A telomere relative length detection kit

Abstract

The present invention relates to a kit for detecting relative telomere length. The kit comprises: telomere gene primer sequences as shown in SEQ ID NOs. 1-2; internal reference gene primer sequences as shown in SEQ ID NOs. 3-4; a probe sequence as shown in SEQ ID NO. 5; an internal reference gene: a multi-copy sequence; and a standard: DNA from an immortalized cell line. The kit is stable for telomere length detection, uses inexpensive and readily available raw materials, and is simple and easy to perform.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology and medical detection, and particularly relates to a telomere relative length detection kit. Background Art

[0002] Telomeres are specialized end-capping structures at the ends of eukaryotic chromosomes, composed of the repeating sequence TTAGGG and various proteins, such as shelterin and telomerase. Telomeres ensure chromosome stability and protect the ends from degradation and fusion with other chromosomes. Because DNA polymerase cannot replicate DNA to the ends of linear chromosomes, they prevent the loss of important genetic information during DNA replication.

[0003] Eukaryotic telomeres typically terminate with a 3' single-stranded DNA overhang, which is crucial for telomere maintenance and capping. This 3' single-stranded DNA repeat loops back and anneals to a double strand, forming a large physical loop structure called the telomeric loop (T-loop). The T-loop may provide a general mechanism to shield the telomere ends from cellular activities that can act on the DNA ends and regulate telomere lengthening and shortening. In the T-loop structure, the 3' overhang is proposed to invade the double-stranded telomeric DNA to form a D-loop (displacement loop), based on pairing with the C-strand and displacing the G-strand. Strand invasion occurs at a certain distance from the physical end of the telomere, thus resulting in a large T-loop structure.

[0004] Currently, commonly used methods for telomere length detection include: telomere terminal restriction fragment analysis (TRF), quantitative fluorescence PCR (qPCR), quantitative fluorescence in situ hybridization (Q-FISH), and flow cytometry fluorescence in situ hybridization (Flow-FISH). The TRF method uses restriction endonuclease digestion of genomic DNA without cleaving telomere fragments. The digestion product is then used to measure telomere length using Southern blotting. Although TRF analysis is considered the "gold standard" for TL measurement, it measures the intensity of telomere smears, which generally determines the average TL and can image various telomere sizes on Southern blots, but the shortest telomeres are not visible. Both Q-FISH and FLOW FISH use a fluorescently labeled probe (CCCTAA)3 to hybridize with cell suspensions and analyze telomere length by detecting telomere fluorescence signals. FLOW-FISH analyzes telomere fluorescence signals using flow cytometry, accurately determining the average telomere length for each cell and exhibiting good sensitivity and reproducibility. However, due to limitations in probe hybridization, the Q-FISH method cannot detect fluorescent signals at telomeres at the ends of chromosomes where the telomere repeats are below the hybridization threshold of the PNA probe (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 far from the ends of chromosomes in vertebrates, thereby producing a certain amount of false positive results. There is also concern that some very bright Q-FISH signals may represent clusters of several telomeres in close proximity, and it is unknown how they will be reflected in quantitative analysis.

[0005] qPCR methods are particularly well-suited for telomere length analysis due to their advantages, including low DNA usage, simple operation, and readily available instrumentation. Existing qPCR methods employ primer design to amplify telomere gene fragments and reference gene fragments, calculating relative telomere length using the T / S ratio. However, due to the use of different personnel or different batches of reagents during the experiment, batch-to-batch variability in test results can be a challenge, making continuous dynamic monitoring and accurate comparative analysis of telomere length difficult. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a telomere relative length detection kit to overcome the defects of the prior art in that the accuracy of telomere length detection is not high.

