JP2007185101A - Biological sample-analyzing apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To carry out precise temperature control in analysis of biological samples in a simple constitution. <P>SOLUTION: A system of temperature control is changed in each step in biological sample analysis. A biological sample-analyzing apparatus is equipped with a rotatable structure having a biological sample-retaining area for retaining a biological sample, a reagent-retaining area for retaining a reagent and a reaction area for reacting the biological sample with the reagent, a rotation control mechanism for controlling rotation of the structure, a first temperature control mechanism capable of topically carrying out temperature control of a part of the structure and a second temperature control mechanism capable of carrying out temperature control of the whole structure. In the biological sample-analyzing apparatus, temperature control of the whole structure is carried out while rotating the structure in an extraction step and the structure is kept in a stationary state and topical temperature control of a part of the structure is carried out in a thermal denaturation step and temperature control of the whole structure is carried out while rotating the structure in an amplification step. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a biological sample analyzer. For example, the present invention relates to a biological sample inspection apparatus that detects the presence or absence of a nucleic acid having a specific base sequence by extracting, amplifying, and detecting a nucleic acid contained in a biological sample.

In general, in order to examine a small amount of nucleic acid extracted from a biological sample, an extraction step for extracting the nucleic acid from the biological sample, an amplification step for amplifying the extracted nucleic acid to a detectable concentration, and a nucleic acid to be detected It comprises a detection step in which a fluorescent label is labeled to detect the presence and concentration of the target nucleic acid by irradiating with excitation light and detecting fluorescence. Among these, especially in the amplification process and the detection process, the temperature range of the chemical reaction required in the process is often narrow, and strict temperature control may be required. Nucleic Acid, a method of nucleic acid amplification at a constant temperature
In the Sequence-Based Amplification (NASBA) method, after heat denaturation of nucleic acids at 65 ° C or higher, amplification and detection are performed simultaneously at a constant temperature. At this time, amplification is performed while maintaining a constant temperature between 40 and 42 ° C. Need to do.

  Japanese Patent Application Laid-Open No. 7-75544 discloses that a sample is circulated through a capillary loop passing through two thermostatic liquid baths or a capillary is connected to two heat exchangers in order to perform the thermal cycle necessary for the amplification reaction. And route the sample in a single pass through the tube, move the sample between heat exchangers that are kept stationary, or pass the sample contained in the capillary A technique for automatically moving or rotating a heat exchanger is disclosed.

JP-A-7-75544

  In the NASBA method, enzyme reagents are required for nucleic acid amplification at a constant temperature, and these reagents rapidly deteriorate in a temperature range of 40 ° C. or higher. The temperature needs to be kept below 40 ° C. On the other hand, during the amplification process at a constant temperature of 40 to 42 ° C., the allowable range of the holding temperature is smaller than that during heat denaturation, and strict temperature control is required.

  Moreover, in the technique disclosed in JP-A-7-75544, the polymerase chain reaction is premised as a nucleic acid amplification method, and a series of amplification steps can be performed only by local temperature control, but between a plurality of set temperatures. Requires a thermal cycle and requires a more complex temperature control system.

  An object of the present invention relates to performing strict temperature control in biological sample analysis with a simple configuration.

  The present invention relates to switching a temperature control system in each step in biological sample analysis. For example, a rotatable structure including a biological sample holding area for holding a biological sample, a reagent holding area for holding a reagent, a reaction area for reacting the biological sample and the reagent, and rotation for controlling the rotation of the structure A biological sample analyzer comprising: a control mechanism; a first temperature control mechanism capable of locally controlling the temperature of a part of the structure; and a second temperature control mechanism capable of controlling the temperature of the entire structure. The temperature of the whole structure is controlled while rotating the structure in the extraction process, the structure is kept stationary in the thermal transformation process, the temperature of a part of the structure is locally controlled, and the structure is rotated in the amplification process. The entire structure is temperature controlled.

  According to the present invention, strict temperature control in biological sample analysis can be performed with a simple configuration.

  For example, in the heat denaturation process, the temperature of a part of the structure is locally controlled, thereby preventing the reagent from being deteriorated by the heat of the heat denaturation process. Further, in the amplification process, by controlling the temperature of the entire structure, strict temperature control can be performed with a simple configuration. This makes it possible to perform automatic genetic testing using the NASBA method on a rotatable structure with a simple apparatus configuration.

