Primer composition and kit for detecting human cell telomerase activity

Abstract

The invention discloses a primer composition and a kit for detecting telomerase activity of human cells. The primer composition for detecting PCR (Polymerase Chain Reaction) comprises the following base sequences: tele-F (SEQ ID NO.1) and tele-R (SEQ ID NO.2), and the base sequence of a starting primer for telomerase reaction: tele-TS (SEQ ID NO.3). The kit comprises (1) telomerase cell lysis buffer; (2) telomerase reaction liquid; (3) a telomerase activity detection RT-PCR reaction solution; and (4) RT-PCR (Reverse Transcription-Polymerase Chain Reaction) reaction primers tele-F and tele-R, and a telomerase reaction starting primer tele-TS. The detection kit disclosed by the invention is high in sensitivity, good in repeatability and simple and convenient to operate; and the detection result determination method is simple. A powerful technical support is provided for rapidly detecting the activity of the human cell telomerase.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biology detection, and in particular to a primer composition and a kit for detecting telomerase activity of human cells. Background Art

[0002] Since the 21st century, the research and development and clinical application of cell drugs have received great attention at home and abroad. Human stem cells, somatic cells and immune cells have strong biological and clinical medical effects. Human cell drugs have good therapeutic effects in the treatment of cancer, organ damage and immune system diseases. Because cell drugs have biological activity, their safety has also attracted great attention. Cell tumorigenicity is one of the important indicators for evaluating their safety.

[0003] Telomeres are special protein-DNA complex structures at the ends of eukaryotic chromosomes. Telomere sequences of human cells are composed of multiple sets of the same sequence "5'-TTAGGG-3'". Telomeres gradually shorten during normal cell division. When they reach a critical length, cells stop dividing and trigger apoptosis.

[0004] Telomerase is a ribonucleoproteinase composed of RNA and protein. Telomerase can replicate and synthesize telomere sequences using its own RNA as a template. Telomerase activity is present in actively proliferating cells, but not in normal mature somatic cells. It is also found that the vast majority of tumor cells are telomerase-positive, while the positive rate in normal tissues is very low. Therefore, telomerase can be used as a broad tumor marker, and telomerase activity has also become an important indicator of cell tumorigenicity.

[0005] In recent years, with the launch of a large number of human cell products, the tumorigenicity of the products has also become an important quality control item.

[0006] At present, there is no standard kit for detecting telomerase activity in human cells. Commonly used detection kits include direct detection kits and indirect detection kits. The principle of the direct detection kit is to amplify and detect the telomere repeat DNA sequence. It is mainly based on the improved method of TRAP (Telomeric repeat amplification protocol, TRAP). Its kit also needs to be subjected to enzyme-linked immunosorbent assay (ELISA) after PCR operation, which takes a long time to detect and is complicated to operate. The principle of the indirect detection kit is that the expression of the telomerase catalytic subunit TERT is positively correlated with the level of telomerase activity. Telomerase activity can be indirectly detected by detecting the mRNA expression of TERT. The method has the disadvantage that mRNA needs to be extracted and reverse transcribed before amplification detection. Finally, the amplification product needs to be detected by agarose gel electrophoresis and the results need to be analyzed by gel image. The detection time is long and the operation is not simple. It is not suitable for the production and quality inspection of cell drugs.

[0007] The above detection kits have various defects, and there is an urgent need for an accurate, rapid, specific and convenient detection kit to solve the problem of detecting telomerase activity in human cells during the production and quality inspection of cell drugs. Summary of the invention

[0008] The purpose of the present invention is to find a primer combination and a kit for detecting telomerase activity in human cells, based on a direct detection method without the need for enzyme-linked immunosorbent assay (ELISA), and a kit for rapidly detecting telomerase activity in human cells using RT-PCR.

[0009] The present invention is implemented in the following way: using telomerase cell lysis solution to lyse cells to obtain high-concentration telomerase, using telomerase reaction solution and telomerase reaction initiation primer tele-TS SEQ ID NO.3 to carry out telomere extension reaction, using telomerase reaction product and telomerase activity detection PCR reaction solution, PCR reaction primer combination tele-F SEQ ID NO.1 and PCR reaction primer combination tele-R SEQ ID NO.2 to carry out RT-PCR, detecting the expression amount of cell telomeres, and determining the telomerase activity according to the Ct value of RT-PCR.

