A calibration device and calibration method for a PCR instrument temperature calibration device

Abstract

The present invention relates to a calibration device and calibration method for a temperature calibration device used in a PCR instrument. The device comprises a base and a top cover, wherein a fixing device is arranged on a side of the base pointing to the top cover, so as to fix the temperature sensor probe of the temperature calibration device of the PCR instrument; the top cover is installed above the base, and a storage space for accommodating the temperature calibration device of the PCR instrument is provided between the top cover and the opposite surface of the base; the top cover has a wire through hole on the opposite surface of the base, so as to allow the data line of the temperature calibration device of the PCR instrument to pass through the storage space. The present invention provides a device for calibrating the temperature sensor probe of the temperature calibration device of the PCR instrument, which ensures the accuracy of the temperature calibration device of the PCR instrument itself, thereby ensuring the temperature accuracy of the PCR instrument calibrated by the temperature calibration device of the PCR instrument, and the device has a simple structure, is easy to use, has strong versatility, and is easy to promote and use.

Description

Technical Field

[0001] The invention belongs to the field of temperature detection and control, and in particular relates to a correction device for a temperature calibration device (equipment) used in a PCR instrument, and further relates to a correction method using the correction device. Background Art

[0002] Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments in vitro in a short time. It is widely used in life science research, food hygiene, medical treatment, forensics and environmental monitoring. The working principle of the PCR instrument (gene amplifier) ​​is to create different temperature environments for DNA amplification, so that DNA denaturation, annealing and primer extension can be achieved. In essence, it is a precise temperature controller. The accuracy, consistency and volatility of its temperature directly affect the amplification quality of gene fragments during the three stages of the DNA reaction. In the process of optimizing the annealing temperature, temperature accuracy is particularly important. This process requires the PCR instrument heating module to have accuracy and consistency. It can be said that in addition to the sample itself, the accuracy of the PCR instrument temperature is a key factor in determining the success or failure of the PCR reaction, and it is also a key factor in determining the success or failure of subsequent genetic testing.

[0003] In order to ensure the temperature accuracy of the PCR instrument, the prior art has proposed a variety of devices for temperature calibration of PCR (Zhang Liping, Research on Temperature Calibration Device of Gene Amplifier (PCR Instrument), Master's Degree Thesis of Tianjin University, 2012; Zeng Ying, Research on Temperature Calibration of PCR Instrument, "Measurement and Testing Technology") 2016, 43(10); CN203855583U). These calibration devices can be summarized as: a plurality of temperature sensor probes (generally thermal resistor type) are evenly arranged on an array PCB substrate to form a temperature sensor module of the PCR instrument temperature calibration device; when in use, the temperature sensor module is placed in the sample rack of the PCR instrument, each temperature sensor probe is inserted into a plurality of test tube slots of the sample rack, the PCR instrument is started, and the temperature of each probe is collected using a data acquisition device, and the data is processed using relevant system operating software and the PCR instrument is temperature calibrated.

[0004] It can be seen that the accuracy of the temperature calibration of the PCR instrument is closely related to the accuracy of the temperature sensor probe of the temperature sensor module. The working principle of these temperature sensor probes is to convert the change of thermal energy into an electrical signal, and the back-end equipment collects the change of the electrical signal, and calculates the corresponding temperature value according to the formula. However, whether it is a thermocouple type probe, a thermal resistor type probe, or a composite probe, its temperature-potential relationship is not linear, but a fitting curve, and with the long-term use of the probe and the inevitable hardware subtle changes caused thereby, the actual temperature-potential relationship is caused to deviate from the preset fitting curve, causing the distortion of these temperature sensor probes themselves, and finally causing the whole set of calibration devices to be unable to reflect the real temperature situation, and it is even more impossible to calibrate the temperature of the PCR instrument. Therefore, the prior art is in urgent need of equipment and methods that can calibrate these PCR instrument temperature calibration devices (rather than the PCR instrument itself) Summary of the invention

[0005] In order to solve the problems existing in the prior art, an object of the present invention is to provide a calibration device for calibrating a temperature calibration device of a PCR instrument. The calibration device can be applicable to temperature calibration devices of PCR of various models, is easy to use and produces reliable results.