  The above and other novel features and effects of the present invention will be described below with reference to the drawings.

  This embodiment will be described with reference to FIGS. The biological sample analyzer includes a cartridge 4 that is a rotatable structure including a biological sample container, a reagent container, and a reaction container in a temperature-controllable thermostat 1 and a rotation control mechanism that controls the rotation of the structure. It is composed of a certain centrifugal rotor 2. The centrifugal rotor 2 is rotatably supported by a motor 3. The motor 3 is connected to the centrifugal rotor 2 via a rotation transmission mechanism 3a, and the motor 3 and the rotation transmission mechanism 3a are suspended on a support base 3b. The cartridges 4 are radially arranged at six locations on the centrifugal rotor 2 as shown in FIG.

  As shown in FIG. 3, the cartridge 4 includes a plurality of sample holding ports 4a, reagent holding ports 4b, and reaction cells 5 formed in a concave portion on a transparent plate-like base material, which are provided on the base material surface. They are connected to each other by a liquid channel 4c which is a groove. The cartridge surface is coated with a non-permeable resin film to prevent liquid leakage from the biological sample and reagent ports 4a and 4b, the reaction cell 5 and the flow path 4c.

  The centrifugal rotor 2 rotates at a rotational speed of 1 to 20 rpm, stops at an arbitrary position, and performs fluorescence measurement by the fluorescence measurement unit 6 and local temperature control on the sample and reagent inside the reaction cell built in the cartridge 4. Make the operation ready. Further, it has a function of controlling the flow of the reagent and the sample by rotating at a rotational speed of 1 to 10,000 rpm and generating a centrifugal force in the cartridge 4. When the sample requires a separation operation based on density, such as blood, the centrifugation operation can be performed by the rotation operation.

  The thermostat is connected to an air circulation duct 7 as shown in FIGS. The air circulation duct 7 is connected to the heat radiating duct 11 through the Peltier element 10 provided with the heat sink / heat sinks 8 and 9, and the exhaust heat from the heat sink / heat sinks 8 and 9 is removed from the apparatus by the heat radiating fans 14 and 15. And the air controlled to a preset temperature in the thermostatic chamber is circulated by the air circulation fans 12 and 13. When the measured temperature in the thermostat is higher than the outside air temperature, 8 is heat absorption and 9 is heat dissipation. On the contrary, when the measured temperature in the thermostat is lower than the outside air temperature, the setting is switched to 8 for heat dissipation and 9 for heat absorption. Thus, the temperature controlled airflow supplied by the air circulation duct 7 uniformly controls the interior of the thermostatic chamber 1 to an arbitrary constant temperature of 20 ° C. to 50 ° C.

  In the reaction cell in the cartridge, in order to heat only the liquid inside the cell by electromagnetic induction heating, as shown in FIGS. 6 (a) and 6 (b), heating composed of a magnetic material so as to surround the reaction cell 5 A ring 16 is provided, and the heating ring 16 is fixed to each reaction cell. On the other hand, when the centrifugal rotor 2 is stationary at the initial position, an electromagnetic induction coil 17 for inducing an induction current to the heating ring 16 at the bottom of the thermostatic chamber at six positions corresponding to the cartridge 4, and a reaction cell A radiation temperature sensor 18 for measuring the internal temperature is arranged. As the radiation temperature sensor, a pyroelectric effect element, a thermopile, or the like can be used as a sensor element. However, a thermopile type radiation temperature sensor is suitable for this apparatus because it can respond at high speed and can simplify the configuration. The electromagnetic induction heating device is disposed on the elevator 19 and is configured so that it can be raised when driven relative to the stand-by state and be close to the lower portion of the heating ring 16. The electromagnetic induction coil is energized with an alternating current having a frequency of 0 to 500 kHz by a drive circuit, and thereby an induced current is induced in the heating ring to generate heat.

  The fluorescence measurement unit 6 includes a fluorescence detection unit 6a using a photomultiplier tube as a detection element, a light emitting diode 6b as an excitation light source, and a condensing lens system 6c. In this embodiment, two fluorescence measurement units are provided, and two types of phosphors can be detected. An amplification reagent containing two fluorescent reagents corresponding to each nucleic acid is reacted with a nucleic acid quantified in advance or a nucleic acid extraction solution to be examined and a nucleic acid extraction solution to be examined. By measuring the fluorescence intensity, the nucleic acid concentration to be examined can be quantified by comparison with the amplification curve of the internal control.

