JP4896564B2 - Method for confirming operation of crushing device, accuracy control method for pretreatment device for genetic testing, and sample analysis system - Google Patents

Description

  The present invention relates to an operation confirmation method for a crushing apparatus, an accuracy control method for a pretreatment apparatus for genetic testing, and a sample analysis system.

  In recent years, genetic testing has rapidly spread in the field of clinical diagnosis. Genetic testing is the analysis of nucleic acids, chromosomes, and the like to test for the presence or absence of mutations or karyotypes associated with genetic diseases. As an example of a genetic test, there is a test for determining whether or not a nucleic acid derived from cancer cells is present in a tissue excised from a living body. The inspection process mainly comprises three steps of pretreatment, nucleic acid amplification, and detection.

There are various methods for the pretreatment process. As an example, a pretreatment reagent (buffer solution) is first added to cell masses such as lymph nodes, tumor masses, and tissue fragments excised from a living body for examination purposes. Next, the excised tissue to which the reagent has been added is crushed (homogenized), and the supernatant obtained by centrifuging the homogenate is used as a measurement sample.
In the nucleic acid amplification step, the measurement sample is accommodated in a sample container, a reagent such as an enzyme or a primer is added thereto, and the target nucleic acid is amplified by a nucleic acid amplification reaction.
In the detection step, the presence / absence of the target nucleic acid in the excised tissue is determined or the concentration is calculated by measuring the fluorescence of the fluorescently stained target nucleic acid or measuring the turbidity of a byproduct produced in proportion to the amplification.
In the genetic test as described above, various factors in each process affect the measurement result. For this reason, in the field of genetic testing, it is important to enhance accuracy control for ensuring accuracy and reliability of each process.
With respect to the nucleic acid amplification step and the detection step, accuracy control has been conventionally performed using positive controls and negative controls.

However, the current situation is that accuracy control is not performed for the pretreatment process. When performing homogenization manually, the degree of homogenization of the excised tissue may vary depending on the skill of the person who performs the pretreatment. In addition, when homogenization is automatically performed by an automatic crushing device or the like, there may be a difference in the degree of homogenization of the excised tissue due to variation in setting of homogenization conditions such as the number of rotations of the blender or wear of the blender due to continuous use. From these facts, it is necessary to manage the accuracy of preprocessing such as homogenization conditions both when the homogenization is performed manually and when it is performed by an automatic crushing apparatus. Therefore, it is desired to develop an accuracy management method for managing the accuracy of the preprocessing device.
The present invention has been made in view of such circumstances, and confirms the operation of a crushing apparatus that can improve the reliability of genetic test data by improving the reliability of the crushing process, which is a pretreatment of genetic testing. It is an object of the present invention to provide a method, a quality control method for a pretreatment device for genetic testing, and a sample analysis system.

In order to achieve the above object, the present invention takes the following technical means.
The present invention related to the operation confirmation method of the crushing device is a method for confirming the operation of the crushing device for crushing a sample containing cells collected from a living body in a buffer solution,
The quality control material encapsulating pyrophosphate was crushed in by Ri buffer solution to the crushing device, as engineering of the pyrophosphate Ru liberated in the buffer solution from the quality control material,
A step of mixing the buffer solution from which pyrophosphate has been released and a divalent cation,
A step of detecting a change in turbidity of the mixed liquid obtained in the step, and a step of determining whether or not the crushing operation is appropriate based on the detection result;
It is characterized by including.
According to the operation confirmation method of this crushing apparatus, since the appropriateness of operation is determined based on the change in turbidity caused by crushing of the quality control substance, the operation can be easily confirmed. This increases the reliability of the crushing process.

Further, the present invention related to the operation confirmation method of the crushing apparatus is a method of confirming the operation of the crushing apparatus that crushes a sample containing cells collected from a living body in a buffer solution in another aspect,
The step of changing the physical properties of the buffer solution by crushing the quality control material encapsulating the labeling material in the buffer solution by the crushing device, and releasing the labeling material from the quality control material into the buffer solution,
Detecting the change in physical properties; and
A step of determining whether or not the crushing operation was appropriate based on the detection result;
Including
Before SL quality control material, the inner layer that contains a labeling substance, and covering the outside of the inner layer, and has a protective layer to protect the inner layer, and said inner layer and said protective layer It is characterized in that an interface is formed between them .
In the above method, it is preferable that the labeling substance and the inner layer are hydrophilic and the protective layer is hydrophobic.
Furthermore, the present invention related to the operation confirmation method of the crushing apparatus is a method for confirming the operation of the crushing apparatus that crushes a sample containing cells collected from a living body in a buffer solution in another aspect,
The step of changing the physical properties of the buffer solution by crushing the quality control material encapsulating the labeling material in the buffer solution by the crushing device, and releasing the labeling material from the quality control material into the buffer solution,
Detecting the change in physical properties; and
A step of determining whether or not the crushing operation was appropriate based on the detection result;
Including
The quality control material is characterized in that the strength is set so as to be crushed by a crushing operation capable of crushing the sample or a crushing operation having a strength higher than that.
Further, the present invention related to the operation confirmation method of the crushing apparatus is a method of confirming the operation of the crushing apparatus that crushes a sample containing cells collected from a living body in a buffer solution in another aspect,
The step of changing the physical properties of the buffer solution by crushing the quality control material encapsulating the labeling material in the buffer solution by the crushing device, and releasing the labeling material from the quality control material into the buffer solution,
Detecting the change in physical properties; and
A step of determining whether or not the crushing operation was appropriate based on the detection result;
Including
On the surface of the quality control material, the hardness by absorbing liquid is characterized by a layer decrease is formed.

