PDRN detection method capable of correcting measurement error of ultraviolet spectrophotometry

Abstract

The invention relates to a PDRN detection method capable of correcting measurement errors of an ultraviolet spectrophotometric method, and belongs to the technical field of PDRN detection. The formula is as follows: Cc = C * {1-K1 * CP-K2 * (R-1.9)}. The invention also provides a PDRN detection method capable of correcting the measurement error of the ultraviolet spectrophotometry. According to the invention, the anti-intuition law that'protein residue and the PDRN measured value of the ultraviolet spectrophotometry are positively correlated 'is found for the first time, the A260 / A280 ratio is innovatively used as a second variable to be introduced into the correction model, and a PDRN content calculation formula capable of correcting the measurement error of the ultraviolet spectrophotometry is provided. The calculation formula can effectively eliminate the systematic interference of protein residues and ultraviolet absorption impurities on the determination result, corrects the determination error of the ultraviolet spectrophotometry, and greatly improves the detection accuracy.

Description

Technical Field

[0001] The present invention relates to a PDRN detection method for correcting errors in ultraviolet spectrophotometry, belonging to the technical field of PDRN detection. Background Art

[0002] Polydeoxyribonucleotide (PDRN) is a DNA fragment of a specific specification extracted from the germ cells of the testes of male salmon. It is composed of four deoxynucleotides, A, T, C, and G, polymerized into a deoxynucleotide chain through phosphodiester bonds. Two deoxynucleotide chains are coiled into a double helix structure according to the base complementary principle of A-T and C-G. It is an essential key genetic material for humans, animals, plants, and microorganisms, and has the functions of anti-inflammatory and promoting tissue repair. Clinical studies have confirmed that PDRN of different molecular weights has different pharmacological effects. Small molecular weight (<162 kDa) has a significant effect on timely anti-inflammatory and reducing tissue inflammation; medium molecular weight (160 kDa < PDRN < 650 kDa) has a significant effect on increasing the synthesis of FGF, collagen, and elastin and promoting tissue repair; large molecular weight (>1000 kDa) has the functions of water retention, physical support, and shielding, and is mainly used for tissue filling.

[0003] Ultraviolet spectrophotometry (UV method) is a commonly used method for determining the nucleic acid content of PDRN. Its basis is that nucleic acids have a maximum absorption value at a wavelength of 260 nm. However, in actual production quality control, this method has significant defects. Its measurement results are easily interfered by impurities in the sample, resulting in poor accuracy. Especially for PDRN products, impurities such as protein residues, salts, and solvents introduced during the production process will seriously interfere with the absorbance value of A260, causing the measured value to deviate significantly from its true value. Detection methods such as high-performance liquid chromatography can be used as alternative solutions to ultraviolet spectrophotometry, but due to their expensive equipment, complex operation, and long time consumption, they are not suitable for rapid quality control at the production site.

[0004] At present, there is a lack of an economical and simple method in this field to correct the influence caused by these interferences, resulting in production manufacturers being unable to accurately control the product quality and even more unable to identify defective batch products. Therefore, it is very necessary to develop a PDRN detection method that can only use ultraviolet spectrophotometry to measure and correct errors to improve the accuracy and reliability of PDRN product quality control. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a PDRN detection method for correcting errors in ultraviolet spectrophotometry.

[0006] The technical solution of the present invention is as follows: A calculation formula for the content of PDRN that can correct the errors in ultraviolet spectrophotometry: Formula: Cc = C × {1 - K1 × C} P -K2×(R-1.9)}; Where Cc represents the corrected PDRN content (%), C represents the apparent PDRN content of the sample determined by ultraviolet spectrophotometry (%), and Cp represents the PDRN content of the sample. P The percentage represents protein content (%). R is the ratio of the UV absorbance of the sample at 260 nm to that at 280 nm. 1.9 is the ratio of the UV absorbance of the PDRN standard at 260 nm to that at 280 nm. K1 and K2 are empirical coefficients determined by fitting historical data.

