Primer combination and kit for quantitatively detecting activity of cell telomerase and application

Abstract

The invention discloses a primer combination for quantitatively detecting cell telomerase activity, a kit and application, and relates to the technical field of telomerase activity detection. According to the primer combination for detecting telomerase activity and the detection method provided by the invention, the primer TS and the primer ACX-UP1 with specific sequences are selected, and the primer ACX-UP1 is provided with a stem-loop structure containing a fluorophore and a quenching group, so that not only can the functions of the primer be realized, but also the functions of a probe can be realized; therefore, the problems that a probe cannot be designed on a telomere repetitive sequence and the specificity of a dye method is poor are solved. The primer combination and the detection method provided by the invention have the characteristics of reliability, stability, convenience, rapidness and accuracy, so that the manpower, material resources and time cost required by telomerase activity detection are greatly reduced, and the accuracy is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of telomerase activity detection, in particular to a primer combination for quantitatively detecting cell telomerase activity, a kit and application thereof. BACKGROUND

[0002] The human telomere DNA sequence is a highly conserved short tandem repeat sequence "TTAGGG" at the end of the chromosome, which, together with the telomere binding protein, forms the telomere and can protect the chromosome from degradation and avoid loss of genetic information. In human cells, the telomere is shortened with each cell division, leading to accumulation of DNA damage and triggering cell aging.

[0003] The length of the telomere is mainly maintained by two mechanisms, telomerase and the alternative lengthening of telomeres (ALT), of which telomerase occupies a dominant position. Telomerase is a reverse transcriptase mainly composed of two parts, telomerase RNA component (TERC) and telomerase reverse transcriptase (TERT). Telomerase synthesizes telomere DNA repeats using its own TERC as a template under the action of TERT, and adds them to the end of the telomere to maintain the stability of the telomere length.

[0004] Telomerase has low activity in most somatic cells, but high activity in some continuously dividing cells such as stem cells, cancer cells and germ cells. By detecting the activity of telomerase, the risk of cancer can be indirectly judged. Therefore, telomerase has important significance in the early prevention and diagnosis of cancer. In addition, in the field of cell therapy, the activity of telomerase can be used to judge whether the treatment cells have the risk of tumorigenicity, so as to avoid adverse reactions caused by cell reinfusion into the human body. Therefore, it is of great significance to develop a fast, sensitive, reliable and easy-to-operate method for detecting telomerase activity.

[0005] Up to now, there are many methods for detecting telomerase activity. Among them, the most classic one is the TRAP method (Telomeric Repeat Amplification Protocol) proposed by Kim et al. in 1995 (Piatyszek, M. A., Kim, N. W., Weinrich, S. L. et al. Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP). Methods Cell Sci 17, 1-15 (1995). https: / / doi.org / 10.1007 / BF00981880; Kim NW, Wu F. Advances in quantification and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP). Nucleic Acids Res. 1997 Jul 1;25(13):2595-7. doi: 10.1093 / nar / 25.13.2595. PMID: 9185569; PMCID: PMC146790.). The principle of the telomeric repeat amplification protocol is that telomerase can add "TTAGGG" repeat sequences downstream of the special primer TS to form TS extension products of varying lengths. Then, the TS extension products are amplified by PCR technology using primer TS and another special primer as upstream and downstream primers, respectively. Finally, the amount of PCR product obtained reflects the size of telomerase activity.

[0006] However, this method is too complicated, requiring PCR, electrophoresis, development, etc., and is time-consuming, and is prone to false negative and false positive results. Therefore, researchers have improved a series of methods based on this method, such as two-primer TRAP method, TRAP-fluorescence resonance energy transfer method, TRAP-proximity assay method, TRAP-hybridization protection assay method, TRAP-enzyme-linked immunosorbent assay (ELISA), TRAP-electrochemical method, TRAP-qPCR method, TRAP-microchip method, and in situ TRAP method (Skvortsov DA, Zvereva ME, Shpanchenko OV, Dontsova OA. Assays for detection of telomerase activity. Acta Naturae. 2011 Jan;3(1):48-68. PMID: 22649673; PMCID:PMC3347595.).

[0007] In addition, researchers have also developed many detection methods that are not based on TRAP, such as chemiluminescence, electrochemistry, fluorescence analysis, colorimetry, surface-enhanced Raman detection, etc. (Zheng TT, Feng ED, Tian Y. Research progress in detection of telomerase activity [J]. Journal of Applied Technology, 2018, 18 (01): 1-13.; Guo Y, Wu X, Xia F, Lou XD. Research progress in detection of telomerase activity [J]. Journal of Analysis and Testing, 2021, 40 (12): 1819-1826.). However, these methods still have certain limitations in practical application. For example, the enzyme in chemiluminescence is easily degraded and is easily disturbed by the environment, and the detection cost is high; electrochemical method for detecting telomerase activity has problems such as unstable signal probe and the need to add additional redox medium; the signal probe in fluorescence analysis often needs complex synthesis steps and has poor biological compatibility, and the detection sensitivity is low; the detection phenomenon of colorimetry is obvious, but the sensitivity is generally low; the cost of surface-enhanced Raman detection is high, and the operation is complex, etc.

[0008] In the prior art, Chinese patent application CN119552947A discloses a method for detecting telomerase activity by colloidal gold color development and fluorescence detection, which can realize rapid double visualization detection of telomerase activity, but cannot accurately quantify telomerase activity. Chinese patent application CN118638905A discloses a SYBR Green real-time fluorescence quantitative method for detecting telomerase activity, which has low detection sensitivity and a minimum detection limit of only 1000 cells. Chinese patent CN111269960B discloses a quantum dot molecular beacon detection method, which has a minimum detection limit of 1 cell, but cannot effectively quantify telomerase activity, which is not conducive to distinguishing samples without telomerase activity and samples with weak telomerase activity.

[0009] Therefore, there is an urgent need to develop a specific, sensitive, rapid and convenient method for detecting telomerase activity, which can provide a research basis for the detection of telomerase activity in the fields of cell drug treatment, cell aging and tumor detection. SUMMARY

[0010] The present application provides a primer combination, a kit and an application for quantitatively detecting cell telomerase activity. The primer combination and the detection method for detecting telomerase activity provided by the present application can realize the functions of primers and probes by selecting primers TS and primers ACX-UP1 with a "stem-loop structure" containing a fluorescent group and a quencher group, thereby solving the problem of being unable to design probes on the telomere repeat sequence and the problem of poor specificity of the dye method. The primer combination and the detection method provided by the present application are reliable, stable, convenient, rapid and accurate, greatly reducing the manpower, material resources and time cost required for telomerase activity detection and greatly improving the accuracy. The following technologies are used to achieve the above-mentioned effects.

[0011] In a first aspect, the present application provides a primer combination for quantitatively detecting cell telomerase activity, which comprises primer TS, primer ACX-UP1, internal reference primer NT-UP1 and internal reference standard; the nucleotide sequence of the primer TS is shown in SEQ ID NO. 1, the nucleotide sequence of the primer ACX-UP1 is shown in SEQ ID NO. 2; the nucleotide sequence of the internal reference primer NT-UP1 is shown in SEQ ID NO. 5, and the nucleotide sequence of the internal reference standard is shown in SEQ ID NO. 7.

