CN112410400A - Telomerase activity detection kit and telomerase activity detection method - Google Patents
Telomerase activity detection kit and telomerase activity detection method Download PDFInfo
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- CN112410400A CN112410400A CN201910776813.2A CN201910776813A CN112410400A CN 112410400 A CN112410400 A CN 112410400A CN 201910776813 A CN201910776813 A CN 201910776813A CN 112410400 A CN112410400 A CN 112410400A
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Abstract
The invention provides a telomerase activity detection kit, which comprises a telomerase extension system component, a magnetic bead system component, a signal generation system of signal enzyme and a substrate, and also provides a method for detecting the telomerase activity of a sample by using the detection kit. The detection kit and the detection method are a triple-mode system for visualizing and portable detection of the activity of telomerase based on a magnetic bead-enzyme system, and combine the advantages of magnetic separation of magnetic beads and amplification of enzymatic reaction signals. By using two different enzymes of glycosidic bond hydrolase and phosphoester bond hydrolase and three corresponding different enzyme substrates, the same sample can be simultaneously and parallelly detected by three methods based on different detection principles, namely a fluorescence detection method, a colorimetric detection method and an ATP detection method, so that a false positive result and a false negative result can be avoided, 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
The invention relates to the technical field of biochemical detection, in particular to a telomerase activity detection kit and a method for detecting telomerase activity in a sample to be detected by using the telomerase activity detection kit.
Background
Telomerase can add telomere repeat (TTAGGG) n to the end of telomere, and is crucial to maintenance and protection of telomere1-4. Telomerase has been found to be overexpressed in approximately 85-90% of human cancer cells, such as bladder, uterine, gastric, esophageal, breast, thyroid, colorectalSquamous cell carcinoma of larynx5-8Whereas telomerase activity is absent in normal tissues. Thus, telomerase is generally recognized as an important biomarker for human malignancies9-12. The simple, rapid and sensitive detection of the telomerase activity has important significance for early diagnosis of cancer.
Various strategies have been developed in the industry for telomerase activity detection. The Polymerase Chain Reaction (PCR) based Telomere Repeat Amplification Protocol (TRAP) is the most widely used assay with satisfactory sensitivity and specificity13-15. However, it still has some limitations, such as time-consuming operation, expensive equipment and false positive results. Other methods that do not employ an enzymatic amplification step, such as electrochemical, fluorescence, colorimetric, surface plasmon resonance, and electrochemiluminescence, have been commonly used for telomerase activity detection16-19. Among these methods, the fluorescence method is sensitive, convenient to operate, and suitable for real-time detection, but the fluorophore is susceptible to interference from the actual sample. Colorimetric assays are visible to the naked eye, but are limited by the color of the actual sample. Single mode assays are at risk of obtaining false positive and false negative results. Accordingly, a variety of detection systems based on dual mode signal readout have been developed to improve reliability and accuracy. To further simplify the testing process, portable sensors such as personal blood glucose meters, blood pressure meters, and pH meters have also been developed to quantitatively monitor telomerase activity, which is known as point-of-care testing (POCT)20-24. POCT is a quick-response, easy-to-operate, low-cost and portable method, can detect targets in complex samples, and is very suitable for preliminary diagnosis of diseases.
Despite the advances made in telomerase activity detection technology, there is still room for improvement.
Disclosure of Invention
The invention aims to improve the existing single-mode and double-mode telomerase activity detection strategies, provide a novel triple-mode for detecting the telomerase activity, avoid false positive results and false negative results and enable the detection results to be more credible.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the present invention provides a telomerase activity detection kit, which comprises the following components:
(1) telomerase extension system components, including telomerase substrate primers and dntps, for generation of telomeric repeat sequences;
(2) a magnetic bead system component comprising a magnetic bead, a first oligonucleotide, and a second oligonucleotide, the first oligonucleotide being attachable to the magnetic bead, the second oligonucleotide being at least partially hybridizable to the first oligonucleotide, and the second oligonucleotide being at least partially hybridizable to the telomere repeat sequence;
(3) a signal producing system component comprising a signaling enzyme and a substrate for the signaling enzyme, wherein the signaling enzyme can be linked to the second oligonucleotide.
The first oligonucleotide may be linked to the magnetic bead by means of a linkage known in the art. The biotin-streptavidin linking system is a linking system commonly used in the art. In some embodiments of the invention, the first oligonucleotide is linked to the magnetic bead via a biotin-streptavidin linking system.
In some preferred embodiments of the invention, the first oligonucleotide is a biotin-modified first oligonucleotide and the magnetic bead is a streptavidin-modified magnetic bead, whereby the first oligonucleotide and the magnetic bead are linked by a biotin-streptavidin linking system. The method of modifying the first oligonucleotide with biotin and the method of modifying the magnetic beads with streptavidin are well known in the art.
As used herein, a "signaling enzyme" refers to an enzyme that is capable of catalyzing its substrate to produce a detectable signal, which may be a fluorescence change signal, a color change signal, or an ATP change signal. The fluorescence change signal can be detected, for example, visually or by a fluorometer; the color change signal may be detected, for example, by colorimetry; the ATP change signal may be detected, for example, by an ATP meter.
The signal enzyme may be linked to the second oligonucleotide by means of a linkage known in the art. The biotin-streptavidin linking system is a linking system commonly used in the art. In some embodiments of the invention, the signaling enzyme is linked to the second oligonucleotide via a biotin-streptavidin linkage system.