[0007] The present invention provides a telomere relative length detection kit, the kit comprising:

[0008] Telomere gene primer sequences are shown in SEQ ID NO.1-2:

[0009] Forward primer: GTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT,

[0010] Reverse primer: GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT;

[0011] The internal reference gene primer sequence is shown in SEQ ID NO.3-4, and the internal reference gene is YH-1:

[0012] Forward primer: CGCACAGAGTAGTAAG-GAAAGTGAAGTAGGCCGGGC,

[0013] Reverse primer: GTGCTGGGATTACAGGCGTGAG;

[0014] The probe sequence is shown in SEQ ID NO.5:

[0015] VIC-ATGGACAGTGAGATCTGTCCAT-BHQ1-CGCACAGAGTAGTAAG;

[0016] Internal reference gene: multiple copies of the sequence are used as the internal reference gene sequence;

[0017] Standard: DNA from immortalized cell lines (e.g. 293T, HeLa cells, etc.).

[0018] The telomere gene is detected by SYBRgreen, and the internal reference gene is detected by a molecular beacon probe method. The telomere gene and the internal reference gene are detected in the same tube.

[0019] Screening of the internal reference gene: 200 BP multi-copy sequences were screened from the human whole genome sequence by Blast to serve as the internal reference gene sequence.

[0020] The single copy sequence of the internal reference gene is shown in SEQ ID NO.6:

[0021] GCCCAGCTAATTTTTTGTATTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGGGCCTGGC.

[0022] The kit also includes an internal quality control standard and a negative control, wherein the negative control is ultrapure water.

[0023] The kit also includes a calculation system for calculating the T / S value of each test sample.

[0024] The method for establishing the internal quality control system of the kit is as follows:

[0025] (1) Select immortalized cell line DNA with a concentration of 15-20 ng / μL (confirmed) as the standard DNA, complete the internal reference qPCR reaction and telomere qPCR reaction, repeat the experiment at least three times, and record the internal reference CT value and telomere CT value in each experiment.

[0026] (2) Calculate the ΔCT value of the standard sample in each experiment;

[0027] (3) Take the average of the △CT values ​​of all standard samples as the △CT value of the standard sample, that is, △CT (标准品) ;

[0028] (4) The ΔCT value of the sample is CT(telomere)-CT(internal reference), that is, ΔCT (样品) ;

[0029] (5) Calculate T / S = 2 -(△CT(样品)-△CT(标准品)) , where 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 to the standard.

[0030] The qPCR reaction system of the internal reference gene comprises:

[0031] 2×SYBR Green I qPCR Mix 10μL;

[0032] Internal reference gene forward primer 0.1 μL;

[0033] 1 μL of internal reference gene reverse primer; wherein the concentrations of the forward primer and reverse primer of the internal reference gene are both 10 μmol / L;

[0034] 50×ROX Reference DyeII 0.4μL;

[0035] Uniprimer probe 1uL, probe concentration 10umol / L;

[0036] H2O 3.3 μL;

[0037] 4 μL of genomic DNA, where the concentration of genomic DNA is 15 ng / μL.

[0038] The qPCR reaction system of the telomere gene comprises:

[0039] 2×SYBR Green I qPCR Mix 10μL;

[0040] 0.1 μL of telomere gene forward primer;

[0041] 0.1 μL of telomere gene reverse primer; wherein the concentrations of the telomere gene forward primer and reverse primer are both 10 μmol / L;

[0042] 50×ROX Reference DyeII 0.4μL;

[0043] Uniprimer probe 1uL, probe concentration 10umol / L;

[0044] H2O 3.3 μL;

[0045] 4 μL of genomic DNA, where the concentration of genomic DNA is 15 ng / μL.

[0046] The qPCR reaction conditions for the internal reference gene and telomere gene are as follows:

[0047] Pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 50°C for 1 s, extension at 72°C for 45 s, and fluorescence collection during annealing at 50°C.

[0048] The present invention also provides an application of a telomere relative length detection kit in biological age evaluation.

[0049] Beneficial effects

[0050] (1) The present invention adopts the calibrated T / S ratio as the telomere length evaluation standard, which can realize the accurate detection and dynamic evaluation analysis of the relative telomere length.

[0051] (2) In the present invention, two genes are detected in the same tube. Compared with the previous scheme of separate detection of internal reference genes and telomere genes, this scheme can eliminate the interference between wells caused by different gene detection and improve the accuracy of experimental results. At the same time, the use of multi-copy internal reference genes instead of single-copy internal reference genes can reduce the copy number difference between the internal reference gene and the telomere gene, improve the stability of the detection results, and make the measured results more accurate.

