Calibration method of nucleic acid quantitative fluorometer
By using methylene blue aqueous solution to replace the RNA reagent kit, constructing a standard curve and combining it with multi-dimensional judgment logic, the problem of unstable calibration of nucleic acid quantitative fluorometers was solved, realizing a low-cost and efficient calibration method, and improving the reliability and stability of detection results.
Patent Information
- Application Number
- CN202511797782.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-02
- Publication Date
- 2026-02-24
AI Technical Summary
The existing calibration methods for quantitative nucleic acid fluorometers rely on expensive imported RNA reagent kits, which leads to unstable test results and cumbersome operation. Furthermore, the fluorescence intensity of traditional RNA fluorescent staining agents is not linearly related to their concentration, resulting in high relative expanded uncertainty of the test results.
Using methylene blue aqueous solution as a standard substance, the optical performance of the nucleic acid quantitative fluorometer was verified by constructing a linear response relationship of chemical standards. This included preparing methylene blue stock solution and calibration solution, collecting fluorescence signals under light-protected and stable conditions, constructing a standard curve of concentration versus fluorescence unit value, and performing calibration by combining multi-dimensional judgment logic.
A calibration method with high linearity, high stability, and low cost has been achieved, which significantly reduces capital consumption and manpower, improves the reliability of test results, and provides a technical closed loop for fault diagnosis.
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Figure CN121558705A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of metrology and testing technology, specifically to calibration technology for non-amplified nucleic acid quantitative detection equipment, and particularly to a calibration method for a nucleic acid quantitative fluorometer. Background Technology
[0002] Quantitative fluorometers for nucleic acid are essential instruments for determining the concentration of DNA and RNA in life science research. Currently, most manufacturers of quantitative fluorometers for nucleic acid in the domestic market typically use a "comparison method" to determine the accuracy of instrument results. This involves comparing the results (fluorescence curves) of standard RNA reagent kits (such as those using RiboGreen or PicoGreen fluorescent dyes) with those from well-known foreign brands. This is used as a means to determine whether the machine's performance meets standards. However, this existing calibration technique has the following significant drawbacks: After staining RNA with RNA fluorescent staining agents in nucleic acid kits, the fluorescence intensity (RFU) value often does not exhibit a strictly linear relationship with the RNA concentration. This nonlinear characteristic leads to a high relative expanded uncertainty in the detection results, making it difficult to accurately reflect the optical linearity performance of the instrument. Using standard reagent kits for comparative calibration not only requires purchasing expensive imported reagent kits and fluorescent dyes, increasing financial costs, but also involves cumbersome procedures that consume significant manpower. Furthermore, the instability of biological reagents (susceptibility to degradation due to light and heat) or the lack of effective control over environmental variables during the process often leads to unstable calibration results and low confidence levels in the detection results. Therefore, developing a calibration method that can replace expensive biological reagent kits and possesses high linearity, high stability, and low cost is a pressing technical problem that needs to be solved in this field. Summary of the Invention
[0003] The purpose of this invention is to address the deficiencies in the existing technology by proposing a calibration method for a nucleic acid quantitative fluorometer.
[0004] To achieve the above objectives, the present invention adopts the following technical solution: A calibration method for a nucleic acid quantitative fluorometer, wherein the method uses methylene blue aqueous solution as a standard substance to replace biological nucleic acid reagents, and verifies the optical performance of the nucleic acid quantitative fluorometer by constructing a linear response relationship of chemical standards; The method includes the following steps: Step 1: Prepare methylene blue mother liquor and a series of methylene blue calibration solutions, and perform light-protected stabilization treatment on the methylene blue mother liquor and methylene blue calibration solutions; Step 2: Use the nucleic acid quantitative fluorometer to be calibrated to collect fluorescence signals of methylene blue calibration solutions of different concentrations, obtain relative fluorescence unit values, construct a standard curve of concentration versus relative fluorescence unit values, and calculate the correlation coefficient of the standard curve. Step 3: Select a methylene blue solution of known concentration as a verification sample, measure the relative fluorescence unit value of the verification sample on the nucleic acid quantitative fluorescence meter, and substitute the measured relative fluorescence unit value into the standard curve to calculate the measured concentration value. Step 4: Calculate the indication error, channel range, and measurement repeatability data based on the measured concentration value and the known concentration value; Step 5: Compare the correlation coefficient, indication error, channel range, and measurement repeatability data of the standard curve with the preset metrological characteristic thresholds in multiple dimensions, and determine whether the calibration result of the nucleic acid quantitative fluorometer is qualified based on the comparison results.