[0006] Specifically, the calibration device of the present invention includes a base that matches the probe of the temperature calibration device of the PCR instrument and a top cover that can be sealed and covered with the base. The base is used for: a fixing device for the temperature sensor probe is set on the side of the base pointing to the top cover, and the fixing device includes blind holes arranged in a matrix corresponding to the temperature sensor probe. The depth of each blind hole of the same type of calibration device is the same, which is consistent with the size and depth of the temperature sensor probe. The structure ensures that the temperature sensor probe is tightly combined with the base, and the temperature equilibration time is as short and consistent as possible. Different types of PCR instruments have different temperature calibrations, such as 96-well, 384-well, etc.

[0007] The fixing device fixes the temperature sensor probe of the temperature calibration device of the PCR instrument; the base makes the temperature sensor probe fit tightly, avoids air isolation, has good thermal conductivity, greatly shortens the temperature balancing time of the temperature sensor probe, and can quickly reach the set calibration temperature.

[0008] The top cover is installed above the base, and there is an accommodating space for accommodating the temperature calibration device of the PCR instrument between the opposite surfaces of the top cover and the base; the edge design of the top cover is completely consistent with the base, and the accommodating space only allows the temperature sensor probe and the supporting support and information acquisition board to be placed in it, minimizing the reserved space to ensure that the temperature measuring probe can be in close contact with the base and the top cover, and structurally ensuring that the temperature equilibrium time of the temperature sensor probe is as short as possible.

[0009] The top cover has a through hole on the opposite side of the base to allow the data line (inseparable from the probe) of the PCR instrument temperature calibration device to pass through the accommodation space. The reservation of the data hole ensures the effective transmission of the data line, but also considers minimizing the space as much as possible, allowing only the data line to enter and exit.

[0010] Furthermore, in order to reduce the overall thickness of the base, the base corresponding to the fixing device has a protrusion toward the bottom surface (in order to facilitate the arrangement of the blind holes arranged in the matrix), and preferably a plurality of parallel grooves are arranged on the protrusion, wherein each groove is arranged at a position corresponding to each row of blind holes. With such an arrangement, when the calibration device is placed in the oil tank for heating during use, it can ensure that the temperature sensor probe can be as close to the medium oil as possible, reduce the balancing time of the temperature sensor, and increase the sensitivity. Therefore, parallel grooves are designed to ensure that the temperature balancing time is as short and as consistent as possible.

[0011] In one embodiment, the blind holes arranged in a matrix corresponding to the temperature sensor probes on the fixture are realized by a plurality of parallel arranged slide rails and a slider with blind holes slidably connected to the slide rails. The slider with blind holes allows the temperature sensor probes to be in close contact with the blind holes to the greatest extent possible, and then the position of the blind holes is adjusted by the slider to adapt to the arrangement of the temperature sensor probes of temperature calibration devices of different models of PCR instruments.

[0012] Furthermore, the opposite surface of the top cover and the base is a square or a rectangle. Since the PCR temperature sensor probe is generally arranged in 8×12, 6×8, 5×6, 16x24 and other types, it is usually a rectangle.

[0013] Furthermore, the fixing device is aligned along one side of the long side of the base or in the center of the base, preferably in the center.

[0014] The top cover and the base are sealed together when in use, and preferably a sealing strip is provided between the two to ensure that oil cannot penetrate into the interior when in use. The cover between the two can be connected by snap-fit ​​connection, slide groove connection, screw connection, or bolt and nut connection. In one embodiment, the top cover and the base are connected by a hinge on one side.

[0015] Furthermore, the calibration device also includes a hollow wire tube that matches the wire through hole, and the data line passes through the wire through hole from the accommodating space and then passes through the wire tube. Most of the calibration device base and upper cover need to be immersed in the calibration oil tank, but the wires cannot contact the oil. Providing the wire tube can make the outlet of the wire above the oil surface, which is convenient for removal. This design reduces the material used to make the top cover by reducing the thickness of the top cover, but can prevent the data line of the PCR instrument temperature calibration device from contacting the oil in the oil tank during use, so the protruding hollow wire tube is provided.

[0016] Among them, the wire passing tube and the top cover are integrally formed, fixedly connected as separate parts, or detachably connected as separate parts. Preferably, the wire passing tube is detachably connected to the top cover by means of the screws or bolts and nuts.

[0017] Furthermore, the calibration device also includes a wire tube cover that matches the opening of the wire tube and is detachable from the wire tube. The wire tube cover is provided with a wire slot for the data line (usually a flat cable) of the PCR instrument temperature calibration device to pass through. Preferably, the wire slot extends along the long side of the wire tube cover and passes through the wide side of the wire tube cover. The length of the wire tube cover is preferably consistent with the height of the wire tube so as to fill the entire space of the wire tube.

