CN112322710A - Telomere relative length detection kit - Google Patents
Telomere relative length detection kit Download PDFInfo
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Abstract
The invention relates to a telomere relative length detection kit. The kit comprises: telomere gene primer sequence shown in SEQ ID No. 1-2; the primer sequence of the internal reference gene is shown as SEQ ID NO. 3-4; the probe sequence is shown as SEQ ID NO. 5; internal reference gene: taking the multi-copy sequence as an internal reference gene sequence; and (3) standard substance: immortalized cell line DNA. The telomere length detection effect of the kit is stable, the raw materials are cheap and easy to obtain, and the experimental operation is simple and easy to implement.
Description
Technical Field
The invention belongs to the technical field of molecular biology and medical detection, and particularly relates to a telomere relative length detection kit.
Background
Telomeres are special end-capped structures at the end of eukaryotic chromosomes, and consist of TTAGGG repeats and a variety of proteins (e.g., refuelins and telomerase). Telomeres ensure chromosome stability and protect the ends from degradation and fusion with other chromosomes. Since DNA polymerases are unable to replicate DNA to the ends of linear chromosomes, they act as protections against the loss of important genetic information during DNA replication.
Eukaryotic telomeres typically terminate in 3' single-stranded DNA overhangs, which are critical for telomere maintenance and capping. This 3' single-stranded repeat DNA loops back and anneals to a double strand, forming a large physical loop structure called a telomere loop (T-loop). The T-loop may provide a general mechanism to mask telomere ends from cellular activity that may act on DNA ends and regulate lengthening and shortening of telomeres. In the T-loop structure, it is proposed that a 3' overhang intrudes into double-stranded telomeric DNA to form a D-loop (substitution loop) in addition to pairing with the C-strand and substituting the G-strand. Chain invasion occurs at a distance from the physical end of the telomere, thus resulting in a large T-loop structure.
Currently, the commonly used telomere length detection method mainly comprises: telomere end restriction fragment analysis (TRF), fluorescent quantitative PCR (qPCR), quantitative fluorescent in situ hybridization (Q-FISH), Flow fluorescent in situ hybridization (Flow-FISH), and the like. Wherein, the TRF method digests genome DNA by restriction endonuclease without cutting telomere fragment, and the digested product is used for detecting the length of the telomere by Southern blotting method. Although TRF analysis is considered the "gold standard" for TL measurement, the intensity of the telomeric smear can be measured, and thus generally determines the mean TL, various telomere sizes can be imaged on the Southern blot, but the shortest telomere cannot be displayed. Both Q-FISH and FLOW FISH are hybridized with cell suspension by adopting a fluorescence labeling probe (CCCTAA)3, and telomere length analysis is carried out by detecting telomere fluorescence signals. The FLOW-FISH technology is used for telomere fluorescence signal analysis through a FLOW cytometer, can accurately obtain the average telomere length of each cell, and has good sensitivity and repeatability. However, due to the restriction of probe hybridization, the Q-FISH method cannot detect a fluorescent signal at telomeres at chromosome ends where the telomere repeat is below the PNA probe hybridization threshold (so-called telomere-free ends). Another disadvantage of all Q-FISH techniques is that the probe may also bind to some Interstitial Telomeric Sequences (ITS) which consist of telomeric repeats in vertebrates far from the end of the chromosome, thus producing some false positive results. There is also a concern that some very bright Q-FISH signals may represent clusters of very close telomeres and it is unknown how they are reflected in the quantitative analysis.
The qPCR method has the advantages of less DNA consumption, simple operation, easy acquisition of instruments and equipment and the like, and is particularly suitable for detecting and analyzing the telomere length. The existing qPCR method amplifies telomere gene segments and reference gene segments by designing primers, and calculates the relative length of telomeres according to a T/S ratio. In the experiment, different personnel or different batches of reagents are used for operation, so that the batch difference of detection results is caused, and the continuous dynamic monitoring and accurate comparative analysis of the telomere length are difficult to realize.
Disclosure of Invention
The invention aims to solve the technical problem of providing a telomere relative length detection kit to overcome the defects of low accuracy of telomere length detection and the like in the prior art.