[0005] Furthermore, in step one, the method for preparing the methylene blue mother liquor includes: Weigh the methylene blue standard powder using an analytical balance; Pour the methylene blue standard powder into the first container, add purified water (less than the final volume) and stir until dissolved; Transfer the dissolved solution to a brown volumetric flask, rinse the first container and add the rinsing solution to the brown volumetric flask, bring the volume to the mark, and invert and shake well.
[0006] Furthermore, in step one, the specific operation of performing light-protected stabilization treatment on the methylene blue mother liquor and the methylene blue calibration solution is as follows: After the methylene blue stock solution and methylene blue calibration solution are prepared, they are placed in a light-protected environment and left to stand for a preset time, which is 30 minutes. The settling process eliminates the interference of bubbles generated in the early stage of solution dissolution on light scattering and allows solute molecules to reach thermodynamic equilibrium in the solvent.
[0007] Furthermore, in step one, the preparation process of the methylene blue mother liquor and the methylene blue calibration solution must meet the aging control conditions: The preparation of the methylene blue stock solution and the methylene blue calibration solution must be completed within a time frame of 5 to 10 minutes. The methylene blue stock solution and methylene blue calibration solution must be used up on the day of preparation.
[0008] Furthermore, the entire calibration process must be carried out under a constant ambient temperature, which is controlled between 17°C and 30°C. The temperature control is used to prevent the increase in the nonradiative transition probability of methylene blue molecules and the decrease in quantum yield due to temperature rise, while suppressing fluorescence quenching caused by solvent polarity effects.
[0009] Furthermore, in step four, the indication error is calculated according to the following formula: In the formula: For indication error, This is the arithmetic mean of three measurements taken by the instrument. This represents the known concentration value of the standard solution.
[0010] Furthermore, in step four, the channel range and measurement repeatability are calculated according to the following formula: The formula for calculating the channel range is: In the formula: The channel is extremely poor. The maximum concentration was measured for all channels. The minimum concentration was measured for all channels; The formula for calculating the measurement repeatability is: In the formula: The relative standard deviation is the measurement repeatability. It is the arithmetic mean of multiple measurements taken by the instrument. This is a single measurement value. For the number of times measured.
[0011] Furthermore, the preset measurement characteristic threshold in step five is set as follows: The correlation coefficient of the standard curve is greater than or equal to 0.99; The channel range is less than or equal to 16%; The repeatability of the measurement is less than or equal to 10%; The indicated error is within the range of -12% to +12%.
[0012] Furthermore, the determination process in step five employs hierarchical verification logic, specifically including: First-level verification: Determine whether the correlation coefficient of the standard curve meets the threshold requirement. If it does not, the standard curve construction is deemed to have failed and will not proceed to subsequent levels of verification. Second-level verification: If the first-level verification is passed, determine whether the indication error, channel range and measurement repeatability data simultaneously meet the preset metrological characteristic thresholds. If all data are within the specified metrological characteristic threshold range, the nucleic acid quantitative fluorometer is deemed to have passed calibration.
[0013] Furthermore, in the second-level verification, if any data exceeds the measurement characteristic threshold, a corresponding fault prompt message is generated: If only the channel range exceeds the threshold, it suggests checking the difference in optical path between holes or the cleanliness of the cuvette; If only the repeatability measurement exceeds the threshold, it suggests checking the stability of the light source or external stray light interference; If only the indicated value error exceeds the threshold, it suggests checking the accuracy of the standard solution concentration preparation.
[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: A methylene blue standard solution was used instead of the RNA reagent kit. As a small molecule chemical substance, methylene blue exhibits a highly linear and direct relationship between its fluorescence intensity (RFU) and concentration within the detection range, effectively solving the problem of non-linear fluorescence values after RNA staining by traditional RNA fluorescent staining agents. By building The standard curve significantly improves the reliability of nucleic acid fluorescence meter detection results, and can greatly reduce calibration costs and time. Methylene blue reagent is inexpensive and readily available, eliminating the need for expensive imported RNA reagent kits and greatly reducing capital consumption. At the same time, the standardized solution preparation and testing process simplifies the operation steps and significantly reduces the time required for manual calibration. Ensuring result stability through environmental and process control: This invention innovatively introduces the technical means of allowing the solution to stand in the dark for 30 minutes after preparation and constant temperature control at 17℃-30℃. The standing treatment eliminates the interference of microbubbles generated by solution stirring on light scattering and allows the solute to reach thermodynamic equilibrium. The constant temperature control effectively prevents the increase of molecular nonradiative transitions (decreased quantum yield) caused by temperature rise and fluorescence quenching caused by solvent polarity effect.
