Patent Application
20170227558
The present invention relates to a nucleic acid analysis device which analyzes a biological sample by amplifying nucleic acid contained in the biological sample, and a device diagnosis method for the nucleic acid analysis device.
The analysis of nucleic acid contained in a biological sample, such as blood, blood plasma, and a piece of tissue, is carried out not only in academic researches such as biology, biochemistry, and medicine, but also in various fields such as diagnosis, cultivar improvement of crops, and industries of food inspection. The most widely used method as an analysis method for nucleic acid is a technique called a polymerase chain reaction (PCR), by which nucleic acid in the analysis target area is subjected to base sequence-specific amplification. In the PCR, the following cycles are repeated 30 to 40 times. That is, reaction solution containing nucleic acid and reagents for amplifying the nucleic acid is heated to about 95° C. such that the nucleic acid is thermally denatured, and then the solution is cooled to about 60° C. such that annealing and extension reaction of the nucleic acid are promoted. As a means for detecting the amplification of nucleic acid associated with the progress of the reaction, in many cases, it is conducted by mixing the reaction solution with fluorescent labels of which fluorescence intensity changes depending on the amount of PCR products, irradiating the mixed solution with excitation light, and measuring the fluorescence intensity emitted from the fluorescent labels.
PTL 1 discloses a technique in which the malfunction of a temperature adjustment unit is diagnosed by measuring the AC resistance of a thermoelectric element (peltier device). It is empirically determined that a thermoelectric element which shows an increase of about 5% in the AC resistance after temperature cycles of about 20,000 times to about 50,000 times fails in a short period of time. When a device is started up or an operator conducts a dedicated self-diagnosis function, the malfunction diagnosis is performed by equalizing the temperatures of a heating surface and a cooling surface of the thermoelectric element and then measuring the AC resistance.
PTL 1: JP 2008-278896 A
However, in the method of PTL 1, it is necessary to equalize the temperatures of both surfaces of the thermoelectric element (peltier device) to perform the diagnosis. Thus, when failure occurs in the temperature adjustment unit during measurement of specimens (during temperature cycling for amplifying nucleic acid), the diagnosis cannot be performed. In addition, since the diagnosis is conducted on the basis of an increase in the resistance caused by fatigue failure of the peltier device, there is a problem in that it is not possible to diagnose the malfunction of components other than the peltier device in the temperature adjustment unit.
An object of the invention is to provide a method by which failure or performance deterioration of a temperature adjustment unit can be detected while specifying a cause part in a state where a nucleic acid amplification process is being performed.
As the method for achieving the object described above, an initial value of the level of control signals input to a temperature adjustment element during temperature maintenance, temperature rise, and temperature fall is set in advance and an error determination threshold is set on the basis of this value. The monitored current control signal level and the threshold are compared in a state where the nucleic acid amplification process is being performed, whereby failure (malfunction and performance deterioration) of the temperature adjustment unit is diagnosed, and further, a component causing the failure is specified.
Failure or performance deterioration of the temperature adjustment unit can be detected by using the diagnosis method of the invention while specifying a cause part in a state where a nucleic acid amplification process is being performed.
When the PCR reaction is performed, accuracy of the temperature adjustment performance is significantly important. In the step of performing thermal denaturation of nucleic acid, when the temperature becomes excessively high beyond 95° C., there is a concern that a nucleic acid amplification enzyme is deactivated and amplification efficiency is reduced. When the temperature is out of the appropriate temperature during annealing, primers cannot be appropriately annealed to the target sequence, and thus the amount of target amplification products is reduced. In addition, when failure occurs in the temperature adjustment performance after the reaction solution obtained by mixing target nucleic acid to be analyzed with reagents or the like is formulated and the PCR reaction is started by the nucleic acid analysis device, the analysis is invalid and specimens are wasted. For this reason, it is preferable that failure of the temperature adjustment unit in the device be detected before specimens are measured, and it is further preferable that the failure be detected in the stage before the temperature adjustment performance goes completely wrong, that is, at the stage of deterioration. Furthermore, it is preferable that the failed component be specified so as to promptly perform repairing.
Hereinafter, a nucleic acid analysis device according to the invention will be described in detail with reference to the accompanying drawings.
