A temperature calibration method for nucleic acid amplification analyzer

Abstract

The invention relates to a temperature calibration method for a nucleic acid amplification analyzer, and relates to the field of instrument calibration. The method comprises the following steps: placing a temperature probe of a standard temperature measuring instrument in the instrument, setting point temperatures of 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C; turning on a data acquisition instrument, heating the instrument to different target temperature values, calculating the average temperature value of each well position, and calculating the absolute value of the difference from the target temperature value; using a standard concentration Mycoplasma pneumoniae quality control product inspection solution, adding a matching Mycoplasma pneumoniae nucleic acid detection kit, and placing the solution in the instrument to detect a nucleic acid sample; providing a temperature drift compensation module, and correcting deviations caused by circuit changes during long-term use through periodic self-test. The method has the advantages of eliminating differences between target temperatures and actual detection temperature values caused by various factors, solving the problem of temperature drift during long-term use of the instrument, and improving the temperature control accuracy of the instrument during long-term use.

Description

Technical Field

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

[0002] Nucleic acid amplification analyzers can efficiently complete the nucleic acid amplification process in a short period of time. Each well of the analyzer monitors the change in the amount of reaction products by detecting the change in light signal intensity after each reaction cycle of the sample being tested. This monitors the change in the amount of reaction products and generates an amplification curve. During or at the end of nucleic acid amplification, the amplification curve for each well can be used to determine whether the sample contains the corresponding nucleic acid fragment.

[0003] During the nucleic acid amplification process, correctly controlling the temperature of each stage (denaturation, annealing, and extension) is critical for amplifying specific target sequences. Precise temperature control can promote the melting of DNA molecules, the binding of primers, and ensure the activity of DNA polymerase. Usually, when screening the annealing gradient temperature for a certain reagent, the gradient temperature interval is set to 2°C at the beginning. After determining the largest temperature range, the screening is continued with a design of 0.5°C intervals. In order to achieve the best amplification effect of the reagent, a gradient temperature of 0.2°C is finally set. Even a temperature difference of 0.2°C can have a significant impact on the reagent amplification results, resulting in irreversible errors, such as primer binding to non-specific DNA sequences, or the amplification of non-specific products. These errors will seriously affect the accuracy and reliability of the test results, and may even lead to misjudgment or misinterpretation of the experimental results. Therefore, temperature accuracy is very critical to the performance of the instrument.

[0004] Because various temperature sensors are nonlinear devices, the temperature values obtained using a single linear formula have a linear correlation coefficient (R) that deviates significantly from 1, making the measured values at most temperature points throughout the entire measurement temperature range inaccurate. Random differences caused by different electronic components, welding methods, and other factors cannot be compensated for, further increasing the deviation in temperature measurement. Furthermore, the temperature collected by the nucleic acid amplification analyzer is the temperature of the instrument's own temperature sensor, which is located between the circuit board and the heating block. The actual temperature to be controlled is the temperature of the liquid in the PCR tube. These two temperatures differ significantly due to their different locations and thermal conductivity efficiencies. Furthermore, when electronic components are used for extended periods of time and are subject to temperature fluctuations, their electrical characteristics will change with temperature. This temperature difference, combined with the additional temperature differences, makes it impossible to accurately calibrate using traditional calibration devices and methods that only target temperature differences.

[0005] Therefore, in view of the above shortcomings, it is necessary to provide a temperature calibration method for a nucleic acid amplification analyzer. Summary of the Invention

[0006] (1) Technical issues to be resolved

[0007] The technical problem to be solved by the present invention is to solve the problem that temperature cannot be accurately controlled by a single linear formula.

[0008] (2) Technical solution

[0009] In order to solve the above technical problems, the present invention provides a temperature calibration method for a nucleic acid amplification analyzer, comprising the following steps:

[0010] Ⅰ. Place the temperature probe of a standard temperature measuring instrument into the instrument to simulate the temperature of the reagents in the tube during the actual nucleic acid amplification process. 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 the calibration.

[0011] II. Turn on the data acquisition instrument and heat the test well to different target temperatures. Using the temperature fluctuation of less than 0.05°C as the timing reference point, collect temperature values every 10 seconds after holding the temperature for 10 seconds. 10 consecutive times for each test well. Calculate the average temperature of each well and the absolute difference between the average temperature and the target temperature.