[0012] The primer ACX-UP1 and the internal reference primer NT-UP1 are provided with stem loop structures, and the stem loop structure of the primer ACX-UP1 is marked with a first fluorescent group and a quenching group at two ends, respectively; the stem loop structure of the internal reference primer NT-UP1 is marked with a second fluorescent group and a quenching group at two ends, respectively; the first fluorescent group and the second fluorescent group are different types of fluorescent groups.

[0013] Further, the first fluorescent group and the second fluorescent group are FAM, Texas Red, CY3, CY5, VIC, TET, NED, ROX, JOE or HEX, and the quenching group is TAMRA, NFQ, ECLIPSE, DABCYL, MGB, BHQ1 or BHQ2.

[0014] Still further, the first fluorescent group is a FAM group, the second fluorescent group is a CY5 group, and the quenching group is Dabcyl.

[0015] In a second aspect of the present application, a product for quantitatively detecting cell telomerase activity is provided, which comprises the primer combination described above.

[0016] Further, the product further comprises negative control cells, positive control cells, target quantitative standard samples; the nucleotide sequence of the target in the target quantitative standard sample is shown in SEQ ID NO. 4.

[0017] Still further, the product further comprises a PCR reaction solution, RNase free H2O, a cell telomerase lysis solution and a reference fluorescent dye; the negative control cells are MSC cells and / or MRC5 cells, and the positive control cells are any one or more of A549 cells, 293 cells, iPSC cells and Hela cells.

[0018] Still further, the PCR reaction solution is TaqMan multiplex qPCR master mix, which contains a PCR buffer, dNTPs, a hot-start Taq polymerase and Mg 2+ .

[0019] Still further, the cell telomerase lysis solution is prepared from DEPC water, Tris-HCL (pH 7.5), CHAPS, anhydrous magnesium chloride, EGTA, B-mercaptoethanol, glycerol, AEBSF, an RNAase inhibitor and the like.

[0020] Still further, the reference fluorescent dye is obtained by diluting the fluorescent dye 10 times with a diluent.

[0021] For example, the ROX reference fluorescent dye can be diluted 10 times with a 50x ROX Reference Dye diluent. The ROX diluent is prepared from Tris-HCl (pH 8.0), EDTA, Tween-20, and the like.

[0022] In a third aspect, the present application provides the use of the product as described above for quantitatively detecting the activity of a telomerase in a cell of a species having a telomere repeat sequence of "TTAGGG".

[0023] Further, the cell is a cell of any one species belonging to the phylum Chordata.

[0024] Further, the cell is a human cell.

[0025] In a fourth aspect, the present application provides a method for quantitatively detecting the activity of a telomerase in a cell, which comprises the following steps:

[0026] A predetermined amount of the cell to be tested is collected, and the cell to be tested, negative control cells, and positive control cells are treated with a cell telomerase lysis solution, and then centrifuged to obtain supernatants for standby use;

[0027] A predetermined amount of the supernatant of the cell to be tested is heated to inactivate, as a heat inactivation control;

[0028] The target quantitative standard is gradient-diluted to obtain target quantitative standard working solutions of various concentrations, and then subjected to PCR amplification. The logarithm of the concentration of the target quantitative standard working solution with a base of 10 is taken as the abscissa, and the CT value is taken as the ordinate, to draw a target standard curve;

[0029] The reference fluorescent dye, the primer TS, the primer ACX-UP1, the internal standard, and the internal standard primer NT-UP1 are prepared to form a reaction system;

[0030] The cell telomerase lysis solution, the target quantitative standard of various concentrations, the supernatant of the cell to be tested, the supernatant of the positive control cells, the supernatant of the negative control cells, and the heat inactivation control are added to the reaction system, respectively, to perform PCR amplification and detection, so as to obtain the CT values of the system negative control group, the standard curve group, the sample to be tested group, the positive control group, the negative control group, and the heat inactivation control group, respectively. The positive control group is recorded as a positive quality control, and the negative control group and the heat inactivation control group are recorded as a negative quality control;

[0031] It is confirmed that the positive quality control, the negative quality control, and the system negative control group are normal. According to the difference between the CT value of the internal standard channel of the cell to be tested and the average CT value of the standard curve group and the system negative control group, it is determined whether the telomerase activity detection system of the cell to be tested is inhibited. The telomerase activity of the cell to be tested is calculated according to the target standard curve.

[0032] Further, in the method for quantitatively detecting the telomerase activity of cells, the nucleotide sequence of the target in the target quantitative standard is shown in SEQ ID NO. 4.

[0033] Further, in the method for quantitatively detecting the telomerase activity of cells, the method for judging whether the detection system of the telomerase activity of the cells to be detected is inhibited by comparing the CT values is:

[0034] When the CT value of the internal reference channel of the cell sample to be detected is not more than 0.5 cycles different from the average CT value of the standard curve group and the system negative control group, the cell sample to be detected has no inhibitory effect on the reaction system.

[0035] When the CT value of the internal reference channel of the cell sample to be detected is more than 0.5 cycles different from the average CT value of the standard curve group and the system negative control group, the supernatant of the cell to be detected has an inhibitory effect on the reaction system, and the extraction solution needs to be diluted and detected again.

[0036] Further, in the method for quantitatively detecting the telomerase activity of cells, the method for calculating the telomerase activity of the cells to be detected by the target standard curve is: the CT value of the target of the cells to be detected is substituted into the target standard curve, and the telomerase activity (TPG number) of the cells to be detected can be calculated.

[0037] Further, in the method for quantitatively detecting the telomerase activity of cells, the standard curve quality control parameters: the amplification efficiency is 90-110%, and R 2 ≥0.990.

[0038] Further, in the method for quantitatively detecting the telomerase activity of cells, the method for confirming whether the positive quality control, the negative quality control and the system negative control group are normal is:

[0039] When the CT value of the internal reference channel is not more than 0.5 cycles different from the average CT value of the standard curve group, the negative quality control and the system negative control group, and the CT value of the target channel is ≤26, the positive quality control is normal.

[0040] When the CT value of the internal reference channel is not more than 0.5 cycles different from the average CT value of the standard curve group, the negative quality control and the system negative control group, and the CT value of the target channel is >32, the negative quality control and the system negative control group (NTC) are normal; if the CT value of the target channel is ≤32, it indicates that the reaction system is contaminated.

[0041] In the above primer combination and the corresponding method for detecting the telomerase activity of cells provided by the application, (1) the primer TS can be used as a substrate of the telomerase in cells, as an upstream primer of a target, and as an upstream primer of an internal reference; (2) the primer ACX-UP1 containing a stem loop structure, a first fluorescent group and a quenching group can be used as a downstream primer and a probe of a target; and (3) the primer NT-UP1 containing a stem loop structure, a second fluorescent group and a quenching group can be used as a downstream primer and a probe of an internal reference.

