Light intensity calibration method of constant-temperature nucleic acid amplification analyzer

Abstract

The invention relates to a light intensity calibration method of a constant-temperature nucleic acid amplification analyzer, which relates to the field of testing and comprises the following steps: preparing calibration solutions with a plurality of gradients, filling detection holes of the constant-temperature nucleic acid amplification analyzer to be calibrated with the calibration solutions, and calibrating the light intensity of the constant-temperature nucleic acid amplification analyzer by utilizing signal value data of the solutions with the plurality of concentrations; obtaining a fitting relational expression in the coordinate system by using a least square method; obtaining calibration parameters according to the fitting relational expression; the calibration solution is put into a constant-temperature nucleic acid amplification analyzer to be tested, the actual light intensity value of the actual calibration solution of each tube is calculated according to the real-time light intensity signal value detected by each channel and the stored calibration parameters, and performance tests of repeatability, consistency and accuracy are carried out. Therefore, the light intensity value of the calibration solution collected between each instrument and each channel is in an allowable range, the signal detection range reaches a consistent level, and calibration is completed, and the method and the device have the advantage that the light intensity signal values collected between different instruments and different channels can reach the consistent level.

Description

Technical Field

[0001] The invention relates to the field of testing technology, and in particular to a light intensity calibration method for a constant temperature nucleic acid amplification analyzer. Background Art

[0002] Isothermal nucleic acid amplification is a nucleic acid amplification technology performed under constant temperature conditions, which can complete the entire nucleic acid amplification process in a short time. The optical module of the isothermal nucleic acid amplification analyzer can monitor the nucleic acid amplification in the reaction product by regularly monitoring the intensity changes of the light signal in the sample to be tested, and generate an amplification curve graph, thereby determining whether the sample to be tested contains the corresponding nucleic acid fragment, and can quantify the content of the initial nucleic acid fragment in the sample to be tested.

[0003] The constant temperature nucleic acid amplification analyzer can test multiple samples and multiple target genes in one experiment, with large test throughput and multiple measurement channels. Due to the large differences in circuit parameters, optical parameters and mechanical assembly between different instruments and different measurement channels, the fluorescence signal values ​​collected between different instruments and different channels will be greatly different. This is an unavoidable problem. If calibration is not performed, different numerical results will be obtained between instruments and instrument channels even when testing the same tube of reagents, which has a direct impact on the detection accuracy of the instrument.

[0004] In addition, there are two situations: 1. The detection value of some channels is too low, and low-concentration samples cannot be tested, especially when the nucleic acid has not been amplified or in the early stage of amplification, the detection value will be submerged in the background, and the true concentration of the nucleic acid cannot be detected; 2. The detection value of some channels is too high. When the light intensity value of the nucleic acid increases exponentially during the amplification process, the test result quickly exceeds the detection limit and reaches saturation, and the true concentration of high-concentration samples cannot be tested. The above situations will cause the instrument to be unable to fully and effectively collect the exponentially growing light intensity signal during the amplification process, the detection dynamic range is small, and the instrument's detection range is smaller than the actual required range, resulting in distorted measurement results.

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

[0006] 1. Technical issues to be resolved The technical problem to be solved by the present invention is to solve the problem of inconsistent fluorescence signal values ​​collected by different instruments and different channels.

[0007] (II) Technical solution In order to solve the above technical problems, the present invention provides a light intensity calibration method for a constant temperature nucleic acid amplification analyzer, comprising the following steps: Ⅰ. Prepare several gradient calibration solutions and calibrate the calibration solutions; Ⅱ. Fill the detection well of the constant temperature nucleic acid amplification analyzer to be calibrated with the calibration solution of each gradient, and generate a curve by fitting the least square method in the coordinate system by using the signal value data of multiple concentration solutions, and obtain the fitting relationship: in, K is the light intensity calibration coefficient; Y is the target light signal value; B is a constant; X is the actual light signal value collected; The calibration parameters are obtained according to the fitting relationship and written into the constant temperature nucleic acid amplification analyzer for storage; III. Place any one PCR reaction tube of each gradient calibration solution or all the PCR reaction tubes of the gradient into the detection hole of the constant temperature nucleic acid amplification analyzer for testing. The analyzer calculates the actual light intensity value of each actual calibration solution according to the real-time light intensity signal value detected by each channel and the saved calibration parameters, and performs performance tests on the repeatability, consistency and accuracy of the light intensity values ​​of all channels in each instrument, so that the light intensity values ​​of the calibration solution collected by each instrument and each channel are within the allowable range, the signal detection range reaches a consistent level, and the calibration is completed.

