Telomerase activity digital pcr detection kit and detection method

Abstract

The invention discloses a telomerase activity digital pcr detection kit and a detection method, and particularly relates to the field of gene detection.The telomerase activity digital pcr detection kit comprises a PCR reaction solution, a telomerase primer, a telomerase reverse transcription buffer solution and a magnetic bead system component, and the PCR reaction solution comprises dNTPs, a probe, a signal enzyme and a signal enzyme substrate. According to the invention, a PCR (polymerase chain reaction) detection technology is utilized, and the same sample can be simultaneously subjected to parallel detection by virtue of two different enzymes, namely glucosidic bond hydrolase and phosphate bond hydrolase, and three corresponding different enzyme substrates through three methods, namely a fluorescence detection method, a colorimetric detection method and an ATP (adenosine triphosphate) detection method based on different detection principles; false positive results and false negative results can be avoided, so that the detection result is more credible, and the method can be used for rapid determination of telomerase activity and determination of single cell level.

Description

Technical Field

[0001] The invention relates to the technical field of gene detection, and more specifically to a telomerase activity digital PCR detection kit and a detection method. Background Art

[0002] Telomerase is a reverse transcription DNA synthase that is responsible for the extension of telomeres and plays an important role in cells. Telomeres are special structures at the ends of eukaryotic chromosomes, consisting of 6 base repeat sequences and binding proteins. They have important biological functions, such as stabilizing chromosome function, preventing chromosome DNA degradation and terminal fusion, protecting chromosome structural genes, and regulating normal cell growth. Telomerase can extend and shorten telomeres, thereby enhancing the proliferation capacity of cells in vitro. The activity of telomerase is inhibited in normal human tissues and reactivated in tumors, which may be involved in malignant transformation. Therefore, the study of telomerase is of great significance to the occurrence and development of diseases such as cell aging and carcinogenesis.

[0003] However, in the existing telomerase activity detection process, a one-way method is used for detection, which is prone to false positive results and false negative results, making the detection results less reliable. Therefore, a telomerase activity digital PCR detection kit and a detection method are proposed. Summary of the invention

[0004] In order to overcome the above-mentioned defects of the prior art, the present invention provides a telomerase activity digital PCR detection kit and a detection method to solve the problems raised in the above-mentioned background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a telomerase activity digital PCR detection kit, comprising a PCR reaction solution, a telomerase primer, a telomerase reverse transcription buffer and a magnetic bead system component.

[0006] Preferably, the PCR reaction solution includes dNTPs, a probe, a signal enzyme and a signal enzyme substrate.

[0007] Preferably, the magnetic bead system components include magnetic beads, a first oligonucleotide and a second oligonucleotide, and the signal enzyme can be linked to the second oligonucleotide.

[0008] Preferably, the sequence of the telomerase substrate primer is 5'-AATCCGTCGAGCAGAGTT-3' (SEQ ID NO: 1); the sequence of the first oligonucleotide is 5'-TTAGGGTTAGGGTTAGGG-3' (SEQ ID NO: 2), and the sequence of the second oligonucleotide is 5'-CCCTAACCCTAACCCTAACCCTAACCCTAA-3' (SEQ ID NO: 3).

[0009] Preferably, the signal enzyme is a glycosidic bond hydrolase, and the substrate is a non-fluorescent fluorescein derivative containing a glycosidic bond.

[0010] Preferably, the glycosidic bond hydrolase is galactosidase, and the substrate is fluorescein 2-β-D-galactopyranoside, or the glycosidic bond hydrolase is cellulase, and the substrate is fluorescein 2-β-D-cellobioside.

[0011] Preferably, the signal enzyme is a phosphate bond hydrolase, the substrate is a compound containing a phosphate bond, the phosphate bond hydrolase is alkaline phosphatase, and the substrate is p-nitrophenyl phosphate or adenosine triphosphate.