[0012] III. Use a standard concentration of Mycoplasma pneumoniae quality control solution, add the matching Mycoplasma pneumoniae nucleic acid detection kit, and place it in the instrument to test the nucleic acid sample. Repeat the test in 8 detection wells and calculate the average CT value. Place the same batch of samples on a standard PCR instrument and calculate the average CT value. Finally, calculate the accuracy, specifically:

[0013]

[0014] in,

[0015] For accuracy;

[0016] The average CT value detected when the nucleic acid sample is placed on the test instrument;

[0017] It is the average CT value detected when the nucleic acid sample is placed on a standard PCR instrument;

[0018] IV. Set up a temperature drift compensation module to correct the deviation of circuit changes during long-term use through periodic self-test.

[0019] As a further illustration of the present invention, preferably, the measured temperature value of the standard temperature measuring instrument and the target temperature value are fitted by least square method in multiple intervals within the temperature calibration range, the instrument temperature is calibrated twice, and the linear correlation coefficient is calculated.

[0020] As a further illustration of the present invention, preferably, the initial contact resistance is recorded and a reference temperature residual distribution is established, and the temperature drift mechanism is triggered when the contact resistance change rate is greater than 5% or the KL divergence is higher than a threshold.

[0021] As a further illustration of the present invention, preferably, the KL divergence calculation formula is:

[0022]

[0023] in,

[0024] For time The current residual distribution of ;

[0025] is the baseline residual distribution.

[0026] As a further illustration of the present invention, preferably, the threshold is dynamically updated based on historical data, and its calculation formula is:

[0027]

[0028] in,

[0029] is the threshold;

[0030] is the forgetting factor, ranging from 0.1 to 0.3;

[0031] For the current value.

[0032] As a further illustration of the present invention, preferably, the contact resistance change rate is calculated as follows:

[0033]

[0034] in,

[0035] is the contact resistance change rate;

[0036] is the current contact resistance;

[0037] is the initial contact resistance.

[0038] As a further illustration of the present invention, preferably, the calibration calculation formula is:

[0039]

[0040] in,

[0041] is the temperature value after calibration;

[0042] For the Slope calibration parameter for the interval;

[0043] is the current sensor contact resistance value;

[0044] For the Intercept calibration parameter for the interval.

[0045] As a further illustration of the present invention, preferably, when the temperature drift mechanism is triggered:

[0046]

[0047]

[0048] in,

[0049] is the learning rate, ranging from 0.1 to 0.3;

[0050] is the residual value, and the calculation formula is:

[0051]

[0052] in,

[0053] The current temperature sensor measurement value;

[0054] and Calibrate the parameters for the current segment;

[0055] is the target temperature value.

[0056] As a further illustration 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 set respectively to fit the calibration parameters by the least squares method, and the calculation formula is:

[0057]

[0058]

[0059] in, and is the average value of temperature and resistance in this range.

[0060] As a further illustration of the present invention, preferably, the value of the learning rate is selected through dynamic adjustment, and the calculation formula is as follows:

[0061]

[0062] in,

[0063] is the initial learning rate;

[0064] is the regulating factor.

[0065] (3) Beneficial effects

[0066] The above technical solution of the present invention has the following advantages:

[0067] The present invention designs a new calibration method, performs least square fitting on the target temperature value and the actual measured temperature value in intervals within the temperature calibration range, refines the local area, and makes the linearity more accurate; after integrating all errors (sensor nonlinearity, temperature difference between the instrument sensor and the tube, circuit component difference, etc.), temperature drift compensation is performed before calibration, the relationship between the instrument target temperature value and the actual measured temperature value of the standard temperature measuring instrument is found, and the temperature calibration is performed on the temperature sensor measurement value of the instrument itself, ensuring that the temperature of the instrument and the actual measured value of the standard temperature measuring instrument are completely consistent, ensuring that the temperature of the instrument is consistent with the temperature of the actual reagent liquid when the instrument is performing a biological experiment, thereby eliminating the difference between the target temperature value and the actual detected temperature value caused by factors such as the nonlinear characteristics of the temperature sensor, the difference of circuit components between different wells of different equipment, the location of the temperature sensor arrangement, etc., and improving the temperature control accuracy of the instrument. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 It is the installation effect diagram of the testing equipment;

[0069] Figure 2 This is the curve of temperature sensor resistance changing with temperature under the traditional calibration method;

[0070] Figure 3 It is the fitting curve between the test temperature and the target temperature value under the traditional calibration method;

[0071] Figure 4 It is a nucleic acid amplification curve chart detected when the nucleic acid sample is placed on the instrument under the traditional calibration method;