The configuration of a nucleic acid analysis unit 31 of
A plurality of (twelve in this example) temperature adjustment blocks 1 is disposed along the outer circumference of the carousel 2 around the central axis thereof and rotationally driven around a rotation shaft 3. The peltier devices (temperature adjustment element) 4 are respectively disposed between the plurality of temperature adjustment blocks 1 and the carousel 2. The temperature of the temperature adjustment block 1 is adjusted by controlling the peltier device 4 while monitoring the temperature of the temperature adjustment block 1 using the temperature sensor 5 installed in the temperature adjustment block 1. A set of the peltier device 4 and the temperature sensor 5 is disposed corresponding to each of the plurality of temperature adjustment blocks 1, and thus the temperatures of the plurality of temperature adjustment blocks 1 are independently adjusted.
The photometer 6 is disposed on the outer circumference of the carousel 2. In this case, for example, two photometers 6, which use light of different wavelengths, are illustrated. However, one or three or more photometers 6 may be disposed as long as it is disposed on the outer circumference of the carousel 2. All of the temperature adjustment blocks 1 move on the same circumference by rotational driving. Therefore, when the temperature adjustment blocks 1 pass through the front of the photometer 6, the relative positions between the photometer 6 and the temperature adjustment blocks 1 are the same for all of the temperature adjustment blocks 1.
The plurality of temperature adjustment blocks 1 is covered, together with the carousel 2, by the shielding plate 7 so as to reduce the optical disturbance at the time of analyzing the temperature adjustment blocks 1 by the photometer 6. When the analysis is conducted, a tube (reaction container) 10 containing reaction solution (sample) obtained by mixing nucleic acid with reagents or the like is held by the temperature adjustment block (holding member) 1. An excitation light irradiation window 8 for receiving excitation light from the photometer 6 and a fluorescence detection window 9 through which the photometer 1 captures fluorescence are provided in each temperature adjustment block 1. In this case, the excitation light irradiation window 8 is disposed on the lower surface of the temperature adjustment block 1 and the fluorescence detection window 9 is disposed on the side surface of the temperature adjustment block 1. However, the arrangement of the windows can be freely set in accordance with the structure of a photometer.
It is preferable that a shielding plate inner portion 11 be maintained at the same temperature so as to minimize an influence on the temperature adjustment block 1 by changes in the external air temperature outside the shielding plate. Therefore, a temperature sensor (not illustrated) and the heater 12 are installed in the inner portion of the shielding plate 7. In this case, the heater 12 is disposed on the side surface on the inner side of the shielding plate 7. However, the heater 12 may be disposed at any position of the shielding plate inner portion 11 in accordance with the structure. Furthermore, various heat sources such as peltier devices and a heat radiator such as a fan and a fin can be used in addition to a heater, and a plurality of these may be combined.
It is preferable that, when temperature cycling for nucleic acid amplification is performed, the speed of temperature rise/temperature fall be set to a constant value (for example, 1° C./sec, etc.) in advance. The reason for this is as follows. The same temperature cycling is always performed, and thus variations in the results between the measurements can be reduced and, when the nucleic acid amplification process is performed, the process can be finished on the expected finish time schedule. Therefore, it is possible to easily plan the analysis schedule.
For the same reason, not only the control signal level of temperature rise/temperature fall but also the control signal level for heating/cooling at the time of constant temperature maintenance changes from the initial value. For example, when the temperature of the shielding plate inner portion 11 is 60° C., the peltier device 4 is heated when the temperature of the temperature adjustment block 1 is maintained at 95° C. and the peltier device 4 is cooled when the temperature of the temperature adjustment block 1 is maintained at 45° C. Similarly to the case of temperature rise/temperature fall, when the peltier device 4 is deteriorated, the control signal level for heating at the time of maintaining the temperature higher than that of the shielding plate inner portion 11 is smaller than that of the initial state and the control signal level for cooling at the time of lower temperature maintenance is greater than that of the initial state.
When changes in the control signal level described above are used, deterioration or failure of the peltier device 4 can be diagnosed. First, the level of control signals input to the peltier device 4 at each stage of temperature cycling is tuned for each peltier device 4 before device shipment and the initial values are stored into the device. Since the temperature of the shielding plate inner portion 11 changes even when the temperature adjustment is performed, an error diagnosis threshold is set by giving likelihood to the initial values, and it is diagnosed as failure when a value deviates from this range.
