Temperature calibration method of nucleic acid amplification analyzer

Abstract

The invention relates to a temperature calibration method of a nucleic acid amplification analyzer, and relates to the field of instrument calibration, a temperature probe of a standard temperature measuring instrument is placed on the instrument, and point value temperatures of 40 DEG C, 50 DEG C, 60 DEG C, 70 DEG C, 80 DEG C, 90 DEG C and 100 DEG C are set; starting the data acquisition instrument, respectively operating and heating to temperatures of different target temperature values, calculating an average temperature value of each hole site, and calculating an absolute value of a difference value between the average temperature value and the target temperature value; a mycoplasma pneumoniae quality control product inspection solution with a standard concentration is used, a matched mycoplasma pneumoniae nucleic acid detection kit is added and then put into an instrument to detect a nucleic acid sample, a temperature drift compensation module is arranged, and the deviation of circuit change in long-term use is corrected through periodic self-inspection; the method has the advantages that the difference between the target temperature value and the actual detection temperature value caused by various factors is eliminated, the temperature drift of the instrument in the long-time use process is solved, and the temperature control accuracy of the instrument in the long-time use process is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of instrument calibration, and particularly to a temperature calibration method for a nucleic acid amplification analyzer. Background Art

[0002] A nucleic acid amplification analyzer can efficiently complete the nucleic acid amplification process in a short time. Each well of the nucleic acid amplification analyzer can monitor the change amount of the reaction product by detecting the change in the optical signal intensity after each reaction cycle of the test sample, and generate an amplification curve graph. During or at the end of the nucleic acid amplification process, it can be judged whether there is a corresponding nucleic acid fragment in the test sample according to the amplification curves of each well.

[0003] During the nucleic acid amplification process, correctly controlling the temperature at each stage (denaturation, annealing, extension) is crucial for amplifying a specific target sequence. Precise temperature control can promote the denaturation of DNA molecules, the binding of primers, and ensure the activity of DNA polymerase. Usually, when performing annealing gradient temperature screening for a certain reagent, the initial gradient temperature interval is set to 2°C. After determining a large temperature range, the interval is designed to be 0.5°C for further screening. In order to achieve the best amplification effect of the reagent, a gradient temperature of 0.2°C needs to be set finally. Even a temperature difference of 0.2°C will have a great impact on the reagent amplification result, resulting in irreversible errors, such as the binding of primers to non-specific DNA sequences, or the amplification of non-specific products, etc. These errors will seriously affect the accuracy and reliability of the detection result, and may even lead to misjudgment or wrong judgment of the experimental result. Therefore, the accuracy of temperature is very crucial for the performance of the instrument.

[0004] Since various temperature sensors are non-linear devices, the temperature values obtained by a single linear formula method have a large deviation of the linear correlation coefficient R relative to 1, and the measured values of most temperature points in the entire measurement temperature range are inaccurate; the random differences caused by different electronic components, welding methods, etc. cannot be compensated, further increasing the temperature measurement deviation. Moreover, the temperature collected by the nucleic acid amplification analyzer is the temperature of the temperature sensor of the instrument itself. The position of the temperature sensor is arranged between the circuit board and the heating block, while the actual temperature to be controlled is the temperature of the liquid in the PCR tube. There is a large difference between these two temperatures due to different positions and heat conduction efficiencies. At the same time, the electrical characteristics of each electronic device will change with the temperature during long-term use under temperature changes. At this time, adding the temperature difference, the traditional calibration devices and calibration methods only for temperature differences cannot accurately calibrate.

[0005] Therefore, in view of the above deficiencies, a temperature calibration method for a nucleic acid amplification analyzer needs to be provided. Summary of the Invention

[0006] (I) Technical Problems to be Solved The technical problem to be solved by the present invention is to solve the problem that accurate temperature control cannot be achieved through a single linear formula.