[0018] Another object of the present invention is to provide a method for using the aforementioned calibration device, comprising the following steps:

[0019] S01, placing the PCR instrument temperature calibration device into the base of the calibration device of the present invention, so that the temperature sensor probe of the PCR instrument temperature calibration device is fixed by the fixing device;

[0020] S02, passing the data line of the PCR instrument temperature calibration device through the line through hole of the top cover of the calibration device of the present invention, and buckling the top cover on the base to ensure a sealed cover;

[0021] S03, placing the calibration device into the oil tank of the precision temperature measuring device, placing a standard platinum thermometer into the oil tank, and connecting the PCR instrument temperature calibration device and the standard platinum thermometer to a computer with calibration software via a reading device;

[0022] S04. Set the precision temperature measuring device to the specified temperature T n After the temperature is balanced, read the reading T of the standard platinum thermometer. 0 As the temperature measured by the precision temperature measuring device, read the reading T of each temperature sensor probe of the PCR instrument temperature calibration device x As the temperature of the temperature sensor probe before calibration;

[0023] S05. According to T 0 and T x, calibrate each temperature sensor probe of the PCR instrument temperature calibration device through the calibration software.

[0024] Furthermore, in step S02, the calibration device further includes a hollow wire-passing tube matched with the wire-passing through hole, and the data line passes through the wire-passing through hole from the accommodating space and then passes through the wire-passing tube.

[0025] Furthermore, in step S04, the specified temperature T n Includes multiple temperature values, preferably T n The value range is 0℃≤T n ≤120℃.

[0026] Furthermore, in step S04, the specified temperature T n It is a plurality of temperature values ​​unevenly distributed from low to high.

[0027] Furthermore, in step S04, the specified temperature T n It is 0℃, 30℃, 50℃, 70℃, 90℃, 120℃.

[0028] Furthermore, the method of use also includes:

[0029] S06. After step S05, the precision temperature measuring device is set to the verification temperature T n ', after the temperature is balanced, read the reading T of the standard platinum thermometer 0 'As the temperature of the precision temperature measuring device, read the reading T of each temperature sensor probe of the PCR instrument temperature calibration device after calibration x ' as the temperature of the temperature sensor probe after correction; according to T 0 and T x ', check the temperature calibration results of each temperature sensor probe.

[0030] Furthermore, in step S06, the verification temperature T n ' includes multiple temperature values, preferably the T n ' is the specified temperature T n Multiple temperature values ​​in reverse order.

[0031] The beneficial effects of the present invention are: providing a device for calibrating the temperature sensor probe of the PCR instrument temperature calibration device, filling the gap in the prior art, and ensuring the accuracy of the PCR instrument temperature calibration device itself by calibrating the temperature sensor probe itself, thereby ensuring the temperature accuracy of the PCR instrument calibrated using the PCR instrument temperature calibration device, and meeting the requirements of DNA amplification for accurate control of the environmental temperature.

[0032] Furthermore, the present invention reduces the use of materials through a reasonably designed calibration device, while ensuring that the probe is heated uniformly and uniformly from a structural perspective, ensuring that the temperature equilibrium time is as short and as consistent as possible, and can also be applied to temperature calibration devices of PCR instruments of different specifications from various manufacturers and models, and has strong versatility. Therefore, the calibration device of the present invention has a simple structure, is easy to use, and is easy to promote and use. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a front view of an embodiment of the correction device of the present invention.

[0034] Figure 2 1 is a top view of a base in one embodiment of the correction device of the present invention.

[0035] Figure 3 1 is a top view of a top cover in one embodiment of the correction device of the present invention.

[0036] Figure 4 It is a front view of another embodiment of the correction device of the present invention.

[0037] Figure 5 It is a top view of a wire tube cover in another embodiment of the correction device of the present invention. DETAILED DESCRIPTION

[0038] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0039] Example 1

[0040] like Figure 1 As shown, the calibration device of the present invention comprises a base 1 and a top cover 2 matched with the base 1. Preferably, the facing surfaces of the top cover and the base are rectangular, and the two are detachably connected by screws.

[0041] See also Figure 2 , a fixing device 3 for the temperature sensor probe is arranged on the side of the base 1 pointing to the top cover 2, so as to fix the temperature sensor probe of the PCR instrument temperature calibration device. As a preferred solution, in order to ensure the best temperature conduction effect, the fixing device is a blind hole 4 arranged in a matrix, and the spacing of the blind holes 4 can be determined according to actual needs. For example, for a certain type of PCR instrument temperature calibration device, the spacing of the blind holes 4 is set to be the same as the spacing of each temperature sensor probe of the calibration device to achieve optimal matching. The depth of each blind hole can also be determined according to the length of the temperature sensor probe.