In some preferred embodiments of the invention, the second oligonucleotide is a biotin-modified second oligonucleotide and the signaling enzyme is a streptavidin-modified signaling enzyme, whereby the second oligonucleotide and the signaling enzyme are linked by a biotin-streptavidin linking system. Methods for modifying the second oligonucleotide with biotin and methods for modifying the signaling enzyme with streptavidin are well known in the art.
In a specific embodiment of the invention, the telomerase substrate primer has the sequence 5'-AATCCGTCGAGCAGAGTT-3' (SEQ ID NO:1), the first oligonucleotide has the sequence 5'-TTAGGGTTAGGGTTAGGG-3' (SEQ ID NO:2), and the second oligonucleotide has the sequence 5'-CCCTAACCCTAACCCTAACCCTAACCCTAA-3' (SEQ ID NO: 3).
In some embodiments of the invention, the signaling enzyme may be a glycosidic bond hydrolase and the substrate may be a non-fluorescent fluorescein derivative comprising a glycosidic bond. Glycosidic bond hydrolases are a class of enzymes that hydrolyze the glycosidic bond of a substrate. In some preferred embodiments of the invention, the glycosidic bond hydrolase is a galactosidase and the substrate is fluorescein 2- β -D-galactopyranoside (FDG). FDG releases fluorescein upon catalytic hydrolysis by galactosidase, causing a change in fluorescence, producing a fluorescent signal that can be visualized or detected. In other preferred embodiments of the present invention, the glycosidic bond hydrolase is a cellulase and the substrate is fluorescein 2- β -D-cellobioside (FCB). FCB releases fluorescein upon catalytic hydrolysis of cellulase, thereby causing a change in fluorescence, producing a fluorescent signal that can be visualized or detected.
In other embodiments of the invention, the signaling enzyme may be a phosphoester bond hydrolase and the substrate may be a compound comprising a phosphoester bond. Phosphoester bond hydrolases are a class of enzymes that hydrolyze the phosphoester bonds of a substrate. In some preferred embodiments of the invention, the phosphoester bond hydrolase is alkaline phosphatase and the substrate is p-nitrophenyl phosphate (pNPP). pNPP produces p-nitrophenol (pNP) in a deep yellow color upon catalytic hydrolysis by alkaline phosphatase, causing a color change that produces a signal that can be detected colorimetrically. In other preferred embodiments of the invention, the phosphoester bond hydrolase is alkaline phosphatase and the substrate is Adenosine Triphosphate (ATP). ATP produces Adenosine Monophosphate (AMP) upon catalytic hydrolysis by alkaline phosphatase, causing a reduction in adenosine triphosphate, producing a signal that can be detected by an ATP meter.
In a second aspect, the present invention provides a method for detecting telomerase activity by the telomerase activity detection kit of the first aspect of the present invention, the method comprising the steps of:
(1) providing a sample to be detected, and carrying out telomerase extension reaction on the sample to be detected and the telomerase extension reaction component to generate a telomere repetitive sequence;
(2) ligating the magnetic beads of the components of the magnetic bead system with the first oligonucleotide to form first oligonucleotide-functionalized magnetic beads, hybridizing the first oligonucleotide-functionalized magnetic beads with the second oligonucleotide to form first and second oligonucleotide-functionalized magnetic beads, and ligating the first and second oligonucleotide-functionalized magnetic beads with the signaling enzyme to ligate the signaling enzyme with the second oligonucleotide to form second oligonucleotide-signaling enzyme linkers to form magnetic bead system-signaling enzyme complexes;
(3) carrying out competitive hybridization reaction on the magnetic bead system-signal enzyme compound and the telomere repetitive sequence to form a telomere repetitive sequence-second oligonucleotide-signal enzyme connector hybrid and form the magnetic bead system-signal enzyme compound without the corresponding second oligonucleotide-signal enzyme connector;
(4) separating the telomere repeat-second oligonucleotide-signaling enzyme linker hybrid from the bead system-signaling enzyme complex without the corresponding second oligonucleotide-signaling enzyme linker to form a separated telomere repeat-second oligonucleotide-signaling enzyme linker hybrid;
(5) enzymatically reacting the isolated telomeric repeat-second oligonucleotide-signaling enzyme linker hybrid with a substrate for the signaling enzyme, and determining a signal generated by the enzymatic reaction, wherein the magnitude of the signal reflects the magnitude of the telomerase activity.
The term "test sample" refers to any sample, preferably a biological sample, such as but not limited to, tissue cytosol, blood, body fluids, urine, etc., in which telomerase activity is to be measured. Methods for obtaining such samples are well known in the art, for example, by disrupting tissue or cells to obtain tissue cellular fluid, or by obtaining blood, body fluid, or urine by aspiration.
Telomerase is an enzyme that adds telomeric repeats (TTAGGG) n to the ends of telomeres. In step (1), the telomerase extension system including the telomerase substrate primer and dNTP is subjected to telomerase substrate primer extension in the presence of telomerase to generate telomere repetitive sequences, which is well known in the art, and relevant reaction conditions are also well known to those skilled in the art or obtained by limited experiments, such as adding KCl and buffer solution in the reaction system.
