JP2011007642A - Nucleic acid analyzer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid analyzer that eliminates aging variation of a measuring device and difference between devices using a reference sample and performs accurate fluorescent measurement.SOLUTION: In this nucleic acid analyzer, a standard reaction vessel having a fluorescent dye is installed in a sample holder of a disk-like carousel, fluorescence emitted by the reaction vessel during passing a detection unit is detected by a fluorescence detecting element while the carousel is rotating in the circumferential direction in an always lit state of a light emitting diode as a light source, and continuously analyzes a plurality of samples. Whenever the reference sample passes the detection unit, the current value of the light emitting diode is adjusted so that the fluorescence signal output of a standard fluorescence sample becomes a reference value.

Description

  The present invention relates to a nucleic acid analyzer having a detection unit for detecting fluorescence from a sample, and using a reference sample having a fluorescent dye, corrects the detection sensitivity due to instrumental error of the apparatus and changes over time of optical system components, and provides high accuracy. Realize fluorescence measurement.

  In general, in a nucleic acid amplification method, a fluorescent dye is used for labeling a nucleic acid, and a sample containing nucleic acid is analyzed by tracking changes in fluorescence intensity over time. Nucleic acid analyzers for measuring such changes in fluorescence intensity over time have been developed.

  In an analyzer, when a light emitting diode is used as a light source, it is generally considered that a constant current circuit is provided in order to keep the luminance at a set value. However, the light emitting diode has a deterioration rate with time depending on an operating current and an environmental temperature. However, the luminance decreases with time.

  Further, in order to suppress a decrease in luminance, a part of the excitation light from the light emitting diode is detected by a detection element, and the current supplied to the light emitting diode is adjusted using a current proportional to the amount of received light in the detection element, It is known to keep the brightness constant.

JP 2003-344266 A

  Nucleic acid analyzers need to have a large dynamic range and a sensitivity that can sufficiently detect a minute fluorescence signal of an extremely low concentration sample.

  The object of the present invention is to provide a standard for a constant concentration having a fluorescent dye, including the influence of the optical filter, lens, and reaction vessel over time, which are factors other than the deterioration of the light emitting diode as the light source over time. By using the sample to adjust the amount of fluorescence emitted from the reference sample to a constant level, it is possible to correct a decrease in detection sensitivity due to changes with time of components constituting the optical system and to realize highly accurate measurement. In addition, by using the reference sample, it is possible to obtain the measurement result of the same measurement sample without instrumental differences. Another object of the present invention is to stabilize the amount of fluorescent light.

  In the present invention, a reaction vessel containing a reference sample having a fluorescent dye is installed in a sample holder of a disk-shaped carousel, and the carousel rotates in the circumferential direction while a light-emitting diode as a light source is always turned on. However, in the nucleic acid analyzer for detecting the fluorescence emitted when the reaction container passes through the detection unit with a fluorescence detection element and continuously analyzing a plurality of samples, each time the reference sample passes the detection unit, the reference sample The current of the light emitting diode is adjusted so that the fluorescent light amount signal output becomes constant at a set reference value.

  ADVANTAGE OF THE INVENTION According to this invention, the nucleic acid analyzer and method which can perform a highly accurate fluorescence measurement are implement | achieved by preventing the fall of the detection sensitivity by the time-dependent change of the component of an optical system, and correct | amending the instrumental difference between apparatuses. be able to.

  By extending the life of the light emitting diode, the time to satisfy the sensitivity specification is extended.

It is a schematic block diagram which shows the 1st example of the reading unit which is the principal part of the analyzer by this invention. It is a schematic block diagram which shows the 2nd example of the reading unit which is the principal part of the analyzer by this invention. It is a figure which shows the example of the temperature control mechanism of the carousel of the analyzer by this invention. It is a figure which shows the measuring method of the optical characteristic of the sample of the analyzer by this invention. It is an internal block diagram of the data processing part of the analyzer by this invention. It is a flowchart of fluorescence light quantity signal processing and fluorescence light quantity correction of the analyzer according to the present invention. It is lifetime data according to the forward current of a light emitting diode. It is a forward current-relative luminous intensity characteristic figure of a light emitting diode. It is an ambient temperature-relative luminous intensity characteristic view of a light emitting diode.

  Embodiments of the present invention will be described below with reference to the accompanying drawings.

  FIG. 1 is a schematic configuration diagram of a reading unit that is a main part of a nucleic acid analyzer according to an embodiment of the present invention. In FIG. 1, the light emitted from the light emitting diode 2 is converted into parallel light by the first lens 3, and after the extra wavelength region is cut by the excitation band pass filter 4, the second lens 7 forms a sample holder of the carousel 8. The sample is condensed on the sample in the reaction vessel 9 which is a transparent vessel supported. Further, a part of the excitation light is irradiated onto the detection element 6 for excitation light monitoring by the half mirror 5 arranged at the subsequent stage of the excitation bandpass filter 4.

  The reaction vessel 9 is housed in a disk-shaped carousel 8, and a motor 25 for rotating the carousel 8 is driven by a motor drive circuit 26. The motor drive circuit 26 has a rotation speed designated by the data processing unit 19 and drives the motor so that the designated rotation speed is obtained. When the carousel 8 starts to rotate at the specified rotation speed, the reaction vessel 9 emits fluorescence when passing the excitation light collected from the second lens 7.