The present invention related to the accuracy control method of the pretreatment device for genetic testing is a method of performing accuracy control of the pretreatment device for genetic testing that crushes a sample containing cells collected from a living body in a buffer solution,
The quality control material encapsulating pyrophosphate were disrupted in buffer by the pretreatment device, the quality control material the buffer crushing step that is released into the pyrophosphate from,
After performing the crushing step, a mixing step of mixing the buffer solution from which the pyrophosphate is liberated and a divalent cation,
A detection step of detecting the turbidity of the liquid mixture obtained in the mixing step; and
A determination step of determining whether or not the crushing operation was appropriate based on the detected turbidity ;
It is characterized by including.
According to this quality control method, the appropriateness of the operation is determined based on the change in turbidity caused by the crushing of the quality control material, so that the quality control of the pretreatment device can be easily performed. As a result, the reliability of the pretreatment device is increased, and as a result, the reliability of genetic test data can be increased.

In the determination step of the above method,
If the detected turbidity is less than a predetermined value, determine that the crushing operation was not appropriate,
When the detected turbidity is not less than a predetermined value, it may be determined that the crushing operation is appropriate.

Further, the present invention relating to a sample analysis system is a sample analysis system used in the above-described accuracy control method ,
A measurement unit for measuring the turbidity of a mixture of the buffer solution from which pyrophosphate is liberated and a divalent cation ;
Based on the turbidity measured by the measurement unit, determination means for determining whether or not the crushing process was appropriate,
It is characterized by having.
The sample analysis system may further include a crushing unit that crushes the quality control substance in the buffer solution.

  ADVANTAGE OF THE INVENTION According to this invention, the operation | movement confirmation method of a crushing apparatus, the quality control method of a pretreatment apparatus for gene tests, and a sample analysis system can be provided. According to this, it is possible to improve the reliability of the data of the genetic test by improving the reliability of the crushing process that is a pre-process of the genetic test.

The method for confirming the operation of the crushing apparatus of the present invention is a method for confirming the operation of the crushing apparatus for crushing a sample containing cells collected from a living body in a buffer solution. Step of changing physical properties of the buffer solution by crushing in the buffer solution and releasing the labeling substance from the quality control material into the buffer solution (step of crushing), detecting a change in the physical properties (Detection step) and a step of determining whether or not the crushing operation was appropriate based on the detection result (determination step).
The crushing apparatus for checking the operation by the method of the present invention crushes a sample containing cells collected from a living body in a buffer solution. Here, examples of the sample containing cells include tissues such as lymph nodes collected from animals such as humans. In addition, water can be used as the buffer solution, and an appropriate buffer may be added to water in order to adjust the pH.

In the crushing step, a quality control substance encapsulating the labeling substance is mixed with a buffer solution and crushed (homogenized) by a crushing process. As a result, the labeling substance is released from the quality control substance into the buffer solution.
Release of the labeling substance into the buffer changes the physical properties of the buffer. Preferably, the labeling substance is added to the buffer during the crushing process and / or after the crushing process, and the physical properties are changed by binding with the labeling substance. Further, this label binding substance may be contained in the buffer in advance.
Examples of changes in physical properties include changes in optical state and changes in viscosity. Examples of the change in the optical state include turbidity, scattered light intensity, absorbance, fluorescence intensity, transmitted light intensity, emission intensity, and color intensity. A change in viscosity can be detected by measuring a change in torque of a motor that operates the crushing device.

  Examples of the change in the optical state include turbidity change, change in absorbance, change in scattered light intensity, change in fluorescence intensity, change in transmitted light intensity, change in emission intensity, change in color intensity, and the like. The change in the optical state is preferably caused by the binding between the label-binding substance and the labeling substance released in the buffer solution. Examples of the combination of the labeling substance and the label binding substance include a combination of pyrophosphate and a divalent cation, a combination of an enzyme and a substrate, and the like. For example, when the labeling substance is pyrophosphate and the label binding substance is a divalent cation, such as magnesium ion, insoluble magnesium pyrophosphate is produced by combining pyrophosphate and magnesium ion, and the buffer solution becomes cloudy. To do. This change in the turbidity of the buffer solution can be detected as a change in the optical state. During the binding reaction between pyrophosphate and magnesium ions, it is preferable to warm the buffer. This promotes the binding reaction between pyrophosphate and magnesium ions.

The labeling substance encapsulated in the quality control substance is not particularly limited as long as the quality control substance is crushed and leaks into the buffer solution to change the physical properties of the buffer solution. In addition, fluorescent substances, luminescent substances, coloring substances, enzymes and the like can be used.
When an enzyme is used as a labeling substance, for example, glucose 6-phosphate dehydrogenase (G6PDH) is enclosed in a quality control substance, and a coenzyme (for example, NAD or NADP) corresponding to this enzyme and glucose are used as a label-binding substance. Hexaphosphate (G6P) can be used. In this case, when the quality control substance is crushed and G6PDH leaks, and G6P and a coenzyme are added to the buffer solution from which G6PDH has leaked, they react to reduce the coenzyme. When the coenzyme is reduced, the absorbance of the buffer solution changes, so that this change in absorbance can be detected as a change in optical state.
Also, peroxidase (POD) is encapsulated in a quality control substance, and a chromogen such as a coupler (for example, 4-aminoantipyrine) or a developer (phenolic compound, etc.) and hydrogen peroxide are used as a label binding substance. Can do. In this case, when the quality control substance is crushed and POD leaks, when the chromogen and hydrogen peroxide are added to the buffer solution from which POD has leaked, they react to produce a coloring substance. Therefore, the color development of the buffer solution can be detected visually or by an absorbance measuring device as a change in the optical state.

  The quality control substance has an inner layer containing a labeling substance, and a protective layer that covers the outer side of the inner layer and protects the inner layer, and is provided between the inner layer and the protective layer. It is preferable that an interface is formed. By protecting the inner layer with a protective layer that does not mix with the inner layer (forms an interface), the labeling substance can be made difficult to leak outside.

Since an interface is formed between the inner layer and the protective layer, when the labeling substance is a water-soluble substance, the inner layer is an aqueous layer and the protective layer is an oil layer. On the other hand, when the labeling substance is a fat-soluble substance (for example, a dye), the inner layer is an oil layer and the protective layer is an aqueous layer.
Here, water, physiological saline, buffer solution, culture solution and the like can be used for the water layer, and various oils and fats, fatty acids, sugar fatty acid esters and the like can be used for the oil layer. Preferably, animal oils and fats, vegetable oils and the like are used.