[0008] As a further illustration of the present invention, preferably, the calibration solution is a phenol red solution, and the concentration gradient of the calibration solution is 2 to 10 gradients.

[0009] As a further illustration of the present invention, preferably, phenol red powder is placed in a beaker, less than 30 ml of purified water is added, and then the volume is adjusted to 50 ml using a volumetric flask to prepare a mother liquor, and then different amounts of the mother liquor are extracted into different centrifuge tubes, and different amounts of pure water are added to each centrifuge tube to fully dissolve and mix to obtain calibration solutions with different gradients.

[0010] As a further illustration of the present invention, preferably, different amounts of calibration solutions with different gradients are added to several PCR reaction tubes, and 40 μl of mineral oil is added to each tube for oil sealing to prevent the solution from volatilizing.

[0011] As a further illustration of the present invention, preferably, the prepared gradient calibration solutions are dispensed into several PCR reaction tubes, and are placed in the standard detection holes of a constant temperature nucleic acid amplification analyzer standard machine for repeatability, consistency and accuracy testing, and the average value of multiple measurements of each tube is taken as the light intensity value of the tube, and the data is recorded to complete the calibration.

[0012] As a further illustration of the present invention, preferably, the prepared gradient calibration solutions are dispensed into several PCR reaction tubes, and one tube is selected from each gradient calibration solution to calibrate all detection channels of a standard constant temperature nucleic acid amplification analyzer. The repeatability, consistency and accuracy of the calibration solution in the tube are tested, and the average value of multiple measurements of each tube is taken as the light intensity value of the tube, and the data is recorded to complete the calibration.

[0013] As a further illustration of the present invention, preferably, the repeatability test comprises the following steps: The calibration solutions of different concentrations were placed on the constant temperature nucleic acid amplification analyzer to collect signal value data. The same test position was selected for each concentration. The test was performed every 30 seconds at an instrument temperature of 65°C. The test was repeated 10 times. The average value and standard deviation of the measurement results of the test solutions of each concentration were obtained respectively. The coefficient of variation was calculated using the formula: in, is the coefficient of variation of the signal measurement value; is the standard deviation of the signal measurement values ​​for each concentration of solution; is the average of the signal measurements for each concentration of solution.

[0014] As a further illustration of the present invention, preferably, the consistency test comprises the following steps: Place calibration solutions of different concentrations into multiple detection wells of the constant temperature nucleic acid amplification analyzer, test every 30 seconds when the instrument temperature reaches 65°C, repeat the test 10 times, calculate the average value of the signal measurement value of each concentration solution in each detection well, calculate the average value of each concentration calibration solution, and calculate the standard deviation of each calibration solution. Finally, calculate the coefficient of variation between different tubes of each calibration solution, which is: in, is the coefficient of variation of the signal measurement value; is the standard deviation of the signal measurement value of each concentration solution; specifically: It is the average value of the signal measurement value of each concentration solution in each detection well; is the average of the signal measurements for each concentration of solution; is the number of test wells for each concentration of solution.

[0015] As a further illustration of the present invention, preferably, the accuracy test comprises the following steps: Place calibration solutions of different concentrations into multiple detection holes of the constant temperature nucleic acid amplification analyzer. When the instrument temperature reaches 65°C, test once every 30 seconds. Repeat the test 10 times. Calculate the average signal measurement value of each concentration solution in each detection hole, and then calculate the accuracy, specifically: in, For accuracy; It is the average value of the signal measurement value of each concentration solution in each detection well; is the target signal value corresponding to each concentration solution.