  The fluorescence emitted from the reaction vessel 9 is converted into parallel light by the third lens 10, and after the excess wavelength region is cut by the fluorescence bandpass filter 11, the fluorescence detection element 13 is irradiated by the fourth lens 12. By detecting the fluorescence by the fluorescence detection element 13, the optical characteristic of the sample is measured.

  The detection signal from the fluorescence detection element 13 is amplified by the fluorescence AMP 15. The multiplexer 17 selects either the excitation light AMP 16 signal or the fluorescence AMP 15 signal, and the ADC 18 performs AD conversion. The ADC 18 performs AD conversion at a constant period from a signal from the timer. The period of the timer is specified by the data processing unit.

  Data from the ADC 18 is read by the data processing unit 19 as excitation light data for monitoring the excitation light amount and fluorescence data for monitoring the fluorescence light amount.

  The fluorescent light amount data read by the data processing unit 19 is subjected to DA conversion by the DAC 20 by setting a current value to be passed through the light emitting diode 2 after data processing. The DAC 20 performs DA conversion at a constant period according to a signal from the timer. The period of the timer is specified by the data processing unit 19.

  The DA-converted output is connected to the input of the comparator 21 and applied to the detection resistor 24 for the current flowing in the light-emitting diode 2 as a reference voltage 23 for supplying the power supply voltage of the light-emitting diode 2 input to the other side of the comparator 21. Compared with the voltage, a voltage corresponding to the difference is output to the control terminal of the transistor (FET) 22 directly connected to the light emitting diode 2 to amplify the current flowing through the light emitting diode 2.

  An example of a reading unit 1 according to the present invention will be described with reference to FIG. The reading unit 1 of this example includes a disc-shaped carousel 8, a rotation mechanism 27 for rotating the carousel 8, at least one detection unit 14 arranged along the circumference of the carousel 8, and a light source of the detection unit 14 A reaction container 34 containing a reference sample having a fluorescent dye for adjusting the luminance of the light emitting diode 2, a photo interrupter 31 for detecting the rotational position of the carousel 8, and a gripper 32 for conveying the reaction container 9. And have.

  The carousel 8 is constituted by a disk-shaped aluminum alloy disk, and is rotatable around a central axis. On the carousel 8, a large number of sample holders 28 and light shielding plates 30 are provided along a circumferential edge. The sample holder 28 holds the reaction container 9 in which the sample solution to be analyzed is stored, and the same number of light shielding plates 30 as the sample holder 28 are provided. The sample holder 28 and the light shielding plate 30 are arranged at equal intervals (equal angles) along the edge of the carousel 8. As illustrated, the light shielding plate 30 is disposed between the sample holders 28 so as to face the sample holders 28.

  The light shielding plate 30 and the photo interrupter 31 constitute a rotational position detection mechanism for detecting the rotational speed and rotational angle of the carousel 8. The photo interrupter 31 has a light emitting element and a light receiving element facing each other. When the light shielding plate 30 passes, the light from the light emitting element is blocked by the light shielding plate 30. The light shielding plate 30 is provided between the sample holders 28 of the carousel 8 so that the rotational speed and rotational position of the carousel 8 can be known. Based on the output of the photo interrupter 31, the timing of the start of sampling for fluorescence measurement is measured.

  The detection units 14 are arranged at equal intervals along the circumference of the carousel 8. In the example of FIG. 2, five detection units 14 are provided, but a number other than five detection units 14 may be provided. The detection unit 14 is replaceable and can be freely attached and detached.

  The rotation mechanism 27 has a stepping motor 25, transmits the rotation of the stepping motor 25 to the carousel 8 by a belt, and rotates the carousel 8. By using the stepping motor 25, the carousel 8 can be rotated at a constant speed. Further, by using the stepping motor 25, it is possible to rotate it by a desired rotation angle. When the number of reaction vessels 9 held by the sample holder 28 is n, the carousel 8 can be rotated by 1 / n rotation (360 / n degrees).

  The carousel 8 of the present embodiment includes at least one reaction vessel 34 having a reference sample for adjusting the luminance of the light emitting diode 2 of the detection unit 14.

  This reference sample has a known concentration. Fluorescence measurement is performed every time the carousel 8 rotates at a constant speed and passes through the detection unit 14, that is, every time it rotates once. The data processor compares the amount of fluorescence obtained from the measurement result with the set amount of fluorescence, and sets the current flowing through the light emitting diode. The current flowing through the light emitting diode is corrected by adjusting the luminance so that the amount of fluorescent light emitted from the reference sample becomes a specified value. The current of the light emitting diode whose luminance is adjusted by the reference sample 34 maintains the set value until the carousel 8 makes one rotation and then passes through the detection unit 14. While the reaction container 9 containing the sample solution to be analyzed passes through the detection unit 14, the luminance adjustment of the light emitting diode 2 is not performed.

  The gripper 32 conveys the reaction container 9 containing the sample solution to be analyzed to the sample holder 28, and conveys the reaction container that has been analyzed to the disposal hole 33.