The quality control material is preferably set to have a strength so as to be crushed by a crushing operation capable of crushing a sample such as a cell mass or a crushing operation having a higher strength. The hardness (elastic modulus (Young's modulus)) of cells (tissues) is, for example, several tens of kPa for fat, several tens of kPa for breast tissue, 120 to 300 kPa for fibrous tissue, and 150 to 300 for non-invasive ductal carcinoma. 350 kPa, 300 to 750 kPa for invasive ductal carcinoma, 40 to 80 kPa for prostate (see TA Krpiskop et al, Ultrasonic Imaging, 1998), and the strength of quality control substances depends on the target sample By selecting the material so that it is above or above, the crushing condition of the quality control substance becomes equal to or more severe than the crushing condition of the sample. It becomes possible to do.
Furthermore, it is preferable that the surface of the quality control material is a layer whose hardness decreases by absorbing the liquid. By forming a further layer (outermost layer) on the surface of the quality control substance, the labeling substance is less likely to leak to the outside. Further, the outermost layer absorbs the liquid and gradually becomes soft, so that crushing occurs at an appropriate time. The outermost layer is a natural polymer such as gelatin, agar, pectin, alginic acid, carrageenan, curdlan, starch, gellan gum, glucomannan or a mixture thereof, or, if necessary, protein, glycoprotein, mucopolysaccharide. It is formed from a substance to which sugar, sugar alcohol, polyhydric alcohol or the like is added. By appropriately adjusting the composition of these substances, it is possible to adjust the hardness of the outermost layer, the water absorption, and the like.

For example, the operation of the crushing device can be guaranteed as follows.
When a crushing operation is performed to sufficiently crush the cell mass, a buffer solution is added to the quality control material designed to leak out the labeling substance (pyrophosphate) enclosed inside, and the specified number of rotations with a blender. Homogenize the rotation time. If the crushing operation is appropriate, pyrophosphate leaks from the quality control substance into the buffer solution, and the buffer solution containing pyrophosphate leaks into the detection cell to which a buffer solution containing magnesium ions has been added in advance. Transfer and pyrophosphate combine with magnesium ions to produce magnesium pyrophosphate, and the buffer becomes cloudy. However, when the crushing operation is insufficient, there is substantially no leakage of pyrophosphate, and the buffer solution does not become cloudy. Therefore, the turbidity is measured, and from this measurement data, it is determined whether the crushing operation is proper or not based on whether or not the buffer solution becomes clouded within a predetermined time (for example, whether the turbidity is 0.1 or more). can do.
When the turbidity of the buffer solution after crushing is less than 0.1, it is determined that the crushing operation was insufficient. In this case, it may be due to incorrect setting of crushing operation conditions (rotation time or number of rotations) or wear of the blade of the blender, etc. Therefore, based on the result of accuracy control, check the setting of the blade or crushing operation conditions, etc. The pretreatment device can be brought into a state suitable for homogenization of a biological sample such as a cell mass.

Hereinafter, the internal accuracy control of the pretreatment apparatus for genetic testing using the above-described operation guarantee method for the crushing apparatus will be described with reference to the drawings.
[Nucleic acid detection system]
The nucleic acid detection system (sample analysis system) 1 of the present embodiment can output a target nucleic acid concentration contained in this specimen as measurement data using a resected tissue from a living body (human body) such as a lymph node. is there.
More specifically, the nucleic acid detection system 1 is used as a genetic diagnosis system for breast cancer lymph node metastasis. A pretreatment (homogenization, extraction processing, etc.) is performed on a lymph node (specimen) excised from a human body to obtain a nucleic acid. A solubilized extract is prepared as a measurement sample for detection, and a cDNA corresponding to the target nucleic acid (mRNA) present in the measurement sample is amplified by the LAMP method (Loop-mediated Isothermal Amplification: see US6410278B1). The concentration of the target nucleic acid (copy number per unit volume) is determined by measuring the turbidity of the solution generated with amplification in real time.

  The nucleic acid detection system 1 is used for rapid diagnosis during surgery, and specifically used during surgery such as breast cancer. For example, the system 1 obtains the concentration of a cancer marker in the lymph node from lymph nodes excised during surgery, and a doctor diagnoses the presence or absence and / or the degree of metastasis of cancer cells during surgery with reference to this. And determine the extent of lymph node dissection. Therefore, high reliability and quickness are required for the output of the system 1.

As shown in FIG. 1, the nucleic acid detection system 1 is a pretreatment device for preparing a measurement sample (measurement sample) by performing pretreatment such as crushing (homogenization) on a sample obtained from a human body or the like. It has a main body 5 and a nucleic acid detection apparatus main body 101 that performs detection processing of a target nucleic acid contained in a measurement sample.
The nucleic acid detection system 1 has a personal computer 6 as a data processing apparatus for performing data processing or data communication. The data processing device 6 also has a function as a control device that receives measurement data from the nucleic acid detection device main body 101 and transmits operation instruction signals to the preprocessing device main body 5 and the nucleic acid detection device main body 101. Yes. That is, the pretreatment device body 5 functions as a pretreatment device, and the nucleic acid detection device body 101 and the data processing device 6 function as a nucleic acid detection device.
The data processing device 6 can also be connected to a network, and transmits measurement data transmitted from the transmission unit of the nucleic acid detection device main body 101 to the server by a data transmission / reception function with a server (not shown). can do.

[Pretreatment equipment]
As shown in FIG. 2, the preprocessing apparatus main body 5 mainly includes a preprocessing unit 50 that performs preprocessing on a sample to obtain a measurement sample.
The preprocessing unit 50 includes a sample setting unit 51 for setting a container containing a sample, a blender (homogenizing unit) 52 for homogenizing the sample, and a pipette for dispensing a homogenized (preprocessed) measurement sample. (Dispensing part) 53 and a transfer part (not shown) for transferring the pipette 53 to the nucleic acid detection device main body 101.