[0072] Figure 5 It is the nucleic acid expansion curve detected when the nucleic acid sample is placed on a standard PCR instrument under the traditional calibration method;

[0073] Figure 6 is a fitting curve of the test temperature and the target temperature value of the present invention;

[0074] Figure 7 It is a nucleic acid amplification curve diagram detected when the nucleic acid sample of the present invention is placed on the instrument. DETAILED DESCRIPTION

[0075] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0076] A temperature calibration method for a nucleic acid amplification analyzer, such as Figure 1 As shown, the following steps are included:

[0077] Ⅰ. Place the temperature probe of a standard temperature measuring instrument into the reagent tube hole on the instrument and connect the other end to the data acquisition device. Use this probe to simulate the temperature of the reagents in the tube during the actual nucleic acid amplification process. Since the normal temperature range for PCR amplification is generally 60℃ to 95℃, the nucleic acid amplification analyzer must be able to achieve accurate temperature measurement within a range of at least 40℃ to 100℃. Therefore, set the point values of 40℃, 50℃, 60℃, 70℃, 80℃, 90℃, and 100℃ to determine the temperature accuracy of the calibration.

[0078] Ⅱ. Turn on the data acquisition instrument and run the temperature to different target temperature values. Take the temperature fluctuation less than 0.05℃ as the timing reference point. After keeping the temperature constant for 10 seconds, collect the temperature value every 10 seconds. Collect 10 times for each detection hole continuously; calculate the average temperature value of each hole , and calculate the target temperature value according to the following formula Absolute value of the difference :

[0079]

[0080] The test results of each temperature point at each hole position should not be greater than 0.2℃.

[0081] III. Use a standard concentration of Mycoplasma pneumoniae quality control solution, add the matching Mycoplasma pneumoniae nucleic acid detection kit, and place it in the instrument to test the nucleic acid sample. Repeat the test in 8 detection wells and calculate the average CT value. Place the same batch of samples on a standard PCR instrument and calculate the average CT value. Finally, calculate the accuracy according to the following formula:

[0082] in,

[0083] For accuracy;

[0084] The average CT value detected when the nucleic acid sample is placed on the test instrument;

[0085] It is the average CT value detected when the nucleic acid sample is placed on a standard PCR instrument;

[0086] IV. 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. Preferably, set the six intervals of 40℃~50℃, 50℃~60℃, 60℃~70℃, 70℃~80℃, 80℃~90℃, and 90℃~100℃ to perform secondary calibration on the instrument temperature and calculate the linear correlation coefficient. The calibration calculation formula is:

[0087]

[0088] in,

[0089] is the temperature value after calibration;

[0090] For the The slope calibration parameter of the interval is calculated as:

[0091]

[0092] is the current sensor contact resistance value;

[0093] For the The intercept calibration parameter of the interval is calculated as:

[0094]

[0095] in, and is the average value of temperature and resistance in this range.

[0096] The present invention also has a temperature drift compensation module, which corrects the deviation of circuit changes during long-term use through periodic self-test. The detection cycle adopts a combination of equidistant sampling and unequal sampling:

[0097]

[0098] in,

[0099] The basic detection cycle is set;

[0100] is a custom constant;

[0101] is the base interval, preferably 24 hours;

[0102] is the disturbance amplitude, preferably 2 to 4 hours;

[0103] The disturbance frequency is preferably recorded once a week, or once every 100 times the sensor is used.

[0104] Record the initial contact resistance of the temperature sensor before testing And establish the baseline temperature residual distribution , the calculation formula is:

[0105]

[0106]

[0107] in,

[0108] is the residual value;

[0109] is the window size, which is 8 in this solution;

[0110] is a Gaussian function;

[0111] is the bandwidth parameter, preferably 0.02°C;

[0112] The current temperature sensor measurement value;

[0113] and Calibrate the parameters for the current segment;

[0114] is the target temperature value.

[0115] 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. The contact resistance change rate is calculated as follows:

[0116]

[0117] in,

[0118] is the contact resistance change rate;

[0119] is the current contact resistance;

[0120] is the initial contact resistance.

[0121] The KL divergence calculation formula is:

[0122]

[0123] in,

[0124] For time The current residual distribution of , namely:

[0125]

[0126] is the baseline residual distribution.

[0127] Threshold Dynamically updated based on historical data, the calculation formula is:

[0128]

[0129] in,

[0130] is the threshold;

[0131] is the forgetting factor, ranging from 0.1 to 0.3;

[0132] For the current value.