Similarly, deterioration of the temperature sensor 5 and the heat conductive sheet 13, in addition to the peltier device 4, can also be diagnosed from changes in the level of control signals input to the peltier device 4.
The diagnosis method described above can also be used for detecting failure other than deterioration of a component.
Since the success or failure of the installation of the tube 10 significantly affects the PCR results, it is normally monitored by a dedicated sensor. However, it is undeniable that there is also a possibility of malfunction of the sensor. To prepare for such a case, dual monitoring can be performed by this diagnosis.
It is necessary to carefully perform failure diagnosis of the temperature adjustment performance. Therefore, to increase the accuracy of diagnosis, diagnosis is performed on the basis of the control signal levels of not only one cycle but a plurality of cycles. As a result, the accuracy can be increased.
In addition, by performing diagnosis by comparing the control signal levels of a plurality of (up to twelve in this embodiment) temperature adjustment units 14, the accuracy of diagnosis can be further increased.
Hereinbefore, the method of diagnosis for each temperature adjustment unit 14 by comparing the current control signal level to the peltier element 4 with the initial value has been described. However, the failure occurring in a common temperature adjustment mechanism other than each temperature adjustment unit 14 can also be diagnosed by using the diagnosis results of the plurality of (up to twelve in this embodiment) temperature adjustment units 14 illustrated in
For example, it is assumed that the diagnosis result similar to that of the case of deterioration of the peltier device 4 of
Since the device diagnosis method described above can be performed even in the middle of nucleic acid amplification, it is possible to perform diagnosis without a special time for diagnosis. However, the device diagnosis method can also be performed not only in the middle of nucleic acid amplification but also in the preparation period of waiting for analysis after the power is turned on.
When nucleic acid analysis is not performed, temperature cycling is performed in a state where the temperature of the shielding plate inner portion 11 reaches a predetermined temperature, the level of control signals for heating/cooling input to the peltier device 4 is monitored over all stages, and the monitored level is compared to the threshold. In this case, since the tube 10 is not installed in the temperature adjustment block 1, the heat capacity of the temperature adjustment block 1 is small and the behavior of the temperature is different from that at the time of measurement. Thus, a threshold other than that for determining failure in the nucleic acid amplification may be set. The relationship shown in
The nucleic acid extraction unit 33 includes a specimen installation portion 41, a centrifuge portion 42, a retreat chamber 43, a tube installation portion 44, an extraction reagent storage 45, a consumable part storage 46, and the like. Although detailed description of the nucleic acid extraction unit 33 is omitted, it has a function of removing unnecessary components from a specimen and extracting only nucleic acid necessary for analysis. The reagent mixing unit 34 includes an analysis reagent storage 47, a consumable part storage 48, a mixer 49, and the like. Although detailed description of the reagent mixing unit 34 is omitted, it has a function of mixing the nucleic acid extracted by the nucleic acid extraction unit 33 with reagents for analysis. The configuration of the nucleic acid analysis unit 35 is similar to that of the nucleic acid analysis unit 31 illustrated in
In the case of the nucleic acid analysis device 32 in which preprocessing units such as the nucleic acid extraction unit 33 and the reagent mixing unit 34 are connected to the nucleic acid analysis unit 35 as described in this embodiment, diagnosis can be performed in the middle of each process such as a preparation stage, a preprocessing stage, and an analyzing stage after the device is turned on.
An analyzer turns on the nucleic acid analysis device 32, sets up specimens, reagents, and consumable parts such as tubes, and starts analysis. At this time, in the case of the nucleic acid analysis device 32 having the nucleic acid extraction unit 33 and the reagent mixing unit 34 as illustrated in
In a case where failure is not detected when the power is turned on, the device proceeds to a normal operation and starts preprocessing for analysis by the nucleic acid extraction unit 33 and the reagent mixing unit 34. Thereafter, even at the time after a nucleic acid amplification process is started, the diagnosis of the temperature adjustment unit can be performed at any time because it does not require special operations. Asa result, when failure is detected, the analysis is immediately stopped, and thus it is possible to prevent erroneous analysis results from being displayed. In addition, when a user wants to diagnose the performance of the temperature adjustment unit, diagnosis can be manually carried out after the device starts up as long as the device is in a state where the analysis operation is not performed.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2014-121972 | Jun 2014 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2015/064396 | 5/20/2015 | WO | 00 |