The user mixes the quality control substance and the buffer solution and sets them in the sample setting unit 51. When the user issues a measurement start instruction using the keyboard or mouse of the data processing device 6, the measurement start instruction is transmitted from the data processing device 6 to the preprocessing device body 5.
When the preprocessing device main body 5 receives the measurement start instruction signal from the data processing device 6, it homogenizes the sample by the blender 52 and creates a measurement sample (homogenization processing).
Then, a measurement specimen (hereinafter also simply referred to as “sample”) is aspirated by the pipette 53, and in the case of normal nucleic acid detection, the pipette 53 moves to the nucleic acid detection apparatus main body 101 and is set in the nucleic acid detection apparatus main body 101. A sample is injected into the prepared sample container 22.
In the case of quality control, the pipette 53 that sucks the quality control sample for measurement created by pre-processing the quality control material for pretreatment moves to the nucleic acid detection device main body 101 and detects nucleic acid as in the case of the sample. A quality control sample for measurement is injected into the sample container 22 set in the apparatus main body 101.

[Nucleic acid detector]
The nucleic acid detection device main body 101 is configured as shown in FIGS. 3 and 4, and details of this device are described in JP-A-2005-98960. Here, the configuration and operation of the nucleic acid detection apparatus main body 101 will be briefly described.
First, the pipette moved from the pretreatment device main body 5 injects the pretreated sample or the quality control sample for measurement into the sample container 22 set in the sample container setting hole 21 a of the sample container base 21.

  The primer reagent container set hole 31a and the enzyme reagent container set hole 31b on the left side of the front of the reagent container set unit 30 have a primer reagent container 32a containing a primer reagent of CK19 (cytokeratin 19) and an enzyme containing an enzyme reagent. A reagent container 32b is set.

  In addition, two racks 42 each containing 36 disposable pipette tips 41 are fitted in the recesses (not shown) of the chip set section 40. Further, the two cell portions 66a of the detection cell 65 are set in the two detection cell set holes of the reaction portion 61 of each reaction detection block 60a.

  In this state, when the operation of the nucleic acid detection device main body 101 starts, first, the arm unit 11 of the dispensing mechanism unit 10 is moved from the initial position to the chip set unit 40, and then the dispensing mechanism unit in the chip set unit 40 Ten syringe parts 12 are moved downward. As a result, the tip of the nozzle portion of the syringe portion 12 is press-fitted into the upper opening of the pipette tip 41, so that the pipette tip 41 is automatically attached to the tip of the nozzle portion of the syringe portion 12. Then, after the syringe part 12 is moved upward, the arm part 11 of the dispensing mechanism part 10 is directed upward of the primer reagent container 32a in which the primer reagent of CK19 set on the reagent container setting base 31 is accommodated. It is moved in the X-axis direction. Then, when the syringe part 12 is moved downward, the tip of the pipette tip 41 attached to the nozzle part of the syringe part 12 is inserted into the surface of the primer reagent of CK19 in the primer reagent container 32a. Then, the primer reagent of CK19 in the primer reagent container 32a is sucked by the pump part of the syringe part 12.

  After the primer reagent is aspirated, after the syringe part 12 is moved upward, the arm part 11 of the dispensing mechanism part 10 is moved above the reaction detection block 60a located on the innermost side (the back side of the apparatus front side). . In this case, the arm part 11 of the dispensing mechanism part 10 is moved so as not to pass above the second to fifth other reaction detection blocks 60a from the back. In the innermost reaction detection block 60a, the syringe part 12 is moved downward, so that the pipette tip 41 attached to the nozzle part 12a of the syringe part 12 is inserted into the cell part 66a of the detection cell 65. Is done. And using the pump part of the syringe part 12, two primer reagents of CK19 are discharged to the cell part 66a (primer reagent dispensing process).

Thereafter, the pipette tip 41 is discarded, and two new pipette tips 41 are automatically attached to the tip of the nozzle portion of the syringe portion 12, and then the enzyme reagent in the enzyme reagent container 32b is detected in substantially the same operation as described above. It is discharged into the cell portion 66a of the cell 65 (enzyme reagent dispensing process).
Thereafter, similarly, the sample (specimen for measurement) in the sample container 22 is discharged to the cell portion 66a of the detection cell 65 (sample dispensing process).
Thereby, a sample for detecting CK19 is adjusted in the cell portion 66a of the detection cell 65.

After the primer reagent, enzyme reagent, and sample are discharged into the cell part, the lid closing operation of the detection cell 65 is performed.
After the lid closing operation is completed, the temperature of the liquid in the detection cell 65 is heated from about 20 ° C. to about 65 ° C. using the Peltier module of the reaction unit 61, so that the target nucleic acid (mRNA of CK19) is obtained by the LAMP method. Is amplified (hereinafter referred to as CK19 cDNA).
And the cloudiness by the magnesium pyrophosphate produced | generated with amplification is detected. Specifically, light having a diameter of about 1 mm from the LED light source unit 62a of the turbidity detection unit 62 is detected through the light irradiation groove of the reaction unit 61, and a detection cell 65 (measurement data acquisition unit) at the time of amplification reaction. The cell portion 66a is irradiated. The irradiated light is received by the photodiode light receiving unit 62b.
Thereby, the liquid turbidity in the cell part 66a of the detection cell 65 during the amplification reaction is detected (monitored) in real time (detection process).
The measurement data of CK19 measured by the photodiode light receiving unit 62b (measurement data acquisition means) is transmitted to the data processing device 6 by a transmission unit (not shown) of the nucleic acid detection device main body 101.

As a result, in the data processing device 6, when time is taken on the horizontal axis and turbidity is taken on the vertical axis, measurement data of CK19 as shown in FIG. 5 is obtained. Then, the time (amplification rise time) until the amplification product of CK19 cDNA in the sample increases rapidly is measured.
Then, based on a calibration curve prepared in advance from the measurement result of the CK19 calibrator as shown in FIG. 6, the concentration (copy number / μL) of CK19 cDNA is calculated from the amplification rise time of CK19. Here, the calibration curve shown in FIG. 6 is a curve in which the horizontal axis represents the amplification rise time and the vertical axis represents the target nucleic acid copy number [copy number / μL]. The shorter the amplification rise time, the lower the target nucleic acid concentration. Is high.