[0133] When the temperature drift mechanism is triggered:

[0134]

[0135]

[0136] in,

[0137] The learning rate can be fixed in the range of 0.1 to 0.3. Alternatively, it can be dynamically adjusted and selected. The calculation formula is as follows:

[0138]

[0139] in,

[0140] is the initial learning rate;

[0141] is the regulating factor.

[0142] like Figure 2 As shown, the blue curve shows the resistance of the instrument's temperature sensor changing with temperature. Using the traditional method, a single linear fit is performed on the temperature and resistance values. The red curve is the fitted curve. The figure shows that the linear correlation coefficient R of the fitted curve deviates significantly from 1, indicating poor linearity. The instrument temperature is calibrated using the traditional linear fitting method. After calibration, the instrument is tested for temperature accuracy and for the deviation in accuracy of the nucleic acid sample CT value.

[0143] The temperature accuracy test results are as follows:

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151] As can be seen from the table above, the temperature accuracy of each temperature point is very poor, the temperature difference is between 0.4 and 6.4 ° C, and all temperatures do not meet the requirement that the temperature deviation test result is no more than 0.2 ° C. The total mean of the test temperature values of all well positions at different temperature points is quadratically fitted with the target temperature value to obtain the fitting curve as shown below: Figure 3 As shown in the figure, it can be seen that the linear correlation coefficient R value between the calibrated test temperature value and the target temperature value still has a large deviation from 1, and the linearity is poor.

[0152] The nucleic acid sample is placed on the instrument for testing, and the CT value results are shown in the following table:

[0153]

[0154] Nucleic acid amplification curve Figure 4 shown.

[0155] The same batch of samples were tested on a standard PCR instrument, and the CT values were shown in the following table:

[0156]

[0157] Nucleic acid amplification curve Figure 5 shown.

[0158] The calculated accuracy deviation of the CT value of the nucleic acid sample is D=5.29.

[0159] The test results show that the instrument calibrated by traditional methods has poor temperature accuracy at different temperature points, large CT values in nucleic acid sample accuracy deviation tests, low nucleic acid amplification efficiency, and inaccurate quantitative judgment of initial nucleic acid concentration. The reasons are analyzed as follows:

[0160] 1. Due to the nonlinear characteristics of the sensor, the accuracy of each temperature point varies greatly;

[0161] 2. The location of the temperature sensor is different from the actual temperature measurement location, resulting in a difference between the actual temperature measurement value and the temperature value detected by the sensor;

[0162] 3. Temperature differences between detection holes caused by different parameters of other circuit components;

[0163] 4. The sensor experiences temperature drift after temperature changes.

[0164] In order to reduce the measurement inaccuracy caused by the above reasons, the present invention obtains 6 groups of K and B values at each hole position through the above method, performs secondary calibration on the instrument respectively, and calculates the linear correlation coefficient R value.

[0165] The data is shown in the following table:

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172] After calibration, the instrument was tested for temperature accuracy and nucleic acid sample accuracy deviation.

[0173] The temperature accuracy test results are as follows:

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181] As can be seen from the table above, the temperature differences of all temperature points are concentrated around 0.01 to 0.1°C, and all temperatures meet the requirement that the temperature deviation test result is no more than 0.2°C. The total mean of the test temperature values of all well positions at different temperature points is quadratically fitted with the target temperature value to obtain the fitting curve as shown below: Figure 6 As shown in the figure, it can be seen that the linear correlation coefficient R value between the calibrated test temperature value and the target temperature value is 1, and the linearity is very good. The nucleic acid sample is placed on the instrument for detection, and the CT value results are as follows:

[0182]

[0183] Nucleic acid amplification curve Figure 7 As shown, the calculated CT value accuracy of the nucleic acid sample is D=0.11.

[0184] In summary, the temperature accuracy test results of the two calibration methods are summarized and compared, and the data are as follows:

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192] The nucleic acid amplification CT value test results after temperature calibration using the two calibration methods were summarized and compared, and the data are as follows:

[0193]

[0194] By comparing the test results, it can be seen that under the same test conditions, the calibration method provided by the present invention has significantly improved the temperature accuracy of each temperature point compared with the traditional method. The CT value results in the CT value accuracy deviation test of the nucleic acid sample are closer to the amplification results 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 more accurately measured.

