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CN-122012737-A - Primer pair for evaluating cell senescence of large yellow croaker based on relative telomere length and method thereof

CN122012737ACN 122012737 ACN122012737 ACN 122012737ACN-122012737-A

Abstract

The invention discloses a primer pair for evaluating cell senescence of large yellow croaker based on relative telomere length and a method thereof, belonging to the field of molecular genetics. The specific method comprises the steps of 1) taking DNA of a large yellow croaker cell genome, 2) carrying out qPCR amplification on the DNA obtained in the step S1 by using a first qPCR amplification system containing a telomere universal primer to obtain a telomere copy number Ct tel , 3) carrying out qPCR amplification on the obtained DNA by using a second qPCR amplification system containing a forward primer as shown in SEQ ID No.1 and a reverse primer as shown in SEQ ID No.2 to obtain an internal reference single copy gene copy number Ct actb2 , and 4) calculating relative telomere length T/S=2 ‑ΔCt ,ΔCt=Ct tel -Ct actb2 . The primer for detecting the length of the opposite telomeres actb provided by the invention can specifically amplify the DNA of the yellow croaker, and can realize accurate detection by adopting a trace sample.

Inventors

  • HU BAOLAN
  • ZHAO JUNXIAN
  • LIU ZISHU
  • NIU RUIHAO
  • CHEN QIHE
  • LIU DONGHONG

Assignees

  • 浙江大学

Dates

Publication Date
20260512
Application Date
20260304

Claims (10)

  1. 1. A primer pair for evaluating the aging of cells of large yellow croaker based on relative telomere length, which is characterized in that actb primer pair is used for specifically amplifying actb gene fragment and comprises a forward primer shown as SEQ ID No.1 and a reverse primer shown as SEQ ID No. 2.
  2. 2. A method for evaluating cell senescence in large yellow croaker by calculating telomere length using the primer set of claim 1, comprising the steps of: S1, extracting total DNA of large yellow croaker cells to obtain a DNA template; s2, performing qPCR amplification on the DNA template obtained in the step S1 by using a first qPCR amplification system containing a telomere universal primer to obtain a telomere copy number Ct tel ; s3, performing qPCR amplification on the DNA template obtained in the step S1 by using a second qPCR amplification system comprising a forward primer shown in SEQ ID No.1 and a reverse primer shown in SEQ ID No.2 to obtain the copy number Ct actb2 of the reference single-copy gene; S4, calculating the relative telomere length T/S based on the telomere copy number Ct tel and the internal reference single copy gene copy number Ct actb2 , and evaluating the aging condition of the large yellow croaker cells based on the relative telomere length T/S.
  3. 3. The method for evaluating the aging of cells of large yellow croaker as recited in claim 1, wherein the DNA template is extracted in step S1 using TRIzol cleavage method or kit.
  4. 4. The method for evaluating the aging of cells of large yellow croaker of claim 1, wherein the first qPCR amplification system employed in step S2 comprises qPCR premix, telomere universal primer, DNA template, and ultrapure water; The second PCR amplification system used in step S3 comprises qPCR premix, forward primer shown as SEQ ID No.1, reverse primer shown as SEQ ID No.2, DNA template and ultrapure water.
  5. 5. The method of claim 4, wherein the qPCR premix is 2 x TB Green Premix Ex Taq II.
  6. 6. The method according to claim 4, wherein the final concentration of the telomere universal primer in the first qPCR amplification system is 0.2-0.4. Mu.M, and the final concentration of the DNA template is 0.5-5 ng/. Mu.L.
  7. 7. The method according to claim 4, wherein the final concentration of the forward primer in the second qPCR amplification system is 0.2 to 0.4. Mu.M, the final concentration of the reverse primer is 0.2 to 0.4. Mu.M, and the final concentration of the DNA template is 0.5 to 5 ng/. Mu.L.
  8. 8. The method for evaluating the aging of cells of large yellow croaker as recited in claim 1, wherein the qPCR amplification conditions in step S2 are that the pre-denaturation is performed for 1 minute at 95 ℃, the denaturation is performed for 10 seconds at 95 ℃, the annealing is performed for 30 seconds at 55 ℃, the elongation is performed for 45 seconds at 72 ℃, and the denaturation, annealing and elongation processes are cycled 35 to 40 times.
  9. 9. The method for evaluating the aging of cells of large yellow croaker as recited in claim 1, wherein the qPCR amplification conditions in step S3 are that the pre-denaturation is performed for 1 minute at 95 ℃, the denaturation is performed for 10 seconds at 95 ℃, the annealing is performed for 30 seconds at 55 ℃, the elongation is performed for 45 seconds at 72 ℃, and the denaturation, annealing and elongation processes are cycled 35 to 40 times.
  10. 10. The method of assessing the aging of cells of large yellow croaker of claim 1, wherein the relative telomere length T/S = 2 -ΔCt ;ΔCt=Ct tel -Ct actb2 .