The calculated target nucleic acid concentration data (measurement data) is displayed on the screen by the display device of the data processing device 6 or other display devices. Further, the data processing device 6 obtains a qualitative determination result for diagnosis support from quantitative measurement data (amplification rise time, copy number), and displays it on the screen of the data processing device display device or other display device. To do.
This determination is made, for example, when the copy number is 250 or less, or when the turbidity does not reach the threshold value even after a predetermined time in the measurement data shown in FIG. if 5 × 10 ³ in a range "+" determines if the copy number is greater than 5 × 10 ³ as "++". Here, “ND” is “metastasis is not detected”, “+” is “few metastases”, “++” is “metastasis is observed”, and so on. The system 2 seeks and displays qualitative results that are useful for supporting definitive diagnosis from quantitative measurement data (amount of cancer cells). The clearing range can be determined.

[Quality control for preprocessing]
When quality control of the pretreatment device 5 is performed, a quality control sample is prepared by preprocessing the quality control material in the buffer solution by the pretreatment device main body 5, and the nucleic acid detection device main body 101 turbidity in the same manner as the sample. Measure.
The quality control sample contains a quality control substance (capsule) containing pyrophosphoric acid as a known amount of labeling substance and a buffer solution, and has been subjected to pretreatment (crushing treatment). It is preferable that the buffer containing the crushed quality control substance is further centrifuged, and the resulting supernatant is used as a quality control sample. Thereby, the quality control sample which does not contain the fragment | piece of the quality control material which was crushed can be obtained.
Here, the quality control substance has a three-layer structure including an inner layer (water layer), a protective layer (oil layer), and an outermost layer, and the inner layer contains pyrophosphoric acid as a labeling substance. And intensity | strength is set so that it may be crushed by the crushing operation | movement which is a grade which can crush the structure | tissue which contains a cancer cell etc. or the strength beyond it. The outermost layer is configured such that the hardness decreases by absorbing the liquid. For this reason, a predetermined reference value (display value) is obtained when a predetermined crushing process (crushing process at a rotation speed of 25 krpm and a rotation time of 90 seconds) is performed by the pretreatment device body 5 and the turbidity is measured.

7 to 8 are flowcharts showing processing of the personal computer 6 and the preprocessing device main body 5 for quality control of the preprocessing.
The quality control process is performed periodically.
In the quality control process, a user (hospital staff or the like) mixes the quality control substance and the buffer solution and sets them in the sample setting section 51 of the pretreatment device main body 5.
When a user inputs a start instruction by an input unit such as a keyboard / mouse of the personal computer 6 of the system 1 in order to start accuracy control of the preprocessing device main body 5, the personal computer 6 receives the instruction ( Step S1-1: See FIG. 7), the personal computer (control device) 6 transmits a pre-processing measurement start instruction to the pre-processing device body 5 (step S1-2).

When the pretreatment device main body 5 receives the measurement start instruction signal (step S1-3: see FIG. 8), the pretreatment unit 50 performs a homogenization process (step S1-4) on the quality control material, create.
The quality control sample is dispensed by the pipette 53 into the sample container 22 of the nucleic acid detection device main body 101. In this sample container 22, a solution containing magnesium ions is dispensed in advance, and when the quality control sample and this solution are mixed, pyrophosphate and magnesium ions react, and the buffer solution becomes cloudy. In the nucleic acid detection device main body 101, the turbidity of this buffer solution is measured (step S1-5). Then, the nucleic acid detection device main body 101 transmits the measurement data of the measured turbidity to the personal computer 6 side (step S1-6).
The personal computer 6 receives turbidity measurement data from the nucleic acid detection device main body 101 (step S1-7: see FIG. 7).

When receiving the turbidity measurement data, the personal computer 6 compares the turbidity at a predetermined time with a preset threshold value, and determines whether or not the crushing process for the quality control substance is appropriate (step S1- 8: Determination (QC (Quality Control) result determination)).
For example, when the turbidity at a predetermined time shows a value approximate to a predetermined value (threshold value) (preferably within a threshold value ± 20%, more preferably within a threshold value ± 10%), the crushing treatment for the quality control substance is performed. It is determined that it was done properly. This indicates that the pretreatment device is in a state in which a tissue mass such as a lymph node can be appropriately crushed.
Conversely, when the value is significantly lower than the turbidity at a predetermined time (for example, less than 80% of the threshold value), it is determined that the crushing treatment for the quality control material is insufficient. This indicates that an abnormality has occurred in the pretreatment device, and it is not in a state in which cell clusters such as lymph nodes can be appropriately crushed.
In the above description, the case of measuring the turbidity of the buffer solution has been described. However, it is possible to determine whether or not the crushing treatment for the quality control substance is appropriate by measuring the transmitted light intensity of the buffer solution. Specifically, when the transmitted light intensity falls below a threshold value (for example, less than 80% of the transmitted light intensity at the start of measurement) within a predetermined time (for example, 5 minutes) from the start of measurement of the transmitted light intensity. It is determined that the crushing process has been appropriately performed. This indicates that the pretreatment device is in a state in which a tissue mass such as a lymph node can be appropriately crushed.
Conversely, if the transmitted light intensity is close to the threshold value within a predetermined time (for example, 80% or more of the transmitted light intensity at the start of measurement), the crushing treatment for the quality control substance is insufficient. It is determined that This indicates that an abnormality has occurred in the pretreatment device, and it is not in a state in which cell clusters such as lymph nodes can be appropriately crushed.