[0007] (II) Technical solution To solve the above technical problem, the present invention provides a temperature calibration method for a nucleic acid amplification analyzer, comprising the following steps: I. Place the temperature probe of the standard temperature measuring instrument into the instrument, use the probe to simulate the temperature of the reagent in the tube during the actual nucleic acid amplification process, and set the point temperature values of 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C to determine the calibration temperature accuracy; II. Turn on the data collector, and respectively run the temperature to different target temperature values. When the temperature fluctuation is less than 0.05°C, use it as the timing reference point. After maintaining the temperature for 10 s, collect the temperature value every 10 s, and continuously collect 10 times for each detection hole; calculate the average temperature value of each hole position, and calculate the absolute value of the difference from the target temperature value; III. Use the standard concentration Mycoplasma pneumoniae quality control product inspection solution, add the supporting Mycoplasma pneumoniae nucleic acid detection kit, and then put it into the instrument to detect the nucleic acid sample. Repeat the test for 8 detection hole positions, and calculate the average value of its CT value; the same batch of samples are placed on a standard PCR instrument for detection, and calculate the average value of its CT value. Finally, calculate the accuracy, specifically: Wherein, is the accuracy; is the average value of the CT values detected when the nucleic acid sample is placed on the instrument to be tested; is the average value of the CT values detected when the nucleic acid sample is placed on a standard PCR instrument; IV. Set up a temperature drift compensation module, and correct the deviation of the circuit change during long-term use through periodic self-checking.

[0008] As a further description of the present invention, preferably, the measured temperature value of the standard temperature measuring instrument and the target temperature value are subjected to least squares fitting in multiple intervals within the temperature calibration range to perform secondary calibration on the instrument temperature, and calculate its linear correlation coefficient.

[0009] As a further description of the present invention, preferably, record the initial contact resistance and establish a reference temperature residual distribution. When the contact resistance change rate is greater than 5%, or the KL divergence is higher than the threshold, the temperature drift mechanism is triggered.

[0010] As a further description of the present invention, preferably, the KL divergence calculation formula is: wherein, is the time of the current residual distribution; is the reference residual distribution.

[0011] As a further description of the present invention, preferably, the threshold is dynamically updated according to historical data, and its calculation formula is: wherein, is the threshold; is the forgetting factor, taking a value of 0.1 to 0.3; is the current value.

[0012] As a further description of the present invention, preferably, the calculation formula of the contact resistance change rate is: wherein, is the contact resistance change rate; is the current contact resistance; is the initial contact resistance.

[0013] As a further description of the present invention, preferably, the calibration calculation formula is: wherein, is the calibrated temperature value; is the slope calibration parameter of the interval; is the current sensor contact resistance value; is the intercept calibration parameter of the interval.

[0014] As a further description of the present invention, preferably, when the temperature drift mechanism is triggered, there is: wherein, is the learning rate, and the value range is 0.1 to 0.3; is the residual value, and the calculation formula is: Among them, is the measured value of the current temperature sensor; and are the current segmented calibration parameters; is the target temperature value.

[0015] As a further description of the present invention, preferably, six intervals of 40°C to 50°C, 50°C to 60°C, 60°C to 70°C, 70°C to 80°C, 80°C to 90°C, and 90°C to 100°C are respectively set, and the calibration parameters are fitted by the least squares method. The calculation formula is: Among them, and are the average values of temperature and resistance within this interval.

[0016] As a further description of the present invention, preferably, the value of the learning rate is selected by dynamic adjustment, and the calculation formula is as follows: Among them, is the initial learning rate; is the adjustment factor.

[0017] (III) Beneficial effects The above technical solutions of the present invention have the following advantages: By designing a new calibration method, the present invention fits the target temperature value and the actual measured temperature value within the temperature calibration range by the least squares method in intervals, refines the local part, and the linearity will be more accurate; after compensating for temperature drift after comprehensively considering all errors (sensor non-linearity, temperature difference between the instrument sensor and the temperature in the tube, circuit device differences, etc.), calibration is performed to find the relationship between the target temperature value of the instrument and the actual measured temperature value of the standard temperature measuring instrument, and temperature calibration is performed on the measured value of the temperature sensor of the instrument itself to ensure that the temperature of the instrument completely corresponds to the actual measured value of the standard temperature measuring instrument, and to ensure that the temperature of the instrument is consistent with the temperature of the actual reagent liquid during the biological experiment, so as to eliminate the differences between the target temperature value and the actual detected temperature value caused by factors such as the non-linear characteristics of the temperature sensor, circuit element differences between different devices and different holes, and the position of the temperature sensor arrangement, and improve the temperature control accuracy of the instrument. Brief description of the drawings

[0018] Figure 1 is the installation effect diagram of the detection device; Figure 2 is the curve of the resistance of the temperature sensor changing with temperature under the traditional calibration method; Figure 3 is the fitting curve of the measured temperature and the target temperature value under the traditional calibration method; Figure 4 is the nucleic acid amplification curve detected when the nucleic acid sample is placed on the instrument under the traditional calibration method; Figure 5 is the nucleic acid amplification curve detected when the nucleic acid sample is placed on a standard PCR instrument under the traditional calibration method; Figure 6 is the fitting curve of the measured temperature and the target temperature value of the present invention; Figure 7 is the nucleic acid amplification curve detected when the nucleic acid sample of the present invention is placed on the instrument. Specific Embodiments

