A primer, method and application thereof for detecting content of PDRN based on fluorescence quantification
By designing primers and methods based on real-time PCR, the problems of high cost and low sensitivity in PDRN content detection were solved, achieving low-cost and high-sensitivity detection applicable to a variety of samples and able to accurately track the degradation process of PDRN in animals.
Patent Information
- Application Number
- CN202411151226.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-21
- Publication Date
- 2026-02-17
- Estimated Expiration
- 2044-08-21
AI Technical Summary
Existing methods for detecting PDRN product content are costly, have low sensitivity, and take a long time, and lack unified standards.
The primers and methods based on real-time PCR were designed to detect PDRN content through real-time PCR amplification reaction. The relative content was calculated by combining the standard curve and the ΔΔCt method. Specific primers were used for DNA extraction and detection.
It achieves low-cost, high-sensitivity detection of PDRN content, accurately tracks its degradation and metabolism in animals, and is suitable for samples such as cells, tissues, and blood, with a detection limit as low as 0.01 ng/mL.
Smart Images
Figure CN118792392B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to primers, methods, and applications for quantitative fluorescence detection of PDRN content, belonging to the fields of molecular biology and biomedical technology. Background Technology
[0002] Polydeoxyribonucleotides (PDRNs) are specific DNA fragments extracted from the reproductive cells of male salmon testes. They exhibit anti-inflammatory, anti-apoptotic, anti-osteoporosis, and anti-melanogenesis effects by activating adenosine A2A receptors and salvage pathways. However, there are currently no unified national or industry standards for the detection of PDRN content. Existing detection methods such as liquid chromatography-mass spectrometry (LC-MS) and ELISA are costly, have low sensitivity, and are time-consuming. Therefore, developing a low-cost, highly sensitive detection method is an urgent problem to be solved.
[0003] Quantitative real-time PCR (qPCR) is a method that detects the total amount of product after each polymerase chain reaction (PCR) cycle by using fluorescence signals during PCR amplification. Because there is a linear relationship between the initial copy number of the template and the Ct value of the template during PCR exponential amplification, specific DNA sequences in the test sample can be quantified using internal or external controls.
[0004] Since its inception, quantitative real-time PCR (qPCR) technology has been widely used in various research fields such as microbial assays, tumor gene detection, and immunoassays due to its high sensitivity and specificity. Therefore, designing a simple and easy-to-use quantitative real-time detection product for PDRN content detection has become an urgent need in product development research. Summary of the Invention
[0005] This invention addresses the shortcomings of existing methods for detecting PDRN content, such as high cost, low sensitivity, and long detection time. It provides primers, methods, kits, and applications for quantitative fluorescence detection of PDRN content, thereby solving the technical problems existing in the prior art as described above.
[0006] The technical solution provided by this invention is as follows:
[0007] One objective of this invention is to provide primers for quantitative fluorescence detection of PDRN content. The primers are selected from any one of primers ID1 to ID7. The upstream primer sequence of primer ID1 is shown in SEQ ID NO: 1, and the downstream primer sequence of primer ID1 is shown in SEQ ID NO: 2. The upstream primer sequence of primer ID2 is shown in SEQ ID NO: 3, and the downstream primer sequence of primer ID2 is shown in SEQ ID NO: 4. The upstream primer sequence of primer ID3 is shown in SEQ ID NO: 5, and the downstream primer sequence of primer ID3 is shown in SEQ ID NO: 6. The upstream primer sequence of primer ID4 is shown in SEQ ID NO: 7, and the downstream primer sequence of primer ID4 is shown in SEQ ID NO: 8. The upstream primer sequence of primer ID5 is shown in SEQ ID NO: 9, and the downstream primer sequence of primer ID5 is shown in SEQ ID NO: 10. The upstream primer sequence of primer ID6 is shown in SEQ ID NO: 11, and the downstream primer sequence of primer ID6 is shown in SEQ ID NO: 7. The sequence of the upstream primer of primer ID7 is shown in SEQ ID NO: 12; the sequence of the downstream primer of primer ID7 is shown in SEQ ID NO: 13; and the sequence of the downstream primer of primer ID7 is shown in SEQ ID NO: 14.