  The temperature control mechanism of the carousel 8 will be described with reference to FIG. In this example, a rubber heater 35 that controls the temperature of the sample in the reaction vessel 9 and a thermistor 36 that monitors the temperature of the sample in the reaction vessel are provided. The temperature of the thermistor 36 for temperature monitoring is set on the inner surface of the carousel 8, and the data processing unit 19 processes the output of the temperature by the PI control of the temperature of the entire carousel 8. adjust. By setting the sample temperature in the reaction vessel 9 to a set value, the amount of fluorescent light emitted from the sample can be detected in a stable state. In addition, since the temperature of the detection unit 14 is simultaneously controlled by heat conduction from the temperature-controlled carousel 8, the temperature of the light-emitting diode 2 in the detection unit 14 is also stabilized at a constant temperature, and the fluorescence of the reaction vessel 34 serving as a reference sample. It is possible to keep the light intensity at a specified value. That is, by making the carousel 8 constant temperature, the amount of fluorescent light emitted from the sample is stabilized and the life of the LED is extended.

  With reference to FIG. 4, a method for measuring optical properties of a sample according to the present invention will be described. When the reaction container 9 containing the sample solution to be analyzed is attached to the sample holder 28, the speed of the carousel 8 is rotated at a constant speed. Around the carousel 8, five detection units 14 are arranged. From the detection unit 14, the excitation light from the light emitting diode 2 is constantly applied to the carousel 8. When the carousel 8 rotates and the reaction vessel 9 containing the sample passes overhead, each detection unit 14 is removed from the reaction vessel 9. Measure fluorescence. The timing of the fluorescence measurement is when the light shielding plate 30 does not block the light emitted from the light emitting element of the photo interrupter 31, that is, when the light is collected (1), the light of the light emitting element is collected (1) to the light receiving element of the photo interrupter 31. The output of the photo interrupter 31 shifts from the low level (3) to the high level (4). When the output of the photo interrupter 31 becomes high level (4), the sampling process 41 of the fluorescent light quantity is started for a certain sampling time (6) after the elapse of the waiting time (5). The reaction vessel 9 that passes over the detection unit 14 has a structure that is positioned so as to pass through the detection unit 14 within the sampling time (6).

  Since the reaction vessel 9 is held and rotated by the sample holder 28 of the carousel 8, the sampled data has a fluorescence amount of the sampled data (7) when passing through the center of the light emitted from the detection unit 14. Is captured by the curve of the fluorescence light amount signal that maximizes. After the sampling is completed, the data stored in the memory 40 is processed by the peak detection processing 47 to perform peak detection. The sample holders 28 of the carousel 8 are circumferentially spaced at equal intervals, and since the carousel 8 is rotated at a constant speed, the trigger interval (9) is constant.

  In the embodiment of the present invention, the amount of fluorescence in the reaction vessel 34 of the reference sample is measured every time the reaction vessel 34 in which the reference sample having fluorescence is stored passes through the detection unit 14, and the data processing unit 19 performs FIG. The peak value is calculated as indicated by point A, and the current flowing through the light emitting diode 2 is adjusted so that the calculated peak value of the fluorescent light amount (7) is constant at the reference value, thereby correcting the fluorescent light amount. .

  In the reaction vessel 34 in which the reference sample is stored, after the fluorescence measurement, a process of adjusting the amount of fluorescence in the reaction vessel of the reference sample to a reference value is performed. This will be described later with reference to FIGS. I do.

  The operation cycle of the carousel 8 includes a container setting period in which the carousel 8 is stopped, the reaction container 9 is discharged, transported, and installed, and a fluorescence measurement period in which fluorescence is measured while rotating the carousel at a constant speed. The length of the container setting period and the fluorescence measurement period is constant, and is repeated at a predetermined cycle. In the embodiment of the present invention, the container setting period is 10 seconds, the fluorescence measurement period is 20 seconds per rotation, and continuous measurement is performed in one cycle 30 seconds.

  More specifically, the following operation is repeated. (A) Rotation / detection → (b) Discharge / conveyance → (c) Adjustment of current flowing in the light emitting diode 2 and (a) → (b) → (c) → (a) → (b) → (c) → Repeat (a).

  Each time the carousel 8 makes one revolution, the reaction vessel 9 passes through all the detection units 14 arranged circumferentially. Each detection unit 14 is assigned fluorescence of a wavelength to be measured. Each detection unit 14 independently detects the fluorescence of the wavelength assigned to itself. Therefore, when the carousel 8 rotates once, five components included in each sample can be analyzed. It is assumed that the same specimen is not collected in each reaction container 9. Data measured by each detection unit 14 is accumulated in the external computer 29 as a change with time of the reaction solution, and further output to the outside as a quantitative analysis result. The data processing unit 19 according to the embodiment of the present invention has the data processing function shown in FIG. 5 and performs an operation according to the flowchart shown in FIG. Again, in the embodiment of the present invention, fluorescence measurement is performed each time the reaction container 34 containing the reference sample passes through each detection unit 14, and the amount of fluorescence in the reaction container 34 of the reference sample is the reference value. The value of the current flowing through the light emitting diode 2 is adjusted and corrected so as to be constant.

  First, data processing of the fluorescence light amount signal will be described. The data processing unit 19 selects the output of the fluorescence AMP 15 from the multiplexer 17 in order to obtain the fluorescence light quantity signal of the reaction vessel 9 (step (a)), and then sets the motor drive circuit 26 of the carousel 8 next. The carousel 8 starts to rotate (step (b)). Next, along with the rotation of the carousel 8, the shielding plate 30 detects a change from the low level to the high level when passing through the photo interrupter 31, and outputs to the data processing unit 19 (step (c)). Next, in order to provide a waiting time until the start of data measurement of the fluorescence light amount signal, the timer 53 is controlled to wait for a set time.