[0019] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0020] A temperature calibration method for a nucleic acid amplification analyzer, as Figure 1 shown, includes the following steps: Ⅰ. Place the temperature probe of the standard temperature measuring instrument at the reagent tube hole position on the instrument, and connect the other end to the data acquisition instrument. Use this probe to simulate the temperature of the reagent in the tube during the actual nucleic acid amplification process. Since the temperature set for PCR amplification is generally in the range of 60°C to 95°C, the nucleic acid amplification analyzer is required to achieve accurate temperature measurement at least in the range of 40°C to 100°C. Therefore, set the point values of 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C to determine the temperature accuracy of calibration.

[0021] Ⅱ. Turn on the data acquisition instrument, and respectively run the temperatures rising to different target temperature values. Take the time when the temperature fluctuation is less than 0.05°C as the timing reference point. After maintaining a constant temperature for 10 s, collect the temperature value every 10 s, and continuously collect 10 times for each detection hole; calculate the average temperature value of each hole position , and calculate the absolute value of the difference from the target temperature value according to the following formula :

[0022] The test result of each hole position at each temperature point should not be greater than 0.2°C.

[0023] III. Use the standard concentration Mycoplasma pneumoniae quality control product to check the solution. After adding the supporting Mycoplasma pneumoniae nucleic acid detection kit, put it into the instrument to detect the nucleic acid sample. Repeat the test for 8 detection hole positions, and calculate the average value of their CT values; put the same batch of samples on a standard PCR instrument for detection, calculate the average value of their CT values, and finally calculate the accuracy according to the following formula: Among them, is the accuracy; is the average value of the CT values detected by the nucleic acid sample on the instrument to be tested; is the average value of the CT values detected by the nucleic acid sample on the standard PCR instrument; IV. Use the least squares method to fit the measured temperature value and the target temperature value of the standard temperature measuring instrument in multiple intervals within the temperature calibration range. It is preferably set to perform secondary calibration on the instrument temperature in 6 intervals of 40°C - 50°C, 50°C - 60°C, 60°C - 70°C, 70°C - 80°C, 80°C - 90°C, and 90°C - 100°C, and calculate their linear correlation coefficients. The calibration calculation formula is: Among them, is the calibrated temperature value; is the slope calibration parameter of the interval, and the calculation formula is: is the current sensor contact resistance value; is the intercept calibration parameter of the interval, and the calculation formula is: Among them, and are the average temperature and resistance within this interval.

[0024] The present invention also has a temperature drift compensation module, which corrects the deviation of circuit changes during long-term use through periodic self-checks. The detection period adopts a combination of equally spaced sampling and unequally spaced sampling: Among them, is the set basic detection period; is a custom constant; is the basic interval, preferably 24 hours; is the perturbation amplitude, preferably 2 - 4 hours; is the perturbation frequency. Preferably, it is recorded once a week, or once after every 100 detections of the sensor.

[0025] Record the initial contact resistance of the temperature sensor before detection and establish the reference temperature residual distribution , and the calculation formula is: where, is the residual value; is the window size, which is selected as 8 in this solution; is the Gaussian function; is the bandwidth parameter, preferably 0.02 °C; is the current measured value of the temperature sensor; and are the current segment calibration parameters; is the target temperature value.

[0026] When the contact resistance change rate is greater than 5%, or the KL divergence is higher than the threshold then trigger the temperature drift mechanism. The calculation formula for the contact resistance change rate is: where, is the contact resistance change rate; is the current contact resistance; is the initial contact resistance.

[0027] The calculation formula for the KL divergence is: where, is the time of the current residual distribution, that is: is the reference residual distribution.

[0028] Threshold value Dynamically updated according to historical data, and its calculation formula is: Wherein, is the threshold value; is the forgetting factor, taking values from 0.1 to 0.3; is the current value.

[0029] When the temperature drift mechanism is triggered: Wherein, is the learning rate, and its value range can be artificially fixed at 0.1 to 0.3. Or it can also be selected by dynamic adjustment, and the calculation formula is as follows: Wherein, is the initial learning rate; is the adjustment factor.

[0030] As Figure 2 shown, the blue curve is the curve of the resistance value of the instrument temperature sensor changing with temperature. According to the traditional method, a single linear fitting is performed on the temperature value and the resistance value. The red curve is the fitted curve. It can be seen from the figure that the linear correlation coefficient R value of the fitted curve has a large deviation from 1, and the linearity is poor. Now, the traditional linear fitting method is used to fit and calibrate the instrument temperature. After the calibration is completed, the temperature accuracy test of the instrument and the CT value accuracy deviation test of the nucleic acid sample are carried out.