The invention provides a telomere relative length detection kit, which comprises:
the telomere gene primer sequence is shown as SEQ ID NO. 1-2:
a forward primer: GTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT the flow of the air in the air conditioner,
reverse primer: GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT, respectively;
the primer sequence of the internal reference gene is shown as SEQ ID NO.3-4, and the internal reference gene is YH-1:
a forward primer: CGCACAGAGTAGTAAG-GAAAGTGAAGTAGGCCGGGC of the formula (I),
reverse primer: GTGCTGGGATTACAGGCGTGAG, respectively;
the probe sequence is shown as SEQ ID NO. 5:
VIC-ATGGACAGTGAGATCTGTCCAT-BHQ1-CGCACAGAGTAGTAAG；
internal reference gene: taking the multi-copy sequence as an internal reference gene sequence;
and (3) standard substance: DNA from an immortalized cell line (e.g., 293T, Hela cells, etc.).
The telomere gene is detected by SYBRgreen, the reference gene is detected by a molecular beacon probe method, and the telomere gene and the reference gene are detected in the same tube.
Screening of the reference gene: a200 BP multicopy sequence is screened out in a human whole genome sequence through Blast to serve as an internal reference gene sequence.
The single copy sequence of the internal reference gene is shown as SEQ ID NO. 6:
GCCCAGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGGGCCTGGC。
the kit also comprises an internal quality control standard substance and a negative control, wherein the negative control is ultrapure water.
The kit also comprises a calculation system for calculating the T/S value of each detection sample.
The method for establishing the internal quality control system of the kit comprises the following steps:
(1) and (3) selecting the DNA of the immortalized cell line with the concentration of 15-20 ng/mu L (confirmed) as the standard DNA, completing the internal reference qPCR reaction and the telomere qPCR reaction, repeating the experiment at least three times, and recording the internal reference CT value and the telomere CT value in each experiment.
(2) Respectively calculating the delta CT value of the standard sample in each experiment;
(3) taking the average value of the delta CT values of all the standard sample as the delta CT value of the standard sample, namely the delta CT(Standard substance)；
(4) The Delta CT value of the sample is CT (telomere) -CT (internal reference), namely Delta CT(sample)；
(5) Calculating T/S2- (. DELTA.CT (sample) -. DELTA.CT (Standard))Wherein T is a sample to be detected, S is a standard substance, and T/S is the relative length ratio of telomeres of the sample to be detected and the standard substance.
The qPCR reaction system of the reference gene comprises:
2×SYBR Green I qPCR Mix 10μL；
0.1 mu L of forward primer of the reference gene;
1 mu L of internal reference gene reverse primer; wherein the concentrations of the forward primer and the reverse primer of the reference gene are both 10 mu mol/L;
50×ROX Reference DyeII 0.4μL；
1uL of Uniprimer probe with a probe concentration of 10 umol/L;
H2O 3.3μL；
4 μ L of genomic DNA, wherein the concentration of genomic DNA was 15 ng/. mu.L.
The qPCR reaction system of the telomere gene comprises:
2×SYBR Green I qPCR Mix 10μL；
0.1 mu L of telomere gene forward primer;
telomere gene reverse primer 0.1 uL; wherein the concentrations of the forward primer and the reverse primer of the telomere gene are both 10 mu mol/L;
50×ROX Reference DyeII 0.4μL；
1uL of Uniprimer probe with a probe concentration of 10 umol/L;
H2O 3.3μL；
4 μ L of genomic DNA, wherein the concentration of genomic DNA was 15 ng/. mu.L.
The qPCR reaction conditions of the reference gene and the telomere gene are as follows:
pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing at 50 ℃ for 1s, extension at 72 ℃ for 45s, and fluorescence collection at 50 ℃ annealing. The invention also provides application of the telomere relative length detection kit in biological age assessment.
Advantageous effects
(1) The invention adopts the calibrated T/S ratio as the telomere length evaluation standard, and can realize accurate detection and dynamic evaluation analysis of the relative length of the telomere.