In step (2), the magnetic beads of the components of the magnetic bead system may be linked to the first oligonucleotide by methods well known to those skilled in the art, for example, the magnetic beads may be linked to the first oligonucleotide by a biotin-streptavidin linking system to form first oligonucleotide functionalized magnetic beads as described in the first aspect. Because the second oligonucleotide can at least partially hybridize to the first oligonucleotide, the first oligonucleotide-functionalized magnetic bead can undergo a hybridization reaction with the second oligonucleotide to form a first oligonucleotide and a second oligonucleotide-functionalized magnetic bead, under conditions that are well known to those skilled in the art or obtained by limited experimentation. Furthermore, the first oligonucleotide and the second oligonucleotide functionalized magnetic beads may be linked to a signaling enzyme by methods well known to those skilled in the art, for example, the second oligonucleotide of the first oligonucleotide and the second oligonucleotide functionalized magnetic beads may be linked to the signaling enzyme by a biotin-streptavidin linking system to form a second oligonucleotide-signaling enzyme linker, as described in the first aspect, thereby forming a magnetic bead system-signaling enzyme complex.
It should be noted that the above step (1) and step (2) do not specify a strict order of precedence. In the actual detection operation, it is possible to perform step (1) and then step (2), or perform step (2) and then step (1).
In step (3), since the second oligonucleotide can at least partially hybridize with the telomere repeat sequence, the telomere repeat sequence will compete with the first oligonucleotide in the magnetic bead system-signal enzyme complex for binding with the second oligonucleotide, i.e., a competitive hybridization reaction occurs, forming a telomere repeat sequence-second oligonucleotide-signal enzyme linker hybrid, and forming a magnetic bead system-signal enzyme complex with the corresponding second oligonucleotide-signal enzyme linker removed. The conditions for performing a competitive hybridization reaction of nucleic acid sequences are well known to those skilled in the art or obtained by limited experimentation.
In step (4), the magnetic bead system-signalase complex may be magnetically attracted, thereby separating the telomere repeat sequence-second oligonucleotide-signalase linker hybrid from the magnetic bead system-signalase complex from which the corresponding second oligonucleotide-signalase linker has been removed, to form a separated telomere repeat sequence-second oligonucleotide-signalase linker hybrid. Methods and conditions for performing magnetic separation are well known to those skilled in the art or obtained by limited experimentation.
In step (5), the signal enzyme in the separated telomere repeat sequence-second oligonucleotide-signal enzyme connector hybrid reacts with the substrate of the signal enzyme in an enzymatic reaction, and the size of the telomerase activity in the sample to be detected can be measured by measuring the size of the signal generated by the enzymatic reaction. The conditions of the enzymatic reaction and the detection of the signal are well known to the person skilled in the art or obtained by limited experimentation.
The signaling enzyme may be a glycosidic bond hydrolase, the corresponding substrate is a non-fluorescent fluorescein derivative comprising a glycosidic bond, the product of the enzymatic reaction is fluorescein, and the resulting signal is a change in fluorescence. Preferably, the glycosidic bond hydrolase is galactosidase and the substrate is fluorescein 2-beta-D-galactopyranoside, or the glycosidic bond hydrolase is cellulase and the substrate is fluorescein 2-beta-D-cellobioside.
The signal enzyme may also be a phosphoester bond hydrolase, preferably alkaline phosphatase, and the substrate is a compound containing a phosphoester bond. The signal is a change in color when the substrate is p-nitrophenyl phosphate, or a decrease in adenosine triphosphate content when the substrate is adenosine triphosphate.
The invention has the beneficial effects that:
the telomerase activity detection kit and the telomerase activity detection method using the telomerase activity detection kit are a triple-mode system for visualizing and portable detection of telomerase activity based on a magnetic bead-enzyme system, combine the advantages of magnetic bead separation and enzymatic reaction signal amplification, and can realize telomerase detection at a single cell level.
Although the telomerase activity assay method of the present invention can be carried out in conventional manner using reaction conditions which are conventional in the art in each step in the telomerase activity assay method of the present invention, the telomerase activity assay method of the present invention as a whole is uniquely innovative in that a triple assay mode is employed, which is different from the conventional single-mode or dual-mode assay modes. Specifically, by providing two different signaling enzymes, namely glycosidic bond hydrolase (e.g., galactosidase or cellulase) and phosphoester bond hydrolase (e.g., alkaline phosphatase), and correspondingly providing three different substrates, namely non-fluorescent fluorescein derivatives (e.g., fluorescein 2- β -D-galactopyranoside and fluorescein 2- β -D-cellobioside), nitrophenyl phosphate and adenosine triphosphate, in the same telomerase activity assay kit, the same sample can be tested simultaneously in parallel by three different detection principles, namely, fluorescence detection (visual detection or fluorometric detection), colorimetric detection and ATP detection (ATP measurement), respectively, thereby avoiding false positive results and false negative results and making the detection result more reliable.
Furthermore, two different enzymes and three different substrates are provided in the same telomerase activity detection kit, and detection flexibility is provided, i.e., triple detection mode (three substrates, three detection principles), double detection mode (two substrates and two detection principles) and single detection mode (one substrate and one detection principle) can be adopted as required, and the results of fluorescence measurement, colorimetric measurement and adenosine triphosphate measurement can be used alone or in combination to achieve optimal detection performance and make the results more convincing. In addition, if necessary, more than three kinds of signal enzymes can be designed, so that the detection means are enriched, and the flexibility and the convenience of detection are improved.
In addition, the telomerase activity detection method is suitable for rapid determination of telomerase activity. The telomerase activity detection method based on the fluorescence detection method and the colorimetric detection method is suitable for developing visual detection sensors, adopts an ATP counter, is convenient to carry and operate, is low in cost, has higher response sensitivity, and is suitable for developing bedside detection (POCT).