  Next, the timer 53 is controlled to set the AD conversion cycle time. As a result, the timer outputs an AD conversion start signal to the ADC 18. In the embodiment of the present invention, for example, 16 bits and the conversion time are 4 μs to 50 μs. From the AD conversion start signal, the ADC 18 performs AD conversion of the fluorescence light amount signal, and notifies the data processing unit 19 that AD conversion has been completed. When the data processing unit 19 receives the AD conversion end signal from the ADC 18, the data processing unit 19 reads the converted data (step (f)). The read data is stored in the memory 40 for each AD conversion (step (g)), and the AD conversion is repeated until the set sampling number is completed (step (h)).

  After completion of the sampling in step (h), in order to calculate the measured fluorescence light quantity of the sample in the reaction vessel 9, the sampled data is read from the memory 40, and the averaging process 54 is performed so that the set sampling period is obtained. In the present invention, in the embodiment, the data can be averaged so that the data interval after averaging is 2 ms (step (i)). For data averaging, the average number can be set arbitrarily before the start of measurement. Next, a smoothing process 55 is performed to smooth the averaged data waveform (step (j)). In the present invention, the Box-Jenkins method or the Savitzky-Golay smoothing method is used in the embodiment.

  Next, the fluorescent light quantity value of the sample is obtained. In order to obtain the peak value of the fluorescence light amount waveform in FIG. 4, the maximum value of the data subjected to the fitting process 56 with the Gaussian waveform, that is, the peak detection process 57 is performed. In the example of the present invention, Gaussian function fitting is used (steps (k) and (l)). The procedure up to the fluorescence measurement is the same in both the reaction vessel 9 for analysis and the reference sample reaction vessel 34 for adjusting the current flowing in the light emitting diode 2. In the reaction vessel 9 that performs the analysis, it is accumulated in the external computer 29 after the peak detection, and is further output to the outside as a quantitative analysis result.

  Next, a method for adjusting the value of the current flowing through the light emitting diode 2 in the correction of the amount of fluorescent light in the reaction vessel 34 in which the reference sample is stored will be described. The correction of the amount of fluorescent light of the reference sample is performed by performing control so that the amount of fluorescent light of the reference sample is always constant at a set reference value, thereby realizing a highly accurate analysis result calculation. The amount of fluorescent light in the reaction vessel 34 containing the reference sample obtained in step (l) is compared with the set reference value of the amount of fluorescent light to calculate the amount of change in the amount of fluorescent light. The data processing unit 19 calculates the amount of change by 1- (PFL / reference value) (step (m)). Thus, the reference current value * K (1 + REV) is calculated in order to output the setting of the current value flowing through the light emitting diode 2 to the DAC 20 (step (n)).

  The calculation result is compared with the predetermined value 1 (step (o)), and when the set current value calculated in step (n) is equal to or less than the predetermined value 1, the current calculated in step (n) The set value is output to the DAC 20 (step (p)). In the embodiment of the present invention, the DAC 20 has 8 bits. While being output to the DAC 20 in step (q), for example, it is used in a stable state of the T period in FIG. 7, and there is no significant fluctuation, but in particular in the environment where the device is installed, It is effective for changing the temperature at room temperature.

  In step (o), when the current set value calculated in step (n) exceeds the predetermined value 1, the current set value is compared with the predetermined value 2 (step (q)). When the current set value calculated in step (n) exceeds the predetermined value 1, since there is no relative relationship as in the characteristic of FIG. 8, the amount of change is corrected with respect to the current set value set immediately before, Output to the DAC 20 (step (r)). The predetermined value 1 can be a time when the luminous intensity starts to decrease due to deterioration with time, for example, point B in FIG.

  Next, when the current set value calculated in step (n) in step (q) exceeds a predetermined value, the predetermined value 2 warns an alarm before the fluorescence detection sensitivity decreases and becomes out of specification (step (s )), That is, the life of the light emitting diode 2 is notified.

  In the present embodiment, it has a function of discriminating a decrease in the amount of fluorescent light from the detected amount of fluorescent light and a function of discriminating an increase in the set current value to the DAC 20. For example, when the point B in FIG. As for the alarm to the user, the data processing may shift from step (o) to step (q) at point B to warn the user of an early alarm. Further, since it is possible to infer from the life characteristics of the light emitting diodes used to warn the alarm of step (s), the transition from step (o) to step (q) is detected, and thereafter the timer It is also possible to issue an alarm with the set value. In another embodiment, when the fluctuation of the amount of fluorescent light in the stable state of the T period in FIG. 7 is within the range with respect to the required specifications and does not cause any problem, the T period is determined by the step (p ) May be a set fixed value stored in the memory 40. The processing after step (q) may be performed after catching the change with time of the optical system components, particularly the decrease in the amount of fluorescent light due to the deterioration of the light emitting diode 2, that is, after the fixed value T period.

  As an effect of the present invention, description will be given of extending the life of the light-emitting diode 2 that is a light source. FIG. 7 shows a life curve by using the current value If flowing in the light emitting diode 2 at a constant value. It can be seen that the luminous intensity decreases from an earlier time when the current value If is 20 mA than when the current value If is 10 mA. This is because, as shown in FIG. 8, when the forward current If increases, the luminous intensity increases, and the deterioration of the sealing resin is accelerated. The lifetime of the light emitting diode 2 is considerably longer than that of an incandescent bulb, and the device itself can be used almost permanently. In most cases where the light-emitting diode 2 becomes unusable, the internal gold wire is broken due to metal oxidation / degradation, overheating or impact in the electrode portion. The product life refers to a point in time when the translucency is lowered due to the deterioration of the sealing resin and the light emission amount becomes below a certain level. In general, the lifetime of a light emitting diode is defined as the time until the total luminous flux decreases to 70% of the initial total luminous intensity, or the luminous intensity decreases to 70% of the initial luminous intensity.