(2) Compared with the traditional separate-tube detection scheme of the reference gene and the telomere gene, the scheme can eliminate the inter-pore difference interference caused by the detection of different genes and improve the accuracy of an experimental result; meanwhile, the multi-copy reference gene is used for replacing a single-copy reference gene, so that the copy number difference of the reference gene and the telomere gene can be reduced, the stability of a detection result is improved, and the detected result is more accurate.
(3) In the selection of the standard substance, the invention selects the DNA of the immortalized cell line as the standard substance, and the telomere length of the cell line is not shortened and stabilized along with the passage of cells.
(4) The telomere length detection method has the advantages of stable telomere length detection effect, cheap and easily-obtained raw materials, and simple and feasible experimental operation.
Drawings
FIG. 1 shows the results of stability tests of reference genes at different concentrations in example 1 of the present invention.
FIG. 2 shows the result of detecting the telomere length of DNA of immortalized cell lines and normal differentiated cells along with the passage of cells in example 1 of the present invention.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
This embodiment provides a kit for telomere length detection, including:
the telomere gene primer sequence is shown as SEQ ID NO. 1-2:
a forward primer: GTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT the flow of the air in the air conditioner,
reverse primer: GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT, respectively;
the primer sequence of the internal reference gene is shown as SEQ ID NO.3-4, and the internal reference gene is YH-1:
a forward primer: CGCACAGAGTAGTAAG-GAAAGTGAAGTAGGCCGGGC of the formula (I),
reverse primer: GTGCTGGGATTACAGGCGTGAG, respectively;
the probe sequence is shown as SEQ ID NO. 5:
VIC-ATGGACAGTGAGATCTGTCCAT-BHQ1-CGCACAGAGTAGTAAG；
establishing a two-color fluorescent PCR system:
telomere gene is detected by SYBRgreen, reference gene is detected by molecular beacon probe method, and the two kinds of gene detection are carried out in the same tube.
Screening of internal reference genes: screening about 200BP multicopy sequence as reference gene sequence in human whole genome sequence by Blast, wherein the gene single copy sequence is shown as SEQ ID NO. 6:
GCCCAGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGGGCCTGGC；
screening of standard products: immortalized cell line (293T) DNA was selected as a standard for this kit.
The kit of the embodiment is used for detecting the telomere length of 1 human leukocyte and calculating the biological age:
(1) after the whole blood genome DNA is extracted, the concentration is detected by a spectrophotometer, and the Sample 1 concentration is 51.14 ng/mu L; diluting the sample DNA concentration to 15 ng/uL;
(2) preparing qPCR reaction liquid, and respectively carrying out internal reference qPCR reaction and telomere qPCR reaction on the sample DNA and 3 quality control products, wherein each reaction is repeated for 3 times;
wherein, the qPCR reaction system of the reference gene comprises: 2 × SYBR Green I qPCR Mi × 10 μ L; 0.1 mu L of forward primer of the reference gene; 1 mu L of internal reference gene reverse primer; wherein the concentrations of the internal reference gene forward primer and the internal reference gene reverse primer are both 10 mu mol/L; 50 XROX Reference DyeII 0.4. mu.L; 1uL of Uniprimer probe with a probe concentration of 10 umol/L; h2O3.3 μ L; 4 μ L of genomic DNA;
the qPCR reaction conditions for the reference gene were as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing at 50 ℃ for 1s, and extension at 72 ℃ for 45 s. Fluorescence was collected upon annealing at 50 ℃.
The qPCR reaction system of telomere gene comprises: 2 × SYBR Green I qPCR Mix10 μ L; 0.1 mu L of telomere gene forward primer; telomere gene reverse primer 0.1 uL; wherein the concentrations of the telomere gene forward primer and the telomere gene reverse primer are both 10 mu mol/L; 50 XROX Reference DyeII 0.4. mu.L; 1uL of Uniprimer probe with a probe concentration of 10 umol/L; h2O3.3 μ L; 4 μ L of genomic DNA;
qPCR reaction conditions for telomere genes were as follows: pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing at 50 ℃ for 1s, and extension at 72 ℃ for 45 s. Fluorescence was collected upon annealing at 50 ℃. And recording the internal reference CT value and the telomere CT value in each experiment.