[0031] The temperature accuracy test results are as follows:

[0032] As can be seen from the above table, the temperature accuracy at each temperature point is very poor, and the temperature difference is between 0.4 °C and 6.4 °C. All temperatures do not meet the requirement that the temperature deviation test result is not greater than 0.2 °C. The total average value of the measured temperature values at all hole positions at different temperature points is quadratically fitted with the target temperature value, and the fitted curve is as shown in Figure 3 shown. It can be seen from the figure that the linear correlation coefficient R value between the calibrated measured temperature value and the target temperature value still has a large deviation from 1, and the linearity is poor.

[0033] The nucleic acid sample was placed on the instrument for detection, and the CT value results are shown in the following table: The nucleic acid amplification curve is as shown in Figure 4 shown.

[0034] Samples of the same batch were placed on a standard PCR instrument for detection, and the CT value results are shown in the following table: The nucleic acid amplification curve is as shown in Figure 5 shown.

[0035] The accuracy deviation D of the CT value of the nucleic acid sample was calculated to be 5.29.

[0036] From the test results, it can be found that for the instrument calibrated by the traditional method, the temperature accuracy at different temperature points is poor, the accuracy deviation of the CT value of the nucleic acid sample test is large, the nucleic acid amplification efficiency is low, and the quantitative judgment of the initial concentration of the nucleic acid is inaccurate. The reasons are analyzed as follows: 1. Due to the non-linear characteristics of the sensor, the accuracy differences at each temperature point are large; 2. There is a difference between the position where the temperature sensor is arranged and the position where the actual temperature is measured, resulting in a difference between the actual temperature measurement value and the temperature value detected by the sensor; 3. The temperature differences between the detection hole positions caused by different parameters of other circuit elements; 4. The sensor has a temperature drift after the temperature changes.

[0037] To reduce the measurement inaccuracy caused by the above reasons, in the present invention, 6 groups of K and B values are obtained at each hole position by the above method, the instrument is calibrated twice respectively, and the linear correlation coefficient R value is calculated.

[0038] The data is shown in the following table:

[0039] After calibration, temperature accuracy test and nucleic acid sample accuracy deviation test are carried out on the instrument.

[0040] The results of the temperature accuracy test are as follows:

[0041] As can be seen from the above table, the temperature differences at all temperature points are concentrated around 0.01 - 0.1 °C, and all temperatures meet the requirement that the temperature deviation test result is not greater than 0.2 °C. The total mean value of the measured temperature values at all hole positions at different temperature points is quadratically fitted with the target temperature value, and the obtained fitting curve is as Figure 6 shown. It can be seen from the figure that the linear correlation coefficient R value between the calibrated measured temperature value and the target temperature value = 1, and the linearity is very good. The nucleic acid sample is placed on the instrument for detection, and the measured CT value results are as follows:

[0042] The nucleic acid amplification curve is as Figure 7 shown, and the calculated accuracy D of the CT value of the nucleic acid sample is 0.11.

[0043] To sum up, the temperature accuracy test results of the two calibration methods are summarized and compared, and the data are as follows:

[0044] The nucleic acid amplification CT value test results after temperature calibration of the two calibration methods are summarized and compared, and the data are as follows:

[0045] From the comparison of test results, it can be seen that under the same test conditions, compared with the traditional method, the calibration method provided by the present invention significantly improves the temperature accuracy at each temperature point. In the accuracy deviation test of the CT value of the nucleic acid sample, the CT value result is closer to the amplification result of the standard PCR instrument, the nucleic acid amplification efficiency is improved, and the original nucleic acid concentration in the nucleic acid sample can be measured more accurately.