[0016] As a further illustration of the present invention, preferably, the coefficient of variation in the repeatability test is not greater than 1%, the coefficient of variation in the consistency test is not greater than 2%, and the percentage deviation in the accuracy test is not greater than 3%.

[0017] (III) Beneficial effects The above technical solution of the present invention has the following advantages: The present invention designs a calibration method to eliminate the differences in collected light intensity data caused by differences in circuit elements (such as resistors, capacitors, LED lights, laser lights, photoelectric receivers and amplifiers, etc.), optical elements (such as light guides, light holes, lenses, filters, etc.) and mechanical assembly between instruments or instrument channels, so that the light intensity signal values ​​collected between different instruments and different channels reach a consistent level. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a light path diagram of the constant temperature nucleic acid amplification analyzer of the present invention; Figure 2 This is a graph of the concentration 1 test result before calibration of machine No. 1 of the present invention; Figure 3 This is a graph of the concentration 2 test result before calibration of machine No. 1 of the present invention; Figure 4 is a graph of the concentration 3 test result of the No. 1 machine before calibration of the present invention; Figure 5 This is a graph of the concentration 1 test result before calibration of the No. 2 unit of the present invention; Figure 6 is a graph of the concentration 2 test result before calibration of the No. 2 unit of the present invention; Figure 7 is a graph of the concentration 3 test result of the No. 2 unit before calibration of the present invention; Figure 8This is a graph of the concentration 1 test result before calibration of the No. 3 unit of the present invention; Fig. 9 This is a graph of the concentration 2 test result of the No. 3 unit before calibration of the present invention; Fig.10 is a graph of the concentration 3 test result before calibration of the No. 3 unit of the present invention; Fig.11 This is the amplification curve diagram of the No. 1 machine before light intensity calibration of the present invention; Fig.12 This is the amplification curve diagram of the No. 2 machine before light intensity calibration of the present invention; Fig.13 This is the amplification curve diagram of the No. 3 machine before light intensity calibration of the present invention; Fig.14 is a graph of the concentration 1 test result after calibration of machine No. 1 of the present invention; Fig.15 is a graph of the concentration 2 test result after calibration of machine No. 1 of the present invention; Fig.16 is a graph of the concentration 3 test result after calibration of machine No. 1 of the present invention; Fig.17 This is a graph of the concentration 1 test result after calibration of the No. 2 machine of the present invention; Fig.18 is a graph of the concentration 2 test result before calibration of the No. 2 unit of the present invention; Fig.19 is a graph of the concentration 3 test result after calibration of the No. 2 machine of the present invention; Fig. 20 This is a graph of the concentration 1 test result after calibration of the No. 3 unit of the present invention; Fig.21 This is a graph of the concentration 2 test result after calibration of the No. 3 unit of the present invention; Fig. 22 is a graph of the concentration 3 test result after calibration of the No. 3 unit of the present invention; Fig.23 This is the amplification curve diagram of the No. 1 machine after light intensity calibration of the present invention; Fig.24 This is the amplification curve diagram of the No. 2 machine after light intensity calibration of the present invention; Fig.25 This is the amplification curve diagram of the No. 3 machine after light intensity calibration of the present invention.

[0019] In the figure: 1. Circuit board; 2. Lamp beads; 3. PCR tube; 4. Light guide; 5. PD sensor. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the 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 creative work are within the scope of protection of the present invention.

[0021] like Figure 1 As shown, the working principle of the constant temperature amplification analyzer applicable to the present invention is: when the instrument reaches the set temperature, the eight LED lamp beads 2 on the circuit board 1 assembly will light up in sequence, and the LED light will be vertically incident from the bottom of the PCR tube 3, and then pass through the through hole of the reaction tube bracket to enter the light guide 4. After entering the light guide 4, the light reaches the 45° reflecting inclined surface, then passes through the through hole of the filter fixing seat, and finally reaches the PD sensor 5, thereby obtaining a light intensity signal.