[0042] The working principle of the primer combination of the application is as shown in the following figure: Figure 1 As shown in the figure, the telomerase in cells can recognize the TS sequence at 30℃ and continuously add the telomere repeat sequence "TTAGGG" to the 3' end of the TS sequence; 10 min of incubation at 95℃ inactivates the telomerase; then, the product extended by the telomerase is used as a template, the TS sequence which is not recognized and extended by the telomerase is used as an upstream primer of a target and an upstream primer of an internal reference, and the primer ACX-UP1 is used as a downstream primer and a probe of a target, so as to amplify the product extended by the telomerase through qPCR; finally, the amount of the amplified product is positively correlated with the activity of the telomerase. On the other hand, the method adds a fixed amount of an internal reference standard into the system, and sets specific internal reference primers (the upstream primer TS and the downstream primer NT-UP1) for detection; if the qPCR reaction is inhibited, the CT value of the internal reference will increase. Therefore, the degree of inhibition of the qPCR reaction can be determined according to the increase.

[0043] In the application, the introduction of the primer ACX-UP1 enables the PCR process to reflect the amount of the product through the change of the fluorescent signal, without the need of electrophoretic identification of the product as in the traditional TRAP, which facilitates the operation, saves time, and avoids the pollution caused by opening the cover of the product.

[0044] Specifically, the method for detecting the telomerase activity of cells provided by the application comprises the following steps:

[0045] (1) Culturing and collecting a certain amount of cells to be tested.

[0046] (2) Treating the collected cells to be tested, negative control cells and positive control cells with a cell telomerase lysis solution, and collecting the lysis supernatant for standby; taking 20 μL of the supernatant and heating it at 95℃ for 10 min as a heat inactivation control for standby.

[0047] (3) Diluting the target quantitative standard (for example, TSR8 sequence, as shown in SEQ ID NO. 4) in a gradient (one concentration gradient every 10 times) to a concentration range of 20000 TPG / μL~0.2 TPG / μL to make a target standard curve.

[0048] (4) Prepare the reaction system, add sample, calculate the telomerase activity of the corresponding to-be-measured cell according to the target standard curve result, and compare it with the negative control cell and the positive control cell to determine the activity of the to-be-measured cell telomerase. At the same time, the difference between the internal reference channel CT value of the to-be-measured cell sample and the average CT value of the internal reference channel of the standard curve group and the system negative control group is compared to determine whether the to-be-measured cell telomerase activity detection system is inhibited.

[0049] Compared with the prior art, the present application has the advantages that:

[0050] 1、The present application selects special primers TS, primer ACX-UP1, target quantitative standard, internal reference primer NT-UP1 and internal reference standard, constructs a double fluorescence qPCR reaction system, and makes it possible to detect telomerase activity by qPCR probe method, and the result is more accurate than that of the traditional SYBR dye method.

[0051] 2、The cumbersome steps of opening the cover for detection and the possible pollution of the original TRAP technology are avoided.

[0052] 3、Compared with the time-consuming of the original method for a whole day, the detection method of the present application is convenient and fast, and the detection result can be obtained in 3.5 h at the fastest.

[0053] 4、By introducing the internal reference gene, it is evaluated whether there is a PCR inhibitor in the to-be-measured cell sample, and false negative results are excluded.

[0054] 5、High sensitivity, at least 10 A549 cells in each reaction can be detected. DETAILED DESCRIPTION

[0055] Figure 1 The working principle of the present application for quantitatively detecting cell telomerase activity by using a primer combination is provided.

[0056] Figure 2 The amplification spectrum for target primer and internal reference primer screening is provided. Wherein A is the amplification curve of different target primers for qPCR at a temperature gradient between 50 DEG C and 60 DEG C; B is the standard curve amplification spectrum of different internal reference primers at four concentrations of 2E1 amol / μL, 2E0 amol / μL, 2E-1 amol / μL and 2E-2 amol / μL.

[0057] Figure 3 The amplification spectrum and standard curve of the target and the internal reference are provided. Wherein, A and B are the target standard curve amplification curve and the standard curve; C and D are the internal reference standard curve amplification curve and the standard curve.

[0058] Figure 4qPCR-TRAP precision and accuracy amplification map. Among them, A, B, C represent 3 repeated experiments, D represents 1 repeated experiment of different operators, and the concentration of TSR8 quantitative standard (target quantitative standard) used is (from left to right of target curve): 2E1 amol / μL, 2E-1 amol / μL, 2E-3 amol / μL, 2E-4 amol / μL. In the figure, the blue line represents the target, and the red line represents the internal reference.

[0059] Figure 5 qPCR-TRAP minimum quantification limit amplification map. Among them, A, B, C represent 3 repeated experiments, and the concentration of TSR8 quantitative standard (target quantitative standard) used is (from left to right of target curve): 2E-3 amol / μL, E-3 amol / μL, 2E-4 amol / μL, E-4 amol / μL. In the figure, the blue line represents the target, and the red line represents the internal reference.

[0060] Figure 6 qPCR-TRAP robustness amplification map. Among them, A, B are amplification maps obtained by fluorescence quantitative PCR instrument (ABI Q1 instrument), A is Bioneer primer, and B is Shengong primer; C and D represent amplification maps obtained by fluorescence quantitative PCR instrument (Bole CFX96 instrument), C is Bioneer primer, and D is Shengong primer. The concentration of TSR8 quantitative standard (target quantitative standard) used is (from left to right of target curve): E-3 amol / μL, 5E-4 amol / μL, 2E-4 amol / μL. In the figure, the blue line represents the target, and the red line represents the internal reference.

[0061] Figure 7 qPCR-TRAP detection sensitivity amplification map. Among them, A, B, C, D represent 4 repeated experiments respectively, and the concentration of A549 telomerase extract used is (from left to right of target curve): 1E6 cells of cell extract is diluted by 10 times, 100 times, 1000 times, 10000 times, 20000 times, and 100000 times. In the figure, the blue line represents the target, and the red line represents the internal reference. Among them, the curves of dilution of 10000 times and higher dilution times cannot be distinguished.

[0062] Figure 8 qPCR-TRAP specificity amplification map. Among them, A, B, C, D, E, F represent the amplification curves of the supernatant after lysis of A549, 293, iPSC, Hela, MSC, MRC5 and the heat inactivated control. In the figure, the blue line represents the target, and the red line represents the internal reference.

[0063] Figure 9 The monitoring accuracy of the qPCR-TRAP internal reference is shown in the amplification map. In the figure, A represents A549 cells, and B represents iPSC cells. The concentration of the telomerase extract solution of the A549 cells is (from left to right on the target curve) 1E6 cell extract solution, 0-fold dilution, 10-fold dilution, 20-fold dilution, and 40-fold dilution. In the figure, the blue line represents the target, and the red line represents the internal reference. DETAILED DESCRIPTION

[0064] The technical solutions of the present application will be described below. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0065] In some embodiments of the present application, a set of primer combinations is provided, including primer TS, primer ACX-UP1, internal reference primer NT-UP1, and internal reference standard; the nucleotide sequence of the primer TS is shown in SEQ ID NO. 1, the nucleotide sequence of the primer ACX-UP1 is shown in SEQ ID NO. 2; the nucleotide sequence of the internal reference primer NT-UP1 is shown in SEQ ID NO. 5, and the nucleotide sequence of the internal reference standard is shown in SEQ ID NO. 7.