Drawings
FIG. 1 shows a schematic diagram of the detection principle of the triple mode of the telomerase activity detection kit and the telomerase activity detection method using the same according to the present invention;
fig. 2 shows experimental test results of feasibility of a triple detection mode of telomerase activity of the present invention, in which fig. 2A shows fluorescence spectra and photographs of fluorescein before (a) and after (b) hydrolysis of galactosidase (Gala); FIG. 2B shows UV/VIS spectra and photographs of p-nitrophenol (pNP) before (a) or after (B) hydrolysis of alkaline phosphatase (ALP); FIG. 2C shows ATP values read using an ATP meter;
FIG. 3 shows experimental test results of the effectiveness of the triple mode detection of telomerase activity of the present invention, in which FIG. 3A shows a linear relationship between fluorescein fluorescence intensity and the logarithm of the number of HeLa cells in the fluorometric mode, with an excitation wavelength of 480nm, and inset shows digital photographs of different telomerase concentrations (expressed as the number of cells) under a 365nm ultraviolet lamp; FIG. 3B shows the evolution of the fluorescence spectrum of fluorescein with increasing telomerase concentration (in cell number)Changing; FIG. 3C shows in the colorimetric assay mode, A311/A400Linear relationship between ratio and number of HeLa cells, inset shows digital photographs of different HeLa cell numbers; FIG. 3D shows the corresponding UV/VIS spectra at increasing telomerase concentrations; FIG. 3E shows a linear relationship between ATP values and the logarithm of the number of HeLa cells in the ATP assay mode;
fig. 4 shows experimental test results of the reliability of the triple detection mode of telomerase activity of the present invention, in which fig. 4A shows the detection results of telomerase activity of extracts of heat-inactivated HeLa cells in a heat-treatment control experiment; figure 4B shows the fluorescence intensity measurements in the presence of different concentrations of curcumin in the curcumin inhibitor treated control experiment;
FIG. 5 shows the results of telomerase activity detection in 12 urine samples from bladder cancer patients and normal individuals, where the upper panel shows the results of fluorescence assays of urine samples, the lower panel shows the results of colorimetric assays of urine samples, from left to right, from patient 1 to patient 10 and two normal individuals, all fluorescence assay photographs taken under a 365nm ultraviolet lamp.
Detailed Description
The present invention will be described in further detail below with reference to specific embodiments and accompanying drawings.
Fig. 1 shows a schematic diagram of the detection principle of the triple mode of the telomerase activity detection kit and the telomerase activity detection method using the telomerase activity detection kit of the present invention, and the detection principle is briefly described below with reference to fig. 1.
The Magnetic Beads (MB) are modified with Streptavidin (SA) to form streptavidin-modified magnetic beads (MB-SA). The first oligonucleotide sequence is modified with biotin (biotin) to form a biotin-modified first oligonucleotide (bio-oligo 1). The second oligonucleotide sequence is modified with biotin (biotin) to form a biotin-modified second oligonucleotide (bio-oligo 2). The second oligonucleotide sequence is designed to at least partially hybridize to the first oligonucleotide sequence. Galactosidase (Gala) is modified with Streptavidin (SA) to form streptavidin-modified galactosidase (SA-Gala). Alternatively, alkaline phosphatase (ALP) is modified with Streptavidin (SA) to form streptavidin-modified alkaline phosphatase (SA-ALP).
The MB-SA is subjected to a ligation reaction with the bio-oligo 1 to form a first oligonucleotide-functionalized magnetic bead. The first oligonucleotide-functionalized magnetic bead is subjected to a hybridization reaction with the bio-oligo 2 to hybridize the bio-oligo 1 with the bio-oligo 2, forming the first oligonucleotide-and second oligonucleotide-functionalized magnetic beads. Subjecting the first oligonucleotide-and second oligonucleotide-functionalized magnetic beads to a ligation reaction with SA-Gala to ligate the second oligonucleotide in the magnetic beads to galactosidase to form a second oligonucleotide-galactosidase conjugate, thereby forming a magnetic bead system-galactosidase complex; alternatively, the first oligonucleotide-and second oligonucleotide-functionalized magnetic beads are subjected to a ligation reaction with SA-ALP to ligate the second oligonucleotide in the magnetic beads to alkaline phosphatase to form a second oligonucleotide-alkaline phosphatase conjugate, thereby forming a magnetic bead system-alkaline phosphatase complex.
And (3) carrying out telomerase extension reaction on the to-be-detected sample containing telomerase in the presence of telomerase substrate primers (TS primers) and dNTP to form a telomerase extension reaction product. It is noted that the second oligonucleotide sequence described above is also designed to at least partially hybridize to the telomerase extension reaction product. And then, telomerase in a sample to be detected can be detected by adopting three modes.
In the first mode, the telomerase extension reaction product is subjected to competitive hybridization with the bead system-galactosidase complex, the telomerase extension reaction product competes with bio-oligo 1 for binding to bio-oligo 2, the second oligonucleotide-galactosidase linker is detached from the magnetic bead, forming a telomere repeat-second oligonucleotide-galactosidase linker hybrid, and forming a bead system-galactosidase complex with the corresponding second oligonucleotide-galactosidase linker removed. The amount of the formed telomeric repeat-second oligonucleotide-galactosidase linker hybrid depends on the concentration of the telomerase extension reaction product, i.e., on the concentration of telomerase in the sample to be tested. And separating the magnetic bead system-galactosidase complex without the corresponding second oligonucleotide-galactosidase connector from the telomere repetitive sequence-second oligonucleotide-galactosidase connector hybrid by magnetic force, wherein the separated magnetic bead system can be recycled. Adding fluorescein 2-beta-D-galactopyranoside (FDG) into the obtained telomere repetitive sequence-second oligonucleotide-galactosidase connector hybrid, wherein the galactosidase can catalyze the FDG to form fluorescein, thereby emitting fluorescence. By detecting the intensity of fluorescence, the concentration of telomerase in the sample to be detected can be reflected. Thus, this mode is a fluorescence measurement mode.