[0052] (3) In the selection of standard products, the present invention selected the DNA of immortalized cell lines as standard products. The telomere length of the cell lines does not shorten with cell passage and is stable.

[0053] (4) The present invention has a stable telomere length detection effect, the raw materials are cheap and easily available, and the experimental operation is simple and easy. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 These are the stability test results of the internal reference gene at different concentrations in Example 1 of the present invention.

[0055] Figure 2 These are the test results of DNA telomere length of the immortalized cell line and normal differentiated cells during cell passage in Example 1 of the present invention. DETAILED DESCRIPTION

[0056] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0057] Example 1

[0058] This embodiment provides a kit for telomere length detection, comprising:

[0059] Telomere gene primer sequences are shown in SEQ ID NO.1-2:

[0060] Forward primer: GTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT,

[0061] Reverse primer: GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT;

[0062] The internal reference gene primer sequence is shown in SEQ ID NO.3-4, and the internal reference gene is YH-1:

[0063] Forward primer: CGCACAGAGTAGTAAG-GAAAGTGAAGTAGGCCGGGC,

[0064] Reverse primer: GTGCTGGGATTACAGGCGTGAG;

[0065] The probe sequence is shown in SEQ ID NO.5:

[0066] VIC-ATGGACAGTGAGATCTGTCCAT-BHQ1-CGCACAGAGTAGTAAG;

[0067] Establishment of dual-color fluorescence PCR system:

[0068] Telomere genes were detected by SYBRgreen, and internal reference genes were detected by molecular beacon probe method. Both gene detections were performed in the same tube.

[0069] Screening of internal reference genes: Using Blast, a multi-copy sequence of about 200 bp was screened from the human genome sequence as the internal reference gene sequence. The single-copy sequence of this gene is shown in SEQ ID NO.6:

[0070] GCCCAGCTAATTTTTTGTATTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCACCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGGGCCTGGC;

[0071] Screening of standard products: Immortalized cell line (293T) DNA was selected as the standard product of this kit.

[0072] The kit of this example was used to detect telomere length of human leukocytes and calculate biological age:

[0073] (1) After whole blood genomic DNA was extracted, the concentration was measured using a spectrophotometer. The concentration of Sample 1 was 51.14 ng / μL. The sample DNA concentration was diluted to 15 ng / μL.

[0074] (2) Prepare qPCR reaction solution, perform internal reference qPCR reaction and telomere qPCR reaction with sample DNA and three quality control products, and perform three replicates for each reaction;

[0075] The qPCR reaction system for the internal reference gene contains: 2×SYBR Green I qPCR Mix × 10 μL; internal reference gene forward primer 0.1 μL; internal reference gene reverse primer 1 μL (the concentration of both internal reference gene forward primer and internal reference gene reverse primer is 10 μmol / L); 50×ROX Reference DyeII 0.4 μL; Uniprimer probe 1 μL, probe concentration is 10 μmol / L; H2O 3.3 μL; genomic DNA 4 μL.

[0076] The qPCR reaction conditions for the internal reference gene were as follows: pre-denaturation at 95°C for 2 min, denaturation at 95°C for 15 s, annealing at 50°C for 1 s, and extension at 72°C for 45 s. Fluorescence was collected during annealing at 50°C.

[0077] The telomeric gene qPCR reaction system includes: 2×SYBR Green I qPCR Mix 10 μL; telomeric gene forward primer 0.1 μL; telomeric gene reverse primer 0.1 μL (the concentration of both telomeric gene forward primer and telomeric gene reverse primer is 10 μmol / L); 50×ROX Reference Dye II 0.4 μL; Uniprimer probe 1 μL (the probe concentration is 10 μmol / L); H2O 3.3 μL; genomic DNA 4 μL.

[0078] The qPCR reaction conditions for telomere genes were as follows: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 15 seconds, annealing at 50°C for 1 second, and extension at 72°C for 45 seconds. Fluorescence was collected during the annealing period at 50°C. The internal reference CT value and the telomere CT value were recorded for each experiment.

[0079] (3) Calculate the △CT value of the standard sample in three repeated experiments.