[0066] The stem-loop structure is provided on the primer ACX-UP1 and the internal reference primer NT-UP1, the first fluorescent group and the quenching group are labeled at the two ends of the stem-loop structure of the primer ACX-UP1, respectively; the second fluorescent group and the quenching group are labeled at the two ends of the stem-loop structure of the internal reference primer NT-UP1, respectively; the first fluorescent group and the second fluorescent group are different types of fluorescent groups.

[0067] Alternatively, in the above primer combination, the first fluorescent group and the second fluorescent group are FAM, Texas Red, CY3, CY5, VIC, TET, NED, ROX, JOE, or HEX, and the quenching group is TAMRA, NFQ, ECLIPSE, DABCYL, MGB, BHQ1, or BHQ2.

[0068] In the following detailed description, the first fluorescent group on the primer ACX-UP1 is specifically selected as the FAM group, the second fluorescent group on the internal reference primer NT-UP1 is specifically selected as the CY5 group, and the quenching groups on the primer ACX-UP1 and the internal reference primer NT-UP1 are both selected as Dabcyl.

[0069] In some other embodiments of the present application, a product comprising the primer combination is provided. The product can be in the form of a kit or the like, which is capable of quantitatively detecting the activity of the cellular telomerase.

[0070] Optionally, the product further comprises negative control cells, positive control cells, and a target quantification standard. The nucleotide sequence of the target in the target quantification standard is shown in SEQ ID NO. 4.

[0071] Optionally, the product further comprises a PCR reaction solution, RNase free H2O, a cellular telomerase lysis solution, and a reference fluorescent dye.

[0072] Specifically, the PCR reaction solution is TaqMan multiplex qPCR master mix, which contains a PCR buffer, dNTPs, a hot-start Taq polymerase, and Mg 2+ .

[0073] Specifically, the cellular telomerase lysis solution is prepared from DEPC water, Tris-HCL (pH 7.5), CHAPS, anhydrous magnesium chloride, EGTA, B-mercaptoethanol, glycerol, AEBSF, and an RNAase inhibitor.

[0074] Further, the reference fluorescent dye is diluted 10 times with a diluent.

[0075] In the following detailed description, the negative control cells are specifically selected from MRC5 cells, and the positive control cells are specifically selected from A549 cells.

[0076] In some other embodiments of the present application, a method for quantitatively detecting the activity of the cellular telomerase using the primer combination is provided. The steps include:

[0077] A predetermined amount of the cells to be tested is collected, and the cells to be tested, negative control cells, and positive control cells are treated with a cellular telomerase lysis solution, and centrifuged to obtain supernatants for standby use;

[0078] A predetermined amount of the supernatant of the cells to be tested is heated to inactivate, as a heat inactivation control;

[0079] The target quantification standard is diluted by 10 times to obtain a quantification standard working solution at each concentration, and PCR amplification is performed. The concentration of the quantification standard working solution is used as the abscissa, and the CT value is used as the ordinate to draw a target standard curve;

[0080] The reference fluorescent dye, the primer TS, the primer ACX-UP1, the internal standard, and the internal standard primer NT-UP1 are prepared to configure a reaction system;

[0081] The telomerase lysis solution, target quantitative standard, supernatant of the cell to be detected, negative control cell supernatant, positive control cell supernatant and heat-inactivated control group are added into the reaction system respectively, PCR amplification is carried out, detection is carried out, and CT values of the system negative control group, the standard curve group, the sample to be detected group, the positive control group, the negative control group and the heat-inactivated control group are obtained respectively; the positive control group is recorded as the positive quality control, and the negative control group and the heat-inactivated control group are recorded as the negative quality control;

[0082] The positive quality control, the negative quality control and the system negative control group are confirmed to be normal; the telomerase activity of the cell to be detected is judged according to the difference between the CT value of the internal reference channel of the cell to be detected and the average CT value of the standard curve group and the system negative control group.

[0083] Optionally, in the target quantitative standard of the method, the nucleotide sequence of the target (oligonucleotide) is as shown in SEQ ID NO. 4.

[0084] Optionally, in the method, the method for judging whether the telomerase activity detection system of the cell to be detected is inhibited by comparing the CT values is:

[0085] (1) When the CT value of the internal reference channel of the cell to be detected is not more than 0.5 cycles different from the average CT value of the standard curve group and the system negative control group, the cell sample to be detected has no inhibitory effect on the reaction system;

[0086] (2) When the CT value of the internal reference channel of the cell to be detected is more than 0.5 cycles different from the average CT value of the standard curve group and the system negative control group, the supernatant of the cell to be detected has an inhibitory effect on the reaction system, and the extraction solution needs to be diluted and detected again.

[0087] Optionally, in the method, the method for calculating the telomerase activity of the cell to be detected through the target standard curve is: the target CT value of the cell to be detected is substituted into the target standard curve, and the telomerase activity (TPG number) of the cell to be detected can be calculated.

[0088] Optionally, in the method, the standard curve parameters are: the amplification efficiency is 90-110%, R 2 ≥ 0.990.

[0089] Optionally, in the method, the method for confirming whether the positive quality control, the negative quality control and the system negative control group are normal is:

[0090] (1) When the CT value of the internal reference channel is not more than 0.5 cycles different from the average CT value of the standard curve group, the negative quality control and the system negative control group, and the CT value of the target channel is ≤26, the positive quality control is normal;

[0091] (2) When the CT value of the internal reference channel is not more than 0.5 cycles different from the average CT value of the standard curve group, the negative quality control group and the system negative control group, and the CT value of the target channel is greater than 32, the negative quality control group and the system negative control group are normal; if the CT value of the target channel is less than or equal to 32, it indicates that the reaction system is contaminated.

[0092] It should be noted that the "cell" claimed by the present application can specifically select a cell of any species of Chordata. For example, a human cell can be selected.

[0093] The following specific embodiments of the present application use TSR8 as a quantitative reference to accurately quantify the telomerase activity of the cell sample to be measured; use TPG as a telomerase activity unit to achieve the purpose of high sensitivity and accurate quantification of telomerase activity; the protein extraction and quantitative analysis of the cell sample to be measured can be completed within 3.5 hours, realizing the rapid detection of the cell sample to be measured; the sensitivity of the present application is high, and the minimum detection limit of the telomerase activity of A549 cells is 10 cells; the specificity is strong, and the telomerase activity in pluripotent stem cells, cancer cells and immortalized cells can be accurately detected.

[0094] Figure 1 The working principle of the qPCR-TRAP stem loop primer provided by the present application is as follows:

[0095] (1) Telomerase can specifically recognize the TS sequence and continuously add a repeated sequence of "TTAGGG" of 6 bp to the 3' end of the TS sequence.