In the second mode, the telomerase extension reaction product is subjected to competitive hybridization with the magnetic bead system-alkaline phosphatase complex, the telomerase extension reaction product competes with bio-oligo 1 for binding to bio-oligo 2, the second oligonucleotide-alkaline phosphatase linker is detached from the magnetic beads, a telomere repeat-second oligonucleotide-alkaline phosphatase linker hybrid is formed, and the magnetic bead system-alkaline phosphatase complex from which the corresponding second oligonucleotide-alkaline phosphatase linker is detached is formed. The amount of formed telomeric repeat-second oligonucleotide-alkaline phosphatase linker hybrid is dependent on the concentration of the telomerase extension reaction product, i.e., on the concentration of telomerase in the sample to be tested. And separating the magnetic bead system-alkaline phosphatase complex from which the corresponding second oligonucleotide-alkaline phosphatase conjugate is removed from the telomere repeat sequence-second oligonucleotide-alkaline phosphatase conjugate hybrid by magnetic force, wherein the separated magnetic bead system can be recycled. P-nitrophenyl phosphate (pNPP) was added to the isolated telomere repeat-second oligonucleotide-alkaline phosphatase linker hybrid, and the alkaline phosphatase catalyzed the formation of p-nitrophenol (pNP) in a dark yellow color by the pNPP. The absorbance of the reaction system is measured by a colorimetric assay method, and the concentration of the telomerase in the sample to be detected can be reflected. Thus, this mode is a colorimetric assay mode.
In a third mode, operating as in the second mode, the telomere repeat-second oligonucleotide-alkaline phosphatase linker hybrid is isolated and then, except that ATP is added thereto, the alkaline phosphatase hydrolyzes the ATP to AMP, and the decrease in ATP is read using an ATP meter, which reflects the concentration of telomerase in the sample to be tested. Therefore, this mode is an ATP measurement mode.
Experiments prove that the telomerase activity detection kit and the telomerase activity detection method using the telomerase activity detection kit have feasibility, effectiveness, reliability and clinical applicability.
Materials and instruments
Magnetic beads (Dynabeads)TMMyOneTMStreptavidin C1) and ATP were obtained from Thermo Fisher Scientific (Invitrogen). Streptavidin-modified beta-galactosidase (SA-Gala) was obtained from Creative Enzymes. Streptavidin-modified alkaline phosphatase (SA-ALP) was obtained from Bioss Antibodies (Beijing biosyntheses Biotechnology Co., Ltd.). Fluorescein 2- β -D-galactopyranoside (FDG) was purchased from Sigma-Aldrich. P-nitrophenyl phosphate (pNPP) obtained from J&K Scientific。
The sequence of the telomerase substrate primer (TS primer) is 5'-AATCCGTCGAGCAGAGTT-3'. The sequence of the first oligonucleotide is 5 '-TTAGGGTTAGGGTTAGGG-biotin-3'. The sequence of the second oligonucleotide is 5 '-CCCTAACCCTAACCCTAACCCTAACCCTAA-biotin-3'.
The urine sample is provided by the second people hospital in Shenzhen city.
The ultraviolet-visible absorption spectrum was obtained on a UV-2550 spectrophotometer (Shimadzu, Japan). Fluorescence spectra were obtained on an RF-6000PC spectrophotometer (Shimadzu, Japan). ATP detection was performed using GDYQ-131SA ATP bioluminescence detector (university of Jilin-little swan).
Second, Experimental methods
1. Telomerase extraction from cultured cells
Hela cells were cultured in DMEM containing 10% FBS and collected by trypsin. Will be 1 × 106The individual cells were transferred to a 1.5mL EP tube and centrifuged at 1000rpm for 10 minutes to collect the cell pellet. The cell pellet was washed with PBS (pH 7.4), centrifuged again, and dispensed into 200. mu.L of ice-cold 1 × CHAPS lysis buffer. The mixture was incubated on ice for 30 minutes and centrifuged at 12000rpm for 20 minutes at 4 ℃. The supernatant was transferred to a new 1.5mL EP tube and stored at-80 ℃.
2. Telomerase extracted from urine sample
Fresh urine samples (200mL) were collected and centrifuged at 1000rpm for 10 minutes at 4 ℃. The precipitate was washed with PBS (pH 7.4) and centrifuged at 1800rpm for 5 minutes at 4 ℃. The pellet was then resuspended in 2mL of ice-cold 1 × CHAPS lysis buffer. After incubation on ice for 30 min and centrifugation at 12000rpm for 20 min at 4 ℃, the supernatant was transferred to a new 1.5mL EP tube and stored at-80 ℃.
3. Telomerase extension reaction
mu.L of telomerase extract obtained from Hela cells or urine samples and 9. mu.L of extension reaction buffer containing 63mM KCl, 0.04mM dNTP and 200nM telomerase substrate primer (TS primer) were mixed in a tube, and the mixture was incubated at 37 ℃ for 60 minutes to obtain telomerase extension reaction product.