Next, FIG. 7 shows the configuration of the control system of the present embodiment excluding the fluorescence measuring section. The electromagnetic induction coil 17 is connected to an inverter circuit including a DC power supply 28, a switching FET 29, and a pulse generation circuit 30. The pulse signal generated by the pulse generation circuit 30 is a switching FET
29 is turned ON / OFF. At that time, a half-wave alternating current having a frequency corresponding to the pulse signal flows through the electromagnetic induction coil 17. Due to the alternating magnetic field generated thereby, an induced current flows through the heating ring 16, and the solution in the reaction cell 5 is heated by Joule heat generated by the induced current. The pulse generation circuit 30 is connected to the programmable controller 23 via the D / A conversion unit 32. The duty ratio or frequency of the ON time of the pulse signal can be changed according to control from the programmable controller 23. Thereby, increase / decrease in the Joule heat generated in the heating ring can be controlled.

On the other hand, the Peltier element driver circuit 31 controls the heat absorption amount / heat dissipation amount of the Peltier element 10 in accordance with a signal from the programmable controller 23. The temperature control of the entire thermostatic chamber 1 by the air circulation duct is performed by controlling the heat absorption amount / heat radiation amount of the Peltier element. The temperature of the thermostatic chamber is controlled via the Peltier element driver circuit 31. On the other hand, in the temperature control in the reaction cell 5 when the centrifugal rotor 2 is stationary, the heating amount is controlled through the switching FET 29. In the temperature measurement for these controls, the solution temperature in the reaction cell is detected by the radiation temperature sensor 18, the temperature measurement in the thermostatic chamber is detected by the thermocouple 21, and the signal output thereof is sent via the A / D conversion unit 22. Do. When heating the reaction cell by electromagnetic induction heating, a pulse motor connected to the elevator 19 is driven via the motor driver 25, the electromagnetic induction coil 17 fixed on the elevator 19 is brought close to the heating ring 16, and the centrifugal rotor 2 Control is made to return to the standby position during rotation. The motor driver 25 is connected to the programmable controller 23 via the pulse controller 27 together with the motor driver 26 for controlling the rotation of the centrifugal rotor 2. The programmable controller 23 implements a program describing a process described below, and controls each part including the fluorescence measuring part to carry out a genetic test.

  Next, a genetic test process according to this example will be described.

  The biological sample test apparatus described in the present embodiment is for determining whether or not a bacterial or viral gene to be tested is present in a biological sample, and measuring the concentration when it is present.

  A biological sample testing device extracts nucleic acid molecules from a sample, performs an amplification process using a primer corresponding to the base sequence of a bacterial or viral gene to be tested, and measures whether or not to amplify To determine the presence or absence of the target gene. When amplified, the concentration of the target gene is measured by measuring the amplification curve of the target gene. As an amplification method, a NASBA method which is a kind of constant temperature amplification method is adopted. The genetic testing process consists of a thermal transformation process and an amplification process.

  The inside of the thermostatic chamber including the cartridge is maintained at a constant temperature of 41 ° C. ± 0.5 ° C. while the nucleic acid solution first extracted from the biological sample is moved and held in the reaction cell.

Next, the reagent is caused to flow from the reagent port to the reaction cell by a centrifugal force generated by the rotation of the centrifugal rotor, the reagent is mixed with DNA and RNA, and the thermal transformation treatment of the DNA and RNA in the reaction cell is performed.
In RNA thermal denaturation treatment, the liquid temperature needs to be maintained at 65 ° C. ± 1 ° C., and in DNA thermal denaturation treatment, it needs to be kept at 95 ° C. ± 1 ° C., whereas it is required for amplification treatment performed after thermal denaturation treatment. The liquid temperature is 41 ° C. ± 0.5 ° C., and the reagent used in the amplification process has a property of rapidly deteriorating at temperatures higher than 41 ° C. For this reason, only the temperature inside the reaction cell is locally controlled to 65 ° C. ± 1 ° C. or 95 ° C. ± 1 ° C. at the time of thermal transformation treatment, and in the amplification process at a constant temperature after thermal transformation treatment, It is desirable to set the temperature to 41 ° C ± 0.5 ° C, which is isothermal with that in the thermostat. As described above, the temperature control required at the time of thermal transformation does not require accuracy as compared with the control temperature in amplification. In addition, since the concentration change of the fluorescence-labeled nucleic acid is detected by measuring the fluorescence intensity during the amplification process, the centrifugation is performed so that six cartridges are sequentially arranged at the positions where the two fluorescence measurement units are arranged. The rotor is rotated at 1-20 rpm. In order to perform these temperature controls, the electromagnetic induction coil provided at the bottom of the thermostatic chamber is brought close to the reaction cell by an elevator and heated to 65 ° C ± 1 ° C or 95 ° C ± 1 ° C for local temperature control. In addition, after completion of the thermal transformation treatment, the electromagnetic induction heating is stopped and the electromagnetic induction coil is lowered, and the temperature of the cartridge including the reaction cell is controlled by the thermostatic bath.