[0007] According to a preferred embodiment of the present invention, K1 = 0.05 and K2 = 0.025; the coefficients are determined by fitting and analyzing data from representative PDRN samples. In a specific, preferred embodiment, this set of coefficients is particularly suitable for error correction of PDRN products derived from salmon testes. Those skilled in the art will understand that, without departing from the principles of the present invention, appropriate adjustments and optimizations to the coefficients based on subtle differences in the specific product source and process still fall within the scope of protection of the present invention.

[0008] A method for detecting PDRN that can correct for errors in ultraviolet spectrophotometry includes the following steps: (1) The apparent PDRN content (C) of the sample was determined by ultraviolet spectrophotometry. (2) The ultraviolet absorbance A260 at 260 nm and the ultraviolet absorbance A280 at 280 nm were measured by ultraviolet spectrophotometry, and the A260 / A280 value (R) was calculated. (3) The protein content in the sample was determined using the Folin-Ciocalteu method (C p ); (4) Calculate the corrected PDRN content (Cc) in the sample to be tested according to the formula; Wherein, the formula is: Cc=C×{1-K1×C P -K2×(R-1.9)}; In the formula, K1=0.05 and K2=0.025.

[0009] The above-mentioned formula for calculating PDRN content that can correct for errors in ultraviolet spectrophotometry, or the PDRN detection method that can correct for errors in ultraviolet spectrophotometry, is applied in PDRN production and quality monitoring.

[0010] Technical features and beneficial effects of the present invention: 1. This invention is the first to discover the counterintuitive finding that "protein residue is positively correlated with PDRN levels measured by ultraviolet spectrophotometry." It innovatively introduces the A260 / A280 ratio as a second variable into the correction model, providing a formula for calculating PDRN content that can correct for errors in ultraviolet spectrophotometry. This formula effectively eliminates the systematic interference of protein residue and ultraviolet absorption impurities on the measurement results, corrects for errors in ultraviolet spectrophotometry, and significantly improves detection accuracy.

[0011] 2. The PDRN detection method provided by this invention, which can correct for errors in ultraviolet spectrophotometry, does not require expensive equipment such as liquid chromatography-mass spectrometry, resulting in extremely low cost and suitability for large-scale production quality control. Furthermore, this method not only corrects errors, but its residual analysis can also inversely indicate deficiencies in the production process, enabling quality monitoring in PDRN production and providing a clear direction for continuous improvement of the PDRN production process. Attached Figure Description

[0012] Figure 1 This is a comparison chart of PDRN content before and after correction.

[0013] Figure 2 The correlation plot between the absolute value of the correction amplitude ΔC, |ΔC|, and the protein content Cp (|ΔC| = |Cc - C|). Detailed Implementation

[0014] The present invention will now be described in detail with reference to specific embodiments. The embodiments described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0015] Unless otherwise specified, all experimental methods used in the following examples are conventional methods. All reagents used in the examples are commercially available products.

[0016] Example 1: Establishing a formula for calculating PDRN content that can correct for errors in ultraviolet spectrophotometry. 1. Measurement of experimental data Twenty batches of PDRN products manufactured by the applicant were selected, with batch numbers PDRN240625-1, PDRN240625-2, PDRN241113-1, PDRN241113-2, PDRN241202-1, PDRN241202-2, PDRN241202-3, PDRN241202-4, PDRN241202-5, PDRN241204-1, PDRN241204- 2. PDRN241204-3, PDRN250627-1, PDRN250627-2, PDRN250627-6, PDRN250708-1, PDRN250708-2, PDRN250708-6, PDRN250715-1, and PDRN250715-2 were used to determine and collect data such as DNA content (C), A260 / A280 ratio (R), and protein content (Cp) for model construction.

[0017] The specific method is as follows: The apparent PDRN content (C) of 20 batches of PDRN products was determined by ultraviolet spectrophotometry.

[0018] The ultraviolet absorbance (A260) at 260 nm and the ultraviolet absorbance (A280) at 280 nm of 20 batches of PDRN products were determined by ultraviolet spectrophotometry, and the A260 / A280 value (R) was calculated.