  FIG. 7 can be considered as a life curve of the light emitting diode 2 in the constant current control. In the embodiment of the present invention, since the reaction vessel 9 for the analysis sample and the reaction vessel 34 for the reference sample are controlled at a constant temperature, the detection unit is also stable at a constant temperature at the same time. During the T period in FIG. 7, the current is controlled in the same manner as the constant current. Therefore, since the operation time for starting the decrease in luminous intensity can be calculated when the current flowing through the diode as shown in FIG. 7C is 10 mA and when the current flowing through the diode as shown in D is 20 mA, the reference current to be set is set. 7 is set as the predetermined value 1 in step (o) of FIG. 6, and the lighting time of the light emitting diode 2 is measured and stored, thereby comparing and determining the usage time. It is also possible to start step (q) or (o).

  In addition, the time for the user to perform analysis per day can be easily calculated in consideration of the processing time of the process until the analysis result is calculated. It is also possible to compare and determine the number of times of analysis as a predetermined value and perform the processing of step (o) or step (q).

  In the embodiment of the present invention, it is necessary to detect a very weak amount of fluorescent light, and in order to obtain the lowest sensitivity, the light intensity is required to be higher than the light intensity determined to be a general lifetime. In the present invention, the amount of fluorescent light in the reference sample reaction vessel 34 is corrected by the time-dependent change of the components constituting the optical system. Therefore, when the light emitting diode 2 begins to deteriorate and the amount of fluorescent light in the reference sample reaction vessel 34 decreases, the amount of fluorescence decreases. Since the forward current If is increased by the amount and the process of step (r) is performed, it is possible to lengthen the time during which the amount of fluorescent light is always kept constant, so that the time during which the light emitting diode 2 can be used at a constant luminous intensity is increased. . That is, the lifetime of the light emitting diode 2 can be extended by increasing the time for satisfying the performance as a nucleic acid analyzer.

  FIG. 9 shows the luminous intensity characteristics of the light emitting diode 2 with respect to the ambient temperature. It can be seen that the change in the ambient temperature has a great influence on the luminous intensity. Therefore, although the temperature control of the reaction vessel has been described with reference to FIG. 3, in the embodiment of the present invention, the temperature of the carousel is controlled so that the sample reaction vessel 9 to be analyzed and the reference sample reaction vessel 34 are kept at a set temperature. By doing so, the ambient temperature of the light emitting diode 2 is also stabilized and effective. In addition, the carousel rotates at the time of measurement, and the amount of fluorescent light of the reference sample is corrected every time it is rotated. Therefore, it is possible to correct for the influence of changes in the environmental temperature of the day, and high-accuracy measurement is possible.

  As another example, if the temperature change in the ambient environment of the light source is large, in the embodiment of the present invention, the temperature in the apparatus is also monitored. It is also possible to control 8 to be constant at a reference set temperature.

  In FIG. 6 (o), the current set value is compared with a predetermined value, but the fluorescent intensity set value may be compared with a predetermined value. The same applies to (q).

  The present invention provides a light amount control method related to fluorescence measurement of a nucleic acid analyzer. By using the method of the present invention, it can also be applied in the technical field using an absorbance method for quantitatively measuring the concentration of a target substance in a sample solution, and measuring the concentration of a component of drinking water in the food field or the environment It is also useful for the safety of drinking water and the measurement of metallic elements in rivers and industrial wastewater.

DESCRIPTION OF SYMBOLS 1 Reading unit 2 Light emitting diode 3 1st lens 4 Excitation band pass filter 5 Half mirror 6 Excitation light signal detection element (detection element for excitation light monitoring)
7 Second lens 8 Carousel 9 Analysis sample reaction vessel 10 Third lens 11 Fluorescence band-pass filter 12 Fourth lens 13 Fluorescence signal detection element (fluorescence detection element)
14 Detection unit 15 AMP for fluorescence
16 AMP for excitation light
17 Multiplexer 18 ADC
19 Data processor 20 DAC
21 Comparator 22 Transistor (FET)
23 Reference voltage 24 Detection resistor 25 Stepping motor (motor)
26 Motor drive circuit 27 Rotating mechanism 28 Sample holder 29 External computer 30 Light blocking plate 31 Photo interrupter 32 Gripper 33 Discarding hole 34 Reference sample reaction vessel 35 Rubber heater 36 Thermistor 40 Memory 41 Sampling process 42 Timer control 43 Timer 44 Averaging process 45 Smoothing process 46 Fitting process 47 Peak detection process 48 Peak variation calculation process 49 Current value setting process

Claims (14)