[0195] Nucleic acid amplification instruments calibrated according to traditional methods usually change instrument parameters due to various reasons after being used for a period of time, while the calibration coefficient still maintains the previous fixed value, which will inevitably lead to temperature inaccuracies and deviations in nucleic acid detection results. Therefore, traditional instruments need to be temperature calibrated once every six months to ensure that the instrument can be used normally; however, the calibration method provided by the present invention is adopted, and a temperature drift compensation module is set up, which can automatically correct the deviations caused by various reasons during long-term use through periodic self-tests, and can ensure temperature accuracy even after long-term use, ensuring the accuracy of nucleic acid detection. The following table shows the comparative test results of the nucleic acid amplification CT value and the standard PCR instrument after one year of frequent use of the instrument:

[0196] From the above comparison results, it can be seen that the calibration method provided by the present invention has an extremely small temperature fluctuation range, whether it is just after calibration or after long-term use after calibration, and can maintain temperature accuracy without the need for subsequent frequent calibration. The CT values in the test results are very close to the amplification results 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 more accurately measured.

[0197] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A temperature calibration method for a nucleic acid amplification analyzer, characterized in that: The following steps are involved: Ⅰ. Place the temperature probe of a standard temperature measuring instrument on the instrument to be tested. Use the probe to detect the temperature of the reagents in the tube during the actual nucleic acid amplification process. Set target temperatures of 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and 100°C to determine the temperature accuracy of the calibration. II. Turn on the data acquisition instrument and heat the test well to different target temperatures. Using the temperature fluctuation of less than 0.05°C as the timing reference point, collect temperature values every 10 seconds after holding the temperature for 10 seconds. 10 consecutive times for each test well. Calculate the average temperature of each well and the absolute difference between the average temperature and the target temperature. III. Use a standard concentration of Mycoplasma pneumoniae quality control solution, add the matching Mycoplasma pneumoniae nucleic acid detection kit, and place it in the instrument to be tested to detect the nucleic acid sample. Repeat the test at 8 detection wells and calculate the average CT value. Place the same batch of samples on a standard PCR instrument and calculate the average CT value. Finally, calculate the accuracy, specifically: in, For accuracy; The average CT value detected when the nucleic acid sample is placed on the test instrument; It is the average CT value detected when the nucleic acid sample is placed on a standard PCR instrument; IV. Set up a temperature drift compensation module to correct the deviation of circuit changes during long-term use through periodic self-test.

2. The temperature calibration method of a nucleic acid amplification analyzer according to claim 1, characterized in that: The measured temperature value of the standard temperature measuring instrument and the target temperature value are fitted by the least square method in multiple intervals within the temperature calibration range, the temperature of the instrument to be measured is calibrated twice, and the linear correlation coefficient is calculated.

3. The temperature calibration method of a nucleic acid amplification analyzer according to claim 2, characterized in that: The initial contact resistance is recorded and the baseline temperature residual distribution is established. 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. The temperature calibration method of a nucleic acid amplification analyzer according to claim 3, characterized in that: The KL divergence calculation formula is: in, For time The current residual distribution of ; is the baseline residual distribution.

5. The temperature calibration method of a nucleic acid amplification analyzer according to claim 4, characterized in that: The threshold is dynamically updated based on historical data, and its calculation formula is: in, is the threshold; is the forgetting factor, ranging 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, characterized in that: The contact resistance change rate is calculated as: in, is the contact resistance change rate; 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: in, is the temperature value after calibration; For the Slope calibration parameter for the interval; is the current sensor contact resistance value; For the Intercept calibration parameter for the interval.

8. The temperature calibration method of a nucleic acid amplification analyzer according to claim 7, characterized in that: When the temperature drift mechanism is triggered: in, is the learning rate, ranging from 0.1 to 0.3; is the residual value, and the calculation formula is: in, The current temperature sensor measurement value; and Calibrate the parameters for the current segment slope and intercept; is the target temperature value.

9. The temperature calibration method of a nucleic acid amplification analyzer according to claim 8, characterized in that: The calibration parameters were fitted by the least squares method using six intervals of 40℃~50℃, 50℃~60℃, 60℃~70℃, 70℃~80℃, 80℃~90℃, and 90℃~100℃. The calculation formula is: in, and is the average value of temperature and resistance in this range.

10. The temperature calibration method of a nucleic acid amplification analyzer according to claim 9, characterized in that: The value of the learning rate is selected through dynamic adjustment and is calculated as follows: in, is the initial learning rate; is the regulating factor.

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