  Next, after the amplification reagent is caused to flow from the reagent port to the reaction cell by the centrifugal force generated by the rotation of the centrifugal rotor, the process proceeds to the amplification process. During the amplification process, fluorescence is measured once per minute for each cartridge by rotating the centrifugal rotor at 60 ° once every 10 seconds. The target gene is examined by the above process.

  According to the present embodiment, a simple apparatus configuration can be achieved by applying optimal temperature control procedures in each step of nucleic acid extraction, thermal denaturation, nucleic acid amplification, and detection in genetic testing, and sequentially switching them according to the progress of the process. In this way, it is possible to perform the temperature control of the above process and perform an automated genetic test using the NASBA method, which is a nucleic acid amplification method at a constant temperature.

  Next, the second embodiment will be described with reference to FIGS. Only the main differences from the first embodiment will be described below.

  In this embodiment, the electromagnetic induction heating device is arranged above the cartridge, and when performing local heating during the thermal transformation process, the electromagnetic induction coil is lowered from the standby position, and is raised at the end of the thermal transformation process to stand by. Return to position. When the electromagnetic induction coil 17 is located on the upper surface of the cartridge, since the upper wall surface 4d of the cartridge enters between the heating ring 16 and the electromagnetic induction coil 17, it is difficult to bring them close to each other. Therefore, in this embodiment, as shown in FIG. 7, a protrusion is provided on the heating ring, and the protrusion is arranged so as to be exposed on the cartridge surface, thereby making it possible to bring the electromagnetic induction coil and the heating ring close to each other. Become. Further, in this embodiment, six radiation thermometers are arranged on the bottom of the thermostatic chamber at positions corresponding to the electromagnetic induction coils, and the temperature measurement and heating of each reaction cell are performed simultaneously. In this embodiment, since the electromagnetic induction coil can be arranged on the upper surface side of the cartridge, the position adjustment becomes easier as compared with the case where the electromagnetic induction coil is arranged below the centrifugal rotor 2.

  Next, a third embodiment will be described with reference to FIG. Hereinafter, only main differences from the first embodiment and the second embodiment will be described.

  In the third embodiment, an optical heating device is used to control the local temperature of the reaction cell, and it includes a heating light source 33, an optical shutter 34, and a condenser lens system 35. In the temperature control according to the present embodiment, the heating light source 33 is turned on when the thermal transformation process is performed, and the temperature rise is controlled by opening / closing the optical shutter or controlling the power applied to the light source based on the measurement result of the radiation temperature sensor 18. Although a xenon lamp or a halogen lamp can be used as the heating light source 33, the use wavelength end of the short wavelength side of the thermopile type radiation temperature sensor 18 is around 8 μm. In order to avoid the influence of light, it is necessary to incorporate a filter that blocks wavelengths of 8 μm or more in the heating light source. In this embodiment, when switching to local heating, the heating device itself does not need to be raised or lowered only by opening and closing the optical shutter.