[0096] (2) In the qPCR amplification stage, the TS sequence not recognized and extended by telomerase is used as an upstream primer, and the stem loop primer is used as a downstream primer to perform qPCR amplification on the TS extension product. The amplification process will open the stem loop structure of the stem loop primer, and the quenching group on the stem loop structure will be removed from the quenching effect on the fluorescent group, so that the fluorescent group emits fluorescence.

[0097] (3) The intensity of the detected fluorescence is proportional to the number of amplification products, and the number of amplification products is proportional to the activity of telomerase. Therefore, the size of the telomerase activity can be judged by detecting the intensity of the fluorescence.

[0098] In the following specific implementation cases of the present application, the reagents and raw material sources used are shown in Table 1

[0099] Table 1

[0100] In the following specific implementation cases of the present application, the cells used are all from the cell strains self-preserved by the company. The primers (stem loop primers, standard long primers) are synthesized by Bailingge Biotechnology (Shanghai) Co., Ltd.

[0101] Example 1

[0102] This embodiment adopts a method for quantitatively detecting cell telomerase activity, which is specifically implemented through the following technical route: (1) preparing CHAPS lysis solution; (2) diluting fluorescent dye ROX; (3) extracting telomerase of the cell sample to be tested, negative control cells and positive control cells; (4) performing heat inactivation treatment on the telomerase extract of the cell sample to be tested; (5) preparing target quantitative standard (TSR8 sequence); (6) preparing internal reference standard (TSNT sequence); (7) preparing PCR reaction system and performing PCR amplification; (8) machine detection and result analysis.

[0103] 1. Prepare 50 mL CHAPS lysis solution

[0104] Use 39.85 mL DEPC water as solvent, take 0.5 mL 1M Tris-HCL (pH=7.5) and add to the DEPC water, i.e. dilute to a final concentration of 10 mM; weigh 0.25 g of solid CHAPS and add to the DEPC water, i.e. dilute to a final concentration of 0.5%; weigh 0.0048 g of solid anhydrous magnesium chloride and add to the DEPC water, i.e. dilute to a final concentration of 1 mM; take 0.1 mL of 0.5M EGTA and add to the DEPC water, i.e. dilute to a final concentration of 1 mM; take 4.55 mL of 55 mM β-mercaptoethanol and add to the DEPC water, i.e. dilute to a final concentration of 5 mM; take 5 mL of glycerol and add to the DEPC water, i.e. dilute to a final concentration of 10%, and shake to mix. According to 1 mL, dispense in EP tubes and store at -80 ℃.

[0105] Before use, add 10 μL of 10 mM AEBSF (serine protease inhibitor) to each 1 mL of CHAPS lysis solution, i.e. to a concentration of 0.1 mM.

[0106] Add 5 μL of RNAase inhibitor to each 200 μL of CHAPS lysis solution, i.e. to a concentration of 1 U.

[0107] 2. Dilute reference fluorescent dye ROX

[0108] (1) Dilute 50x ROX Reference Dye 10 times using ROX diluent.

[0109] The formula of ROX diluent is: 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 0.01% Tween-20. The specific preparation method of ROX diluent is: take 1 mL of 1 M Tris-HCl, 10 μL of 0.5 M EDTA, 5 μL of Tween-20 into a 50 mL centrifuge tube, and add HyPure water to 50 mL, shake and mix evenly to obtain the ROX diluent. 50x ROX Reference Dye is diluted 10 times with the above-mentioned ROX diluent to obtain 50x ROX Reference Dye II.

[0110] (2) Prepare the quantitative system: add 0.4 μL of 50x ROX Reference Dye II to each 20 μL qPCR system.

[0111] 3, Extraction of telomerase from the cells to be tested, negative control cells and positive control cells

[0112] In this embodiment, A549 cells are selected as positive controls and MSC cells are selected as negative controls.

[0113] (1) Collect 1E6 cells to be tested / negative control cells / positive control cells, centrifuge at 3000 g for 5 min, and remove the supernatant.

[0114] (2) Resuspend the above cell precipitate in 200 μL of CHAPS lysis buffer (AEBSF and RNAase inhibitor have been added) on ice for 30 min; centrifuge at 16000 g at 4°C for 20 min, and remove the precipitate.

[0115] (3) Take 160 μL of the supernatant for detection or store at -80°C for standby. When detecting, dilute 10 times with CHAPS lysis buffer. Pay attention to prevent contamination during operation.

[0116] 4, Heat inactivation treatment of telomerase extract in the cell sample to be tested

[0117] (1) Dilute the telomerase extract of the cell sample to be tested (i.e. the supernatant obtained in step 3 above) according to the required dilution ratio (usually 10 times) (dilute with CHAPS lysis buffer), and divide 20 μL into a new EP tube.

[0118] (2) Heat in water bath at 95°C for 10 min to complete the heat inactivation treatment. The heat inactivated sample is recommended to be used for detection immediately.

[0119] 5, Preparation of target quantitative standard (oligonucleotide TSR8) working solution

[0120] (1) Take 5 μL TSR8 (10 μM) to 45 μL Easy dilution, mix well, dilute TSR8 to about 1E6 amol / μL.

[0121] Repeat the operation, gradient dilution to 100 amol / μL, continue to take 10 μL TSR8 (100 amol / μL) to 40 μL Easy dilution, mix well, dilute TSR8 to about 20 amol / μL, that is, 20000 TPG / μL.

[0122] (2) Take 5 μL to 45 μL Easy dilution, mix well, dilute TSR8 to about 2000 TPG / μL.

[0123] (3) Perform 10-fold gradient dilution to 0.2 TPG / μL in this way. That is, the target standard curve point concentrations are 20000 TPG / μL, 2000 TPG / μL, 200 TPG / μL, 20 TPG / μL, 2 TPG / μL, 0.2 TPG / μL, respectively, denoted as Std1, Std2, Std3, Std4, Std5, Std6.

[0124] Note: 1 amol TSR8 = 1000 TPG units.

[0125] 6. Preparation of internal standard (TSNT)

[0126] (1) Take 5 μL TSNT (10 μM) to 45 μL Easy dilution, mix well, dilute TSNT to about 1E6 amol / μL.

[0127] (2) Repeat the operation, gradient dilution to 100 amol / μL, continue to take 10 μL TSNT (10 μM) to 40 μL Easy dilution, mix well, dilute TSNT to about 20 amol / μL.

[0128] (3) Take 5 μL to 45 μL Easy dilution, mix well, dilute TSNT to about 2 amol / μL, which is the internal standard.

[0129] 7. Preparation of PCR reaction system and sample addition

[0130] (1) Primer design

[0131] The present embodiment designs several stem-loop structures, as shown in Table 2 below. After screening, ACX-UP1 is confirmed as the optimal primer. The present application also designs a series of internal standard and corresponding internal standard primers, and NT-UP1 is screened as the optimal primer.