[0022] A method for calibrating light intensity of a constant temperature nucleic acid amplification analyzer comprises the following steps: 1. Prepare calibration solution: Prepare a mother solution with phenol red powder (or other chemical dye raw materials) and purified water in a certain proportion, and then dilute the mother solution step by step into 2 to 10 gradients. The number of gradients to be diluted can be appropriately selected according to the accuracy of the calibration required. The present invention uses 3 gradients, which are defined as concentration 1, concentration 2 and concentration 3 respectively. The preparation method is as follows: ① Preparation of mother solution: Take 0.018g of phenol red powder and put it into a beaker. First add 30ml of purified water, and then use a volumetric flask to make up to 50ml to prepare a 1mM phenol red aqueous solution.

[0023] ② Take 25μl of 1mM phenol red aqueous solution and add it to a 0.5ml centrifuge tube, add 275μl of pure water, dissolve and mix thoroughly, then take 30μl of the solution and add it to 8 PCR reaction tubes (multiple tubes of solution can be prepared for each gradient concentration, and the constant temperature nucleic acid amplification instrument used for calibration in the present invention is an 8-channel detection instrument, so 8 tubes of solution are prepared for each gradient concentration), to obtain 8 tubes of 1 / 12mM phenol red aqueous solution, and then add 40μl of mineral oil (other chemical reagents to prevent the solution from volatilizing can also be used) to each tube for oil sealing, as the concentration 1 light intensity calibration calibration tube solution.

[0024] ③ Take 70μl of 1mM phenol red aqueous solution and add it to a 0.5ml centrifuge tube, add 210μl of pure water, dissolve and mix thoroughly, then take 30μl of the solution and add it to 8 PCR reaction tubes to prepare 8 tubes of 1 / 4mM phenol red aqueous solution. Add 40μl of mineral oil to each tube for oil sealing as the concentration 2 light intensity calibration tube solution.

[0025] ④ Take 30 μl of 1 mM phenol red aqueous solution and add it to 8 PCR reaction tubes to prepare 8 tubes of 1 mM phenol red aqueous solution. Then add 40 μl of mineral oil to each tube for oil sealing. This will serve as the concentration 3 light intensity calibration tube solution.

[0026] Among them, all preparation and dilution processes need to be mixed on a mixer to ensure that the data is more accurate when placed on the constant temperature nucleic acid amplification analyzer for signal value collection. After the light intensity calibration tube solution is dispensed into the PCR reaction tube, it needs to be oil-sealed to prevent the liquid from evaporating. And 8 tubes of solution are prepared for each gradient concentration, so that all wells of the constant temperature nucleic acid amplification instrument can be calibrated and tested at the same time, improving efficiency.

[0027] 2. Determine the calibration solution. There are two methods: Method 1: Place 3×8 tubes of each prepared gradient calibration solution into the standard detection holes (such as hole 1) of the constant temperature nucleic acid amplification analyzer standard machine for detection, and perform performance tests such as repeatability and consistency on the 3×8 tube solutions to ensure that the solution is qualified and can be used for the calibration of the constant temperature nucleic acid amplification analyzer. Take the average value of multiple measurements of each tube as the light intensity value of the tube, record the data, and use it for the detection target signal value of the constant temperature nucleic acid amplification analyzer to be calibrated.

[0028] Method 2: To improve the calibration efficiency, one tube of each gradient calibration solution can be selected to calibrate all detection channels of the constant temperature nucleic acid amplification analyzer standard machine, ensuring that all channels of the calibrated standard machine can reach the same level as the standard detection wells when detecting the same tube of calibration solution. Put 3×8 tubes of each gradient calibration solution into each detection well of the constant temperature nucleic acid amplification analyzer standard machine for detection, and perform repeatability, consistency and other performance tests on the 3×8 tube solutions to ensure that the solution is qualified and can be used for the calibration of the constant temperature nucleic acid amplification analyzer. Take the average value of multiple measurements of each tube as the light intensity value of the tube, record the data, and use it for the detection target signal value of the constant temperature nucleic acid amplification analyzer to be calibrated.