[0012] A method for detecting telomerase activity using a telomerase activity digital PCR detection kit comprises the following steps:

[0013] S1: Obtain telomerase samples;

[0014] S2: adding telomerase primers, dNTPs, and telomerase reverse transcription buffer to the telomerase sample to react under the condition of pH 6.0-8.0, adding a stop solution to terminate the extension reaction of telomerase, cooling down to obtain an extension reaction product, thereby obtaining a telomere repeat sequence;

[0015] S3: After hybridization reaction, each group in the magnetic bead system components is connected with the signal enzyme to form a magnetic bead system-signal enzyme complex;

[0016] S4: hybridizing the magnetic bead system-signal enzyme complex with the obtained telomeric repeat sequence to form the magnetic bead system-signal enzyme complex with the corresponding second oligonucleotide-signal enzyme linker removed, and separating;

[0017] S5: Finally, the separated telomeric repeat sequence-second oligonucleotide-signal enzyme linker hybrid is subjected to an enzymatic reaction with the substrate of the signal enzyme, and the signal generated by the enzymatic reaction is measured, wherein the size of the signal reflects the size of the telomerase activity.

[0018] Preferably, during the reaction in S2, the final concentration of the telomerase primer is 10 uM, and the final concentration of dNTPs is 2 mM.

[0019] Beneficial effects of the present invention:

[0020] The present invention provides a digital PCR detection kit and detection method for telomerase activity, which utilizes PCR detection technology, through two different enzymes, glycosidic bond hydrolase and phosphate bond hydrolase, and three different corresponding enzyme substrates, and can respectively perform parallel detection on the same sample through three methods based on different detection principles, namely fluorescence detection method, colorimetric detection method and ATP detection method, thereby avoiding false positive results and false negative results, making the detection results more credible, and can be used for rapid determination of telomerase activity and determination at the single cell level. DETAILED DESCRIPTION

[0021] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0022] The present invention provides a telomerase activity digital PCR detection kit, which is prepared by modifying magnetic beads with streptavidin to form streptavidin-modified magnetic beads. The first oligonucleotide sequence is modified with biotin to form a biotin-modified first oligonucleotide. The second oligonucleotide sequence is modified with biotin to form a biotin-modified second oligonucleotide. The second oligonucleotide sequence is designed to be at least partially hybridizable with the first oligonucleotide sequence. Beta-galactosidase is modified with streptavidin to form streptavidin-modified beta-galactosidase. Alternatively, alkaline phosphatase is modified with streptavidin to form streptavidin-modified alkaline phosphatase;

[0023] Then, the magnetic beads are subjected to a ligation reaction with the first oligonucleotide to form magnetic beads functionalized with the first oligonucleotide, and the magnetic beads functionalized with the first oligonucleotide are subjected to a hybridization reaction with the second oligonucleotide to hybridize the first oligonucleotide with the second oligonucleotide to form magnetic beads functionalized with the first oligonucleotide and the second oligonucleotide. The magnetic beads functionalized with the first oligonucleotide and the second oligonucleotide are subjected to a ligation reaction with β-galactosidase to connect the second oligonucleotide in the magnetic beads with β-galactosidase to form a second oligonucleotide-β-galactosidase linker, thereby forming a magnetic bead system-β-galactosidase complex; or, the magnetic beads functionalized with the first oligonucleotide and the second oligonucleotide are subjected to a ligation reaction with alkaline phosphatase modified with streptavidin to connect the second oligonucleotide in the magnetic beads with alkaline phosphatase to form a second oligonucleotide-alkaline phosphatase linker, thereby forming a magnetic bead system-alkaline phosphatase complex;