[0132] Table 2 Related primer sequences of qPCR-TRAP multiplex fluorescence detection system

[0133] (2) Establishing PCR reaction system

[0134] During detection, the standard curve of the target is drawn by using the standard TSR8, with the range of 40000 TPG / reaction~0.4TPG / reaction. A549 is used as the positive control cell, and MSC is used as the negative control cell. Heat inactivation control is set for each sample, and system negative control (NTC) is set for each experiment. There is a fixed concentration of internal standard (TSNT) in the PCR reaction system, which is 4amol / reaction. If there is Taq enzyme inhibition, the CT value of the internal standard will increase. The detection result of the sample is displayed in the form of TPG, and the size of the telomerase activity of the sample is judged by comparing the TPG value of the positive control and the negative control.

[0135] According to the following Table 3, 20 μL of PCR reaction system is prepared.

[0136] Table 3 PCR reaction system

[0137] After mixing, the reaction solution is divided into eight-tube or 96-well plate, and then 2 μL of standard curve group, sample group, negative / positive control group and heat inactivation control group are added into each tube. Among them, the standard curve group is set with two duplicate holes, and the other groups are set with three duplicate holes, as shown in the following Table 4.

[0138] Table 4

[0139]

[0140] 8. Machine detection and result analysis and determination

[0141] (1) After adding the sample, machine detection can be performed. The PCR reaction program is shown in Table 5.

[0142] Table 5 PCR reaction program

[0143] Set the target channel to FAM, the internal reference channel to CY5, and the reference fluorescence to ROX (select according to the model as needed). Set the standard curve group, system negative control (NTC), positive control, and negative control plates.

[0144] (2) Threshold setting

[0145] Set the threshold line according to the maximum fluorescence increment of the TSR8 quantitative standard curve group and 1 / 15 of the maximum fluorescence increment of the internal reference standard curve group, respectively.

[0146] (3) Result analysis and determination

[0147] ① Positive control result determination

[0148] The internal reference channel (CY5): the CT value differs from the average CT value of the standard curve group, negative control, and NTC by no more than 0.5 cycles.

[0149] The target channel (FAM): CT value ≤ 26.

[0150] If the above conditions are met, the positive control is considered normal.

[0151] ② System negative control (NTC) and negative control result determination (negative control group, heat-inactivated control group)

[0152] The internal reference channel (CY5): the CT value differs from the average CT value of the standard curve group, negative control, and NTC by no more than 0.5 cycles.

[0153] The target channel (FAM): CT value > 32.

[0154] If the above conditions are met, the negative control and NTC are considered normal.

[0155] If the CT value of the target channel (FAM) is ≤ 32, it indicates that the detection system is contaminated, and the operation process needs to be checked.

[0156] ③ Standard curve parameters: the amplification efficiency should be within 90-110%, and R 2 ≥ 0.990.

[0157] ④ Judgment of the detection results of the test cell sample

[0158] Under the premise that the positive control, NTC, and negative control meet the experimental requirements, in the internal reference channel (CY5) of the test cell sample: if the CT value differs from the average CT value of the standard curve group and NTC by no more than 0.5 cycles, it is considered that the test cell sample does not have an inhibitory effect on the reaction system; if the CT value exceeds 0.5 cycles, it is considered that the test cell sample may have an inhibitory effect on the reaction system, and the extract needs to be diluted and retested.

[0159] In the target channel (FAM) of the cell sample, the formula for calculating the number of cells per reaction of the cell sample is as follows:

[0160] Cell count / reaction = total number of cells / 200 μL (cell lysis buffer) / dilution factor × 2 μL (template volume).

[0161] For example, if 1E6 A549 cells are added to 200 μL of CHAPS cell lysis buffer and diluted 10 times, then the number of cells per reaction in the cell sample to be tested is 1000, that is, 1000 cells / reaction.

[0162] The detection value (TPG) of the target channel (FAM) of the cell sample to be tested is obtained by the analysis software, which is the telomerase activity corresponding to the number of cells in each reaction of the cell sample to be tested.

[0163] For example, if the detection value of the A549 target channel (FAM) is 150 TPG per 1000 cells / reaction, it means that the telomerase activity corresponding to each 1000 A549 cells is 150 TPG, the telomerase activity corresponding to each 10000 A549 cells is 1500 TPG, and so on.

[0164] Note: If the target CT value is <32 when the sample is known to be negative cells (such as MSCs), it indicates that the system may be contaminated and the cause needs to be analyzed and the test repeated.

[0165] Example 2: Primer Screening

[0166] The designed target primers ACX-UP1 and ACX-UP2, and internal control primers NT-UP1 and NT-UP2 were screened. For the target primers, qPCR was performed at a temperature gradient of 50-60℃ with a template concentration of 2E1 amol / μL. For the internal control primers, TSNT standard solutions at concentrations of 2E1 amol / μL, 2E0 amol / μL, 2E-1 amol / μL, and 2E-2 amol / μL were used as points on the internal control standard curve for qPCR.

[0167] like Figure 2 As shown, for the target primers, ACX-UP1 exhibits an earlier amplification curve, a smaller CT value, and higher sensitivity. For the internal control primers, although the CT values ​​at various points on the standard curve do not differ significantly, NT-UP1 shows a better linear shape during the plateau phase, while the NT-UP2 amplification curve shows a downward trend during the plateau phase and has a poor linear shape. In conclusion, ACX-UP1 and NT-UP1 were selected as the primers used in this method.

[0168] Example 3: Linear range of target standard and standard curve of internal reference

[0169] Take 2E1 amol / μL~2E-4 amol / μL target quantitative standard (TSR8 oligonucleotide) solution as each point of target standard curve, and take 2E1 amol / μL~2E-2 amol / μL TSNT internal reference standard solution as each point of internal reference standard curve, and take RNase free H2O as a system negative control, and use the PCR reaction system and reaction procedure in Table 3 and Table 5 above for amplification.

[0170] The amplification curves and standard curves of the target and the internal reference are shown in Figure 3 , and the amplification efficiency, R 2 , slope, CT value and other data are shown in Table 6 below.

[0171] Table 6: Linear range of qPCR-TRAP of target and internal reference

[0172] The results show that the amplification efficiency of the target and the internal reference is all greater than or equal to 90%, and R 2 is greater than or equal to 0.99, indicating that the qPCR-TRAP multiplex fluorescence detection system provided in the application has a good linear relationship within the range of 2E1 amol / μL~2E-4 amol / μL of the target TSR8 quantitative standard and 2E1 amol / μL~2E-2 amol / μL of the internal reference TSNT standard.

[0173] Example 4: Precision and accuracy verification

[0174] Take 4 concentration points of the target quantitative standard (TSR8 oligonucleotide), i.e. 2E1 amol / μL, 2E-1 amol / μL, 2E-3 amol / μL and 2E-4 amol / μL, for precision detection; 6 repeated holes are made for each concentration point, and 3 repeated experiments are made in total; meanwhile, the 4th repeated experiment is made by different operators to verify the intermediate precision in the method. Each plate experiment is accompanied by a standard curve group, and RNase free H2O is used as a system negative control, and the reaction system and reaction procedure in Table 3 and Table 5 above are used for amplification.

[0175] The amplification spectrum of the 4 repeated experiments is shown in Figure 4 , and the CT value, recovery rate and RSD value of the 4 repeated experiments are shown in Table 7 below.