The front view of a biological sample test | inspection apparatus. The bottom view of arrangement | positioning of the fluorescence measurement part and cartridge on a centrifugal rotor. FIG. The side view of a biological sample test | inspection apparatus. The rear view of an air circulation duct and a heat radiating duct. Sectional drawing which shows the structure of a centrifugal rotor and an electromagnetic induction heating part. The block diagram of a control system. Sectional drawing which shows the structure of the centrifugal rotor in a 2nd Example, and an electromagnetic induction heating part. Sectional drawing which shows the structure of the centrifugal rotor and optical heating part in a 3rd Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Constant temperature bath, 2 ... Centrifugal rotor, 3 ... Motor, 3a ... Rotation transmission mechanism, 3b ... Support stand, 4 ... Cartridge, 4a ... Sample holding port, 4b ... Reagent holding port, 4c ... Liquid flow path, 5 ... Reaction Cell 6 Fluorescence measuring unit 6 a Fluorescence detecting unit 6 b Light emitting diode 6 c, 35 Condensing lens system 7 Air circulation duct 8, 9 Absorption / heat dissipation plate 10 Peltier element 11 Radiation duct, 12, 13 ... Air circulation fan, 14, 15 ... Radiant fan, 16 ... Heating ring, 17 ... Electromagnetic induction coil, 18 ... Radiation temperature sensor, 19 ... Elevator, 20 ... Sensor amplifier, 21 ... Thermocouple 22 ... A / D conversion unit, 23 ... programmable controller,
24 ... Pulse motor, 25, 26 ... Motor driver, 27 ... Pulse controller, 28 ... DC power supply, 29 ... Switching FET, 30 ... Pulse generation circuit, 31 ... Peltier element driver circuit, 32 ... D / A conversion unit, 33 ... Heating light source, 34... Optical shutter.

Claims (9)

A rotatable structure comprising a biological sample container for holding a biological sample, a reagent container for holding a reagent, and a reaction container for reacting the biological sample and the reagent;
A rotation control mechanism for controlling the rotation of the structure;
A thermostatic chamber in which the structure is disposed;
A first temperature control mechanism for controlling the temperature of a part of the structure;
A biological sample analyzer comprising a second temperature control mechanism for controlling the temperature inside the thermostatic chamber,
The structure is stationary, and the temperature of a part of the structure is controlled by the first temperature control mechanism.
A biological sample analyzer characterized in that the structure is rotated and the temperature inside the thermostatic chamber is controlled by a second temperature control mechanism. The biological sample analyzer according to claim 1,
When extracting nucleic acid from a biological sample, the structure is rotated and the temperature inside the thermostatic chamber is controlled by the second temperature control mechanism.
A biological sample analyzer characterized in that, when a nucleic acid is thermally denatured, the structure is stationary and a part of the structure is temperature-controlled by the first temperature control mechanism. The biological sample analyzer according to claim 1,
When extracting nucleic acid from a biological sample, the structure is rotated and the temperature inside the thermostatic chamber is controlled by the second temperature control mechanism.
A biological sample analyzer characterized in that when a nucleic acid is amplified, the structure is rotated and the temperature inside the thermostat is controlled by a second temperature control mechanism. The biological sample analyzer according to claim 1,
It has an analysis mechanism for analyzing nucleic acids,
A biological sample analyzer characterized in that, when analyzing a nucleic acid, the structure is rotated and the temperature inside the thermostatic chamber is controlled by a second temperature control mechanism. The biological sample analyzer according to claim 1,
A biological sample analyzer, wherein the first temperature control mechanism controls the temperature of the reaction vessel. The biological sample analyzer according to claim 1,
The first temperature control mechanism includes a magnetic material provided in the structure and an electromagnetic induction coil provided outside the structure,
A biological specimen inspection apparatus characterized by heating a magnetic material by an electromagnetic induction method. The biological sample analyzer according to claim 1,
The second temperature control mechanism includes a heat exchanger with the outside of the apparatus, piping connected to the thermostat and the heat exchanger, and an air supply mechanism in the piping,
A biological sample inspection apparatus that performs temperature control inside a thermostatic chamber by exchanging heat with the outside of the apparatus. A rotatable structure comprising a biological sample holding region for holding a biological sample, a reagent holding region for holding a reagent, and a reaction region for reacting the biological sample with the reagent;
A rotation control mechanism for controlling the rotation of the structure;
A first temperature control mechanism capable of locally controlling the temperature of a part of the structure;
A biological sample analyzer comprising: a second temperature control mechanism capable of controlling the temperature of the entire structure;
When extracting nucleic acid from a biological sample, rotate the structure, control the temperature of the entire structure,
When the nucleic acid is heat denatured, the structure is kept stationary, and the temperature of a part of the structure is locally controlled.
A biological sample analyzer characterized in that when a nucleic acid is amplified, the structure is rotated and the temperature of the entire structure is controlled. The biological sample analyzer according to claim 8, wherein
It has an analysis mechanism for analyzing nucleic acids,
A biological sample analyzer characterized in that when a nucleic acid is analyzed, the structure is rotated and the temperature of the entire structure is controlled.

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