[0010] A primer combination for detecting telomerase activity in human cells, wherein the primer combination for detecting PCR reaction includes the following base sequence:

[0011] tele-F: 5'-GGCAATCCGTACGAGCAGAGTTAAG-3'SEQ ID NO.1

[0012] tele-R: 5'-TTAAGCGCGGCTTACCCTTACCCTTACCCTAACCGGCC-3' SEQ ID NO. 2.

[0013] The base sequence of the initiating primer for telomerase reaction:

[0014] tele-TS: 5'-GGCAATCCGTACGAGCAGAGTTAAG-3'SEQ ID NO.3.

[0015] A kit for detecting telomerase activity in human cells, comprising the following components:

[0016] 1) Telomerase cell lysate;

[0017] 2) Telomerase reaction solution;

[0018] 3) RT-PCR reaction solution for telomerase activity detection;

[0019] 4) The above-mentioned RT-PCR reaction primers tele-F and tele-R and the above-mentioned telomerase reaction initiation primer tele-TS.

[0020] Furthermore, the kit for detecting telomerase activity in human cells comprises the following separately packaged components:

[0021] Component A, for performing cell lysis, including telomerase cell lysis solution;

[0022] Component B, used for telomerase reaction, including telomerase reaction solution, 100 mM dNTP, 0.01 mM telomerase reaction initiation primer tele-TS base sequence SEQ ID NO.3;

[0023] Component C is used for telomerase-catalyzed extension reaction, including telomerase activity detection PCR reaction solution, PCR reaction primer combination 0.01mM tele-F base sequence SEQ ID NO.1, 0.01mM tele-R base sequence SEQ ID NO.2.

[0024] The telomerase cell lysis solution contains: 0.2% of 1.5M Tris-HCl at pH 8.8, 1.6mM MgCl2·6H2O, 1.1mM EDTA, 1% NP-40, 0.8mM sodium deoxycholate, 10% glycerol, 43mM NaCl, 5mM β-mercaptoethanol, 65mM CHAPS, and the remainder is DEPC water, and the percentages are volume percentages.

[0025] The telomerase reaction solution contains: 12% by volume of 1.5M Tris-HCl with a pH of 8.8, 67mM KCl, 14mM MgCl2·6H2O, 10mM EGTA, and the remainder is DEPC water.

[0026] The telomerase activity detection PCR reaction solution contains: 0.01% PCR-grade SYBR Green I 10000×, 1.3% 1.5M Tris-HCl at pH 8.8, 1.3mM EDTA, 0.14M KCl, 9% glycerol, 26mM MgCl2·6H2O, 1M betaine, 2% formamide, 129mM DTT, 5% DMSO, 39mM ammonium sulfate, 0.1% 2.5mM dNTP, 5U / μl hot start enzyme, the remainder is sterile water, and the percentages are volume percentages.

[0027] The advantages and technical effects of the present invention are as follows: Different from the traditional telomerase activity detection, the kit of the present invention detects the telomerase activity of multiple generations and batches of Hela cells and MRC-5 cells by using PCR to obtain the Ct values ​​of a large number of samples. After repeated experiments, according to the characteristics of the telomerase activity of Hela cells and MRC-5 cells and the experimental results, it is decided to use Hela cells as a positive control and MRC-5 cells as a negative control, and the judgment standard of the telomerase activity of human cells in the present invention is obtained based on statistical software. The old Trap method finally requires agarose gel electrophoresis detection of the amplified product and gel image analysis of the result, which takes a long time to detect and is not easy to operate, and is not suitable for the production and quality inspection of cell drugs. The kit of the present invention can intuitively judge the strength of telomerase activity only by the Ct value. Compared with the same type of kits in the market (Telomerase Activity Quantification qPCR Assay Kit manufacturer: ScienCell article number: 8928), the telomerase reaction time of the kit of the present invention is 1 hour, and the Telomerase Activity Quantification qPCR Assay Kit is 3 hours, and the sensitivity of PCR detection of telomerase expression is higher than that of the same products in the market. The kit of the present invention is accurate, fast, highly sensitive, reproducible, easy to operate, and the method for determining the test results is simple. It provides a new detection technology for mass production and later quality control of cell drug products. It can be used for the detection of telomerase activity of human cells in cell drug production and quality inspection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 RT-PCR amplification curves for telomerase activity detection in Hela cells, HuH-7 (human liver cancer cells), MRC-5 cells and blank control.