Description

Primer pair for evaluating cell senescence of large yellow croaker based on relative telomere length and method thereof Technical Field The invention belongs to the field of molecular genetics, and particularly relates to a primer pair for evaluating cell senescence of large yellow croaker based on relative telomere length and a method thereof. Background Cell culture meat is now becoming an emerging technology, which is an alternative to some of the traditional animal meat industry. Cell culture meat is a technology for forming usable muscle tissue by in vitro culture and differentiation of animal stem cells, and the core principle is to simulate tissue development and formation in a stable in vitro environment by using cells with self-renewal and differentiation potential. As an important marine economic fish, the cell culture meat-forming method of large yellow croaker (LARIMICHTHYS CROCEA) is mature. The muscle cell number in the natural fish meat of the large yellow croaker accounts for about 60 percent, and is also an important factor of the cell culture meat of the large yellow croaker. The myoblast cells can generate cell aging phenomenon in the long-term in-vitro culture process, induce the change of gene expression and substance metabolism, influence proliferation and differentiation capacity of the myoblast cells, further lead to the reduction of the meat forming capacity of the cells, and even secrete health risk substances. Monitoring myoblast senescence status is therefore critical for maintenance of yield and quality control of cultured rhein meat. Cell senescence is closely related to shortening of telomeres, and telomere length is considered as an important molecular marker of the degree of cell senescence. Telomeres (telomere) are special DNA repeated sequences at the tail end of chromosome in eukaryotic cells, repeated sequences of different species are not consistent, wherein telomeres of vertebrates are 6-base repeated sequences TTAGGG, and protein complexes formed by combining the telomeres and binding proteins can effectively protect the integrity of chromosome of cells in the process of division. However, since the conventional DNA polymerase cannot replicate the telomere structure of the chromosome end in chromosome replication, the telomere length gradually shortens in the course of cell proliferation and division. If the cells are damaged by DNA or otherwise abnormal, telomeres will shorten rapidly. Shortening of telomeres eventually leads to abnormal growth conditions such as cell senescence and growth arrest. As one of the markers of cell senescence or death, telomere length plays an important role in the overall evaluation of biological senescence and disease. Therefore, it is particularly urgent to establish an accurate, reliable and convenient telomere length measurement method. The real-time quantitative PCR (qPCR) technology has the characteristics of high sensitivity, simple operation, visual result and the like, and is one of the common methods for detecting the telomere length. The main principle of qPCR method for detecting telomere length is to collect DNA sample and telomere specific primer, and amplified telomere copy number (T) and single copy gene copy number (S) obtained by amplifying the primers, and estimate the relative telomere length in the sample by calculating T/S ratio. The method is simple to operate, short in time, and suitable for large-scale rapid detection of telomere length, and the required DNA amount is only 5-20 ng, and the health condition and aging process of cells are evaluated. Although telomere repeats are identical in vertebrates, single copy genes are not identical in different species, and qPCR methods are required to determine specific single copy genes of different species and construct single copy gene primers for telomere length, so a method for qPCR measurement of telomere length applicable to all organisms cannot be established to assess cell senescence and health. Disclosure of Invention The invention aims to solve the defects of the prior art and provides a primer pair for evaluating the cell senescence of large yellow croaker based on relative telomere length and a method thereof. The specific technical scheme adopted by the invention is as follows: In a first aspect, the invention provides a primer pair for evaluating the aging of cells of large yellow croaker based on relative telomere length, which is used for specifically amplifying actb gene fragment, comprising a forward primer as shown in SEQ ID No.1 and a reverse primer as shown in SEQ ID No. 2. In a second aspect, the invention provides a method for evaluating the aging of cells of large yellow croaker by calculating the telomere length by using the primer pair in the first aspect, which comprises the following specific steps: S1, extracting total DNA of large yellow croaker cells to obtain a DNA template; s2, performing qPCR amplification on the DNA template obtained in