[0029] The repeatability test includes the following steps: The calibration solutions of different concentrations were placed on the constant temperature nucleic acid amplification analyzer to collect signal value data. The same test position was selected for each concentration. The test was performed every 30 seconds at an instrument temperature of 65°C. The test was repeated 10 times. The average value and standard deviation of the measurement results of the test solutions of each concentration were obtained respectively. The coefficient of variation was calculated using the formula: in, is the coefficient of variation of the signal measurement value, and its result should not be greater than 1%; is the standard deviation of the signal measurements for each concentration of solution; is the average of the signal measurements for each concentration of solution.

[0030] Conformance testing consists of the following steps: The calibration solutions of different concentrations were placed in multiple detection holes of the constant temperature nucleic acid amplification analyzer. When the instrument temperature reached 65°C, the test was performed every 30 seconds. The test was repeated 10 times. The collected signal value data was recorded as ( =1,2,3, representing 3 calibration solutions) =1,2…8, representing 8 test bits) ( =1,2…10, representing 10 tests). Calculate the average signal measurement value of each concentration solution in each detection well, calculate the average value of each concentration calibration solution, and calculate the standard deviation of each calibration solution. Finally, calculate the coefficient of variation between different tubes of each calibration solution, specifically: in, is the coefficient of variation of the signal measurement value, which should be no greater than 2%; is the standard deviation of the signal measurement value of each concentration solution; specifically: It is the average value of the signal measurement value of each concentration solution in each detection well, that is: is the average value of the signal measurement value of each concentration solution, that is: is the number of test wells for each concentration of solution.

[0031] Accuracy testing consists of the following steps: Place calibration solutions of different concentrations into multiple detection holes of the constant temperature nucleic acid amplification analyzer. When the instrument temperature reaches 65°C, test once every 30 seconds. Repeat the test 10 times. Calculate the average signal measurement value of each concentration solution in each detection hole, and then calculate the accuracy, specifically: in, For accuracy, the deviation percentage is no more than 3%; It is the average value of the signal measurement value of each concentration solution in each detection well; is the target signal value corresponding to each concentration solution.

[0032] The obtained standard machine test data is as follows: Table 1 Concentration 1 test results Table 2 Concentration 2 test results Table 3 Concentration 3 test results It can be seen from the above tables that the data of the prepared calibration solutions tested on the standard machine are all qualified and can be used for instrument calibration.

[0033] 3. Calibrate the constant temperature nucleic acid amplification analyzer: Place the 8 tubes of calibration solution of each gradient into the 8 detection holes of the constant temperature nucleic acid amplification analyzer to be calibrated. Use the fully automatic measurement software to calculate the calibration relationship between the target signal value under multiple gradients and the actual signal value collected by the instrument. Write the calibration parameters of the calibration relationship of each channel into each constant temperature nucleic acid amplification analyzer for storage.

[0034] The method for determining the light intensity calibration relationship comprises the following steps: ①Power on the constant temperature nucleic acid amplification analyzer, do not put in the calibration solution tube, run the nucleic acid amplification process, wait for the temperature to stabilize, collect 8 detection position signal values, collect a signal value data every 30 seconds, collect 10 data for each detection position, and calculate the average value as the background value of each channel. The background value comes from the stray light in the optical path, the circuit background noise and the inherent error of the mechanical assembly. When testing the light intensity data of each concentration in the subsequent test, the background value is subtracted and used as the effective light signal value of the corresponding channel.