[0046] After a period of use, the parameters of the nucleic acid amplification instrument calibrated by the traditional method usually change due to various reasons, while the calibration coefficient still maintains the previous fixed value, which will inevitably lead to inaccurate temperature and deviation in nucleic acid detection results. Therefore, traditional instruments need to be temperature-calibrated once every six months to ensure normal use. By adopting the calibration method provided by the present invention and setting a temperature drift compensation module, the deviation caused by various reasons during long-term use can be automatically corrected through periodic self-checks. Even after long-term use, the temperature accuracy can be ensured, and the accuracy of nucleic acid detection can be guaranteed. The following table shows the comparison test results of the nucleic acid amplification CT value of the instrument after one year of frequent use and the standard PCR instrument:

[0047] From the above comparison results, it can be seen that for the calibration method provided by the present invention, whether it is just after calibration or after long-term use, the temperature fluctuation range is extremely small. Without subsequent frequent calibration, the temperature accuracy can be maintained, the CT value in the detection results is very close to the amplification result of the standard PCR instrument, the nucleic acid amplification efficiency is improved, and the original nucleic acid concentration in the nucleic acid sample can be measured more accurately.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A temperature calibration method for a nucleic acid amplification analyzer, characterized in that: Including the following steps: Ⅰ. Place the temperature probe of the standard temperature measuring instrument on the instrument, use the probe to simulate the temperature of the reagent in the tube during the actual nucleic acid amplification process, and set the point temperature values of 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C to determine the calibrated temperature accuracy; Ⅱ. Turn on the data acquisition instrument, and run the temperature to different target temperature values respectively. When the temperature fluctuation is less than 0.05°C, use it as the timing reference point. After maintaining a constant temperature for 10 s, collect the temperature value every 10 s, and continuously collect 10 times for each detection hole; calculate the average temperature value of each hole position, and calculate the absolute value of the difference from the target temperature value; Ⅲ. Use the standard concentration Mycoplasma pneumoniae quality control product inspection solution, add it to the supporting Mycoplasma pneumoniae nucleic acid detection kit and then put it into the instrument to detect the nucleic acid sample. Repeat the test for 8 detection hole positions, and calculate the average value of its CT value; the same batch of samples is placed on a standard PCR instrument for detection, and the average value of its CT value is calculated. Finally, calculate the accuracy, specifically: Wherein, For accuracy; is the average CT value detected when the nucleic acid sample is placed on the instrument to be tested; is the average CT value detected when the nucleic acid sample is placed on a standard PCR instrument; Ⅳ. Set the temperature drift compensation module, and correct the deviation of the circuit change during long-term use through periodic self-checking.

2. The temperature calibration method of a nucleic acid amplification analyzer according to claim 1, characterized in that: Perform least squares fitting on the measured temperature value of the standard temperature measuring instrument and the target temperature value in multiple intervals within the temperature calibration range to perform secondary calibration on the instrument temperature, and calculate its linear correlation coefficient.

3. A temperature calibration method for a nucleic acid amplification analyzer according to claim 2, characterized in that: Record the initial contact resistance and establish the reference temperature residual distribution. When the contact resistance change rate is greater than 5%, or the KL divergence is higher than the threshold, the temperature drift mechanism is triggered.

4. A temperature calibration method for a nucleic acid amplification analyzer according to claim 3, characterized in that: The calculation formula of KL divergence is: Wherein, is the time current residual distribution; is the reference residual distribution.

5. A temperature calibration method for a nucleic acid amplification analyzer according to claim 4, characterized in that: The threshold is dynamically updated according to historical data, and its calculation formula is: Wherein, is a threshold value; is the forgetting factor, taking values from 0.1 to 0.3; For the current value.

6. The temperature calibration method of a nucleic acid amplification analyzer according to claim 5, wherein: The calculation formula of the contact resistance change rate is: Wherein, is the change rate of contact resistance; is the current contact resistance; is the initial contact resistance.

7. A temperature calibration method for a nucleic acid amplification analyzer according to claim 6, characterized in that: The calibration calculation formula is: Wherein, is the calibrated temperature value; is the slope calibration parameter for the interval; is the current sensor contact resistance value; For the intercept calibration parameter of the interval.

8. A temperature calibration method for a nucleic acid amplification analyzer according to claim 7, characterized in that: When the temperature drift mechanism is triggered, there is: Wherein, is the learning rate, and its value range is 0.1 to 0.3; is the residual value, and the calculation formula is: Wherein, is the measured value of the current temperature sensor; and are the current sectional calibration parameters; is the target temperature value.

9. A temperature calibration method for a nucleic acid amplification analyzer according to claim 8, characterized in that: Set 6 intervals of 40°C to 50°C, 50°C to 60°C, 60°C to 70°C, 70°C to 80°C, 80°C to 90°C, and 90°C to 100°C respectively, and fit the calibration parameters by the least squares method. The calculation formula is: Among them, and are the average values of temperature and resistance within this interval.

10. A temperature calibration method for a nucleic acid amplification analyzer according to claim 9, characterized in that: The value of the learning rate is selected by dynamic adjustment, and the calculation formula is as follows: Wherein, is the initial learning rate; is a regulatory factor.

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