The present invention is not limited to the above embodiment.
The labeling substance enclosed in the quality control substance is not limited to water-soluble pyrophosphate, and for example, a fat-soluble dye may be used. In this case, the inner layer becomes an oil layer and the protective layer becomes an aqueous layer.
In the above embodiment, turbidity or absorbance is measured as optical information. However, the present invention is not limited thereto, and scattered light intensity or fluorescence intensity can also be measured. The scattered light intensity can be measured with a flow cytometer after the crushing treatment using a quality control substance that forms particles of an appropriate size by crushing (homogenization), for example. Also. With fluorescence intensity, for example, it is possible to crush (homogenize) a quality control material encapsulating a fluorescent dye in a solution containing the material to be stained, and measure the fluorescence intensity of the fluorescent dye released from the quality control material. is there.

  The entire pretreatment device body 5 may be omitted, and the absorbance of the manually pretreated sample may be measured using the detection unit 62 of the nucleic acid detection device body 101. As described above, when the external quality control processing in the nucleic acid detection device main body 101 is configured so that both the measurement of the preprocessed specimen and the measurement of the control solution can be performed, the function of switching both measurements is provided. It is preferable to be included.

  When the preprocessing unit 50 is omitted as described above, the user can recognize how much preprocessing (homogenization) is necessary based on the quality control result of the preprocessing. In the sample pretreatment, it is understood that homogenization may be performed similarly to the homogenization of the pretreatment quality control substance.

The pre-processing unit 50 of the pre-processing device is not limited to the one disclosed as the embodiment, and an apparatus that executes the method described in JP-T-2001-518284 may be used.
In addition, the pretreatment device main body 5 and the nucleic acid detection device main body 101 do not need to be integrally formed, and may be separate devices. When both the apparatus main bodies 5 and 101 are configured as separate apparatuses, the movement of the specimen from the preprocessing apparatus main body 5 to the detection apparatus 101 may be performed manually, or a separate moving mechanism may be provided. .

In the above embodiment, the nucleic acid detection device main body 101 is connected to the data processing device 6. However, the functions of the data processing device 6 may be integrated into the nucleic acid detection device main body 101, or these may be integrated. The device body 5, the nucleic acid detection device body 101, and the data processing device 6 may be integrated.
In the above embodiment, the method for managing the quality using the internal quality management system has been described. However, it is also possible to perform the external quality management by connecting the data processing device 6 to the network.

  Further, after the quality control material is crushed, fragments of the quality control material crushed by centrifugation or the like may be settled, and the supernatant may be used as a quality control sample for turbidity measurement.

  Further, the mixing of the buffer solution and the quality control substance may be automatically performed by the pretreatment device. In this case, the pretreatment apparatus includes a reagent dispensing pipette that dispenses a reagent to the quality control material set in the sample setting unit 51.

  In the following, an example is shown, operation guarantee (Example 1) with a quality control material (hereinafter referred to as “pyrophosphate capsule”) in which water-soluble pyrophosphate is encapsulated as a labeling substance, and a fat-soluble dye is encapsulated as a labeling substance The operation guarantee (Example 2) by the quality control material (hereinafter referred to as “dye ball”) will be described.

Example 1
First, the pyrophosphate capsule will be described. As shown in FIG. 9, the pyrophosphate capsule A is a capsule having a three-layer structure of a hydrophilic inner layer A1, a hydrophobic protective layer A2, and an outermost layer A3. Pyrophosphate capsule A is composed of 39.4 wt% pyrophosphate aqueous solution (inner layer A1), 36.0 wt% vegetable oil (protective layer A2), 20.4 wt% outermost layer (A3) (16.0 wt% gelatin). And 4.4% by weight of glycerin) and 4.2% by weight of others (such as emulsifiers). The average mass is 177.0 mg (CV 5.0%), the average outermost layer thickness is about 270 μm (CV 18%), and the diameter (φ) is 7 mm. This pyrophosphate capsule A was manufactured by consigning to Morishita Jintan Co., Ltd.
The following examination was performed using this pyrophosphate capsule A.

(1) Examination of optimum number of pyrophosphate capsules A used for accuracy control One, two, three or four pyrophosphate capsules A are placed in a 50 mL centrifuge tube, and the homogenizing reagent (Sysmex Corporation) 4 mL of 200 mM glycine, 143 mM cesium trifluoroacetate, 42 mM hydrochloric acid (pH 3) and NP-40 (protein grade) 1%) was added. The pyrophosphate capsule in the homogenizing reagent was crushed (25000 rpm, 90 seconds) using a blender (PHYSCOTRON HANDY MICRO HOMOGENIZER NS-350D) equipped with a blade (generator shaft NS-7, φ7 mm). Thereafter, centrifugation is performed, and 50 to 200 μL of the supernatant is collected, 20 μL of cytokeratin reagent (manufactured by Sysmex) is added to the supernatant, and turbidity is detected with a gene amplification detector (GD-100: manufactured by Sysmex). Was measured. The cytokeratin reagent contains 8.9 mM magnesium ion. The result is shown in FIG.
From FIG. 10, it was found that turbidity could not be detected when the number of pyrophosphate capsules A was 3 or less, but turbidity could be detected when 4 or more pyrophosphate capsules A (equivalent to 14 mM pyrophosphate) were added.

(2) Examination of the influence of magnesium ions on the turbidity of the solution in which the pyrophosphate capsule A was crushed The turbidity obtained when the solution in which the pyrophosphate capsule A was crushed was measured with GD-100 was pyrophosphoric acid eluted by crushing Whether or not it was due to the white turbidity of magnesium pyrophosphate produced by the binding of magnesium ions to magnesium ions.
Four pyrophosphate capsules A and 4 mL of a homogenizing reagent were placed in a 50 mL centrifuge tube, and crushed with the blender (25 krpm, 90 seconds). After centrifugation at 4 ° C. and 1200 rpm for 5 minutes, about 500 μL of the supernatant was transferred to a measurement tube.
Thereafter, 20 μL of a homogenizing reagent added with 8.9 mM magnesium ions or 20 μL of a homogenizing reagent not containing magnesium ions was added to a detection cell of GD-100, and 2 μL of the supernatant was dispensed thereto to obtain turbidity and transmitted light intensity. Was measured. The turbidity results are shown in FIG. 11, and the transmitted light intensity results are shown in FIG.
From FIGS. 11 and 12, pyrophosphoric acid leaked from the pyrophosphoric acid capsule A by the crushing treatment is combined with magnesium ions in the homogenizing reagent, whereby the turbidity of the homogenizing reagent is increased (the transmitted light intensity is reduced). This indicates that the turbidity obtained when the solution obtained by crushing pyrophosphate capsule A is measured with GD-100 is due to white turbidity generated by the combination of pyrophosphate and magnesium ions leaked by crushing.