(3) And respectively calculating the delta CT value of the standard sample in three repeated experiments.
(4) Taking the average value of the delta CT values of the three standard sample samples as the delta CT value of the standard sample, namely the delta CT(Standard substance)The Δ CT values of the standard samples are shown in Table 2.
(5) The Delta CT value of the sample is CT (telomere) -CT (internal reference), and the average value is taken, namely the Delta CT(sample)The sample Δ CT values are shown in Table 1.
(6) Calculating T/S2- (. DELTA.CT (sample) -. DELTA.CT (Standard))Wherein T is the sample to be tested, S is the standard, and T/S is the relative telomere length ratio of the sample to be tested and the standard, and the result is shown in Table 3.
TABLE 1
TABLE 2
TABLE 3
15 blood samples are selected, and the concentration of 20ng/ul and the concentration of 10ng/ul are respectively detected according to the steps (4 ul is loaded, so the sample content is 80ng and 40 ng);
the experimental process comprises the following steps: with different reference genes: YH-1 and 36B4 to determine the stability of the detection result.
Theoretically, the detection results of the same blood sample should be relatively consistent under different sample concentrations, which proves that the detection system has strong stability. The invention finds that when 36B4 is used as an internal reference, the detection result can obviously fluctuate under different concentrations, namely delta CT inconsistency. And YH-1, the detection results are highly consistent, which proves that the stability of the detection results is good, and the detection results are not influenced by the sample concentration. As shown in FIG. 1, YH-1 as an internal reference result is more stable by comparing the stability of the detection results of different template concentrations.
Hela cells and 293T cells are the same kind of cells and are immortalized cells. Namely, the telomere length of the cells does not change along with the cell passage, which is reflected in that the delta CT does not change obviously. The telomere length of Hela cells is about 6.7. 293T cells were around 7.5. The telomere length of 10 generation, 20 generation, 30 generation and 40 generation cells is detected according to the steps, and no obvious change is found, so that the invention selects 2937T cells as a standard substance, Huvec cells are normal differentiated cells and serve as a control group, and the delta CT changes. As shown in fig. 2, Hela and 293T are immortalized cell lines, and Huvec is a normal differentiated cell (control group), and it can be seen that the telomere length of DNA (Δ CT) of the immortalized cell line does not change with cell passage.
The above description is only for the specific embodiments of the present disclosure, but the scope of the present disclosure is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present disclosure, and all the changes or substitutions should be covered within the scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be subject to the protection scope of the claims.
SEQUENCE LISTING
<110> university of east China
<120> telomere relative length detection kit
<130> 1
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 33
<212> DNA
<213> Artificial sequence
<400> 1
gtttgggttt gggtttgggt ttgggtttgg gtt 33
<210> 2
<211> 39
<212> DNA
<213> Artificial sequence
<400> 2
ggcttgcctt acccttaccc ttacccttac ccttaccct 39
<210> 3
<211> 36
<212> DNA
<213> Artificial sequence
<400> 3
cgcacagagt agtaaggaaa gtgaagtagg ccgggc 36
<210> 4
<211> 22
<212> DNA
<213> Artificial sequence
<400> 4
gtgctgggat tacaggcgtg ag 22
<210> 5
<211> 38
<212> DNA
<213> Artificial sequence
<400> 5
atggacagtg agatctgtcc atcgcacaga gtagtaag 38
<210> 6
<211> 141
<212> DNA
<213> Artificial sequence
<400> 6
gcccagctaa ttttttgtat ttttagtaga gacggggttt caccgtgtta gccaggatgg 60
tctcgatctc ctgacctcgt gatccacccg cctcggcctc ccaaagtgct gggattacag 120
gcgtgagcca ccgggcctgg c 141
Claims (10)
1. A relative telomere length detection kit, which is characterized by comprising:
telomere gene primer sequence shown in SEQ ID No. 1-2;
the primer sequence of the internal reference gene is shown as SEQ ID NO.3-4, and the internal reference gene is YH-1;
the probe sequence is shown as SEQ ID NO. 5;
internal reference gene: taking the multi-copy sequence as an internal reference gene sequence;
and (3) standard substance: immortalized cell line DNA.