[0029] Figure 2 RT-PCR amplification curves for telomerase activity detection in Hela cells, hUC-MSC cells, MRC-5 cells and blank control.

[0030] Figure 3 The RT-PCR amplification curve of the control test of simultaneously detecting the telomerase activity of the same number of Hela cells using the kit of the present invention and the kit of the same method in the market (Telomerase Activity Quantification qPCR Assay Kit manufacturer: ScienCell item number: 8928). DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods involved in the following examples are generally conventional conditions unless otherwise specified, and the materials and reagents used in the following examples are commercially available unless otherwise specified. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0032] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. In the examples, cell counting was performed using a cell counter. The data were statistically analyzed using SPSS 22.0, and the measurement data were expressed as mean ± standard deviation. The t-test was used to compare the differences between the two, and P < 0.05 was a significant difference.

[0033] DPBS: Dulbecco phosphate-buffered saline, Hyclone, Catalog No.: SH30028.02. DMSO: Dimethyl sulfoxide, origen, Catalog No.: CP-70. Tris-HCl: (1.5M PH 8.8), Solebao, Catalog No.: T1010. MgCl2·6H2O: Solebao, Catalog No.: M8161. EDTA: Ethylene Diamine Tetraacetic Acid, Solebao, Catalog No.: E8040. NP-40: Nonidet P-40, Solebao, Catalog No.: N8031. Sodium deoxycholate: Solebao, Catalog No.: D8331. Glycerol: Jiahe Biotechnology Co., Ltd. NaCl: Tianjin Fengchuan Chemical Reagent Technology Co., Ltd. β-Mercaptoethanol, Beijing Bailingwei Technology Co., Ltd., Catalog No.: 249096. CHAPS: 3-[(3-Cholanidopropyl)dimethylammonio]-1-pro panesulfonate, Solebao, Catalog No.: IC0290. DEPC water: diethylpyrocarbonate water, Solebao, Catalog No.: R1600. KCl, Tianjin Fengchuan Chemical Reagent Technology Co., Ltd. EGTA: Ethylene Glycol Tetraacetic Acid, Solebao, Catalog No.: E8050. dNTP (100mM): Solebao, Catalog No.: PC2300. SYBR Green I 10000× (PCR grade): Solebao, Catalog No.: SR4110. Betaine: Solebao, Catalog No.: B8230. Formamide: Sigma, Catalog No.: F9037-100ml. BSA: Albumin from bovine serum, Solebao, Catalog No.: A8020. DTT: Dithiothreitol, Solebold, Catalog No.: D8220. Ammonium sulfate: Solebold, Catalog No.: A8821. dNTP (2.5mM): Solebold, Catalog No.: PC2100. Hot start enzyme: Solebold, Catalog No.: PC1010. PMSF: phenylmethanesulfonyl fluoride, Solebold, Catalog No.: P0100.

[0034] Telomerase cell lysis solution: contains 0.2% (volume percentage) Tris-HCl (1.5M pH 8.8), 1.6mM MgCl2·6H2O, 1.1mM EDTA, 1% (volume percentage) NP-40, 0.8mM sodium deoxycholate, 10% (volume percentage) glycerol, 43mM NaCl, 5mM β-mercaptoethanol, 65mM CHAPS, and the balance is DEPC water.

[0035] Telomerase reaction solution: contains 12% (volume percentage) Tris-HCl (1.5M pH 8.8), 67mM KCl, 14mM MgCl2·6H2O, 10mM EGTA, and the balance is DEPC water.

[0036] Telomerase activity detection PCR reaction solution: containing 0.01% (volume percentage) SYBR Green I 10000× (PCR grade), 1.3% (volume percentage) Tris-HCl (1.5M pH 8.8), 1.3mM EDTA, 0.14M KCl, 9% (volume percentage) glycerol, 26mM MgCl2·6H2O, 1M betaine, 2% (volume percentage) formamide, 129mM DTT, 5% (volume percentage) DMSO, 39mM ammonium sulfate, 0.1% (volume percentage) dNTP (2.5mM), 5U / μl hot start enzyme, and the balance is sterile water.

[0037] The PCR reaction primer combination is tele-F, and the base sequence of SEQ ID NO.1 is: 5'-GGCAATCCGTACGAGCAGAGTTAAG-3'.