(3) Examination of the influence of the rotational speed of the blender on the crushing of the pyrophosphoric acid capsule A Four pyrophosphoric acid capsules A are placed in a 50 mL centrifuge tube, and 4 mL of the homogenizing reagent is added thereto. , 15, 20 or 25 krpm for 90 seconds. Thereafter, the same operation as in Example 1 (1) was performed. The result is shown in FIG.
From FIG. 13, it was confirmed that the state of pyrophosphate capsule A crushing tends to depend on the rotational speed of the blender, and that pyrophosphoric acid capsule A is crushed at a rotational speed of 20 krpm or more in a rotational time of 90 seconds.
Here, in order to compare with the crushed state of the tissue mass, 400 mg of porcine lymph node was crushed for 90 seconds at 5, 10, 15, 20 or 25 krpm with the blender, and the concentration of the protein released from the cells of the crushed lymph node was measured. did. The concentration was measured by measuring the absorbance at 280 nm of the supernatant of the homogenate. The result is shown in FIG.
From FIG. 14, in the case of the crushing process for 90 seconds, the protein concentration in the reagent increased when the rotation speed was 15 krpm. In addition, at the rotation speed of 20 krpm or more, a large change in the protein concentration was not seen as compared with the rotation speed of 15 krpm. From the above, it was shown that most of the porcine lymph nodes were crushed under the condition of at least 15 krpm (rotating time: 90 seconds).
From the above, it was found that the rotational speed at which the pyrophosphate capsule A was crushed and the turbidity was detected was larger than the rotational speed at which the lymph node was crushed. That is, this indicates that pyrophosphate capsule A is crushed under severer conditions than lymph nodes.

(4) Examination of influence of blender rotation time on crushing of pyrophosphate capsule A In a 50 mL centrifuge tube, 4 pyrophosphate capsules A were added, 4 mL of the homogenizing reagent was added thereto, and the blender was used at 25 krpm, Crush for 10, 30, 60 or 90 seconds. Thereafter, the same operation as in Example 1 (1) was performed. The result is shown in FIG.
From FIG. 15, the pulverized state of the pyrophosphate capsule A tends to depend on the rotation time of the blender. When the rotation speed is 25 krpm, the pyrophosphate capsule A leaks out from the pyrophosphate capsule A when rotated for 60 seconds or more. The degree was confirmed to rise.
Here, in order to compare with the crushed state of the tissue mass, 400 mg of porcine lymph node was crushed by the above blender at a rotation speed of 25 krpm for 10, 30, 60 or 90 seconds, and the protein concentration was measured. The measurement was performed in the same manner as in Example 1 (3). The result is shown in FIG.
From FIG. 16, in the case of the rotation speed of 25 krpm, the protein concentration in the reagent increased when the rotation time was 60 seconds. In addition, when the rotation time was 90 seconds, no significant change was observed in the protein concentration as compared with the case where the rotation time was 60 seconds. From the above, it was shown that most of the porcine lymph nodes were crushed at least for 60 seconds (rotation speed: 25 krpm).
From these results, it was found that the rotation time when the pyrophosphate capsule A was crushed and the turbidity was detected was the same rotation time as the rotation time when the porcine lymph node was crushed.
From the above measurement results, it is confirmed that the homogenization reagent added with cytokeratin reagent becomes cloudy in the measurement using GD-100 if the homogenization of pyrophosphate capsule A, which is a quality control substance, is performed accurately. It was confirmed that the porcine lymph node, which is a tissue mass, was sufficiently crushed if it was sufficiently crushed so that the reagent became cloudy. From these facts, it was judged that this pyrophosphate capsule A could be used as a quality control substance.

(Example 2)
Quantification of a ball (hereinafter referred to as a dye ball) in which a sodium alginate was used as a raw material and a fat-soluble dye was encapsulated as a labeling substance was examined.
For example, the pigment ball B may be formed by dropping a 1% aqueous solution of sodium alginate 500 to 600 cP (199-09916, manufactured by Wako Pure Chemical Industries, Ltd.) into a 1% aqueous solution of calcium chloride drop by drop. Was made. Using a metering syringe, dissolve the dye (Sudan III (trade name) manufactured by Wako Pure Chemical Industries, Ltd.) in 0.5% oleic acid in the interior so that the concentration is 0.5%. A dye ball B was prepared by enclosing 5 μL. As shown in FIG. 17, this dye ball B has three layers: a hydrophobic inner layer (dye oleic acid solution) B1, a hydrophilic protective layer (sodium alginate aqueous solution) B2, and an outermost layer (gel of sodium alginate aqueous solution) B3. Consists of structure.
Three, five, ten, fifteen, twenty or twenty-five dye balls B were placed in each of six test tubes, and water was added to all the test tubes to make 2 mL. Then, the dye balls B in each tube were completely destroyed with a pestle by visual confirmation. The hydrophobic layer and the hydrophilic layer were separated by centrifugation, and the hydrophilic layer was removed with an aspirator. Uncolored oleic acid was added thereto to prepare 200 μL of a dye solution. This dye solution is diluted 5-fold (dye solution 20 μL + oleic acid 80 μL) or 2-fold diluted (dye solution 30 μL + oleic acid 30 μL), and 50 μL is dispensed into a 96-well plate, and the absorbance at 465 nm is obtained using an absorption plate reader. Was measured. The results are shown in Table 1 and FIG.