2. The kit of claim 1, wherein the telomere gene is detected by SYBRgreen, the reference gene is detected by molecular beacon probe method, and the telomere gene and reference gene detection are performed in the same tube.
3. The kit of claim 1, wherein the screening for the reference gene comprises: a200 BP multicopy sequence is screened out in a human whole genome sequence through Blast to serve as an internal reference gene sequence.
4. The kit of claim 1, wherein the single copy sequence of the reference gene is shown as SEQ ID No. 6; the kit also comprises an internal quality control standard substance and a negative control, wherein the negative control is ultrapure water.
5. The kit of claim 1, further comprising a computing system for computing the T/S value for each test sample.
6. The kit according to claim 5, wherein the method for establishing the quality control system in the kit is as follows:
(1) selecting immortalized cell line DNA with the concentration of 15-20 ng/mu L as standard DNA, completing internal reference qPCR reaction and telomere qPCR reaction, repeating the experiment at least three times, and recording an internal reference CT value and a telomere CT value in each experiment;
(2) respectively calculating the delta CT value of the standard sample in each experiment;
(3) taking the average value of the delta CT values of all the standard sample as the delta CT value of the standard sample, namely the delta CT(Standard substance)；
(4) The Delta CT value of the sample is CT (telomere) -CT (internal reference), namely Delta CT(sample)；
(5) Calculating T/S2- (. DELTA.CT (sample) -. DELTA.CT (Standard))Wherein T is a sample to be detected, S is a standard substance, and T/S is the relative length ratio of telomeres of the sample to be detected and the standard substance.
7. The kit of claim 6, wherein the qPCR reaction system of the reference gene comprises:
2×SYBR Green I qPCR Mix 10μL；
0.1 mu L of forward primer of the reference gene;
1 mu L of internal reference gene reverse primer; wherein the concentrations of the forward primer and the reverse primer of the reference gene are both 10 mu mol/L; 50 XROX Reference DyeII 0.4. mu.L;
1uL of Uniprimer probe with a probe concentration of 10 umol/L;
H2O 3.3μL；
4 μ L of genomic DNA, wherein the concentration of genomic DNA was 15 ng/. mu.L.
8. The kit of claim 6, wherein the qPCR reaction system for telomere genes comprises:
2×SYBR Green I qPCR Mix 10μL；
0.1 mu L of telomere gene forward primer;
telomere gene reverse primer 0.1 uL; wherein the concentrations of the forward primer and the reverse primer of the telomere gene are both 10 mu mol/L;
50×ROX Reference DyeII 0.4μL；
1uL of Uniprimer probe with a probe concentration of 10 umol/L;
H2O 3.3μL；
4 μ L of genomic DNA, wherein the concentration of genomic DNA was 15 ng/. mu.L.
9. The kit according to claim 6, wherein the qPCR reaction conditions of the reference gene and the telomere gene are as follows:
pre-denaturation at 95 ℃ for 2 min; denaturation at 95 ℃ for 15s, annealing at 50 ℃ for 1s, extension at 72 ℃ for 45s, and fluorescence collection at 50 ℃ annealing.
10. Use of the kit of claim 1 for biological age assessment.
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Cited By (2)
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| CN113957136A (en) * | 2021-10-27 | 2022-01-21 | 中科解码(北京)生物技术有限公司 | Telomere length detection primer composition, kit and application thereof |
| CN114262737A (en) * | 2021-12-29 | 2022-04-01 | 成都中创清科医学检验所有限公司 | Primer combination for measuring length of relative telomere, kit and detection method thereof |
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| CN113957136A (en) * | 2021-10-27 | 2022-01-21 | 中科解码(北京)生物技术有限公司 | Telomere length detection primer composition, kit and application thereof |
| CN114262737A (en) * | 2021-12-29 | 2022-04-01 | 成都中创清科医学检验所有限公司 | Primer combination for measuring length of relative telomere, kit and detection method thereof |
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