[0038] The primer combination for the PCR reaction is tele-R, and the base sequence of SEQ ID NO.2 is: 5'-TTAAGCGCGGCTTACCCTTACCCTTACCCTAACCGGCC-3'.

[0039] The telomerase reaction initiation primer tele-TS, SEQ ID NO.3 base sequence is:

[0040] 5'-GGCAATCCGTACGAGCAGAGTTAAG-3'.

[0041] The component A specifically includes: telomerase cell lysate.

[0042] The component B specifically includes telomerase reaction solution, 100mM dNTP (100mM dATP, 100mM dGTP, 100mM dCTP, 100mM dTTP), and 0.01mM telomerase reaction initiation primer tele-TS SEQ ID NO.3.

[0043] The component C specifically includes a telomerase activity detection PCR reaction solution, a PCR reaction primer combination of 0.01 mM tele-F SEQ ID NO.1, and 0.01 mM tele-R SEQ ID NO.2.

[0044] Figure 1The RT-PCR amplification curves for telomerase activity detection of Hela cells, MRC-5 cells, and hUC-MSC cells with the same cell number, where red is the amplification curve of the positive control Hela cells, blue is the amplification curve of the negative control MRC-5 cells, black is the amplification curve of the hUC-MSC cells to be tested, and green is the amplification curve of the blank control sterile water.

[0045] Figure 2 The RT-PCR amplification curves for telomerase activity detection of Hela cells, MRC-5 cells, and HuH-7 cells with the same cell number, where red is the amplification curve of the positive control Hela cells, blue is the amplification curve of the negative control MRC-5 cells, black is the amplification curve of the test sample HuH-7 cells, and green is the amplification curve of the blank control sterile water.

[0046] Figure 3 The RT-PCR amplification curves of Hela cell telomerase activity detected with the same number of cells using different kits are shown in red, and the amplification curve of Hela cells using the kit of the present invention is shown in blue, while the amplification curve of Hela cells using Telomerase Activity Quantification qPCR Assay Kit (manufacturer: ScienCell, catalog number: 8928) is shown in blue.

[0047] Example 1 Detection of telomerase activity in human umbilical cord mesenchymal stem cells (hUC-MSC)

[0048] The present invention uses human umbilical cord mesenchymal stem cells as the object, obtains high-concentration telomerase by lysing the cells, uses a telomerase reaction solution, 100mM dNTP (100mM dATP, 100mM dGTP, 100mM dCTP, 100mM dTTP), and 0.01mM telomerase reaction initiation primer tele-TS SEQ ID NO.3 to perform telomere extension reaction, performs RT-PCR on the telomerase reaction product and a telomerase activity detection PCR reaction solution, a PCR reaction primer combination tele-F SEQ ID NO.1, and a PCR reaction primer combination tele-R SEQID NO.2, detects the telomere expression amount of the human umbilical cord mesenchymal stem cells, and determines the telomerase activity according to the Ct value of the RT-PCR.

[0049] 1. Cell Lysis

[0050] Fresh human umbilical cord mesenchymal stem cells, Hela cells and MRC-5 cells were washed with DPBS buffer, and then about 1.0×10 6Cells were centrifuged at 1500 rpm and 4°C for 5 minutes, and the supernatant was discarded. 100 μl of telomerase cell lysis solution, 2 μl of PMSF (0.1 M) and 2 μl of β-mercaptoethanol were added to the cell pellet, and the mixture was thoroughly mixed by pipetting, and then placed at 4°C for 30 minutes. The supernatant was collected by centrifugation at 12000 rpm and 4°C for 20 minutes, which was the telomerase cell lysis product.

[0051] 2. Telomere Extension

[0052] Prepare telomerase reaction system, add 13μl DEPC water, 5μl telomerase reaction solution, 2μl cell telomerase lysate, 1μl dATP (100mM), 1μl dGTP (100mM), 1μl dCTP (100mM), 1μl dTTP (100mM), 1μl telomerase reaction start primer tele-ts (0.01mM) into a 1.5ml EP tube in sequence, centrifuge at 12000rpm at room temperature for 1 minute, place the telomerase reaction system in a dry heat machine, at 37℃ for 1h, then 85℃ for 10 minutes. This mixed solution is the telomerase reaction product.