[0008] The second objective of this invention is to provide a method for quantitative fluorescence detection of PDRN content, using the aforementioned primers for quantitative fluorescence detection of PDRN content, and comprising the following steps:
[0009] Step A: Plotting the standard curve:
[0010] PDRN pure product was diluted to different concentrations, and then PDRN of different concentrations was used as templates. A reaction system including primers for quantitative PDRN content detection was used for quantitative PCR amplification. Ct values were detected and standard curves were plotted.
[0011] Step B: Quantitative Detection of PDRN Content:
[0012] DNA is extracted from the sample to be tested. Using the DNA from the sample as a template, a reaction system including primers for quantitative PCR amplification of PDRN content is used. The Ct value is detected, and the corresponding PDRN content is calculated based on the standard curve drawn in step A. Then, the concentration of nucleic acid in the sample to be tested is used for correction to reduce the error between different samples caused by DNA extraction.
[0013] Furthermore, in step A, the PDRN concentration is determined by measuring the absorbance value under ultraviolet light at a wavelength of 260 nm.
[0014] Furthermore, the reaction system of the primers for quantitative PCR detection of PDRN content includes: 10 μL of qPCR SYBR Green MasterMix, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, 7.2 μL of RNase-Free H2O, and 2 μL of template.
[0015] Furthermore, in step B, the method for extracting DNA from the sample to be tested is as follows: DNA extraction kit is used to quickly obtain the DNA components in the sample.
[0016] Furthermore, the DNA extraction kit employs phenol-chloroform organic solvent extraction, alcohol precipitation extraction, or silica gel column purification technology for extraction.
[0017] Furthermore, in step B, the relative content of PDRN among different samples can also be calculated using the ΔΔCt method.
[0018] A third objective of this invention is to provide a kit for quantitative fluorescence detection of PDRN content, comprising the aforementioned primers for quantitative fluorescence detection of PDRN content.
[0019] The fourth objective of this invention is to provide an application of the above-mentioned method for quantitative fluorescence detection of PDRN content in the in vitro and in vivo degradation and metabolism of PDRN.
[0020] The technical solution provided by this invention has the following advantages compared with the prior art:
[0021] (1) The primers of the present invention based on fluorescence quantitative detection of PDRN content have high specificity, making it easy to distinguish the host gene sequence in the experiment of determining its degradation and metabolism in animals. At the same time, the high abundance ensures that its degradation and metabolism in animals is traceable and accurately reflects its timeliness in degradation and metabolism.
[0022] (2) The method of the present invention based on fluorescence quantitative detection of PDRN content is simple and easy to implement. Whether it is DNA extraction or fluorescence quantitative detection, the operation is simple and the cost is low.
[0023] (3) The method for detecting PDRN content based on fluorescence quantitative PCR of the present invention has low sample requirements and high detection sensitivity. It can detect samples such as cells, tissues, blood, and saliva, and the content is <5*10. 6 Both cultured cell samples and 1-20 mg animal tissue samples can be used for detection. When using primers with high gene sequence abundance, the detection limit of PDRN content can be as low as 0.01 ng / mL. Attached Figure Description
[0024] Figure 1 The standard curve of PDRN using primer ID1;
[0025] Figure 2 This is a graph showing the in vitro degradation curve of PDRN.
[0026] Figure 3 This is a graph showing the change in the percentage of standardized relative content of each gene over time at different postoperative time points. Detailed Implementation
[0027] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.
[0028] Reagents and biological materials used in the specific embodiments:
[0029] PDRN comes from Ruijiming (Shandong) Biotechnology Co., Ltd.;
[0030] 0.9% sodium chloride injection was purchased from Shandong Kelun Pharmaceutical Co., Ltd.
[0031] Isoflurane for pets was purchased from Jiangsu Hengfengqiang Biotechnology Co., Ltd.
[0032] The FastPure Cell / Tissue DNA Isolation Mini Kit was purchased from Nanjing Novizan Biotechnology Co., Ltd.
[0033] Recombinant DNase I (RNase-free) and Hieff UNICON Universal Blue qPCR SYBR Green MasterMix were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.
[0034] The EDTA solution was purchased from Shanghai Beyotime Biotechnology Co., Ltd.
[0035] SD rats were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.