A disk-shaped carousel, a rotating mechanism for rotating the carousel, at least one detection unit arranged along the circumference of the carousel, and a rotational position detecting mechanism for detecting the rotational position of the carousel; The carousel is configured to support a plurality of reaction containers storing samples at equal intervals along a circumferential direction, and the detection unit is a light source that generates excitation light to irradiate the reaction container And a nucleic acid analyzer comprising a fluorescence detection element for detecting fluorescence emitted from the sample stored in the reaction container,
A nucleic acid analyzer characterized in that the current flowing through the light source is controlled so that the amount of fluorescent light of a fluorescent sample serving as a reference whose concentration is known is constant with a preset reference value. The nucleic acid analyzer according to claim 1,
A nucleic acid analyzer comprising a mechanism for isolating the carousel. The nucleic acid analyzer according to claim 1,
A nucleic acid analyzer characterized by repeatedly performing fluorescence detection performed by rotating a carousel, loading and unloading of a container, and current control of a light source in this order.   2. The nucleic acid analyzer according to claim 1, wherein an alarm is issued when the controlled current value exceeds a maximum standard current. A disk-shaped carousel, a rotating mechanism for rotating the carousel, at least one detection unit arranged along the circumference of the carousel, and a rotational position detecting mechanism for detecting the rotational position of the carousel; Have
The carousel is configured to support n reaction vessels containing samples at regular intervals along the circumferential direction.
The rotational position detection mechanism sets n shielding plates mounted on the carousel at equal intervals along the circumferential direction, detection means for detecting the passage of the shielding plates, and setting the reaction vessel. A heating unit for adjusting the temperature and a temperature sensor for monitoring the temperature of the reaction vessel;
The detection unit includes a light source that generates excitation light that irradiates the reaction container, a unit that irradiates a part of the light source to the excitation light detection element, and a fluorescence that detects fluorescence emitted from a sample stored in the reaction container. A sensing element;
An excitation light signal amplifier for amplifying a signal from the excitation light detection element; a fluorescence signal amplifier for amplifying a signal from the fluorescence detection element; and the excitation light signal widther according to a signal to be measured. Means for switching, an AD converter for AD converting the signal output by switching the signal from the detection element;
Storage means for storing data output from the AD converter; means for calculating a peak value of a sample to be measured based on the stored fluorescence light amount signal data; and storing the calculated peak value in an external computer; Furthermore, in a nucleic acid analyzer that has a means for outputting to the outside as a quantitative analysis result and analyzes a sample containing nucleic acid by tracking changes in fluorescence intensity over time,
As a means for correcting the time-dependent change of the optical components constituting the detection unit, the reference fluorescent sample is controlled so that the fluorescent light amount of the reference fluorescent sample is always set to the set reference value of the fluorescent light amount. Means for supporting at least one contained reaction vessel on the carousel;
The reaction container containing the reference fluorescent sample is measured each time the carousel rotates and passes through the detection unit, and after calculating the amount of change between the calculated fluorescent light amount and the reference value of the fluorescent light amount A data processing unit that adjusts a current value that flows through the light source with respect to a reference current value that is set according to the amount of change from the reference value; and a current value of the light source that is set to adjust the current that flows through the light source And a DA converter for DA-converting the nucleic acid. The nucleic acid analyzer according to claim 5, wherein
In the detection unit, means for condensing the excitation light of the light source that generates the excitation light, means for cutting the condensed excitation light other than a necessary wavelength, and a part of the light source as an excitation light detection element Means for condensing the light that has passed through the means for irradiating to the reaction vessel, means for condensing the fluorescence emitted from the sample, means for cutting the collected fluorescence other than the fluorescence of the required wavelength, and the cut A nucleic acid analyzer comprising: means for condensing the emitted fluorescence to irradiate the fluorescence detection element. The nucleic acid analyzer according to claim 5 or 6,
The nucleic acid analyzer, wherein the shielding plate is disposed between samples so as to face a reaction container holder that supports the reaction container. The nucleic acid analyzer according to claim 5, wherein
Means for comparing the calculated current amount of fluorescence and the reference value, and comparing the set current value of the light source calculated according to the change amount with the reference value with a predetermined value;
If the calculated current set value is smaller than a predetermined value as a result of the comparison by the comparing means, the calculated current set value is output to the DA converter, and if larger than the predetermined value, the previously set value is set. A nucleic acid analyzer comprising: a means for outputting a current value of a light source obtained by multiplying a current set value of the light source by a change amount of the amount of fluorescent light to a DA converter. The nucleic acid analyzer according to claim 8, wherein
After calculating the amount of change between the calculated amount of fluorescence and the reference value, when the calculated current setting value is larger than a predetermined value, and a means for comparing with a predetermined value for informing the life of the light source;
When the predetermined value for notifying the life of the light source is small, the current value of the light source obtained by multiplying the current setting value of the light source previously set by the amount of change in the amount of fluorescent light is output to the DA converter, A nucleic acid analyzer characterized by means for warning an alarm when a predetermined value for informing the life is large. A disk-shaped carousel, a rotating mechanism for rotating the carousel, at least one detection unit arranged along the circumference of the carousel, and a rotational position detecting mechanism for detecting the rotational position of the carousel; Have
The carousel is configured to support n reaction vessels containing samples at regular intervals along the circumferential direction.
The rotational position detection mechanism sets n shielding plates mounted on the carousel at equal intervals along the circumferential direction, detection means for detecting the passage of the shielding plates, and setting the reaction vessel. A heating unit for adjusting the temperature and a temperature sensor for monitoring the temperature of the reaction vessel;