[0053] Telomerase activity detection

[0054] Prepare the PCR reaction system for detecting telomerase activity in human cells. Add 10 μl of telomerase activity detection PCR reaction solution, 7 μl of sterile water, 1 μl of primer tele-F (0.01 mM), 1 μl of primer tele-R (0.01 mM), 1 μl of the sample to be tested / positive control / negative control / blank control to the eight-tube connection in sequence; the PCR reaction conditions are: 95°C for 5 min for 1 cycle, 95°C for 30 s, 54°C for 30 s, and 72°C for 30 s for 40 cycles. The sample to be tested is 1×10 6 The telomerase reaction product of human umbilical cord mesenchymal stem cells to be tested, and the positive control was 1×10 6 Telomerase reaction product of Hela cells, negative control was 1×10 6 The telomerase reaction product of MRC-5 cells was prepared, and the blank control was sterile water. Three parallels were set for each cell type and blank control.

[0055] 4. Determination of telomerase activity

[0056] Table 1 1×10 6 The arithmetic mean of the Ct values ​​of the RT-PCR experiment of telomerase activity in hUC-MSC cells

[0057]

[0058] According to the Ct value of RT-PCR experimental results, the telomerase activity of hUC-MSC cells was weakly positive.

[0059] Example 2 Detection of telomerase activity in human hepatoma cells (HuH-7)

[0060] The present invention takes human liver cancer cells as the research object and uses a kit for detecting the telomerase activity of human-derived cells.

[0061] 1. Cell Lysis

[0062] Fresh human liver cancer cell samples, Hela cells and MRC-5 cells were washed with DPBS buffer, and then about 1.0×10 6 Cells were centrifuged at 1500 rpm and 4°C for 5 minutes, and the supernatant was discarded. 100 μl of component A, 2 μl of PMSF (0.1 M) and 2 μl of β-mercaptoethanol were added to the cell pellet, and the mixture was thoroughly mixed by pipetting, and then placed at 4°C for 30 minutes. The supernatant was collected by centrifugation at 12000 rpm and 4°C for 20 minutes, which was the telomerase cell lysis product.

[0063] 2. Telomere Extension

[0064] Prepare the telomerase reaction system. Add 13 μl DEPC water, 10 μl component B and 2 μl cell telomerase lysis product to a 1.5 ml EP tube in sequence. Centrifuge at 12000 rpm at room temperature for 1 minute. Place the telomerase reaction system in a dry heat oven at 37°C for 1 hour and 85°C for 10 minutes. This mixture is the telomerase reaction product.

[0065] Telomerase activity detection

[0066] Prepare the PCR reaction system for detecting telomerase activity in human cells. Add 12 μl of component C, 7 μl of sterile water, and 1 μl of the sample to be tested / positive control / negative control / blank control to the eight-tube connection in sequence. The PCR reaction conditions are: 95°C for 5 min for 1 cycle, 95°C for 30 s, 54°C for 30 s, and 72°C for 30 s for 40 cycles. The sample to be tested is 1×10 6 The telomerase reaction product of human liver cancer cells to be tested, and the positive control is 1×10 6 Telomerase reaction product of Hela cells, negative control was 1×10 6 The blank control was sterile water, and three replicates were set for each sample and control.

[0067] 4. Determination of telomerase activity

[0068] Table 2 1×10 6 The arithmetic mean of the Ct values ​​of the RT-PCR experiments on telomerase activity in HuH-7 cells

[0069]

[0070] According to the Ct value of RT-PCR experimental results, the telomerase activity of HuH-7 cells was positive.

[0071] Example 3 Hela cell telomerase activity detection control test

[0072] The present invention uses Hela cells as the research object, and uses the kit developed by the present invention and a kit with the same detection method in the market (Telomerase Activity Quantification qPCR Assay Kit manufacturer: ScienCell item number: 8928) to simultaneously detect the telomerase activity of Hela cells.

[0073] Experimental steps of the kit developed by the present invention:

[0074] 1. Cell Lysis

[0075] Take fresh Hela cells as test samples, wash them with DPBS buffer, and then take about 1.0×10 6 Cells were centrifuged at 1500 rpm and 4°C for 5 minutes, and the supernatant was discarded. 100 μl of component A, 2 μl of PMSF (0.1 M) and 2 μl of β-mercaptoethanol were added to the cell pellet, and the mixture was thoroughly mixed by pipetting, and then placed at 4°C for 30 minutes. The supernatant was collected by centrifugation at 12000 rpm and 4°C for 20 minutes, which was the telomerase cell lysis product.