[0035] ② Prepare the light intensity calibration tube solution, add the solution into the PCR reaction tube, place the reaction tube on the detection position of the constant temperature nucleic acid amplification analyzer, run the constant temperature nucleic acid amplification program on the light intensity calibration tube solution, collect the fluorescence signal value data of all channels, and eliminate the background value of the collected data to obtain the channel effective value. The effective value is used for calculation and calibration in subsequent experiments.

[0036] In the calibration method of the light intensity calibration relationship of the constant temperature nucleic acid amplification analyzer provided by the present invention, at least two concentrations of light intensity calibration calibration tube solutions are used to obtain the light signal value data of each channel after eliminating the background value of each concentration solution, and the light signal value of the same concentration solution after eliminating the background value is used as the horizontal coordinate, and the target light signal value is used as the vertical coordinate. Since the concentration is gradient dispersed, between zero and the highest concentration, the target light signal value is 0 to 4095. Using the signal value data of multiple concentration solutions, the least squares method is fitted in the coordinate system to generate a fitting curve, and the relationship of the fitting curve is obtained: in, K is the light intensity calibration coefficient; Y is the target light signal value; B is a constant; X is the actual light signal value collected.

[0037] Three instruments to be calibrated were selected for data comparison test before and after light intensity calibration, numbered as machine 1, machine 2, and machine 3. The test data of these three machines before calibration are shown in the following table: Table 4 Concentration 1 test data table of unit 1 Table 5 Concentration 2 test data table for unit 1 Table 6 Concentration 3 test data table of unit 1 Table 7 Concentration 1 test data table of unit 2 Table 8 Concentration 2 test data table of unit 2 Table 9 Concentration 3 test data table of unit 2 Table 10 Concentration 1 test data of unit 3 Table 11 Concentration 2 test data of unit 3 Table 12 Concentration 3 test data table of unit 3 It can be seen from the above tables that the consistency and accuracy test data of each uncalibrated instrument are very poor and do not meet the test requirements.

[0038] In order to analyze the data more intuitively, it is presented in the form of a curve graph, such as Figure 2-Figure 10 As shown, it can be seen that the discreteness between channels of the calibrated strong instrument is extremely large.

[0039] Amplification curves of 3 instruments Figure 11-13 It can be found that the detection values ​​of some channels are too low. When the nucleic acid has not been amplified or in the first few cycles of amplification, the detection value is submerged in the background, and all test values ​​are 0, and the real-time concentration of the nucleic acid cannot be detected. The detection values ​​of some channels are too high. When the light intensity value of the nucleic acid expands exponentially during the amplification process, the test results quickly exceed the detection upper limit of 4095 and reach saturation, and the post-amplification concentration of high-concentration samples cannot be tested.

[0040] Using the data in the table above, we calculated the deviation between the signal value of each detection position of each instrument and the standard machine. It can be seen that before calibration, there are huge differences between the three instruments and the standard machine data. Some of the differences are close to 40%, which will cause the instrument measurement results to be very inaccurate.

[0041] Place the 3 concentrations of light intensity calibration tube solutions on the 8 detection positions of the 3 constant temperature nucleic acid amplification analyzers to be tested, run the constant temperature nucleic acid amplification analysis program, wait for the instrument temperature to stabilize, collect a signal data every 30 seconds for each detection position, collect 10 data for each channel, and use the least squares method to calculate the K and B values ​​of each channel. The calculation results are shown in the following table: Table 13 Calculation results of machine 1 Table 14 Calculation results of unit 2 Table 15 Calculation results of unit 3 4. To measure the calibrated constant temperature nucleic acid amplification analyzer, the following two methods can be used: Method 1: Take one tube of each gradient calibration solution and put it into the 8 detection holes of the calibrated constant temperature nucleic acid amplification analyzer for separate testing. The analyzer calculates the actual light intensity value of the actual calibration solution in the tube according to the real-time light intensity signal value detected by each channel and the saved calibration parameters. The light intensity values ​​of the 8 channels of each instrument are tested for repeatability, consistency and accuracy to ensure that the light intensity values ​​of the same tube of calibration solution collected by each instrument and each channel are within the allowable range, and the signal detection range and capability reach a consistent level to meet the test index requirements.