[0036] SPF rat and mouse growth and reproduction compound feed was purchased from Keao Xieli (Tianjin) Feed Co., Ltd.
[0037] The ordinary wood shavings bedding material was purchased from Beijing Keao Xieli Feed Co., Ltd.
[0038] Example 1: Construction of qPCR standard curve
[0039] 1. Sample preparation
[0040] Take 2 μL of 1% PDRN and dilute it 10 times with 18 μL of RNase-Free H2O, i.e., 1‰ PDRN. Take 6 μL of 1‰ PDRN and dilute it 4 times with 18 μL of RNase-Free H2O to obtain the highest concentration point of the standard curve ("4-fold dilution" point, i.e., 0.25‰ PDRN). Take 6 μL of 0.25‰ PDRN and dilute it 4 times again with 18 μL of RNase-Free H2O ("4-fold dilution" point). 2 (Multiple point), and then the sample was diluted 4 times in sequence using the same method. 3 4 4 ……4 13 times.
[0041] The standard curve samples were tested for PDRN content (total nucleic acid content) using Nanodrop. When the PDRN content was below 2 ng / μL, it was outside the detection range of Nanodrop. In this case, the PDRN content of the sample was calculated by dividing the concentration of the previous dilution factor by 4.
[0042] 2. Quantitative fluorescence analysis
[0043] Prepare the PCR reaction system by adding 10 μL of Hieff UNICON Universal Blue qPCR SYBR GreenMasterMix, 0.4 μL of 10 μM ID1 upstream primer, 0.4 μL of 10 μM ID1 downstream primer, 7.2 μL of RNase-Free H2O, and 2 μL of template to each tube. The template is diluted DNA template. The negative control group uses RNase-Free H2O instead of template. Each DNA template is tested in triplicate. Perform quantitative PCR according to the procedure shown in Table 1.
[0044] Table 1. Quantitative Real-Time PCR Reaction Procedure
[0045]
[0046] 3. Results of Standard Curve Plotting
[0047] The PDRN content and fluorescence quantification results of the standard curve samples are shown in Table 2. The PDRN content was logarithmed, and the logarithm of the PDRN content was plotted on the x-axis, with the Ct value of the fluorescence quantification on the y-axis to create the standard curve. Figure 1 As shown.
[0048] Table 2 shows the PDRN content and fluorescence quantitative results of the standard curve samples.
[0049]
[0050]
[0051] according to Figure 1 The amplification efficiency E of primer ID1 is calculated using the slope of the standard curve.
[0052] E=10 -1 / 斜率 -1 = 10 -1 / -3.36 -1 = 98.44%
[0053] The amplification efficiency of primer ID1 is between 90% and 110%, R 2 A value greater than 0.98 indicates that the method has high sensitivity, good primer specificity amplification, and is suitable for PDRN detection.
[0054] The amplification efficiency and characteristics of different primers under this method were examined using the same method. A standard curve was plotted and the following data were collected:
[0055] Table 3. Amplification efficiency and linearity of primers with different specificities
[0056] Serial Number upstream primer Downstream primer Amplification efficiency <![CDATA[Calibration curve linearity (R 2 )]]> ID1 SEQ ID No:1 SEQ ID No:2 98.4％ 0.998 ID2 SEQ ID No:3 SEQ ID No:4 100.7％ 0.993 ID3 SEQ ID No:5 SEQ ID No:6 104.9％ 0.998 ID4 SEQ ID No:7 SEQ ID No:8 105.3％ 0.996 ID5 SEQ ID No:9 SEQ ID No:10 103.2％ 0.998 ID6 SEQ ID No:11 SEQ ID No:12 97.8％ 0.994 ID7 SEQ ID No:13 SEQ ID No:14 97.6％ 0.996
[0057] Example 2: In vitro degradation of PDRN
[0058] 1. Sample preparation
[0059] Weigh 220.9 mg of PDRN dry powder and add 22.09 mL of 0.9% sodium chloride injection to dilute the sample to a concentration of 1% (10 mg / mL).