[0176] Table 7: Precision and accuracy of qPCR-TRAP

[0177]

[0178] The results show that the RSD of CT value of target gene in three experiments is less than or equal to 20%, the average value of target recovery rate fluctuates within ± 25%, and the fluctuation of ΔCT value of the internal reference gene detection is within ± 0.5 CT value range of NTC and standard curve group. At the same time, the RSD of CT value of target gene in four experiments of another operator is less than or equal to 20%, the average value of target recovery rate fluctuates within ± 25%, and the fluctuation of ΔCT value of the internal reference gene detection is also within ± 0.5 CT value range of the average value of NTC and standard curve group. This indicates that the method has good accuracy and precision (repeatability and intermediate precision).

[0179] Example 5: Verification of the lowest quantification limit

[0180] Four concentration points were taken near the lowest concentration of the linear range of the target in the method, which were 2E-3 amol / μL, E-3 amol / μL, 2E-4 amol / μL, and E-4 amol / μL, respectively. Six repeated holes were made for each concentration point, and a total of three repeated experiments were made. Each plate experiment was accompanied by a standard curve group, and RNase free H2O was used as a system negative control. The reaction system and reaction procedure in Table 3 and Table 5 above were used for amplification.

[0181] The amplification spectrum of four repeated experiments is shown in Figure 5 The CT value, recovery rate, and RSD value of four repeated experiments are shown in Table 8.

[0182] Table 8: The lowest quantification limit of qPCR-TRAP

[0183]

[0184] As shown in Figure 4 all detection holes can be lined up (4 concentrations, 6 repeated holes for each concentration) in three experiments, and the trend of lining up is consistent. From the recovery rate and RSD value, when the concentration is 0.2 TPG / reaction, the recovery rate is less than 75.00%, and the RSD value exceeds 25%. When the concentration is 0.4 TPG / reaction and above, both the recovery rate and the RSD value are within the effective range. Therefore, the lowest quantification limit of the method is 0.2 TPG / μL, i.e. 2E-4 amol / μL.

[0185] Example 6: Verification of durability

[0186] To verify the applicability of different manufacturers' synthetic primers and different qPCR instrument models to the present method, we selected different primer synthesis manufacturers, Biolline and Shenguo, and different instrument models, ABI QuantStudio 1 plus and Bole CFX96 Touch. Three concentration points near the lowest quantification limit of the target of the present method were taken, i.e., E-3 amol / μL, 5E-4 amol / μL, and 2E-4 amol / μL, and 8 repeated holes were made for each concentration point. Each plate experiment was accompanied by a standard curve group, and RNase free H2O was used as a system negative control, and the reaction system and reaction procedure in Table 3 and Table 5 above were used for amplification.

[0187] The amplification graphs of 2 experiments are shown in Figure 6 Table 9, and the CT values, recovery rates, and RSD values of 2 experiments are shown in Table 9.

[0188] Table 9 qPCR-TRAP robustness

[0189] The results show that, in different instrument models, different manufacturers' primers are used for testing. The RSD value of the target CT value is ≤30%, and the average fluctuation of the target recovery rate is ≤±30%, which meets the requirements. It is proved that the present detection method has good robustness.

[0190] Example 7: detection sensitivity verification

[0191] In the present example, real cell telomerase extraction samples are used to verify the detection sensitivity of the primer combination of the present application. 10 6 cells of A549 cells in the cell bank of the present company are taken, and telomerase is extracted by the same lysis method as described above. After 2 μL of the lysis supernatant is diluted by 10 times, 100 times, 1000 times, 10000 times, 20000 times, and 100000 times, it is added for detection, i.e., equivalent to 1000, 100, 10, 1, 0.5, and 0.1 cells of telomerase extract per reaction. Four repeated holes are set for each concentration, and the corresponding heat inactivation control is set, and a total of 4 repeated experiments are performed. Each plate experiment is accompanied by a standard curve group, and CHAPS lysis solution is used as a system negative control, and the reaction system and reaction procedure in Table 3 and Table 5 above are used for amplification.

[0192] The amplification graphs of 4 experiments are shown in Figure 7 Table 10, and the recovery rates and RSD values of 4 experiments are shown in Table 10.

[0193] Table 10 qPCR-TRAP detection sensitivity

[0194] The results show that the detection method can detect 1 cell of telomerase activity in four experiments, but the recovery rate does not meet the requirements. When there are 10 cells, the RSD value of the target CT value is ≤20%, and the average value of the recovery rate fluctuates ≤±50%, which meets the requirements. Therefore, the detection sensitivity of the method can be as low as 10 A549 cells / reaction.

[0195] Example 8: Verification of specificity

[0196] This example uses known negative samples (MSC and MRC5 cells) and positive samples (A549, 293, iPSC and Hela cells) to verify the specificity of the method. Four repeated holes are set for each sample, and the corresponding heat inactivation control group is also set. The standard curve group is used in the experiment, and CHAPS lysis solution is used as the system negative control. The reaction system and reaction procedure in Tables 3 and 5 above are used for amplification.

[0197] The specificity amplification map is shown in Figure 8 The CT values of the target and the internal reference are shown in Table 11.

[0198] Table 11 qPCR-TRAP specificity

[0199]

[0200] The experimental results show that the target CT value of the telomerase positive sample is <30, the target CT value of the telomerase negative sample is >32, and the ΔCT value of the internal reference gene detection fluctuates within ±0.5 CT value of the average value of the NTC and the standard curve group. At the same time, the CT value of all samples of the target heat inactivation control group is greater than 32, and the ΔCT value of the internal reference gene detection also fluctuates within ±0.5 CT value of the average value of the NTC and the standard curve group. It shows that the specificity of the method is good.

[0201] Example 9: Verification of accuracy of internal reference monitoring

[0202] Because there may be PCR inhibitors in the sample extraction solution, an internal reference is needed to monitor whether there is a qPCR inhibition effect during PCR. A549 and iPSC cells, and the corresponding heat inactivation samples, are taken, and the sample extraction solution is diluted by 0, 10, 20 and 40 times, respectively. Four repeats are set for each sample, and each experiment is performed once. The standard curve group is used in the two experiments, and CHAPS lysis solution is used as the system negative control. The reaction system and reaction procedure in Tables 3 and 5 above are used for amplification.

[0203] The internal reference monitoring accuracy amplification map is shown in Figure 9As shown, the TPG value of the target, the recovery rate and the CT value and the ΔCT value of the internal reference are shown in Table 12.

[0204] Table 12 Accuracy of internal reference monitoring of qPCR-TRAP

[0205]

[0206] The results show that the dilution recovery rate of the A549 sample extract is only 68.23% without dilution, which is far less than 90%, indicating that there is a qPCR inhibition effect, and the ΔCT value of the internal reference detection is greater than 0.5 CT values. The dilution recovery rates of the samples diluted by 10, 20 and 40 times are all within the range of 100%±10%, indicating that there is no qPCR inhibition effect, and the ΔCT value of the internal reference detection is also less than 0.5 CT values. The dilution recovery rate of the A549 sample extract is only 70.18% without dilution, which is far less than 90%, indicating that there is a qPCR inhibition effect, and the ΔCT value of the internal reference detection of the heat inactivated control group is greater than 0.5 CT values. The dilution recovery rates of the samples diluted by 10, 20 and 40 times are all within the range of 100%±10%, indicating that there is no qPCR inhibition effect, and the ΔCT value of the internal reference detection is also less than 0.5 CT values. Therefore, the internal reference monitoring system of the method has good accuracy and can effectively monitor the possible qPCR inhibition effect in the telomerase extract of the sample.