[0019] The protein content (C) of 20 batches of PDRN products was determined using the Folin-Ciocalteu method. p ).

[0020] 2. Patterns in key parameters Data analysis showed that the residual protein content was C. p It is positively correlated with the apparent PDRN content C, i.e., C p The increase in C indicates a systematic high level; and the degree of deviation between the A260 / A280 ratio R and the theoretical pure product ratio (1.9) represents the degree of overall interference from impurities. That is, when protein interference is present in the sample, A280 increases significantly, so a negative coefficient is needed for reverse correction.

[0021] 3. Construction of the theoretical model Based on the above patterns, the theoretical form of the correction model is constructed as: Cc = C × {1 - K1 × C} P -K2×(R-1.9)}.

[0022] Protein correction term (K1): It is known that proteins have ultraviolet absorption at wavelengths close to 260 nm, which directly superimposes on the DNA absorbance value, leading to an artificially high measured value C. The inventors of this application initially assume that 1% protein residue may cause a positive bias of several percent in the PDRN measurement value. Therefore, the theoretical estimate of K1 should be in the range of 0.01 to 0.10.

[0023] Purity Correction Term (K2): The A260 / A280 ratio is a classic indicator of nucleic acid purity; for the PDRN standard, it is 1.9 in this system. A deviation from this baseline indicates the presence of proteins (lowering the ratio) or other impurities. The inventors of this application assume that a correction of several percent may be required for every 0.1 unit deviation in the ratio. Therefore, the theoretical estimate of K2 should be in the range of 0.01 to 0.10.

[0024] C and C P Linear trend analysis was performed using (R² = 1.9). The values ​​of K1 and K2 were adjusted based on the slope of the trend line to ensure that the model systematically corrected any artificially inflated values. Ultimately, K1 was determined to be 0.05, meaning that a 1% increase in protein content had an average impact of approximately 5% on the PDRN measurement value; and K2 was determined to be 0.025, meaning that a 0.1-unit deviation in the A260 / A280 ratio had an impact of approximately 0.25% on the results. 4. Determination of the correction formula The preferred correction formula of this invention is finally established as: Cc = C × {1 - 0.05 × C} P -0.025×(R-1.9)}.

[0025] The innovation of this model lies in establishing, for the first time, a quantitative relationship between protein content, the A260 / A280 ratio, and PDRN measurement error, achieving complex interference elimination through simple mathematical transformations. The coefficient determination process combined statistical analysis and engineering practice, ensuring the model's reliability and practicality. Its excellent correction effect and practicality have been fully verified by the correlation between residuals and protein content in Example 2.

[0026] Example 2: PDRN detection method that can correct for errors in ultraviolet spectrophotometry. 1. Using the PDRN content calculation formula established in Example 1 that can correct for errors in ultraviolet spectrophotometry, 20 batches of PDRN products produced by the applicant (batch numbers detailed in Example 1) were used as the samples to be tested in this example for PDRN detection. The specific steps are as follows: (1) The apparent PDRN content (C) of the sample was determined by ultraviolet spectrophotometry. (2) The ultraviolet absorbance A260 at 260 nm and the ultraviolet absorbance A280 at 280 nm were measured by ultraviolet spectrophotometry, and the A260 / A280 value (R) was calculated. (3) The protein content (Cp) in the sample was determined using the Folin-Ciocalteu method. (4) Calculate the corrected PDRN content (Cc) in the sample to be tested according to the formula; Formula: Cc = C × {1 - K1 × C} P -K2×(R-1.9)}; Where Cc represents the corrected PDRN content (%), C represents the apparent PDRN content of the sample determined by ultraviolet spectrophotometry (%), and Cp represents the PDRN content of the sample. P The percentage represents protein content (%). R is the ratio of the UV absorbance of the sample at 260 nm to that at 280 nm. 1.9 is the ratio of the UV absorbance of the PDRN standard at 260 nm to that at 280 nm. K1 and K2 are empirical coefficients determined by fitting historical data.