[0042] See also Figure 3The top cover 2 is installed on the base 1, and there is a storage space 5 for accommodating the temperature calibration device of the PCR instrument between the top cover 2 and the opposite surface of the base 1; the top cover 2 has a wire through hole 6 on the surface opposite to the base 1, so that the data line of the temperature calibration device of the PCR instrument can pass through the storage space 5. In a preferred case, the wire through hole 6 is opened on one side of the top cover 2 along its long side, and below the wire through hole 6 is a matrix of blind holes 4 as a fixing device.

[0043] In a preferred embodiment, the bottom surface of the base corresponding to the fixing device has a protrusion (not shown in the figure), and a plurality of parallel grooves are arranged on the protrusion, wherein each groove is respectively arranged at a position between each row of blind holes corresponding to the front side of the fixing device.

[0044] Example 2

[0045] like Figure 4 As shown, in order to facilitate the fixing of the data line of the temperature calibration device of the PCR instrument, on the basis of the calibration device shown in Example 1, a hollow wire tube 7 matching the wire through hole is further provided, and the data line passes through the wire through hole 6 from the accommodating space 5 and then passes through the hollow wire tube 7. In order to further prevent the dissipation of heat, the calibration device also includes a wire tube cover 8, and the wire tube cover 8 matches the opening of the hollow wire tube 7. For example, the outer diameter of the lower part of the wire tube cover 8 is shrunk so that it can be inserted into the hollow wire tube 7. A wire seam 9 is provided on the wire tube cover 8 for the data line (usually a cable) of the temperature calibration device of the PCR instrument to pass through. As a preferred embodiment, as Figure 5 As shown, the wire-passing seam 9 extends along the long side of the wire-passing tube cover 8 and passes through the wide side of the wire-passing tube cover 8, so that the placement and removal of the data cable can be facilitated.

[0046] Example 3

[0047] Based on Example 1 or 2, this Example 3 demonstrates a preferred method for calibration using the calibration device of the present invention.

[0048] S01. Place the temperature calibration device of the PCR instrument into the base 1 of the calibration device of the present invention, so that the temperature sensor probe of the temperature calibration device of the PCR instrument falls into the corresponding blind hole 4.

[0049] S02. Pass the data line of the PCR instrument temperature calibration device through the line through hole 6 of the top cover 2 of the calibration device of the present invention. In one embodiment, the data line passes through the line through tube 7. Then, buckle the top cover 2 onto the base 1 and close it.

[0050] S03. Place the calibration device into the oil tank of the precision temperature measuring device, place the standard platinum thermometer into the oil tank, and connect the PCR instrument temperature calibration device and the standard platinum thermometer to a computer with calibration software via a reading device.

[0051] S04, set the specified temperature T of the precision temperature measuring device n Set to T 1 =0℃、T 2 =30℃、T 3 =50℃、T 4 =70℃、T 5 =90℃、T 6 =120℃, for each T n After the temperature is balanced, read the readings T of the standard platinum thermometer in turn. 01 , T 01 , T 03 , T 04 , T 05 , T 06 , as the temperature measured by the precision temperature measuring device, read the reading T of each temperature sensor probe of the PCR instrument temperature calibration device x The temperature of the temperature sensor probe before calibration is shown in the table below.

[0052] Temperature series <![CDATA[T n ]]> <![CDATA[T 0 ]]> <![CDATA[Probe T No. 1 1 > <![CDATA[Probe T No. 2 2 > ...... <![CDATA[Probe T of x number x > 0℃ <![CDATA[T 1 ]]> <![CDATA[T 01 ]]> <![CDATA[T 11 ]]> <![CDATA[T 21 ]]> ...... <![CDATA[T x1 ]]> 30℃ <![CDATA[T 2 ]]> <![CDATA[T 02 ]]> <![CDATA[T 12 ]]> <![CDATA[T 22 ]]> ...... <![CDATA[T x2 ]]> 50℃ <![CDATA[T 3 ]]> <![CDATA[T 03 ]]> <![CDATA[T 13 ]]> <![CDATA[T 23 ]]> ...... <![CDATA[T x3 ]]> 70℃ <![CDATA[T 4 ]]> <![CDATA[T 04 ]]> <![CDATA[T 14 ]]> <![CDATA[T 24 ]]> ...... <![CDATA[T x4 ]]> 90℃ <![CDATA[T 5 ]]> <![CDATA[T 05 ]]> <![CDATA[T 15 ]]> <![CDATA[T 25 ]]> ...... <![CDATA[T x5 ]]> 120℃ <![CDATA[T 6 ]]> <![CDATA[T 06 ]]> <![CDATA[T 16 ]]> <![CDATA[T 26 ]]> ...... <![CDATA[T x6 ]]>

[0053] S05. According to T 0 and T x , calibrate each temperature sensor probe of the PCR instrument temperature calibration device, and use the calibration software to calculate to obtain the calibration result.