For the heat treatment control experiment, the telomerase extract was first heat treated at 95 ℃ for 10 minutes. For curcumin inhibitor treatment control experiments curcumin was added to each tube at 37 ℃ to final concentrations of 0 μ M, 200 μ M, 500 μ M, 1mM, 10mM and 50mM for 60 minutes.
4. Telomerase activity detection
(1) Detection of telomerase Activity based on streptavidin conjugated beta-galactosidase (SA-Gala)
Taking appropriate amount of magnetic beads (Dynabeads)TMMyOneTMStreptavidin C1) was washed three times with 400. mu.L Tris-HCl (20mM, pH 7.40, containing 135mM NaCl). Then, in the first step, the magnetic beads and the first oligonucleotide (5 '-TTAGGGTTAGGGTTAGGG-biotin-3') were added to 400. mu.L of Tris-HCl buffer at a ratio of 1:1, followed by mixing and placing the mixture on a shaker at 37 ℃ for 30 minutes. The liquid was discarded and washed three times with 400. mu.L Tris-HCl. In the second step, the reaction product and the second oligonucleotide (5 '-CCCTAACCCTAACCCTAACCCTAACCCTAA-biotin-3') were added to 400. mu.L of Tris-HCl at a ratio of 3.2:1, followed by mixing and placing the resulting mixture on a shaker at 37 ℃ for 30 minutes. In the third step, the product of the second step and streptavidin-modified beta-galactosidase (SA-Gala) were added to 400. mu.L of Tris-HCl buffer at a ratio of 3.2:1, followed by mixing and mixing the resulting mixtureThe mixture was placed on a shaker at 37 ℃ for 30 minutes. And a fourth step of adding the reaction mixture of the third step and telomerase extension reaction products of different concentrations to Tris-HCl, and placing the resulting mixture on a 37 ℃ oscillator for 30 minutes. In the fifth step, after magnetic separation, the supernatant was collected and divided into four tubes each having 100. mu.L. 200 μ L of Tris-HCl was added to each tube, followed by 5 μ L of FDG (15 μ M) and incubation at 37 ℃ for 30 min. Finally, all fluorescence spectra were collected at an excitation wavelength of 480nm and an emission wavelength of 500-660 nm.
(2) Telomerase activity detection based on streptavidin-conjugated alkaline phosphatase (SA-ALP)
For the SA-ALP based telomerase activity assay, the previous steps were identical to the SA-Gala based telomerase activity assay except that in the third step, the product of the second step and SA-ALP were added at a ratio of 6.6:1 and in the fifth step, two different assay formats were taken. One assay format was to collect the supernatant and divide it into four tubes, 100 μ L each. mu.L Tris-HCl was added to each tube, followed by 12. mu.L pNPP (5mM) and incubation at 37 ℃ for 30 minutes. Finally, the uv-vis spectrum was measured, with a scanning wavelength range of 450 to 260 nm. Another detection mode is to take 100. mu.L of supernatant and add 100. mu.L of LTris-HCl to a final volume of 200. mu.L. Then 20. mu.L ATP (100. mu.M) was added and incubated at 37 ℃ for 30 minutes. ATP was measured by an ATP bioluminescence detector.
Third, result and discussion
1. Feasibility of telomerase Activity detection
In the SA-Gala based telomerase activity detection mode, non-fluorescent FDG is one of the most sensitive substrates for galactosidase, hydrolyzed by galactosidase first to FMG and then to highly fluorescent fluorescein. An increase in fluorescence occurs after galactosidase-mediated hydrolysis of FDG. As shown in FIG. 2A, the fluorescence intensity of fluorescein (curve a) was effectively enhanced after hydrolysis of FDG by galactosidase at an excitation wavelength of 480nm (curve b).
In the mode of telomerase activity detection based on SA-ALP and pNPP, pNPP is a commonly used chromogenic substrate that can produce p-nitrophenol (pNP) in the presence of ALP, and pNP is deepYellow, as determined by colorimetric assays. As shown in FIG. 2B, A is after ALP mediated hydrolysis of pNPP311/A400The ratio increases significantly.
In the telomerase activity detection mode based on SA-ALP and ATP, as shown in fig. 2C, ALP hydrolyzes ATP to AMP in the presence of telomerase, and then reads the decrease in ATP using an ATP meter.
The results in fig. 2 confirm the feasibility of the triple mode of detection of telomerase activity based on fluorescence, colorimetric and ATP assays of the present invention.
2. Telomerase activity detection effectiveness
Telomerase activity of telomerase extracts from Hela cells was quantitatively determined by fluorescence, colorimetric and ATP assays.
FIG. 3A shows that the fluorescence intensity of FDG is linear with the logarithm of the number of cells in the range of 25-2500 HeLa cells in the fluorometric model. The limit of detection (LOD) was 2 cells based on the 3. sigma./S equation. The inset shows a digital photograph of a fluorescent reaction system with a given concentration of telomerase (expressed as number of cells) under a 365nm uv lamp. Fig. 3B shows the evolution of the fluorescence spectrum of fluorescein with increasing telomerase concentration. Fluorescence increased significantly as the number of HeLa cells increased from 12.5 cells to 2500 cells.
FIG. 3C shows A in the colorimetric assay mode311/A400The ratio is linear with the number of cells between 12.5 and 1500 HeLa cells. Based on the 3 σ/S equation, the detection limit was 8 HeLa cells. The inset shows a digital photograph of a chromogenic reaction system with a given concentration of telomerase (expressed as the number of cells). Fig. 3D shows the corresponding uv/vis spectra at increasing telomerase concentrations.