[0042] Method 2: To improve the detection efficiency, the 8 tubes of calibration solution of each gradient can be placed in the 8 detection holes of the calibrated constant temperature nucleic acid amplification analyzer for synchronous testing. The analyzer calculates the actual light intensity value of each tube of actual calibration solution according to the saved calibration parameters based on the real-time light intensity signal value detected by each channel. The light intensity values ​​of the 8 channels of each instrument are tested for repeatability, consistency and accuracy (relative to the corresponding calibration tube) to ensure that the light intensity values ​​of the calibration solution collected by each instrument and each channel are within the allowable range, and the signal detection range and capability are consistent to meet the test index requirements.

[0043] The K and B values ​​were written into the constant temperature nucleic acid amplification analyzer instrument chip through the fully automatic light intensity calibration software, and then the light intensity calibration tube solutions of three concentrations were placed into the constant temperature nucleic acid amplification analyzer to perform repeatability, consistency and other performance tests on the three instruments. The test results are shown in the following table: Table 16 Concentration 1 test results of unit 1 Table 17 Concentration 2 test results of unit 1 Table 18 Concentration 3 test results of unit 1 Table 19 Concentration 1 test results of unit 2 Table 20 Concentration 2 test results of unit 2 Table 21 Concentration 3 test results of unit 2 Table 22 Concentration 1 test results of unit 3 Table 23 Concentration 2 test results of unit 3 Table 24 Concentration 3 test results of unit 3 The comparison data of the deviation values ​​of the three instruments and the standard machine after light intensity calibration are summarized as follows: Table 25 Concentration 1 test comparison table Table 26 Concentration 2 test comparison table Table 27 Concentration 3 test comparison table Combination Figure 14-Figure 25 It can be seen from the test data and amplification curve that under the same test conditions, after light intensity calibration, the signal value differences between different instruments and different channels are effectively reduced, the consistency test results are all less than 2%, the accuracy test results are all less than 3%, and the detection values ​​are all within the detection range of the instrument, which can realize the full collection of the exponentially growing light intensity signal during the amplification process, thereby ensuring the accuracy and consistency of the experimental results.

[0044] 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 embodiments of the present invention.

Claims

1. A method for calibrating light intensity of a constant temperature nucleic acid amplification analyzer, characterized in that: The following steps are involved: Ⅰ. Prepare several gradient calibration solutions and calibrate the calibration solutions; Ⅱ. Fill the detection well of the constant temperature nucleic acid amplification analyzer to be calibrated with the calibration solution of each gradient, and generate a curve by fitting the least square method in the coordinate system by using the signal value data of multiple concentration solutions, and obtain the fitting relationship: in, K is the light intensity calibration coefficient; Y is the target light signal value; B is a constant; X is the actual light signal value collected; The calibration parameters are obtained according to the fitting relationship and written into the constant temperature nucleic acid amplification analyzer for storage; III. Place any one PCR reaction tube of each gradient calibration solution or all the PCR reaction tubes of the gradient into the detection hole of the constant temperature nucleic acid amplification analyzer for testing. The analyzer calculates the actual light intensity value of each actual calibration solution according to the real-time light intensity signal value detected by each channel and the saved calibration parameters, and performs performance tests on the repeatability, consistency and accuracy of the light intensity values ​​of all channels in each instrument, so that the light intensity values ​​of the calibration solution collected by each instrument and each channel are within the allowable range, the signal detection range reaches a consistent level, and the calibration is completed.

2. The light intensity calibration method of a constant temperature nucleic acid amplification analyzer according to claim 1, characterized in that: The calibration solution is phenol red solution, and the concentration gradient of the calibration solution is 2 to 10 gradients.