[0015] In addition, a hierarchical judgment logic that includes linearity validity verification and metrological characteristic sub-item verification has been established. It can not only give a "qualified / unqualified" conclusion, but also provide specific technical diagnosis directions for instrument failures (such as optical path differences, light source aging, liquid preparation errors, etc.) by analyzing specific out-of-specification indicators (channel range, indication error or repeatability) when unqualified, thus forming a complete technical closed loop. Attached Figure Description
[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.
[0017] Figure 1This is a schematic diagram of the overall process of a nucleic acid quantitative fluorometer calibration method according to the present invention; Figure 2 This is a schematic diagram of the process for preparing and stabilizing the methylene blue standard solution in this invention; Figure 3 This is a flowchart of the judgment and fault diagnosis process based on hierarchical verification logic in this invention; Figure 4 This is a schematic diagram of the standard curve of methylene blue concentration versus fluorescence intensity constructed in an embodiment of the present invention. Detailed Implementation
[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.
[0019] Example 1: like Figure 1-4 As shown, this embodiment provides a standardized preparation method for a methylene blue standard solution used as an alternative to nucleic acid reagent kits. This method strictly limits the preparation time, light conditions, and settling time to ensure the optical stability of the solution.
[0020] Experimental equipment and reagents preparation: Methylene Blue (AR) was used as the standard. Accurate weighing was performed using an analytical balance with a sensitivity of 0.00001 g. Use a metrologically calibrated hand pipette (200 μL size) or a high-throughput pipetting workstation for pipetting operations; Use 100mL brown volumetric flasks to avoid photolysis.
[0021] Mother liquor preparation steps: S101: Accurately weigh 0.05g of methylene blue standard powder using an analytical balance; S102: Pour the weighed powder into a clean small beaker, add less than 50mL of purified water, and stir with a glass rod until the powder is completely dissolved. S103: Use a glass rod to transfer the solution in the small beaker to a 100mL brown volumetric flask, and rinse the small beaker several times with a small amount of pure water. Transfer the rinsing solution into the volumetric flask as well. S104: Add water to the brown volumetric flask and bring it to the 100mL mark. Shake well by inverting the flask to obtain the methylene blue stock solution. The steps for preparing the calibration solution are as follows: S201: Invert and shake the prepared mother liquor again to mix it well, and pour some into a small beaker; S202: Calculate the required volume of stock solution according to the required calibration concentration. For example, when preparing a 0.25 ng / μL calibration solution, accurately pipette 50 μL of stock solution into a 100 mL brown volumetric flask; when preparing a 0.4 ng / μL calibration solution, pipette 80 μL of stock solution into another 100 mL brown volumetric flask.
[0022] S203: Add purified water to each of the volumetric flasks above and bring the volume to the 100 mL mark, then invert and shake well. For the 0 ng / μL blank control point (wherein, the 0 ng / μL calibration solution is purified water), use purified water directly as the calibration solution.
[0023] It should be noted that time control is also required for key processes. Specifically, the entire process of preparing the mother liquor and calibration solution must be completed within 5-10 minutes, and the solution must be prepared and used on the same day, and cannot be used on the next day. Light-shielding stabilization treatment is also required. Specifically, after the solution is prepared and shaken well, the time should be recorded, and the container should be placed in a light-shielding cabinet and left to stand for 30 minutes. In this step, the standing step is used to eliminate air bubbles introduced during stirring, preventing air bubbles from scattering light and interfering with fluorescence detection. On the other hand, it allows solute molecules to fully diffuse in the solvent to reach thermodynamic equilibrium, ensuring the stability of the fluorescence signal.