[0024] In the process of fluorescence test, the telomerase extension reaction product and the magnetic bead system-galactosidase complex are subjected to competitive hybridization reaction, the telomerase extension reaction product and the first oligonucleotide compete with the second oligonucleotide for binding, the second oligonucleotide-galactosidase linker is detached from the magnetic bead, and a telomere repeat sequence-second oligonucleotide-galactosidase linker hybrid is formed, and a magnetic bead system-galactosidase complex with the corresponding second oligonucleotide-galactosidase linker removed is formed. The amount of the telomere repeat sequence-second oligonucleotide-galactosidase linker hybrid formed depends on the concentration of the telomerase extension reaction product, that is, depends on the concentration of telomerase in the sample to be tested. The magnetic bead system-galactosidase complex with the corresponding second oligonucleotide-galactosidase linker removed is separated from the telomere repeat sequence-second oligonucleotide-galactosidase linker hybrid by magnetic force, and the separated magnetic bead system can be recycled. Adding fluorescein 2-β-D-pyranogalactoside to the separated telomeric repeat sequence-second oligonucleotide-galactosidase linker hybrid, galactosidase can catalyze fluorescein 2-β-D-pyranogalactoside to form fluorescein, thereby emitting fluorescence. By detecting the intensity of the fluorescence, the concentration of telomerase in the sample to be tested can be reflected;

[0025] In the process of colorimetric testing, the telomerase extension reaction product and the magnetic bead system-alkaline phosphatase complex are subjected to competitive hybridization reaction, the telomerase extension reaction product and the first oligonucleotide compete with the second oligonucleotide for binding, the second oligonucleotide-alkaline phosphatase linker is detached from the magnetic beads, and a telomere repeat sequence-second oligonucleotide-alkaline phosphatase linker hybrid is formed, and a magnetic bead system-alkaline phosphatase complex with the corresponding second oligonucleotide-alkaline phosphatase linker removed is formed. The amount of the telomere repeat sequence-second oligonucleotide-alkaline phosphatase linker hybrid formed depends on the concentration of the telomerase extension reaction product, that is, depends on the concentration of telomerase in the sample to be tested. The magnetic bead system-alkaline phosphatase complex with the corresponding second oligonucleotide-alkaline phosphatase linker removed is separated from the telomere repeat sequence-second oligonucleotide-alkaline phosphatase linker hybrid by magnetic force, and the separated magnetic bead system can be recycled. p-nitrophenyl phosphate is added to the isolated telomeric repeat sequence-second oligonucleotide-alkaline phosphatase linker hybrid, and alkaline phosphatase can catalyze nitrophenyl phosphate to form dark yellow p-nitrophenol. The absorbance of the reaction system is measured by colorimetric determination, which can reflect the concentration of telomerase in the sample to be tested.

[0026] The sequence of the telomerase substrate primer is 5'-AATCCGTCGAGCAGAGTT-3' (SEQ ID NO: 1); the sequence of the first oligonucleotide is 5'-TTAGGGTTAGGGTTAGGG-3' (SEQ ID NO: 2), and the sequence of the second oligonucleotide is 5'-CCCTAACCCTAACCCTAACCCTAACCCTAA-3' (SEQ ID NO: 3).

[0027] The signal enzyme is a glycosidic bond hydrolase, and the substrate is a non-fluorescent fluorescein derivative containing a glycosidic bond.

[0028] The glycosidic bond hydrolase is galactosidase, and the substrate is fluorescein 2-β-D-galactopyranoside, or the glycosidic bond hydrolase is cellulase, and the substrate is fluorescein 2-β-D-cellobiose glycoside.

[0029] The signal enzyme is a phosphate bond hydrolase, the substrate is a compound containing a phosphate bond, the phosphate bond hydrolase is alkaline phosphatase, and the substrate is p-nitrophenyl phosphate or adenosine triphosphate.

[0030] A method for detecting telomerase activity using a telomerase activity digital PCR detection kit comprises the following steps:

[0031] S1: Obtain telomerase samples;

[0032] In the specific implementation, the extraction of telomerase: add 4mL of culture medium to the cell culture dish, take out about 300uL of the cell solution in the centrifuge tube, add it to the culture dish, shake gently to evenly distribute the cells, and put it into the incubator to grow to maintain HeLa, MCF-7 and L02 cells. Collect various cells during the exponential growth phase and wash twice with ice-cold PBS buffer (0.01M, pH 7.4), and then resuspend the cells in 200μL ice-cold CHAPS lysis buffer (10mMpH7.5Tris-HCl, 1mMMgCl2, 1mM EGTA, 0.5% CHAPS, 10% glycerol, 0.1mM PMSF). Incubate the lysate in an ice water bath for 10min, and then centrifuge at 15000rpm for 30min at 4°C. Finally, collect the supernatant and carefully transfer it to a fresh RNase-free test tube and store it at -80°C for later use.