[0027] A total of 20 batches of samples were tested using the above method. The comparison results of PDRN content before and after correction are shown in Table 1 and Table 2. Figure 1 As shown.

[0028] Table 1 Depend on Figure 1 As shown in Table 1, the method provided by this invention can quickly detect the content of PDRN, effectively correct the measurement error of ultraviolet spectrophotometry, eliminate the influence of protein and product purity on DNA content measurement, and the results are closer to the true value.

[0029] 2. To verify the effectiveness of the correction model, the inventors of this application analyzed the correlation between the correction amplitude and the interference factors. The correlation between |ΔC| and Cp is as follows: Figure 2 As shown.

[0030] Depend on Figure 2 As shown in Table 1, |ΔC| is significantly positively correlated with Cp (R0). 2 =0.9998), which proves that the higher the protein content, the larger the correction range, consistent with the model's expectations. This correction model effectively eliminates the influence of protein and product purity on DNA content determination. The corrected PDRN content values ​​are more concentrated, and extremely high values ​​(such as unreasonable values ​​>105%) are effectively corrected. The value range converges to a reasonable range of 85%-101%, which is consistent with the actual level of production process. The consistency, accuracy, and reliability of the test results have been significantly improved, making it fully applicable to high-standard production quality control.

[0031] Example 3: Application of the PDRN content calculation formula / detection method that can correct for errors in ultraviolet spectrophotometry. The PDRN content calculation formula / detection method provided by this invention, which can correct for errors in ultraviolet spectrophotometry, can be used as a guide for PDRN production and quality monitoring, as detailed below: 1. For process products in PDRN production When |ΔC|≤1.5, it indicates that the process is stable, the purification effect is good, and the measurement results are highly reliable.

[0032] When ΔC < -3.0, it indicates that the product is seriously contaminated and the measurement result is falsely high. An early warning response should be initiated immediately, and the production process should be traced back, such as whether the activity of proteinase K has decreased and whether the washing process is thorough. The production process should be adjusted in a timely manner to deal with potential quality risks. 2. For the final product in PDRN production The original DNA content should not be used as the standard; the corrected DNA content should be used instead. This will prevent batches with inflated measurement results from being released as qualified products, and ensure the reliable quality of products leaving the factory.

[0033] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A formula for calculating PDRN content that can correct for errors in ultraviolet spectrophotometry, characterized in that the formula is: Cc = C × {1 - K1 × C} P -K2×(R-1.9)}; Where Cc represents the corrected PDRN content (%), C represents the apparent PDRN content of the sample determined by ultraviolet spectrophotometry (%), and Cp represents the PDRN content of the sample. P The value represents the protein content (%), and R is the ratio of the UV absorbance of the sample at 260 nm to that at 280 nm. 1.9 represents the ratio of the UV absorbance of the PDRN standard at 260 nm to that at 280 nm; K1 and K2 are empirical coefficients determined by fitting historical data.

2. The PDRN content calculation formula for correcting errors in ultraviolet spectrophotometry as described in claim 1, characterized in that, K1=0.05, K2=0.

025.

3. A method for detecting PDRN that can correct for errors in ultraviolet spectrophotometry, characterized in that, The steps include the following: (1) The apparent PDRN content (C) of the sample was determined by ultraviolet spectrophotometry. (2) The ultraviolet absorbance A260 at 260 nm and the ultraviolet absorbance A280 at 280 nm were measured by ultraviolet spectrophotometry, and the A260 / A280 value (R) was calculated. (3) The protein content in the sample was determined using the Folin-Ciocalteu method (C p ); (4) Calculate the corrected PDRN content (Cc) in the sample to be tested according to the formula; Wherein, the formula is: Cc=C×{1-K1×C P -K2×(R-1.9)}; In the formula, K1=0.05 and K2=0.

025.

4. The application of the PDRN content calculation formula with correctable ultraviolet spectrophotometric determination error as described in claim 1 or the PDRN detection method with correctable ultraviolet spectrophotometric determination error as described in claim 3 in PDRN production and quality monitoring.

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