[0080] (4) Take the average of the three △CT values ​​of the standard sample as the △CT value of the standard sample, that is, △CT (标准品) The △CT values ​​of the standard samples are shown in Table 2.

[0081] (5) The ΔCT value of the sample is CT(telomere)-CT(internal reference), and the average value is taken, that is, ΔCT (样品) The △CT values ​​of the samples are shown in Table 1.

[0082] (6) Calculate T / S = 2 -(△CT(样品)-△CT(标准品)) , where 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 to the standard. The results are shown in Table 3.

[0083] Table 1

[0084]

[0085]

[0086] Table 2

[0087]

[0088] Table 3

[0089]

[0090] Fifteen blood samples were selected and tested at concentrations of 20 ng / ul and 10 ng / ul according to the above steps (4 ul was loaded, so the sample content was 80 ng and 40 ng respectively);

[0091] Experimental process: Different internal reference genes: YH-1 and 36B4 were used to determine the stability of the test results.

[0092] Theoretically, the test results of the same blood sample at different sample concentrations should be relatively consistent, proving that the detection system is highly stable. The present invention found that when 36B4 was used as an internal reference, the test results at different concentrations would fluctuate significantly, i.e., ΔCT was inconsistent. However, when YH-1 was used, the test results were highly consistent, proving that the test results were stable and not affected by sample concentration. Figure 1 As shown in the figure, by comparing the stability of the detection results at different template concentrations, it can be seen that the results of YH-1 as an internal reference are more stable.

[0093] Hela cells and 293T cells are the same type of cells, both of which are immortalized cells. That is, the telomere length of the cells does not change with cell passage, which is reflected in the fact that △CT does not change significantly. The telomere length of Hela cells is about 6.7. The telomere length of 293T cells is about 7.5. The telomere lengths of 10th, 20th, 30th and 40th generation cells were tested according to the above steps, and it was found that there was indeed no significant change. Therefore, the present invention selected 2937T cells as the standard, and Huvec cells are normal differentiated cells as the control group, so △CT changes. Figure 2 It can be seen that Hela and 293T are immortalized cell lines, and Huvec is a normal differentiated cell (control group). It can be seen that the DNA telomere length (△CT) of the immortalized cell line does not change with cell passage.

[0094] The above description is merely a specific embodiment of the present disclosure, but the scope of protection of the present disclosure is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this disclosure should be included in the scope of protection of the present disclosure. Therefore, the scope of protection of the present disclosure should be based on the scope of protection of the claims. SEQUENCE LISTING <110> Donghua University <120> A 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<00002​​​​​​​​​​​​​​​​​​​​

Claims

1. A telomere relative length detection kit, characterized in that: The kit comprises: Telomere gene primer sequences, as shown in SEQ ID NO. 1-2; The primer sequences of the internal reference gene are shown in SEQ ID NO.3-4; The probe sequence is shown in SEQ ID NO.5; Internal reference gene: multiple copies of the sequence are used as the internal reference gene sequence; Standard: DNA from immortalized cell lines; The internal reference gene is detected by a molecular beacon probe method, and the telomere gene and the internal reference gene are detected in the same tube.

2. The kit according to claim 1, wherein The method for establishing the internal quality control system of the kit is as follows: (1) Select immortalized cell line DNA at a concentration of 15-20 ng / μL as standard DNA, complete internal reference qPCR reaction and telomere qPCR reaction, repeat the experiment at least three times, and record the internal reference CT value and telomere CT value in each experiment; (2) Calculate the ΔCT value of the standard sample in each experiment; (3) Take the average of the △CT values ​​of all standard samples as the △CT value of the standard sample, that is, △CT (标准品) ; (4) The ΔCT value of the sample is CT(telomere)-CT(internal reference), that is, ΔCT (样品) ; (5) Calculate T / S = 2 -(△CT( 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 to the standard.

3. The kit according to claim 2, wherein The qPCR reaction conditions for the internal reference gene and telomere gene are as follows: Pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 50°C for 1 s, extension at 72°C for 45 s, and fluorescence collection during annealing at 50°C.

4. Use of the kit according to claim 1 in biological age assessment.

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