[0033] S2: adding telomerase primers, dNTPs, and telomerase reverse transcription buffer to the telomerase sample to react under the condition of pH 6.0-8.0, adding a stop solution to terminate the extension reaction of telomerase, cooling down to obtain an extension reaction product, thereby obtaining a telomere repeat sequence;

[0034] In the specific implementation, 20uL of 50uM telomerase primer sequence solution is taken, and the specific telomerase primer sequence is shown in SEQ ID NO: 1, 2uL of 100mM dNTPs solution and 48uL of telomerase reverse transcription buffer (20mM Tris-HCl, pH8.3, 1mM EDTA, 0.05% Tween 20, 63mM KCl, 0.1mg / mL BSA, 1.5mMMgCl2) and 30uL of the telomerase mixture extracted in step 1 are added, mixed evenly, and placed on a shaker at 500rpm and 37°C for reaction for 1h; after the reaction time, 10uL of 1% mass ratio SDS denaturant solution is added to the prepared solution of the extended product to terminate the extension reaction of telomerase, and the product is placed at 4°C for cooling for 30min to obtain the final extension product;

[0035] S3: After hybridization reaction, each group in the magnetic bead system components is connected with the signal enzyme to form a magnetic bead system-signal enzyme complex;

[0036] S4: hybridizing the magnetic bead system-signal enzyme complex with the obtained telomeric repeat sequence to form the magnetic bead system-signal enzyme complex with the corresponding second oligonucleotide-signal enzyme linker removed, and separating;

[0037] In specific implementation, telomerase activity detection based on streptavidin-conjugated β-galactosidase (SA-Gala) was performed. An appropriate amount of magnetic beads (DynabeadsTM MyOneTM streptavidin C1) was washed three times with 400 μL Tris-HCl (20 mM, pH 7.40, containing 135 mM NaCl). Then, in the first step, the magnetic beads and the first oligonucleotide (5'-TTAGGGTTAGGGTTAGGG-biotin-3') were added to 400 μL Tris-HCl buffer at a ratio of 1:1, and then mixed and the mixture was placed on a 37°C shaker for 30 minutes. The liquid was discarded and washed three times with 400 μL Tris-HCl. In the second step, the reaction product and the second oligonucleotide (5'-CCCTAACCCTAACCCTAACCCTAACCCTAA-biotin-3') were added to 400 μL Tris-HCl at a ratio of 3.2:1, and then mixed and the resulting mixture was placed on a 37°C shaker for 30 minutes. In the third step, the product of the second step and streptavidin-modified β-galactosidase (SA-Gala) were added to 400 μL Tris-HCl buffer at a ratio of 3.2:1, and then mixed and the resulting mixture was placed on a 37°C oscillator for 30 minutes. In the fourth step, the reaction mixture of the third step and different concentrations of telomerase extension reaction products were added to Tris-HCl, and the resulting mixture was placed on a 37°C oscillator for 30 minutes. In the fifth step, after magnetic separation, the supernatant was collected and divided into four tubes, each with 100 μL. 200 μL Tris-HCl was added to each tube, followed by 5 μL FDG (15 μM) and incubated at 37°C for 30 minutes. Finally, all fluorescence spectra were collected at an excitation wavelength of 480 nm and an emission wavelength of 500-660 nm;

[0038] S5: finally, subjecting the separated telomere repeat sequence-second oligonucleotide-signal enzyme linker hybrid to an enzymatic reaction with a substrate of the signal enzyme, and determining a signal generated by the enzymatic reaction, wherein the magnitude of the signal reflects the magnitude of the telomerase activity;