[0054] S06. After calibration, set the precision temperature measuring device to the verification temperature T n’ (T 1’ =120℃、T 2’ =90℃、T 3’ =70℃、T 4’ =50℃、T 5’ =30℃、T 6’ = 0°C), after the temperature is balanced, read the reading T of the standard platinum thermometer 0 'As the temperature of the precision temperature measuring device, read the reading T of each temperature sensor probe of the PCR instrument temperature calibration device after calibration x ' as the temperature of the temperature sensor probe after correction; according to T 0 ' and T x ', check the temperature calibration results of each temperature sensor probe.

[0055] The following table shows the 5 =Calibration result at 90°C.

[0056]

[0057]

[0058] It can be seen from the table that before calibration, the temperature range difference of the 9 probes was 0.28°C. After calibration using the calibration device and calibration method of the present invention, the temperature range difference of the 9 probes was reduced to 0.08°C.

[0059] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the invention, which should be covered by the protection scope of the present invention.

Claims

1. A calibration device for a PCR instrument temperature calibration device, characterized in that: It includes a base and a top cover that can be sealed with the base: A fixing device is provided on a side of the base pointing to the top cover, so as to fix the temperature sensor probe of the temperature calibration device of the PCR instrument; The top cover is installed above the base, and there is a receiving space for receiving the temperature calibration device of the PCR instrument between the top cover and the opposite surface of the base; The top cover has a wire through hole on the surface opposite to the base, so that the data wire of the PCR instrument temperature calibration device can pass through the accommodation space; The fixing device comprises a plurality of blind holes arranged in a matrix, and the blind holes are suitable for placing the probes; The fixing device comprises a plurality of slide rails arranged in parallel and a sliding block having a blind hole slidably connected to the slide rails; The wire-passing through hole is a protruding hollow wire-passing tube; There is also a wire passing tube cover matched with the hollow wire passing tube, and the wire passing tube cover has a wire passing gap for the data line to pass through; The bottom surface of the base has a protrusion relative to the fixed position, and a plurality of grooves arranged in parallel are arranged on the protrusion, wherein each groove corresponds to a position between each row of blind holes; The facing surfaces of the top cover and the base are square or rectangular; The sealing connection between the top cover and the base is achieved by means of sealant; The top cover and the base are connected by snap fastening, sliding groove, screws, or bolts and nuts; or the top cover and the base are connected by hinges on one side.

2. The method for using the calibration device according to claim 1, characterized in that: The following steps are involved: S01, placing a PCR instrument temperature calibration device into the base of the calibration device, so that the temperature sensor probe of the PCR instrument temperature calibration device is fixed by the fixing device; S02, passing the data line of the PCR instrument temperature calibration device through the line through hole of the top cover of the calibration device, and buckling the top cover on the base; S03, placing the calibration device into the oil tank of the precision temperature measuring device, placing a standard platinum thermometer into the oil tank, and connecting the PCR instrument temperature calibration device and the standard platinum thermometer to a computer with calibration software through a reading device; S04. Set the precision temperature measuring device to the specified temperature T n After the temperature is balanced, read the reading T0 of the standard platinum thermometer as the measured temperature of the precision temperature measuring device, and read the reading T of each temperature sensor probe of the PCR instrument temperature calibration device. x As the temperature of the temperature sensor probe before calibration; S05, according to T0 and T x , calibrate each temperature sensor probe of the PCR instrument temperature calibration device through the calibration software; Furthermore, after step S05, the precision temperature measuring device is set to the verification temperature T n ’ After the temperature is balanced, read the reading T0 of the standard platinum thermometer. ’ As the measured temperature of the precision temperature measuring device, read the reading T of each temperature sensor probe of the PCR instrument temperature calibration device after calibration. x ’ As the temperature of the temperature sensor probe after correction; according to T0 and T x ’ , check the temperature calibration results of each temperature sensor probe.

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