[0207] In summary, the primer combination provided by the application can accurately, reliably and conveniently detect the telomerase activity in the sample cells, and the exact value is represented in the form of TPG. The method provided by the application can be beneficial to researchers and industry practitioners in the related field to quickly compare the telomerase activity of different cell samples, and then evaluate the potential tumorigenic risk or cancer risk. The method has important significance for the fields of cell aging, tumor diagnosis, stem cell drug research and development, etc. The method is not only suitable for detecting the telomerase activity of human cells, but also suitable for other vertebrates and even all animal samples with the same telomere repeat sequence "TTAGGG" as humans.

[0208] The above specific embodiments describe the implementation of the application in detail, but the application is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concepts of the application, the technical solutions of the application can be modified and changed in many simple ways, and these simple changes all belong to the protection scope of the application.

Claims

1. A primer combination for quantitative detection of cellular telomerase activity, characterized in that, The primer combination comprises primer TS, primer ACX-UP1, internal reference primer NT-UP1 and internal reference standard; the nucleotide sequence of the primer TS is shown as SEQ ID NO. 1, the nucleotide sequence of the primer ACX-UP1 is shown as SEQ ID NO. 2; the nucleotide sequence of the internal reference primer NT-UP1 is shown as SEQ ID NO. 5, and the nucleotide sequence of the internal reference standard is shown as SEQ ID NO. 7; The stem loop structure is arranged on the primer ACX-UP1 and the internal reference primer NT-UP1, and the first fluorescent group and the quenching group are respectively arranged at two ends of the stem loop structure of the primer ACX-UP1; the second fluorescent group and the quenching group are respectively arranged at two ends of the stem loop structure of the internal reference primer NT-UP1; the first fluorescent group and the second fluorescent group are different types of fluorescent groups.

2. The primer combination for quantitatively detecting the activity of a cell telomerase according to claim 1, wherein The first fluorescent group and the second fluorescent group are FAM, Texas Red, CY3, CY5, VIC, TET, NED, ROX, JOE or HEX, and the quenching group is TAMRA, NFQ, ECLIPSE, DABCYL, MGB, BHQ1 or BHQ2. Further, the first fluorescent group is a FAM group, the second fluorescent group is a CY5 group, and the quenching group is Dabcyl.

3. A product for the quantitative detection of cellular telomerase activity, characterized in that, The product comprises the primer combination of claim 1 or 2.

4. The product for quantitatively detecting the activity of cell telomerase according to claim 3, wherein The product further comprises negative control cells, positive control cells and target quantification standard; the nucleotide sequence of the target in the target quantification standard is shown as SEQ ID NO.

4.

5. The product for quantitatively detecting the activity of cell telomerase according to claim 4, wherein The product further comprises PCR reaction solution, RNase free H2O, cell telomerase lysis solution and reference fluorescent dye; the negative control cells are MSC cells and / or MRC5 cells, and the positive control cells are any one or more of A549 cells, 293 cells, iPSC cells and Hela cells.

6. Use of a product according to any one of claims 3 to 5, characterized in that, The product is used for quantitatively detecting cell telomerase activity, and the cells are cells of a species containing telomere repeat sequences of "TTAGGG"; Further, the cells are cells of any one species belonging to the phylum Chordata; Further, the cells are human cells.

7. A method for quantitatively detecting the activity of a cellular telomerase, characterized by, The product of any one of claims 3-5 is used, comprising the following steps: A predetermined number of cells to be tested are collected, and the cells to be tested, negative control cells and positive control cells are treated with cell telomerase lysis solution, centrifuged and the supernatant is reserved; A predetermined amount of the supernatant of the cells to be tested is heated to inactivate, as a heat inactivation control; The target quantification standard is gradiently diluted to obtain target quantification standard working solution at each concentration, PCR amplification is performed, the logarithm of the concentration of the target quantification standard working solution with a base of 10 is taken as the abscissa, and the CT value is taken as the ordinate, and a target standard curve is drawn; The reference fluorescent dye, primer TS, primer ACX-UP1, internal reference standard and internal reference primer NT-UP1 are prepared to configure a reaction system; The cell telomerase lysis solution, each ladder concentration of the target quantitative standard, the supernatant of the cell to be detected, the supernatant of the positive control cell, the supernatant of the negative control cell and the heat inactivated control are added into the reaction system respectively, PCR amplification is carried out, detection is carried out, and the CT values of the system negative control group, the standard curve group, the sample group to be detected, the positive control group, the negative control group and the heat inactivated control group are obtained respectively; the positive control group is recorded as the positive quality control, and the negative control group and the heat inactivated control group are recorded as the negative quality control; It is confirmed that the positive quality control, the negative quality control and the system negative control group are normal; whether the telomerase activity detection system of the cell to be detected is inhibited is judged according to the difference between the CT value of the internal reference channel of the cell sample to be detected and the average CT value of the standard curve group and the system negative control group; and the telomerase activity of the cell to be detected is calculated according to the target standard curve.

8. The method for quantitatively detecting the activity of a cell telomerase according to claim 7, wherein The method for judging whether the telomerase activity detection system of the cell to be detected is inhibited by comparing the CT values is as follows: When the CT value of the internal reference channel of the cell sample to be detected and the average CT value of the standard curve group and the system negative control group differ by no more than 0.5 cycles, the cell sample to be detected does not have an inhibitory effect on the reaction system; When the CT value of the internal reference channel of the cell sample to be detected and the average CT value of the standard curve group and the system negative control group differ by more than 0.5 cycles, the supernatant of the cell to be detected has an inhibitory effect on the reaction system.

9. The method for quantitatively detecting the activity of a cell telomerase according to claim 7, wherein The method for calculating the telomerase activity of the cell to be detected through the target standard curve is as follows: the target CT value of the cell to be detected is substituted into the target standard curve, and the telomerase activity of the cell to be detected can be calculated.

10. The method for quantitatively detecting the activity of a cell telomerase according to claim 7, wherein The method for confirming whether the positive quality control, the negative quality control and the system negative control group are normal is as follows: When the CT value of the internal reference channel and the average CT value of the standard curve group, the negative quality control and the system negative control group differ by no more than 0.5 cycles, and the CT value of the target channel is ≤ 26, the positive quality control is normal; When the CT value of the internal reference channel and the average CT value of the standard curve group, the negative quality control and the system negative control group differ by no more than 0.5 cycles, and the CT value of the target channel is > 32, the negative quality control and the system negative control group are normal; if the CT value of the target channel is ≤ 32, it indicates that the reaction system is contaminated.

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