[0039] In specific implementation, for the SA-ALP-based telomerase activity detection, the previous steps are the same as the SA-Gala-based telomerase activity detection, except that in the third step, the product of the second step and SA-ALP are added in a ratio of 6.6:1, and in the fifth step, two different detection modes are adopted. One detection mode is to collect the supernatant and divide it into four tubes, 100 μL in each tube. 500 μL Tris-HCl is added to each tube, followed by 12 μL pNPP (5 mM) and incubated at 37°C for 30 minutes. Finally, the UV-visible spectrum is measured, with a scanning wavelength range of 450 to 260 nm. Another detection mode is to take 100 μL of supernatant and add 100 μL Tris-HCl to a final volume of 200 μL. Then 20 μL ATP (100 μM) is added and incubated at 37°C for 30 minutes. ATP is measured by an ATP bioluminescence detector.

[0040] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A digital PCR detection kit for telomerase activity, characterized in that: The invention comprises PCR reaction solution, telomerase primer, telomerase reverse transcription buffer and magnetic bead system components.

2. A telomerase activity digital PCR detection kit and detection method according to claim 1, characterized in that: The PCR reaction solution includes dNTPs, a probe, a signal enzyme and a signal enzyme substrate.

3. A telomerase activity digital PCR detection kit according to claim 2, characterized in that: The magnetic bead system components include magnetic beads, a first oligonucleotide and a second oligonucleotide, and the signal enzyme can be connected to the second oligonucleotide.

4. A telomerase activity digital PCR detection kit according to claim 3, characterized in that: The sequence of the telomerase substrate primer is 5'-AATCCGTCGAGCAGAGTT-3' (SEQ ID NO: 1); the sequence of the first oligonucleotide is 5'-TTAGGGTTAGGGTTAGGG-3' (SEQ ID NO: 2), and the sequence of the second oligonucleotide is 5'-CCCTAACCCTAACCCTAACCCTAACCCTAA-3' (SEQ ID NO: 3).

5. A telomerase activity digital PCR detection kit according to claim 4, characterized in that: The signal enzyme is a glycosidic bond hydrolase, and the substrate is a non-fluorescent fluorescein derivative containing a glycosidic bond.

6. A telomerase activity digital PCR detection kit and detection method according to claim 5, characterized in that: The glycosidic bond hydrolase is galactosidase, and the substrate is fluorescein 2-β-D-galactopyranoside, or the glycosidic bond hydrolase is cellulase, and the substrate is fluorescein 2-β-D-cellobiose glycoside.

7. A telomerase activity digital PCR detection kit according to claim 6, characterized in that: The signal enzyme is a phosphate bond hydrolase, the substrate is a compound containing a phosphate bond, the phosphate bond hydrolase is alkaline phosphatase, and the substrate is p-nitrophenyl phosphate or adenosine triphosphate.

8. The method for detecting telomerase activity according to the telomerase activity digital PCR detection kit according to claims 1-7, characterized in that: The following steps are involved: S1: Obtain telomerase samples; S2: adding telomerase primers, dNTPs, and telomerase reverse transcription buffer to the telomerase sample to react under the condition of pH 6.0-8.0, adding a stop solution to terminate the extension reaction of telomerase, cooling down to obtain an extension reaction product, thereby obtaining a telomere repeat sequence; S3: After hybridization reaction, each group in the magnetic bead system components is connected with the signal enzyme to form a magnetic bead system-signal enzyme complex; S4: hybridizing the magnetic bead system-signal enzyme complex with the obtained telomeric repeat sequence to form the magnetic bead system-signal enzyme complex with the corresponding second oligonucleotide-signal enzyme linker removed, and separating; S5: Finally, the separated telomeric repeat sequence-second oligonucleotide-signal enzyme linker hybrid is subjected to an enzymatic reaction with the substrate of the signal enzyme, and the signal generated by the enzymatic reaction is measured, wherein the size of the signal reflects the size of the telomerase activity.

9. The method for detecting telomerase activity using a telomerase activity digital PCR detection kit according to claim 8, characterized in that: During the reaction in S2, the final concentration of the telomerase primer is 10 uM, and the final concentration of dNTPs is 2 mM.