[0060] 2. In vitro degradation by DNase I (deoxyribonuclease I)
[0061] Take 200 μL of 1% PDRN, dilute it 4 times with 600 μL of 0.9% sodium chloride injection, and aliquot it into PCR tubes, 25.5 μL / tube, for a total of 16 tubes; 8 tubes contain 4.5 μL of 0.9% sodium chloride injection as the negative control group, and 8 tubes contain 3 μL of DNase I Reaction Buffer (10×) and 1.5 μL of Recombinant DNase I (RNase-free, 2U / μL) as the experimental group.
[0062] Sixteen tubes of PDRN were placed in a 37°C water bath. At 0, 0.5, 1, 2, 3, 5, 15, and 30 min, one tube each of the negative control group and the experimental group were removed. 3 μL of 50 mM EDTA solution was added and mixed well. The mixture was then inactivated at 65°C for 10 min.
[0063] 6. In vitro DNase I degradation results of PDRN samples
[0064] After PDRN samples were degraded by DNase I in vitro, fluorescence quantification was performed using primer ID1. The results are shown in Table 4. The PDRN content was calculated by substituting the Ct values into the standard curve. Figure 2 As shown.
[0065] Table 4. PDRN content and fluorescence quantitative results of in vitro DNase I-degraded samples.
[0066]
[0067] PDRN is rapidly and completely degraded in vitro under the action of DNase I.
[0068] Example 3: In vivo degradation of PDRN
[0069] 1. Preparation of experimental animals
[0070] Forty-eight SPF-grade 7-week-old SD rats were selected, half male and half female, with males weighing 290g–340g and females weighing 200g–240g. The females were non-pregnant and nulliparous. The SD rats were housed in a barrier environment with a temperature of 18–26℃, humidity of 40–70%, and light intensity ≥200Lux (12h / 12h light / dark alternation), with three rats per cage. After purchase, the rats underwent quarantine and acclimatization for at least 7 days, and only those that passed quarantine were used in the experiment.
[0071] 2. Sample injection
[0072] Eight groups of SD rats were used, with six rats in each group, half male and half female. Before the operation, the skin at the injection site on the back was prepared. After isoflurane anesthesia, the rats were placed prone on the operating table, and a 1% PDRN sample was injected into the intradermal layer of the rat's back. The preparation method of the 1% PDRN was the same as in Example 1.
[0073] 3. Sample DNA extraction
[0074] At each experimental endpoint (0, 2, 4, 8, 12, 24, 72, and 144 hours post-operation), blood was collected from SD rats and placed in EDTA anticoagulant tubes before euthanasia. The injection site tissue was exposed, and changes were visually examined. Any abnormalities observed (such as infection, hematoma, edema, etc.) were recorded. After the injection site was minced, total DNA was extracted from the skin tissue and anticoagulant blood samples using the FastPure Cell / Tissue DNA Isolation Mini Kit.
[0075] 4. Detection of gene content in samples
[0076] DNA samples were diluted 4-fold as templates for quantitative fluorescence detection, using the same method as step 4 in Example 1. The difference was that primer ID7 was used as an internal control for in vivo degradation, and primer ID4 content detection was added.
[0077] 5. Data Processing Methods
[0078] After quantitative fluorescence analysis, the relative gene content was calculated using the ΔΔCt method.
[0079] Primer ID7 is an internal reference gene, and its Ct value for quantitative quantification is represented by Ct(internal reference gene);
[0080] Primers ID1 and ID4 are the target gene, and their quantitative fluorescence Ct values are expressed as Ct(target gene);
[0081] The difference in Ct values between the target gene and the internal reference gene is ΔCt = Ct(target gene) - Ct(internal reference gene);
[0082] The sample taken at 0 hours post-PDRN injection site served as the control group, and its ΔCt value was ΔCt(control group).
[0083] The other samples are the experimental group, and their ΔCt value is ΔCt(experimental group);
[0084] The difference between the experimental group ΔCt and the control group ΔCt is ΔCt = ΔCt(experimental group) - ΔCt(control group), where ΔCt(control group) is the average value of 6 samples.
[0085] The relative content of the target gene RQ(target gene) = 2^(-△△Ct)*100%.
[0086] The relative content of the gene at 0 hours post-PDRN injection site was taken as 100%, and the relative content of the target gene in other samples was standardized. That is, for the same gene, the RQ (target gene) value of the sample was divided by the RQ (target gene) value of the sample at 0 hours post-PDRN injection site.
