor for genetic testing of the present invention, (1) DNA as a specimen, DNA extracted from specimen force, or cDNA synthesized from specimen or RNA extracted from specimen force by reverse transcription reaction, and 5 ′ position The primer modified with biotin is fed from these storage parts to the downstream fine channel. Amplifying the nucleic acid in the flow path in the nucleic acid amplification section, mixing the amplification reaction solution containing the amplified nucleic acid and the denaturing solution in the fine flow path, denaturing the amplified DNA into a single strand, amplification The processed solution obtained by denaturing the resulting DNA into a single strand is sent to the detection section in the microchannel adsorbed with the protein that has affinity for piotins, and the amplified nucleic acid is passed through the step of trapping the amplified gene. A probe DNA whose end is fluorescently labeled with FITC (fluorescein isothiocyanate) is allowed to flow through the detection section in the microchannel where the trap is trapped, and this is hybridized to the immobilized gene. (A pre-amplified gene and fluorescently labeled probe DNA may be trapped at the detector.)

(2) A colloidal gold solution whose surface is modified with an anti-FITC antibody that specifically binds to FITC is allowed to flow into the microchannel, and the gold colloid is adsorbed to the FITC-modified probe hybridized to DNA.

(3) Optically measure the concentration of gold colloid in the fine channel.

[0068] When streptavidin is immobilized in a microchannel formed on a polystyrene substrate, it is not necessary to perform a special chemical treatment. It is only necessary to apply the piotino-affinity protein on the fine channel downstream of the amplification reaction part and to adsorb the piotin-affinity protein on the channel. As a probe DNA for genetic testing, fluorescently labeled oligonucleotides are preferably used. As the DNA base sequence, a sequence that is complementary to a part of the gene base sequence to be detected is selected. By appropriately selecting the base sequence of the probe DNA, it is possible to detect with high sensitivity without being affected by the coexisting DNA and background by specifically binding to the target gene.

[0069] Known fluorescent substances such as FITC, RITC, NBD, Cy3, and Cy5 can be used as the fluorescent dye for labeling the probe. In particular, FITC anti-FITC antibodies, for example, gold colloid anti-FITC anti-mouse IgG are preferable because they are available.

[0070] The ability to measure the fluorescence of the fluorescent dye FITC It is necessary to consider the light fading of the fluorescent dye, the background noise, and the like. A method that can finally measure with high sensitivity by visible light is preferable. This is because the equipment is more versatile than fluorescence photometry, and there are few interference factors and data processing is easy.

[0071] A preferred embodiment of the microreactor for genetic testing of the present invention is as follows:

Within one chip, at least

A reagent storage section for storing reagents used in nucleic acid amplification reaction, probe binding reaction, detection reaction, etc .; A positive control accommodating part for accommodating a positive control;

A negative control accommodating portion for accommodating a negative control;

A probe container that houses a probe (for example, a probe that hybridizes to a gene to be detected amplified by a nucleic acid amplification reaction);

A fine flow path communicating with each of these accommodating portions,

An amplification section for performing a nucleic acid amplification reaction;

A detection part in the downstream flow path and

And a waste liquid storage part,

A flow path communicating with each of these accommodating portions,

A pump connection portion that can be connected to each accommodating portion and a separate micropump for feeding the liquid in the flow path;

A micropump is connected to the chip via a pump connection part, and a predetermined amount of the amplification reagent used for the nucleic acid amplification reaction stored in the reagent storage part is sent to the flow path, and divided into three by the liquid supply dividing means. The divided reagents are then added to the merging section immediately before the nucleic acid amplification section in the flow path.

GO positive control containment force mixture (2) formed by mixing with the sent positive control, and

Get

The mixed solution (1) and the mixed solution (2) are further divided into two by the liquid feeding and dividing means, and then

Only the mixture (1), the mixture of the mixture (1) and the mixture (2), each fluid of the mixture (2) only, and only the mixture (3) are amplified in the amplification section.