Fig. 3E shows that ATP values are linear with the logarithm of HeLa cell number in the ATP assay mode, ranging from 5 to 1000 HeLa cells. The detection limit was 1 cell based on the 3 σ/S equation.
The results show that the triple detection modes of the fluorescence assay, the colorimetric assay and the ATP assay can effectively detect telomerase on a single cell level, and realize accurate and sensitive quantitative detection of the telomerase activity.
3. Reliability of telomerase activity detection
To further ensure the reliability of the telomerase activity triple detection mode of the present invention, a heat treatment control experiment and a curcumin inhibitor treatment control experiment were performed.
In the heat treatment control experiment, a normal HeLa cell extract and a heat-inactivated HeLa cell extract at 95 ℃ were investigated. As shown in fig. 4A, no significant fluorescence enhancement of heat inactivated HeLa cell extracts was recorded, indicating that the determination of telomerase activity using the triple detection mode of telomerase activity of the present invention is reliable and accurate.
The reliability of the triple mode of detection of telomerase activity of the invention was further tested in curcumin inhibitor treatment control experiments. Curcumin is a commonly used inhibitor of telomerase activity. During telomerase extension, curcumin was added to the reaction solution at different concentrations. As shown in fig. 4B, the fluorescence intensity of fluorescein gradually decreased with the increase of curcumin concentration, indicating that curcumin can effectively inhibit telomerase activity, and the telomerase activity triple detection mode of the present invention is reliable.
4. Clinical applicability
Bladder cancer is the second most common malignancy of the urogenital system with a high recurrence rate. To evaluate the applicability of the triple mode of telomerase activity detection of the present invention in clinical diagnosis of bladder cancer, 12 urine samples from 10 bladder cancer patients and 2 normal individuals were studied. Fluorescence measurement (F) and colorimetric measurement (A) of urine of cancer patients311/A400) And ATP measurements (I) were significantly higher than those of healthy human urine (table 1).
TABLE 1 comparison of results obtained with triple mode detection of telomerase Activity of the invention with clinical diagnostic results
aΔF＝F-F0；bΔ(A311/A400)＝A311/A400-(A311/A400)0；cΔI＝I-I0
The results of the 12 urine samples were also visualized as shown in FIG. 5, where the upper panel shows the results of fluorescence measurements of the urine samples and the lower panel shows the results of colorimetric measurements of the urine samples, which from left to right belong to patient 1 to patient 10 and two normal individuals, all fluorescence measurements being taken under a 365nm UV lamp.
Urine-based telomerase activity assays are non-invasive and are considered to be the preferred method for clinical diagnosis of bladder cancer. The result shows that the telomerase activity triple detection mode can detect the telomerase activity in a complex sample environment, is used for clinical diagnosis of bladder cancer, and has good selectivity and accuracy.
Reference documents:
(1)Patolsky,F.；Gill,R.；Weizmann,Y.；Mokari,T.；Banin,U.；Willner,I.J.Am.Chem.Soc.2003,125,13918-13919.
(2)Ou,X.；Hong,F.；Zhang,Z.；Cheng,Y.；Zhao,Z.；Gao,P.；Lou,X.；Xia,F.；Wang,S.Bioiosens.Bioelectron.2017,89,417-421.
(3)Lai,W.；Wei,Q.；Xu,M.；Zhuang,J.；Tang,D.Bioiosens.Bioelectron.2017,89,645-651.
(4)Zhang,Y.；Xue,Q.；Liu,J.；Wang,H.Bioiosens.Bioelectron.2017,87,537-544.
(5)Xu,X.；Wei,M.；Liu,Y.；Liu,X.；Wei,W.；Zhang,Y.；Liu,S.Bioiosens.Bioelectron.2017,87,600-606.
(6)Chen,Y.；Qu,K.；Zhao,C.；Wu,L.；Ren,J.；Wang,J.；Qu,X.Nat.Commun.2012,3,1074.
(7)Lin,B.；Liu,D.；Yan,J.；Qiao,Z.；Zhong,Y.；Yan,J.；Zhu,Z.；Ji,T.；Yang,C.J.ACS Appl.Mater.Interfaces 2016,8,6890-6897.
(8)Shang,J.；Li,Z.；Liu,L.；Xi,D.；Wang,H.ACS Sensors 2018,3,512-518.
(9)Duan,R.；Wang,B.；Zhang,T.；Zhang,Z.；Xu,S.；Chen,Z.；Lou,X.；Xia,F.Anal.Chem.2014,86,9781-9785.
(10)Zhuang,Y.；Shang,C.；Lou,X.；Xia,F.Anal.Chem.2017,89,2073-2079.
(11)Wang,L.；Chen,C.；Huang,H.；Huang,D.；Luo,F.；Qiu,B.；Guo,L.；Lin,Z.；Yang,H.Bioiosens.Bioelectron.2018,121,153-158.
(12)Zhu,X.；Xu,H.；Lin,R.；Yang,G.；Lin,Z.；Chen,G.Chem.Commun.2014,50,7897-7899.
(13)Hong,M.；Xu,L.；Xue,Q.；Li,L.；Tang,B.Anal.Chem.2016,88,12177-12182.
(14)Yang,X.J.；Zhang,K.；Zhang,T.T.；Xu,J.J.；Chen,H.Y.Anal.Chem.2017,89,4216-4222.
(15)He,C.；Liu,Z.；Wu,Q.；Zhao,J.；Liu,R.；Liu,B.；Zhao,T.ACS Sensors 2018,3,757-762.