[0024] Example 2: like Figure 1-4 As shown, based on Example 1, a calibration and testing procedure for a nucleic acid quantitative fluorometer is provided. Specifically, it includes the following steps for performance testing of the nucleic acid quantitative fluorometer using the methylene blue solution prepared in Example 1: (1) Environmental conditions control: The laboratory ambient temperature during the entire testing process must be controlled between 17℃ and 30℃. It should be noted that strict temperature control is used to prevent the increase in the nonradiative transition probability of methylene blue molecules and the decrease in quantum yield due to temperature rise, while suppressing the fluorescence quenching caused by solvent polarity effect and ensuring the accuracy of measurement data. (2) Plotting the standard curve, the specific steps are as follows: S301: Prepare a new microplate or 8-cup, and inject 200 μL of methylene blue standard solution of different concentration gradients (e.g., 0 ng / μL, 0.25 ng / μL, 0.4 ng / μL) into the detection wells; S302: Operate the nucleic acid quantitative fluorometer to be calibrated (select RNA / red light mode, corresponding to an excitation wavelength of approximately 480nm and an emission wavelength of approximately 520nm) to acquire fluorescence signals; S303: The instrument plots a standard curve based on the collected relative fluorescence units (RFU) and concentration data, and automatically calculates the linear correlation coefficient. The technical solution of this invention requires A score of 0.99 or higher (i.e., "two nines" or higher) is required before proceeding to the next stage of testing. (3) Performance verification test: Select methylene blue calibration solution of known concentration as verification sample (preferred concentration points: 0.25ng / μL, 0.3ng / μL, 0.35ng / μL) for instrument test, collect instrument readings, and substitute them into the standard curve to calculate the measured concentration value; (4) Calculation of metrological characteristic parameters: According to JJF 1059.1-2012, the collected data are processed using the following formulas: (401) The formula for calculating the indication error (accuracy) is as follows: In the formula: —Indication error, % —The arithmetic mean of three measurements taken by the instrument, ng / μL; —The known concentration (theoretical value) of the standard solution, ng / μL (402) The formula for calculating the channel range is as follows: In the formula: —The range of measurements across all channels, ng / μL; —Maximum concentration across all channels, ng / μL; —Minimum concentration across all channels, ng / μL.
[0025] (403) The formula for calculating measurement repeatability is as follows: In the formula: —Relative standard deviation (measurement repeatability), % —The arithmetic mean of 10 measurements taken by the instrument, ng / μL; —Single measurement, ng / μL; —Number of measurements (usually n=10).
[0026] Example 3: This embodiment employs a hierarchical verification logic to comprehensively evaluate the performance of the nucleic acid quantitative fluorometer.
[0027] To more accurately evaluate the instrument's performance across different concentration ranges, preset measurement characteristic thresholds were established in this embodiment. These thresholds were based on a tiered pass / fail criterion set according to the concentration gradient of the methylene blue solution. See Table 1 below: Table 1 The concentration points (0.25 / 0.30 / 0.35 ng / μL) in the table above correspond to the concentrations of the validation samples selected in Example 2. As the concentration increases, the instrument signal-to-noise ratio improves, thus the requirements for repeatability and channel range are correspondingly increased (limits are narrowed), reflecting the rigor of the calibration.
[0028] First level of the hierarchical verification process: Linearity validity verification first involves determining the correlation coefficient of the standard curve generated in step S303. Is it greater than or equal to 0.99?
[0029] like If the system determines that "standard curve construction failed", the process will terminate. The system will prompt the user to check whether the solution preparation timed out, whether there is precipitation, or whether there is strong interference in the optical path system, and will require the solution to be prepared again and the curve to be plotted. like The linear fit is deemed satisfactory, and the process proceeds to the second level of verification.
[0030] The second level is the sub-item verification of metrological characteristics. Under the premise of passing the first level of verification, the indication error, channel range and measurement repeatability data calculated in Example 2 are compared with the above-mentioned preset thresholds.
[0031] The overall conclusion output qualification criteria are as follows: the nucleic acid quantitative fluorescence meter is deemed "metrologically qualified" only when the indication error, channel range, and measurement repeatability simultaneously meet the preset threshold requirements. The conditions for non-compliance judgment and fault diagnosis are as follows: if any indicator exceeds the threshold, it is judged as "measuring non-compliance", and a corresponding fault prompt message is generated, as follows: If only the channel range exceeds the limit: it suggests checking the consistency of the optical path between instrument wells or the cleanliness of the cuvette / ELISA plate; If only the repeatability of the measurement exceeds the limit, it suggests checking the stability of the light source (such as LED aging) or external stray light interference. If only the indicated value error exceeds the standard: it suggests checking the accuracy of the methylene blue standard solution concentration preparation or the precision of the pipette.
[0032] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A calibration method for a nucleic acid quantitative fluorometer, characterized in that, The method uses methylene blue aqueous solution as a standard substance to replace biological nucleic acid reagents, and verifies the optical performance of the nucleic acid quantitative fluorometer by constructing a linear response relationship of chemical standards. The method includes the following steps: Step 1: Prepare methylene blue stock solution and multiple methylene blue calibration solutions with different concentration gradients, and perform light-protected stabilization treatment on the methylene blue stock solution and methylene blue calibration solutions; Step 2: Use the nucleic acid quantitative fluorometer to be calibrated to collect fluorescence signals of methylene blue calibration solutions of different concentrations, obtain relative fluorescence unit values, construct a standard curve of concentration versus relative fluorescence unit values, and calculate the correlation coefficient of the standard curve. Step 3: Select a methylene blue solution of known concentration as a verification sample, measure the relative fluorescence unit value of the verification sample on the nucleic acid quantitative fluorescence meter, and substitute the measured relative fluorescence unit value into the standard curve to calculate the measured concentration value. Step 4: Calculate the indication error, channel range, and measurement repeatability data based on the measured concentration value and the known concentration value; Step 5: Compare the correlation coefficient, indication error, channel range, and measurement repeatability data of the standard curve with the preset metrological characteristic thresholds in multiple dimensions, and determine whether the calibration result of the nucleic acid quantitative fluorometer is qualified based on the comparison results.