The detection unit includes a light source that generates excitation light that irradiates the reaction container, a unit that irradiates a part of the light source to the excitation light detection element, and a fluorescence that detects fluorescence emitted from a sample stored in the reaction container. A sensing element;
An excitation light signal amplifier for amplifying a signal from the excitation light detection element; a fluorescence signal amplifier for amplifying a signal from the fluorescence detection element; and the excitation light signal widther according to a signal to be measured. Means for switching, an AD converter for AD converting the signal output by switching the signal from the detection element;
Storage means for storing data output from the AD converter; means for calculating a peak value of a sample to be measured based on the stored fluorescence light amount signal data; and storing the calculated peak value in an external computer; Furthermore, in a nucleic acid analyzer that has a means for outputting to the outside as a quantitative analysis result and analyzes a sample containing nucleic acid by tracking changes in fluorescence intensity over time,
As a means for correcting the time-dependent change of the optical components constituting the detection unit, the reference fluorescent sample is controlled so that the fluorescent light amount of the reference fluorescent sample is always set to the set reference value of the fluorescent light amount. Means for supporting at least one contained reaction vessel on the carousel, and a reaction vessel containing the reference fluorescent sample, each time the carousel rotates and passes through the detection unit, performs fluorescence measurement, Means for comparing the calculated peak value of the amount of fluorescent light with a predetermined value of the amount of fluorescent light determined in order to detect a decrease in the amount of fluorescent light due to deterioration of the light source from the lifetime curve of the light source used;
A unit that outputs a preset fixed current value when the fluorescence light amount is smaller than a predetermined value, and a change amount between the calculated fluorescence light amount and the reference value of the fluorescence light amount when the determination result is greater than the predetermined value, based on the determination result. After the calculation, the data processing means for adjusting the current value flowing through the light source with respect to the set reference current value according to the amount of change from the reference value, and the light source set to adjust the current flowing through the light source A nucleic acid analyzer comprising: a DA converter that converts the current value of DA into a DA converter. A disk-shaped carousel, a rotating mechanism for rotating the carousel, at least one detection unit arranged along the circumference of the carousel, and a rotational position detecting mechanism for detecting the rotational position of the carousel; Have
The carousel is configured to support n reaction vessels containing samples at regular intervals along the circumferential direction.
The rotational position detection mechanism sets n shielding plates mounted on the carousel at equal intervals along the circumferential direction, detection means for detecting the passage of the shielding plates, and setting the reaction vessel. A heating unit for adjusting the temperature and a temperature sensor for monitoring the temperature of the reaction vessel;
The detection unit includes a light source that generates excitation light that irradiates the reaction container, a unit that irradiates a part of the light source to the excitation light detection element, and a fluorescence that detects fluorescence emitted from a sample stored in the reaction container. A sensing element;
An excitation light signal amplifier for amplifying a signal from the excitation light detection element; a fluorescence signal amplifier for amplifying a signal from the fluorescence detection element; and the excitation light signal widther according to a signal to be measured. Means for switching, an AD converter for AD converting the signal output by switching the signal from the detection element;
Storage means for storing data output from the AD converter, means for calculating a peak value of a sample to be measured based on the stored fluorescence light quantity signal data, and storing the calculated peak value in an external computer In addition, in the nucleic acid analyzer that has a means for outputting to the outside as a quantitative analysis result and analyzes a sample containing a nucleic acid by tracking changes in fluorescence intensity over time,
As a means for correcting the time-dependent change of the optical components constituting the detection unit, the reference fluorescent sample is controlled so that the fluorescent light amount of the reference fluorescent sample is always set to the set reference value of the fluorescent light amount. Means for supporting at least one reaction vessel stored in the carousel, means for measuring the time during which the light source is turned on and storing it in the memory, and the life curve of the light source to be used become the reference due to deterioration of the light source A time for starting a decrease in the amount of fluorescent light of the fluorescent sample as a predetermined value, and a means for comparing the lighting time of the measured light source with the predetermined value;
When the measured lighting time does not reach the predetermined value, the means for outputting a fixed current value set in advance, and when the predetermined time is exceeded from the determination result, the calculated amount of fluorescence and fluorescence A nucleic acid, characterized in that, after calculating the amount of change from the reference value of the light amount, means for outputting to the DA converter the current value of the light source obtained by multiplying the current set value of the light source previously set by the amount of change in the amount of fluorescent light Analysis equipment. A disk-shaped carousel, a rotating mechanism for rotating the carousel, at least one detection unit arranged along the circumference of the carousel, and a rotational position detecting mechanism for detecting the rotational position of the carousel; Have
The carousel is configured to support n reaction vessels containing samples at regular intervals along the circumferential direction.
The rotational position detection mechanism sets n shielding plates mounted on the carousel at equal intervals along the circumferential direction, detection means for detecting the passage of the shielding plates, and setting the reaction vessel. A heating unit for adjusting the temperature and a temperature sensor for monitoring the temperature of the reaction vessel;
The detection unit includes a light source that generates excitation light that irradiates the reaction container, a unit that irradiates a part of the light source to the excitation light detection element, and a fluorescence that detects fluorescence emitted from a sample stored in the reaction container. A sensing element;
An excitation light signal amplifier for amplifying a signal from the excitation light detection element; a fluorescence signal amplifier for amplifying a signal from the fluorescence detection element; and the excitation light signal widther according to a signal to be measured. Means for switching, an AD converter for AD converting the signal output by switching the signal from the detection element;