The amplification product generated in the nucleic acid amplification section and the probe stored in the probe storage section are sent to the detection section in the downstream flow path, mixed in the flow path and combined with the probe (or hybridization), Based on this reaction product and detected by each detection unit A waste liquid storage part that is in communication with the specimen pretreatment part and the detection part through a through hole, is a hollow chamber provided at the bottom of the microreactor, and is a sealed waste liquid reservoir that contains waste liquids and the like resulting from the measurement of the specimen. It is comprised so that it may have.

As described above, the genetic test has been described with reference to the drawings shown as an example of a preferred embodiment of the present invention, but the present invention is not limited to a powerful mode and example.

Claims

The scope of the claims

[1] At least in one chip

A positive control accommodating part for accommodating a positive control;

A negative control accommodating portion for accommodating a negative control;

An amplification section for performing a nucleic acid amplification reaction;

A detection part in the downstream flow path and

A flow path communicating with each of these accommodating portions,

Then, the mixture (1) and the mixture (2) were further divided into two parts,

Amplification reaction is performed on the fluid mixture (1) only, the mixture of the mixture (1) and the mixture (2), each fluid of the mixture (2) only, and only the mixture (3) in the amplification section. Is detected by each detection unit.

[2] In addition, a probe housing portion is arranged in communication with the flow path for accommodating a probe to be hybridized to the gene to be detected amplified by the nucleic acid amplification reaction,

Similarly, in the positive control and the negative control, those amplified products are combined or hybridized in the flow path with the probe accommodated in the probe accommodating portion. The genetic test method according to claim 1, wherein the detection is performed based on the reaction product.

At least in one chip

A positive control accommodating part for accommodating a positive control;

A negative control accommodating portion for accommodating a negative control;

An amplification section for performing a nucleic acid amplification reaction;

A detection part in the downstream flow path and

A flow path communicating with each of these accommodating portions,

A microreactor for genetic testing provided with

The amplification unit includes an amplification unit that amplifies only the mixture (1), an amplification unit that amplifies the mixture of the mixture (1) and the mixture (2), and the mixture (2). It consists of four parts: an amplification part that amplifies only the mixture and an amplification part that amplifies only the mixture (3). The detection unit includes a detection unit that detects an amplification product obtained by an amplification reaction in an amplification unit that amplifies only the mixed solution (1), and a mixture of the mixed solution (1) and the mixed solution (2). A detection unit that detects an amplification product obtained by an amplification reaction in an amplification unit that performs an amplification reaction, and a detection that detects an amplification product obtained by an amplification reaction in an amplification unit that performs an amplification reaction of only the mixture (2). Part and the mixed solution

(3) A microreactor for gene detection, characterized in that it comprises four parts: a detection part for detecting an amplification product obtained by an amplification reaction in an amplification part for amplifying only.

[4] In addition, a probe storage unit that is disposed in communication with each of the detection units and stores a probe that is hybridized to the amplification product,

4. The detection range according to claim 3, wherein each of the detection units detects a reaction product in which the amplification product and the probe delivered from the probe storage unit are combined or hybridized. Microreactor for genetic testing.

[5] The first, second and third liquid feeding and dividing means are

Branched microchannels,

A backflow prevention unit for preventing backflow of liquid in the flow path;

Consisting of

5. The microreactor for genetic testing according to claim 3 or 4, wherein the liquid feeding and the amount of the liquid feeding in the branched flow path are controlled.

[6] A device main body in which a micropump and a temperature control device are integrated with a detection device that optically detects nucleic acids, and any one of claims 3 to 5 that can be attached to the device main body. A genetic testing system comprising the microreactor for genetic testing described in 1 above, wherein nucleic acid is measured by mounting the microreactor on the main body of the apparatus.

[7] The micropump

A second flow path in which a ratio of a change in flow path resistance to a change in differential pressure is smaller than the first flow path; A pressurizing chamber connected to the first flow path and the second flow path;

An actuator for changing the pressure inside the pressurizing chamber;

The genetic test system according to claim 6, further comprising: