Primer pair and method for evaluating large yellow croaker cell senescence based on relative telomere length
By designing primer pairs targeting the actb2 gene of large yellow croaker and using real-time quantitative PCR technology, the problem of assessing the senescence status of large yellow croaker cells was solved, achieving efficient and low-consumption detection of relative telomere length, and supporting the assessment of cell health status.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-03-04
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies cannot establish an accurate, reliable, and convenient method for assessing relative telomere length in large yellow croaker, making it difficult to effectively monitor its cellular senescence state and affecting the yield and quality of cultured meat.
A primer pair for specifically amplifying the actb2 gene fragment of large yellow croaker was designed, and combined with real-time quantitative PCR technology, cell senescence was assessed by calculating the ratio of telomere length to the copy number of the internal reference single-copy gene. This included the use of forward and reverse primers, as well as specific PCR amplification and melting reaction conditions.
This method enables precise assessment of the aging state of large yellow croaker cells, reduces sample consumption, improves the specificity and repeatability of the test, and provides a reference indicator for cell health status.
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Figure CN122012737A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular genetics, specifically relating to a primer pair and method for assessing cell senescence in large yellow croaker based on relative telomere length. Background Technology
[0002] As an emerging technology, cultured meat has become an alternative to some traditional livestock meat production methods. Cultured meat is a technique that uses animal stem cells to differentiate into usable muscle tissue in vitro. Its core principle is to simulate tissue development and formation using cells with self-renewal and differentiation potential in a stable in vitro environment.
[0003] Large yellow croaker (Larimichthys crocea) is an important marine economic fish, and its cell culture method for meat production is relatively mature. Myoblasts account for approximately 60% of the natural flesh of large yellow croaker and are also a crucial factor in the culture of large yellow croaker meat. During long-term in vitro culture, myoblasts undergo cellular senescence, leading to changes in gene expression and metabolism, affecting their proliferation and differentiation capabilities, and consequently reducing their meat-forming ability, and even secreting substances that pose health risks. Therefore, monitoring the senescence status of myoblasts is crucial for maintaining the yield and quality control of cultured large yellow croaker meat.
[0004] Cellular senescence is closely related to telomere shortening, and telomere length is considered an important molecular marker of cellular senescence. Telomeres are specialized DNA repetitive sequences at the ends of chromosomes in eukaryotic cells. These repetitive sequences vary among species; in vertebrates, telomeres consist of a 6-base repetition sequence TTAGGG, which, when combined with binding proteins, forms a protein complex that effectively protects chromosome integrity during cell division. However, because ordinary DNA polymerases cannot replicate telomere structures at chromosome ends during chromosome replication, telomere length gradually shortens during cell proliferation and division. If cells suffer DNA damage or other abnormalities, telomere shortening accelerates. Ultimately, telomere shortening leads to abnormal growth conditions such as cellular senescence and growth arrest. As a marker of cellular senescence or death, telomere length plays a crucial role in the comprehensive assessment of overall biological aging and disease. Therefore, establishing accurate, reliable, and convenient methods for measuring telomere length is particularly urgent.
[0005] Quantitative real-time PCR (qPCR) is a commonly used method for detecting telomere length due to its high sensitivity, ease of operation, and intuitive results. The main principle of qPCR for telomere length detection is to collect DNA samples and amplify them with telomere-specific primers and single-copy gene primers, respectively, obtaining the telomere copy number (T) and single-copy gene copy number (S). The relative telomere length in the sample is estimated by calculating the T / S ratio. This method is not only simple to operate and quick, but also requires only 5-20 ng of DNA, making it suitable for large-scale rapid telomere length detection while simultaneously assessing cellular health and aging processes. Although telomere repeat sequences are identical in vertebrates, single-copy genes differ across species. Therefore, qPCR detection of telomere length requires identifying species-specific single-copy genes and constructing single-copy gene primers. Consequently, it is impossible to establish a universally applicable qPCR method for measuring telomere length to assess cellular aging and health. Summary of the Invention
[0006] The purpose of this invention is to overcome the shortcomings of the prior art and to provide a primer pair and method for assessing cell senescence in large yellow croaker based on relative telomere length.
[0007] The specific technical solution adopted in this invention is as follows:
[0008] In a first aspect, the present invention provides a primer pair for assessing cell senescence in large yellow croaker based on relative telomere length. This primer pair is used to specifically amplify the actb2 gene fragment and includes a forward primer as shown in SEQ ID No. 1 and a reverse primer as shown in SEQ ID No. 2.
[0009] Secondly, the present invention provides a method for assessing cellular senescence in large yellow croaker by calculating telomere length using the primer pairs described in the first aspect, the specific steps of which are as follows:
[0010] S1: Extract total DNA from large yellow croaker cells to obtain a DNA template;
[0011] S2: Using the first qPCR amplification system containing universal telomere primers, the DNA template obtained in step S1 was amplified by qPCR to obtain the telomere copy number Ct. tel ;
[0012] S3: Using a second qPCR amplification system containing the forward primer as described in SEQ ID No. 1 and the reverse primer as described in SEQ ID No. 2, perform qPCR amplification on the DNA template obtained in step S1 to obtain the copy number Ct of the internal reference single-copy gene. actb2 ;
[0013] S4: Based on telomere copy number Ct teland the copy number (Ct) of the internal reference single-copy gene actb2 Calculate the relative telomere length (T / S); assess the cellular senescence of large yellow croaker based on the relative telomere length (T / S).
[0014] Preferably, the DNA template in step S1 is extracted using the TRIzol lysis method or a kit.
[0015] As a preferred embodiment, the first qPCR amplification system used in step S2 includes qPCR premix, universal telomere primers, DNA template and ultrapure water;
[0016] The second PCR amplification system used in step S3 includes: qPCR premix, forward primer as described in SEQ ID No. 1, reverse primer as described in SEQ ID No. 2, DNA template, and ultrapure water.
[0017] Furthermore, the qPCR premix solution used was 2×TB Green Premix Ex Taq II.
[0018] Furthermore, in the first qPCR amplification system, the final concentration of the universal telomere primer is 0.2~0.4 μM, and the final concentration of DNA is 0.5~5 ng / μL.
[0019] Furthermore, in the second qPCR amplification system, the final concentration of the forward primer is 0.2~0.4 μM, the final concentration of the reverse primer is 0.2~0.4 μM, and the final concentration of the DNA template is 0.5~5 ng / μL.
[0020] Preferably, the qPCR amplification conditions in step S2 are as follows: first, pre-denaturation at 95℃ for 1 minute, followed by denaturation at 95℃ for 10 seconds, annealing at 55℃ for 30 seconds, and extension at 72℃ for 45 seconds, with the denaturation, annealing, and extension program repeated 35-40 times.
[0021] As a preferred embodiment, the qPCR amplification conditions in step S3 are as follows: first, pre-denaturation at 95°C for 1 minute, then denaturation at 95°C for 10 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 45 seconds, with the denaturation, annealing, and extension program cycled 35-40 times.
[0022] Preferably, the relative telomere length T / S = 2 -ΔCt ; ΔCt=Ct tel -Ct actb2 .
[0023] Compared with the prior art, the present invention has the following advantages:
[0024] (1) The present invention sets up primer pairs for the actb2 cell gene of large yellow croaker (Larimichthys crocea), which excludes the amplification of other species, has specificity, and has good reproducibility.
[0025] (2) The primer pair provided by the present invention can achieve accurate detection with a small amount of sample. Experimental verification shows that only 10 ng of genomic DNA is needed to obtain reliable relative telomere length data in large yellow croaker myoblasts, which significantly reduces the consumption of living tissue or precious samples.
[0026] (3) This invention can assess the degree of cell aging by detecting the relative telomere length of large yellow croaker cells, providing a reference indicator for cell health status and functional capacity. Attached Figure Description
[0027] Figure 1 The melting curve is shown in Example 1.
[0028] Figure 2 The amplification efficiency results are from Example 1;
[0029] Figure 3 Comparison curves of amplification of large yellow croaker DNA and mouse DNA in Example 2 using actb2 primers;
[0030] Figure 4 Comparison curves of amplification of large yellow croaker DNA and zebrafish DNA in Example 2 using actb2 primers;
[0031] Figure 5 Comparison curves of amplification of large yellow croaker DNA using actb2 primers and yeast DNA from Example 2;
[0032] Figure 6 Comparison curves of amplification of large yellow croaker DNA and human DNA in Example 2 using actb2 primers;
[0033] Figure 7 The relative telomere lengths of different samples in Example 3;
[0034] Figure 8 The results of β-galactosidase staining in the 30th generation large yellow croaker myoblasts in Example 4;
[0035] Figure 9 The results of β-galactosidase staining in the 60th generation large yellow croaker myoblasts in Example 4;
[0036] Figure 10 The results of β-galactosidase staining in the 90th generation large yellow croaker myoblasts in Example 4. Detailed Implementation
[0037] The present invention will be further described and illustrated below with reference to the accompanying drawings and specific embodiments. The technical features of each embodiment of the present invention can be combined accordingly, provided that there is no mutual conflict.
[0038] The following examples use actb2 primer pairs that specifically amplify the actb2 gene fragment, including the forward primer shown in SEQ ID No. 1 and the reverse primer shown in SEQ ID No. 2.
[0039] SEQ ID NO.1: 5'-TCGTGCGTGACATCAAGGAG-3'
[0040] SEQ ID NO. 2: 5'-GTTGGGGAACTTACCGAGGAA-3'.
[0041] Example 1
[0042] This embodiment provides a method for verifying the amplification effect of the actb2 primer pair, and the specific steps are as follows:
[0043] (1) Extraction of DNA from large yellow croaker cells using TRIzol lysis method
[0044] Cells were collected from a suspension of large yellow croaker myoblasts cultured to the 34th generation by centrifugation. Subsequently, 3 × 10⁶ cells were collected. 6 ~5×10 6 Add 1 mL of TRIzol lysis buffer to each cell, aspirate and mix well, then incubate at room temperature for 5 minutes. Next, add 200 μL of chloroform, mix well and let stand for 3 minutes, then centrifuge at 12000×g for 15 minutes at 4°C.
[0045] After centrifugation, remove the supernatant, add 0.3 mL of anhydrous ethanol, mix well, and let stand at room temperature for 3 minutes. Then, centrifuge at 2000×g for 5 minutes at 4°C and discard the supernatant. Next, add 1 mL of 0.1M sodium citrate containing 10% ethanol to wash the precipitate, let stand for 30 minutes, inverting and mixing occasionally, and then centrifuge at 2000×g for 5 minutes at 4°C. Discard the supernatant and repeat the washing once.
[0046] After repeated washing, the precipitate was washed with 75% ethanol and incubated at room temperature for 20 minutes, inverting and mixing occasionally. Then, it was centrifuged at 2000×g for 5 minutes at 4°C, and the supernatant was discarded. Finally, the DNA was allowed to air dry at room temperature for 10 minutes, and then dissolved in 200 μL ddH2O to obtain the DNA to be tested.
[0047] (2) PCR reaction amplification
[0048] 1) The concentration of extracted DNA was measured using Nanodrop, and 6 samples were obtained by serial dilution of 10-fold.
[0049] 2) The DNA obtained in the above steps was amplified by PCR using a first PCR amplification system containing telomerase. The first PCR amplification system contained: 10 μL of TB Green Premix Ex Taq (2x), 0.4 μL of telomerase, 1 μL of DNA template, and finally brought to a final volume of 20 μL with ddH2O. The concentration of the DNA template was 10 ng / μL.
[0050] The PCR amplification conditions were as follows: first, pre-denaturation at 95℃ for 1 minute, then denaturation at 95℃ for 10 seconds, annealing at 55℃ for 30 seconds, and extension at 72℃ for 45 seconds, with the denaturation, annealing, and extension program repeated 40 times.
[0051] 3) The PCR amplification product obtained in step 2) is subjected to a melting reaction under the following conditions: the reaction system is gradually heated from 65℃ to 95℃, and held at 95℃ for 10 seconds. During the heating process, the fluorescence signal is monitored in real time, and the fluorescence intensity at each temperature point is recorded. A melting curve is generated based on the changes in the fluorescence signal, as shown below. Figure 1 As shown. Amplification efficiency is as follows. Figure 2 As shown, the slope k of the fitted line is obtained after linear fitting.
[0052] according to Figure 1 The amplification product exhibits a single melting peak, indicating that the actb2 primers designed in this invention have good specificity for large yellow croaker DNA. Primer amplification efficiency calculation formula: E=10 -1 / k -1, according to Figure 2 The amplification efficiency of the actb2 primer pair used in this invention was calculated to be 95.13%, indicating that the primer pair has a good amplification effect.
[0053] Example 2
[0054] This embodiment provides a method for verifying the specificity of actb2 primer pairs, the specific steps of which are as follows:
[0055] (1) Extraction of different types of DNA by TRIzol lysis method
[0056] Centrifugation was used to collect cell suspensions from mice, zebrafish, yeast, and humans, serving as the control group. Centrifugation was used to collect cell suspensions from large yellow croaker myoblasts passaged to the 34th generation, serving as the experimental group. The TRIzol lysis method for DNA extraction followed the same procedure as step (1) in the example.
[0057] (2) PCR reaction amplification
[0058] The concentration of extracted DNA was measured using Nanodrop and diluted to 10 ng / μL with ddH2O.
[0059] The DNA template obtained in the above steps was amplified by PCR using a second PCR amplification system containing the forward primer as described in SEQ ID No. 1 and the reverse primer as described in SEQ ID No. 2. The second PCR amplification system contained: 10 μL of TB GreenPremix Ex Taq (2x), 0.4 μL of forward primer, 0.4 μL of reverse primer, 1 μL of DNA template, and finally brought to a final volume of 20 μL with ddH2O.
[0060] The PCR amplification conditions were as follows: first, pre-denaturation at 95℃ for 1 minute, then denaturation at 95℃ for 10 seconds, annealing at 55℃ for 30 seconds, and extension at 72℃ for 45 seconds, with the denaturation, annealing, and extension program repeated 40 times.
[0061] The obtained DNA amplification results are shown in the following figures. Figures 3-6 As shown in the figure, the actb2-specific primers designed in this invention cannot effectively amplify mouse, zebrafish, yeast, and human DNA, but they do have amplification specificity for large yellow croaker cell DNA.
[0062] Example 3
[0063] This embodiment provides a method for assessing cellular senescence in large yellow croaker based on real-time fluorescence quantitative relative telomere length detection. The specific steps are as follows:
[0064] (1) Collect nine cell samples from every 10 passages of large yellow croaker myoblasts from passage 10 to 90. Extract DNA template from the large yellow croaker myoblasts using the TRIzol lysis method, as described in step (1) of Example 1.
[0065] (2) PCR amplification reaction
[0066] 1) The DNA template obtained in the above steps was amplified by PCR using a first PCR amplification system containing telomerase. The first PCR amplification system contained: 10 μL of TB Green Premix Ex Taq (2x), 0.4 μL of telomerase, 1 μL of DNA template, and finally ddH2O to a final volume of 20 μL. The concentration of the DNA template was 10 ng / μL.
[0067] The PCR amplification conditions were as follows: first, pre-denaturation at 95℃ for 1 minute, followed by denaturation at 95℃ for 10 seconds, annealing at 55℃ for 30 seconds, and extension at 72℃ for 45 seconds. This denaturation, annealing, and extension program was repeated 40 times to obtain the telomere copy number Ct. tel .
[0068] 2) The DNA template obtained in the above steps was amplified by PCR using a second PCR amplification system containing the forward primer as described in SEQ ID No. 1 and the reverse primer as described in SEQ ID No. 2. The second PCR amplification system contained: 10 μL of TBGreen Premix Ex Taq (2x), 0.4 μL of forward primer, 0.4 μL of reverse primer, 1 μL of DNA template, and finally, ddH2O was added to a final volume of 20 μL. The concentration of the DNA template was 10 ng / μL.
[0069] The PCR amplification conditions were as follows: first, pre-denaturation at 95℃ for 1 minute, followed by denaturation at 95℃ for 10 seconds, annealing at 55℃ for 30 seconds, and extension at 72℃ for 45 seconds. This denaturation, annealing, and extension program was repeated 40 times to obtain the copy number Ct of the internal control single-copy gene. actb2 .
[0070] (3) Calculate the relative telomere length T / S using the following formula:
[0071] ΔCt=Ct tel -Ct actb2
[0072] T / S=2 -ΔCt
[0073] The calculated results are as follows Figure 7 As shown. According to Figure 7 It can be seen that after the resuscitation of large yellow croaker myoblasts, the length of the protelomere first shortened and then remained basically consistent after 60 passages in vitro. However, after more than 60 transfers, the relative telomere length of large yellow croaker myoblasts shortened, indicating that the relative telomere length of large yellow croaker myoblasts will shorten after long-term in vitro culture, which weakens their ability to protect chromosome structure and causes cell senescence.
[0074] Example 4
[0075] This embodiment uses β-galactosidase staining to characterize cellular senescence and demonstrate the relationship between relative telomere length and cellular senescence. The specific steps are as follows:
[0076] (1) Collect myoblast samples from the 30th, 60th and 90th generations of large yellow croaker for β-galactosidase staining. The cells were cultured in 6-well plates and the experiment was carried out.
[0077] (2) The specific steps of the β-galactosidase staining experiment are as follows:
[0078] 1) Remove the cell culture medium, wash once with PBS, add 1 mL of β-galactosidase staining and fixing solution, and fix at room temperature for 15 minutes.
[0079] 2) Remove the cell fixative and wash the cells three times with PBS for three minutes each time.