Storage means for storing data output from the AD converter, means for calculating a peak value of a sample to be measured based on the stored fluorescence light quantity signal data, and storing the calculated peak value in an external computer In addition, in the nucleic acid analyzer that has a means for outputting to the outside as a quantitative analysis result and analyzes a sample containing a nucleic acid by tracking changes in fluorescence intensity over time,
As a means for correcting the time-dependent change of the optical components constituting the detection unit, the reference fluorescent sample is controlled so that the fluorescent light amount of the reference fluorescent sample is always set to the set reference value of the fluorescent light amount. Calculate the number of operations by setting the number of operations as a predetermined value from the life curve of the light source to be used, calculating the means for the at least one reaction vessel to be supported by the carousel and the time for performing the daily measurement from the analysis process step. A means of comparing
When the number of operations does not reach a predetermined value, a means for outputting a preset fixed current value, and when the number of times of the predetermined value is exceeded from the determination result, the calculated fluorescence light amount and the reference value of the fluorescent light amount And a means for outputting to the DA converter a current value of the light source obtained by multiplying the current set value of the light source previously set by the amount of change in the amount of fluorescent light. A disk-shaped carousel, a rotating mechanism for rotating the carousel, at least one detection unit arranged along the circumference of the carousel, and a rotational position detecting mechanism for detecting the rotational position of the carousel; Have
The carousel is configured to support n reaction vessels containing samples at regular intervals along the circumferential direction.
The rotational position detection mechanism sets n shielding plates mounted on the carousel at equal intervals along the circumferential direction, detection means for detecting the passage of the shielding plates, and setting the reaction vessel. A heating unit for adjusting the temperature and a temperature sensor for monitoring the temperature of the reaction vessel;
The detection unit includes a light source that generates excitation light that irradiates the reaction container, a unit that irradiates a part of the light source to the excitation light detection element, and a fluorescence that detects fluorescence emitted from a sample stored in the reaction container. A sensing element;
An excitation light signal amplifier for amplifying a signal from the excitation light detection element; a fluorescence signal amplifier for amplifying a signal from the fluorescence detection element; and the excitation light signal widther according to a signal to be measured. Means for switching, an AD converter for AD converting the signal output by switching the signal from the detection element;
Storage means for storing data output from the AD converter, means for calculating a peak value of a sample to be measured based on the stored fluorescence light quantity signal data, and storing the calculated peak value in an external computer In addition, in the nucleic acid analyzer that has a means for outputting to the outside as a quantitative analysis result and analyzes the sample containing the nucleic acid by tracking the change in fluorescence intensity over time, a temperature sensor that monitors the temperature in the apparatus, The processing means for monitoring the temperature in the apparatus, and the temperature control of the heating unit for calculating the fluctuation of the temperature in the apparatus and adjusting the temperature to set the reaction vessel are performed, and the amount of fluorescence of the reference fluorescent sample is calculated. Since the control is performed so that the reference fluorescence amount is always constant, at least one reaction vessel in which the reference fluorescent sample is stored is supported by the carousel. The amount of change between the calculated fluorescent light amount and the reference value of the fluorescent light amount is measured every time the carousel rotates and passes through the detection unit. After the calculation, the data processing means for adjusting the current value flowing through the light source with respect to the set reference current value according to the amount of change from the reference value, and the light source set to adjust the current flowing through the light source A nucleic acid analyzer comprising: a DA converter that converts the current value of DA into a DA converter. A disk-shaped carousel, a rotating mechanism for rotating the carousel, at least one detection unit arranged along the circumference of the carousel, and a rotational position detecting mechanism for detecting the rotational position of the carousel; Have
The carousel is configured to support n reaction vessels containing samples at regular intervals along the circumferential direction.
The rotational position detection mechanism sets n shielding plates mounted on the carousel at equal intervals along the circumferential direction, detection means for detecting the passage of the shielding plates, and setting the reaction vessel. A temperature sensor for monitoring the temperature of the superheater unit for adjusting the temperature and the temperature of the reaction vessel;
The detection unit includes a light source that generates excitation light that irradiates the reaction container, a unit that irradiates a part of the light source to the excitation light detection element, and a fluorescence that detects fluorescence emitted from a sample stored in the reaction container. A sensing element;
An excitation light signal amplifier for amplifying a signal from the excitation light detection element; a fluorescence signal amplifier for amplifying a signal from the fluorescence detection element; and the excitation light signal widther according to a signal to be measured. Means for switching, an AD converter for AD converting the signal output by switching the signal from the detection element;
Storage means for storing data output from the AD converter, means for calculating a peak value of a sample to be measured based on the stored fluorescence light quantity signal data, and storing the calculated peak value in an external computer Furthermore, in the nucleic acid analyzer having a means for outputting to the outside as a quantitative analysis result and analyzing the sample containing the nucleic acid by tracking the change in fluorescence intensity over time, the carousel is rotating, that is, measuring. Means for moving at least one reaction vessel containing a reference fluorescent sample to a position that does not hinder the rotation of the carousel, and the time for discharging, transporting and installing the reaction vessel, that is, the carousel is stopped. The reference fluorescent sample reaction vessel is moved to the carousel sample holder during the period, the amount of fluorescent light is measured, averaged and calculated data and fluorescence A data processing unit that calculates a change amount of the amount with respect to a reference value, and adjusts a current value that flows through the light source with respect to a set reference current value according to the change amount with respect to the reference value; and a current that flows through the light source A nucleic acid analyzer, comprising: a DA converter that DA converts a current value of a light source set to adjust the current.

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