2. The calibration method for a nucleic acid quantitative fluorometer according to claim 1, characterized in that, In step one, the preparation method of the methylene blue mother liquor includes: Weigh the methylene blue standard powder using an analytical balance; Pour the methylene blue standard powder into the first container, add purified water (less than the final volume) and stir until dissolved; Transfer the dissolved solution to a brown volumetric flask, rinse the first container and add the rinsing solution to the brown volumetric flask, bring the volume to the mark, and invert and shake well.
3. The calibration method for a nucleic acid quantitative fluorometer according to claim 2, characterized in that, In step one, the specific operation of stabilizing the methylene blue mother liquor and the methylene blue calibration solution by avoiding light is as follows: After the methylene blue stock solution and methylene blue calibration solution are prepared, they are placed in a light-protected environment and left to stand for a preset time, which is 30 minutes. The static treatment eliminates the interference of bubbles generated in the early stage of solution dissolution on light scattering and allows solute molecules to reach thermodynamic equilibrium in the solvent.
4. The calibration method for a nucleic acid quantitative fluorometer according to claim 3, characterized in that, In step one, the preparation process of the methylene blue mother liquor and the methylene blue calibration solution must meet the aging control conditions: The preparation of the methylene blue stock solution and the methylene blue calibration solution must be completed within a time frame of 5 to 10 minutes. The methylene blue stock solution and methylene blue calibration solution must be used up on the day of preparation.
5. The calibration method for a nucleic acid quantitative fluorometer according to claim 1, characterized in that, The entire calibration process must be carried out under a constant ambient temperature, which is controlled between 17°C and 30°C. The temperature control is used to prevent the increase in the nonradiative transition probability of methylene blue molecules and the decrease in quantum yield due to temperature rise, while suppressing fluorescence quenching caused by solvent polarity effects.
6. The calibration method for a nucleic acid quantitative fluorometer according to claim 1, characterized in that, In step four, the indication error is calculated according to the following formula: In the formula: For indication error, This is the arithmetic mean of three measurements taken by the instrument. This represents the known concentration value of the standard solution.
7. The calibration method for a nucleic acid quantitative fluorometer according to claim 1, characterized in that, In step four, the channel range and measurement repeatability are calculated according to the following formula: The formula for calculating the channel range is: In the formula: The channel is extremely poor. The maximum concentration was measured for all channels. Measure the minimum concentration for all channels; The formula for calculating the repeatability of the measurement is: In the formula: The relative standard deviation is the measurement repeatability. It is the arithmetic mean of multiple measurements taken by the instrument. This is a single measurement value. For the number of times measured.
8. The calibration method for a nucleic acid quantitative fluorometer according to claim 1, characterized in that, The preset measurement characteristic threshold in step five is set as follows: The correlation coefficient of the standard curve is greater than or equal to 0.99; The channel range is less than or equal to 16%; The repeatability of the measurement is less than or equal to 10%; The indicated error is within the range of -12% to +12%.
9. The calibration method for a nucleic acid quantitative fluorometer according to claim 8, characterized in that, The determination process in step five employs hierarchical verification logic, specifically including: First-level verification: Determine whether the correlation coefficient of the standard curve meets the threshold requirement. If it does not, the standard curve construction is deemed to have failed and will not proceed to subsequent levels of verification. Second-level verification: If the first-level verification is passed, determine whether the indication error, channel range and measurement repeatability data simultaneously meet the preset metrological characteristic thresholds. If all data are within the specified metrological characteristic threshold range, the nucleic acid quantitative fluorometer is deemed to have passed calibration.
10. A calibration method for a nucleic acid quantitative fluorometer according to claim 9, characterized in that, In the second-level verification, if any data exceeds the measurement characteristic threshold, a corresponding fault prompt message is generated: If only the channel range exceeds the threshold, it suggests checking the difference in optical path between holes or the cleanliness of the cuvette; If only the repeatability measurement exceeds the threshold, it suggests checking the stability of the light source or external stray light interference; If only the indicated value error exceeds the threshold, it suggests checking the accuracy of the standard solution concentration preparation.