3. The light intensity calibration method of a constant temperature nucleic acid amplification analyzer according to claim 2, characterized in that: Place phenol red powder in a beaker, add less than 30 ml of purified water, and then use a volumetric flask to dilute to 50 ml to make a mother solution. Then extract different amounts of mother solution into different centrifuge tubes, add different amounts of pure water to each centrifuge tube to fully dissolve and mix to obtain calibration solutions with different gradients.

4. The light intensity calibration method of a constant temperature nucleic acid amplification analyzer according to claim 3, characterized in that: Different amounts of calibration solutions of different gradients were added to several PCR reaction tubes, and 40 μl of mineral oil was added to each tube for oil sealing to prevent the solution from volatilizing.

5. The light intensity calibration method of a constant temperature nucleic acid amplification analyzer according to claim 1, characterized in that: The prepared gradient calibration solutions were dispensed into several PCR reaction tubes and placed in the standard detection holes of the constant temperature nucleic acid amplification analyzer standard machine for repeatability, consistency and accuracy testing. The average value of multiple measurements of each tube was taken as the light intensity value of the tube, and the data was recorded to complete the calibration.

6. The method for calibrating light intensity of a constant temperature nucleic acid amplification analyzer according to claim 1, characterized in that: The prepared gradient calibration solutions are dispensed into several PCR reaction tubes. One tube is selected from each gradient calibration solution to calibrate all detection channels of the standard constant temperature nucleic acid amplification analyzer. The repeatability, consistency and accuracy of the calibration solution are tested. The average value of multiple measurements of each tube is taken as the light intensity value of the tube, and the data is recorded to complete the calibration.

7. The light intensity calibration method of a constant temperature nucleic acid amplification analyzer according to claim 1, characterized in that: Repeatability testing consists of the following steps: The calibration solutions of different concentrations were placed on the constant temperature nucleic acid amplification analyzer to collect signal value data. The same test position was selected for each concentration. The test was performed every 30 seconds at an instrument temperature of 65°C. The test was repeated 10 times. The average value and standard deviation of the measurement results of the test solutions of each concentration were obtained respectively. The coefficient of variation was calculated using the formula: in, is the coefficient of variation of the signal measurement value; is the standard deviation of the signal measurements for each concentration of solution; is the average of the signal measurements for each concentration of solution.

8. The light intensity calibration method of a constant temperature nucleic acid amplification analyzer according to claim 7, characterized in that: Conformance testing consists of the following steps: Place calibration solutions of different concentrations into multiple detection wells of the constant temperature nucleic acid amplification analyzer, test every 30 seconds when the instrument temperature reaches 65°C, repeat the test 10 times, calculate the average value of the signal measurement value of each concentration solution in each detection well, calculate the average value of each concentration calibration solution, and calculate the standard deviation of each calibration solution. Finally, calculate the coefficient of variation between different tubes of each calibration solution, which is: in, is the coefficient of variation of the signal measurement value; is the standard deviation of the signal measurement value of each concentration solution; specifically: It is the average value of the signal measurement value of each concentration solution in each detection well; is the average of the signal measurements for each concentration of solution; is the number of test wells for each concentration of solution.

9. The method for calibrating light intensity of a constant temperature nucleic acid amplification analyzer according to claim 8, characterized in that: Accuracy testing consists of the following steps: Place calibration solutions of different concentrations into multiple detection holes of the constant temperature nucleic acid amplification analyzer. When the instrument temperature reaches 65°C, test once every 30 seconds. Repeat the test 10 times. Calculate the average signal measurement value of each concentration solution in each detection hole, and then calculate the accuracy, specifically: in, For accuracy; It is the average value of the signal measurement value of each concentration solution in each detection well; is the target signal value corresponding to each concentration solution.

10. The light intensity calibration method of a constant temperature nucleic acid amplification analyzer according to claim 9, characterized in that: The coefficient of variation in the repeatability test is no more than 1%, the coefficient of variation in the consistency test is no more than 2%, and the percentage deviation in the accuracy test is no more than 3%.

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