[0087] 6. Changes in the relative content of different genes at different PDRN injection sites
[0088] The relative contents of ID1 and ID4 over time were calculated using the ΔΔCt method, and the results are shown in Table 5 below.
[0089] Table 5. Standardized relative contents (%) of ID1 and ID4 at different postoperative time points.
[0090] Time (h) ID1 ID4 0 100.0±48.60 100.0±42.83 2 76.59±37.85 67.47±33.39 4 46.67±39.56 43.58±36.75 8 25.62±34.01 20.63±24.79 12 19.43±19.74 15.07±14.51 24 3.70±3.87 3.67±4.30 72 0.00±0.00 0.00±0.00 144 0.00±0.00 0.00±0.00
[0091] The levels of ID1 and ID4 genes in the skin at the PDRN injection site decreased rapidly within 8 hours post-procedure, decreasing by 74.38% and 79.37%, respectively. By 24 hours post-procedure, the levels of both ID1 and ID4 genes had decreased by more than 96%.
[0092] Based on the above results, Figure 3As shown, the content changes of ID1 and ID4 at the PDRN injection site show the same trend, both of which can represent the degradation of PDRN in SD rats. That is, it degrades rapidly within 8 hours after surgery, the content decreases by more than 96% by 24 hours after surgery, and is basically completely degraded by 72 hours after surgery.
[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A primer for quantitative real-time detection of PDRN content, characterized in that, The primers are selected from primer ID1 or ID4; the upstream primer sequence of primer ID1 is shown in SEQ ID NO: 1, and the downstream primer sequence of primer ID1 is shown in SEQ ID NO: 2; the upstream primer sequence of primer ID4 is shown in SEQ ID NO: 7, and the downstream primer sequence of primer ID4 is shown in SEQ ID NO:
8.
2. A method for quantitative detection of PDRN content based on fluorescence, characterized in that, The primers for detecting PDRN content based on fluorescence quantitative detection as described in claim 1 were used for detection.
3. The method for quantitative detection of PDRN content based on fluorescence according to claim 2, characterized in that, Includes the following steps: Step A: Plotting the standard curve: PDRN pure product was diluted to different concentrations, and then PDRN of different concentrations was used as templates. A reaction system including primers for quantitative PDRN content detection was used for quantitative PCR amplification. Ct values were detected and standard curves were plotted. Step B: Quantitative Detection of PDRN Content: DNA was extracted from the sample to be tested. Using the DNA from the sample as a template, quantitative real-time PCR amplification was performed using a reaction system that included primers for detecting PDRN content based on fluorescence quantitative PCR. The Ct value was detected, and the corresponding PDRN content was calculated based on the standard curve plotted in step A.
4. The method for quantitative detection of PDRN content based on fluorescence according to claim 3, characterized in that, In step A, the PDRN concentration was determined by measuring the absorbance under ultraviolet light at a wavelength of 260 nm.
5. The method for quantitative detection of PDRN content based on fluorescence according to claim 3, characterized in that, The reaction system of the primers for quantitative real-time detection of PDRN content includes: 10 μL of qPCR SYBR Green MasterMix, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, 7.2 μL of RNase-Free H2O, and 2 μL of template.
6. The method for quantitative detection of PDRN content based on fluorescence according to claim 3, characterized in that, In step B, the method for extracting DNA from the sample to be tested is as follows: DNA extraction kit is used to quickly obtain the DNA components in the sample.
7. The method for quantitative detection of PDRN content based on fluorescence according to claim 6, characterized in that, The DNA extraction kit uses phenol-chloroform organic solvent extraction, alcohol precipitation extraction, or silica gel column purification technology for extraction.
8. The method for quantitative detection of PDRN content based on fluorescence according to claim 3, characterized in that, In step B, the relative content of PDRN among different samples is calculated using the ΔΔCt method.
9. A reagent kit for quantitative detection of PDRN content based on fluorescence, characterized in that, It contains primers for quantitative fluorescence detection of PDRN content as described in claim 1.
10. The application of the method for quantitative fluorescence detection of PDRN content as described in any one of claims 2-8 in tracking the in vitro and in vivo degradation and metabolism of PDRN.