[0076] 2. Telomere Extension

[0077] Prepare the telomerase reaction system. Add 13 μl DEPC water, 10 μl component B and 2 μl cell telomerase lysis product to a 1.5 ml EP tube in sequence. Centrifuge at 12000 rpm at room temperature for 1 minute. Place the telomerase reaction system in a dry heat oven at 37°C for 1 hour and 85°C for 10 minutes. This mixture is the telomerase reaction product.

[0078] Telomerase activity detection

[0079] Prepare the PCR reaction system for detecting telomerase activity in human cells. Add 12 μl of component C, 7 μl of sterile water, and 1 μl of the sample to be tested to the eight-tube connection. The PCR reaction conditions are: 95°C for 5 min for 1 cycle, 95°C for 30 s, 54°C for 30 s, and 72°C for 30 s for 40 cycles. The sample to be tested is 1×10 6 The telomerase reaction products of Hela cells were set up in triplicate.

[0080] Telomerase Activity Quantification qPCR Assay Kit (Manufacturer: ScienCell Catalog No.: 8928) was used according to the instructions to take about 1.0×106 The telomerase activity of Hela cells was detected.

[0081] Determination of telomerase activity

[0082] Table 3 1×10 6 The arithmetic mean of the Ct values ​​of RT-PCR experiments on telomerase activity in Hela cells

[0083]

[0084] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the requirements of the attached specification of claims.

Claims

1. A primer composition for detecting telomerase activity in human cells, characterized in that: The primer combination for the detection PCR reaction includes the following base sequences: tele-F: 5'-GGCAATCCGTACGAGCAGAGTTAAG-3'SEQ ID NO.1 tele-R: 5'-TTAAGCGCGGCTTACCCTTACCCTTACCCTAACCGGCC-3' SEQ ID NO.

2.

2. The primer composition for detecting telomerase activity in human cells according to claim 1, characterized in that: The base sequence of the initiating primer for telomerase reaction: tele-TS: 5'-GGCAATCCGTACGAGCAGAGTTAAG-3'SEQ ID NO.

3.

3. A kit for detecting telomerase activity in human cells, characterized in that: Includes the following components: (1) Telomerase cell lysate; (2) Telomerase reaction solution; (3) RT-PCR reaction solution for telomerase activity detection; (4) The RT-PCR reaction primers tele-F and tele-R of claim 1 and the telomerase reaction initiation primer tele-TS of claim 2.

4. The kit for detecting telomerase activity in human cells according to claim 3, characterized in that: Includes the following individually packaged components: Component A, for performing cell lysis, including telomerase cell lysis solution; Component B, used for telomerase reaction, including telomerase reaction solution, 100 mM dNTP, 0.01 mM telomerase reaction initiation primer tele-TS base sequence SEQ ID NO.3; Component C is used for telomerase-catalyzed extension reaction, including telomerase activity detection PCR reaction solution, PCR reaction primer combination 0.01mM tele-F base sequence SEQ ID NO.1, 0.01mM tele-R base sequence SEQ ID NO.

2.

5. The kit for detecting telomerase activity in human cells according to claim 4, characterized in that: The telomerase cell lysis solution contains: 0.2% of 1.5M Tris-HCl at pH 8.8, 1.6mM MgCl2·6H2O, 1.1mM EDTA, 1% NP-40, 0.8mM sodium deoxycholate, 10% glycerol, 43mM NaCl, 5mMβ-mercaptoethanol, 65mM CHAPS, and the remainder is DEPC water, and the percentages are volume percentages.

6. The kit for detecting telomerase activity in human cells according to claim 4, characterized in that: The telomerase reaction solution contains: 12% by volume of 1.5M Tris-HCl with a pH of 8.8, 67 mM KCl, 14 mM MgCl2·6H2O, 10 mM EGTA, and the remainder is DEPC water.

7. The kit for detecting telomerase activity in human cells according to claim 4, characterized in that: The telomerase activity detection PCR reaction solution contains: 0.01% PCR-grade SYBR Green I 10000×, 1.3% 1.5M Tris-HCl at pH 8.8, 1.3mM EDTA, 0.14M KCl, 9% glycerol, 26mM MgCl2·6H2O, 1M betaine, 2% formamide, 129mM DTT, 5% DMSO, 39mM ammonium sulfate, 0.1% 2.5mM dNTP, 5U / μl hot start enzyme, the remainder is sterile water, and the percentages are volume percentages.

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