From FIG. 18, it was confirmed that the absorbance increased depending on the number of the dye balls B.
From the above results, it can be seen that this dye ball B can be used as a quality control substance. For example, when 10 dye balls B are crushed as described above and the resulting dye solution is diluted 2 times, the absorbance is a value close to 0.074 (preferably within ± 20%, more preferably ± 10). %)), It can be determined that the crushing operation performed on the dye ball B was appropriate. On the other hand, when the absorbance is significantly lower than 0.074 (for example, less than 80%), it is determined that the crushing operation is not appropriate and the dye ball B has not been sufficiently crushed. In such a case, it is considered that tissue masses such as lymph nodes cannot be sufficiently crushed, so it is necessary to review the setting of the crushing operation (for example, increasing the rotation speed of the blender, increasing the rotation time, etc.) ).

1 is an overall configuration diagram of a nucleic acid detection system. It is a schematic block diagram of a pre-processing apparatus. It is a perspective view of a nucleic acid detection apparatus. It is a top view of a nucleic acid detection apparatus. It is a graph which shows the relationship between amplification rise time and turbidity. It is the graph in which the calibration curve which shows the relationship between amplification rise time and target nucleic acid copy number was drawn. It is a flowchart which shows the quality control process of the pre-process in a personal computer. It is a flowchart which shows the quality control process of the pre-processing in a pre-processing apparatus main body and a nucleic acid detection apparatus main body. It is sectional drawing which shows a pyrophosphate capsule. It is a graph which shows the relationship between the number of pyrophosphate capsules, and turbidity. It is a graph which shows the relationship between the presence or absence of the magnesium ion of a buffer solution, and turbidity. It is a graph which shows the relationship between the presence or absence of the magnesium ion of a buffer solution, and transmitted light intensity. It is a graph which shows the relationship between the rotation speed of a blender, and turbidity. It is a graph which shows the relationship between the rotation speed of a blender, and the protein concentration of a porcine lymph node. It is a graph which shows the relationship between the rotation time of a blender, and turbidity. It is a graph which shows the relationship between the rotation time of a blender, and the protein concentration of a porcine lymph node. It is sectional drawing which shows a pigment | dye ball | bowl. It is a graph which shows the relationship between the dilution of a pigment | dye ball | bowl, and a light absorbency.

Explanation of symbols

1 Sample analysis system (nucleic acid detection system)
5 Pretreatment device 6 Data processing device 101 Nucleic acid detection device

Claims (9)

A method for confirming the operation of a crushing device for crushing a sample containing cells collected from a living body in a buffer solution,
The quality control material encapsulating pyrophosphate was crushed in by Ri buffer solution to the crushing device, as engineering of the pyrophosphate Ru liberated in the buffer solution from the quality control material,
A step of mixing the buffer solution from which pyrophosphate has been released and a divalent cation,
A step of detecting a change in turbidity of the mixed liquid obtained in the step, and a step of determining whether or not the crushing operation is appropriate based on the detection result;
Operation check method of crushing device, including. A method for confirming the operation of a crushing device for crushing a sample containing cells collected from a living body in a buffer solution,
The step of changing the physical properties of the buffer solution by crushing the quality control material encapsulating the labeling material in the buffer solution by the crushing device, and releasing the labeling material from the quality control material into the buffer solution,
Detecting the change in physical properties; and
A step of determining whether or not the crushing operation was appropriate based on the detection result;
Including
The quality control substance has an inner layer containing a labeling substance, and a protective layer that covers the outer side of the inner layer and protects the inner layer, and between the inner layer and the protective layer. A method for confirming the operation of the crushing apparatus , in which an interface is formed on the surface. The method according to claim 2 , wherein the labeling substance and the inner layer are hydrophilic, and the protective layer is hydrophobic. A method for confirming the operation of a crushing device for crushing a sample containing cells collected from a living body in a buffer solution,
The step of changing the physical properties of the buffer solution by crushing the quality control material encapsulating the labeling material in the buffer solution by the crushing device, and releasing the labeling material from the quality control material into the buffer solution,
Detecting the change in physical properties; and
A step of determining whether or not the crushing operation was appropriate based on the detection result;
Including
The operation checking method of the crushing apparatus, wherein the quality control material is set to have a strength so as to be crushed by a crushing operation capable of crushing the sample or a crushing operation having a higher strength. A method for confirming the operation of a crushing device for crushing a sample containing cells collected from a living body in a buffer solution,
The step of changing the physical properties of the buffer solution by crushing the quality control material encapsulating the labeling material in the buffer solution by the crushing device, and releasing the labeling material from the quality control material into the buffer solution,
Detecting the change in physical properties; and
A step of determining whether or not the crushing operation was appropriate based on the detection result;
Including
The method for confirming the operation of the crushing apparatus, wherein a layer whose hardness decreases by absorbing liquid is formed on the surface of the quality control material. A method for performing accuracy control of a pretreatment device for genetic testing that disrupts a sample containing cells collected from a living body in a buffer solution,
The quality control material encapsulating pyrophosphate triturated with by Ri buffer solution in the pretreatment device, the quality control material the buffer crushing step that is released into the pyrophosphate from,
After performing the crushing step, a mixing step of mixing the buffer solution from which the pyrophosphate is liberated and a divalent cation,
A detection step of detecting the turbidity of the liquid mixture obtained in the mixing step; and
A determination step of determining whether or not the crushing operation was appropriate based on the detected turbidity ;
Control method of pretreatment device for genetic testing including In the determination step,
If the detected turbidity is less than a predetermined value, determine that the crushing operation was not appropriate,
The method according to claim 6 , wherein when the detected turbidity is equal to or greater than a predetermined value, the crushing operation is determined to be appropriate. A sample analysis system used in the quality control method according to claim 6 or 7 ,
A measurement unit for measuring the turbidity of a mixture of the buffer solution from which pyrophosphate is liberated and a divalent cation ;
Based on the turbidity measured by the measurement unit, determination means for determining whether or not the crushing process was appropriate,
A sample analysis system. The sample analysis system according to claim 8 , further comprising a crushing unit that crushes the quality control substance in the buffer solution.

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