[0080] 3) Remove PBS and add 1 mL of staining working solution to each well. Each 1 mL staining working solution system contains: 10 μL of β-galactosidase staining solution A, 10 μL of β-galactosidase staining solution B, 930 μL of β-galactosidase staining solution C, and 50 μL of X-Gal solution.
[0081] 4) Incubate overnight at 37ºC.
[0082] 5) Observe cell staining under a regular optical microscope.
[0083] The staining results of cells from passages 30, 60, and 90 are as follows: Figure 8 , 9 As shown in Figure 10. According to Figure 8 , 9 It can be seen that no obvious blue staining was observed in the large yellow croaker myoblasts after staining during the early in vitro passage process, indicating that telomeres can effectively protect against damage to cell chromosome structure and thus delay cell senescence. According to... Figure 10 It was observed that after more than 90 passages in vitro, the cells showed obvious blue staining under a microscope, indicating senescence in the large yellow croaker myoblasts. This suggests that significantly shortened telomere length leads to loss of chromosome protection and cellular senescence. This demonstrates a clear positive correlation between telomere shortening and cellular senescence.
[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the invention. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all technical solutions obtained through equivalent substitution or transformation fall within the protection scope of the present invention.
Claims
1. A primer pair for assessing cellular senescence in large yellow croaker based on relative telomere length, characterized in that, The actb2 primer pair is used for specific amplification of the actb2 gene fragment, including the forward primer shown in SEQ ID No. 1 and the reverse primer shown in SEQ ID No.
2.
2. A method for assessing cellular senescence in large yellow croaker by calculating telomere length using the primer pair described in claim 1, characterized in that, The specific steps are as follows: S1: Extract total DNA from large yellow croaker cells to obtain a DNA template; S2: Using the first qPCR amplification system containing universal telomere primers, the DNA template obtained in step S1 was amplified by qPCR to obtain the telomere copy number Ct. tel ; S3: Using a second qPCR amplification system containing the forward primer as described in SEQ ID No. 1 and the reverse primer as described in SEQ ID No. 2, perform qPCR amplification on the DNA template obtained in step S1 to obtain the copy number Ct of the internal reference single-copy gene. actb2 ; S4: Based on telomere copy number Ct tel and the copy number (Ct) of the internal reference single-copy gene actb2 Calculate the relative telomere length (T / S); assess the cellular senescence of large yellow croaker based on the relative telomere length (T / S).
3. The method for assessing cellular senescence in large yellow croaker according to claim 1, characterized in that, In step S1, the DNA template is extracted using the TRIzol lysis method or a kit.
4. The method for assessing cellular senescence in large yellow croaker according to claim 1, characterized in that, The first qPCR amplification system used in step S2 includes qPCR premix, universal telomere primers, DNA template, and ultrapure water; The second PCR amplification system used in step S3 includes: qPCR premix, forward primer as described in SEQ ID No. 1, reverse primer as described in SEQ ID No. 2, DNA template, and ultrapure water.
5. The method for assessing cellular senescence in large yellow croaker according to claim 4, characterized in that, The qPCR premix used was 2×TB Green Premix Ex Taq II.
6. The method for assessing cellular senescence in large yellow croaker according to claim 4, characterized in that, In the first qPCR amplification system, the final concentration of the universal telomere primer is 0.2~0.4 μM, and the final concentration of the DNA template is 0.5~5 ng / μL.
7. The method for assessing cellular senescence in large yellow croaker according to claim 4, characterized in that, In the second qPCR amplification system, the final concentration of the forward primer is 0.2~0.4 μM, the final concentration of the reverse primer is 0.2~0.4 μM, and the final concentration of the DNA template is 0.5~5 ng / μL.
8. The method for assessing cellular senescence in large yellow croaker according to claim 1, characterized in that, The qPCR amplification conditions described in step S2 are as follows: first, pre-denaturation at 95℃ for 1 minute, then denaturation at 95℃ for 10 seconds, annealing at 55℃ for 30 seconds, and extension at 72℃ for 45 seconds, and the denaturation, annealing and extension program is cycled 35 to 40 times.
9. The method for assessing cellular senescence in large yellow croaker according to claim 1, characterized in that, The qPCR amplification conditions described in step S3 are as follows: first, pre-denaturation at 95℃ for 1 minute, then denaturation at 95℃ for 10 seconds, annealing at 55℃ for 30 seconds, and extension at 72℃ for 45 seconds, and the denaturation, annealing and extension program is cycled 35 to 40 times.
10. The method for assessing cellular senescence in large yellow croaker according to claim 1, characterized in that, The relative telomere length T / S=2 -ΔCt ; ΔCt=Ct tel -Ct actb2 .