(16)Yang,Y.；Li,C.；Hu,X.；Yang,Y.；Yin,Y.；Wang,Z.Chem.Commun.2017,53,5752-5755.
(17)Qian,R.；Ding,L.；Ju,H.J.Am.Chem.Soc.2013,135,13282-13285.
(18)Li,Y.；Han,H.；Wu,Y.；Yu,C.；Ren,C.；Zhang,X.Bioiosens.Bioelectron.2019,142,111543.
(19)Luo,S.；Zhang,Y.；Situ,B.；Zheng,L.Sensor.Actuat.B-Chem.2018,273,853-861.
(20)Fan,W.；Qi,Y.；Qiu,L.；He,P.；Liu,C.；Li,Z.Anal.Chem.2018,90,5390-5397.
(21)Kato,D.；Oishi,M.ACS Nano 2014,8,9988-9997.
(22)Zeinhom,M.M.A.；Wang,Y.；Song,Y.；Zhu,M.J.；Lin,Y.；Du,D.Bioiosens.Bioelectron.2018,99,479-485.
(23)Chen,W.；Fang,X.；Li,H.；Cao,H.；Kong,J.Bioiosens.Bioelectron.2017,94,169-175.
(24)Guo,Y.；Yang,K.；Sun,J.；Wu,J.；Ju,H.Bioiosens.Bioelectron.2017,94,651-656.
the present invention has been described above using specific examples, which are only for the purpose of facilitating understanding of the present invention, and are not intended to limit the present invention. Numerous simple deductions, modifications or substitutions may be made by those skilled in the art in light of the teachings of the present invention. Such deductions, modifications or alternatives also fall within the scope of the claims of the present invention.
Claims (10)
1. A telomerase activity detection kit is characterized by comprising the following components:
(1) telomerase extension system components, including telomerase substrate primers and dntps, for generation of telomeric repeat sequences;
(2) a component of a magnetic bead system comprising a magnetic bead, a first oligonucleotide, and a second oligonucleotide, the first oligonucleotide being attachable to the magnetic bead, the second oligonucleotide being at least partially hybridizable to the first oligonucleotide, and the second oligonucleotide being at least partially hybridizable to the telomere repeat sequence;
(3) a signal producing system component comprising a signaling enzyme and a substrate for said signaling enzyme, wherein said signaling enzyme can be linked to said second oligonucleotide.
2. The telomerase activity detection kit of claim 1, wherein the first oligonucleotide is linked to the magnetic beads via a biotin-streptavidin linking system.
3. The telomerase activity detection kit of claim 2, wherein said first oligonucleotide is a biotin-modified first oligonucleotide and said magnetic bead is a streptavidin-modified magnetic bead, whereby said first oligonucleotide and said magnetic bead are linked via a biotin-streptavidin linking system.
4. The telomerase activity detection kit of claim 1, wherein said signaling enzyme is linked to said second oligonucleotide via a biotin-streptavidin linkage system.
5. The telomerase activity detection kit of claim 4, wherein said second oligonucleotide is a biotin-modified second oligonucleotide and said signaling enzyme is a streptavidin-modified signaling enzyme, whereby said second oligonucleotide and said signaling enzyme are linked by a biotin-streptavidin linking system.
6. The telomerase activity detection kit of any one of claims 1-5, wherein the telomerase substrate primer has a sequence of 5'-AATCCGTCGAGCAGAGTT-3' (SEQ ID NO:1), the first oligonucleotide has a sequence of 5'-TTAGGGTTAGGGTTAGGG-3' (SEQ ID NO:2), and the second oligonucleotide has a sequence of 5'-CCCTAACCCTAACCCTAACCCTAACC CTAA-3' (SEQ ID NO: 3).
7. The telomerase activity detection kit of any one of claims 1-5, wherein the signal enzyme is a glycosidic bond hydrolase and the substrate is a non-fluorescent fluorescein derivative comprising a glycosidic bond; preferably, the glycosidic bond hydrolase is galactosidase, the substrate is fluorescein 2-beta-D-galactopyranoside, or the glycosidic bond hydrolase is cellulase, and the substrate is fluorescein 2-beta-D-cellobioside.
8. The telomerase activity detection kit of any one of claims 1-5, wherein the signal enzyme is a phosphate ester bond hydrolase, and the substrate is a compound comprising a phosphate ester bond; preferably, the phosphoester bond hydrolase is alkaline phosphatase, and the substrate is p-nitrophenyl phosphate or adenosine triphosphate.
9. A method for detecting telomerase activity using the telomerase activity detection kit of any of claims 1-8, comprising the steps of:
(1) providing a sample to be detected, and carrying out telomerase extension reaction on the sample to be detected and the telomerase extension reaction component to generate the telomere repetitive sequence;
(2) ligating the magnetic beads of the components of the magnetic bead system with the first oligonucleotide to form first oligonucleotide-functionalized magnetic beads, hybridizing the first oligonucleotide-functionalized magnetic beads with the second oligonucleotide to form first and second oligonucleotide-functionalized magnetic beads, and ligating the first and second oligonucleotide-functionalized magnetic beads with the signaling enzyme to ligate the signaling enzyme with the second oligonucleotide to form second oligonucleotide-signaling enzyme linkers, thereby forming magnetic bead system-signaling enzyme complexes;
(3) performing competitive hybridization reaction on the magnetic bead system-signal enzyme complex and the telomere repetitive sequence to form a telomere repetitive sequence-second oligonucleotide-signal enzyme connector hybrid, and forming the magnetic bead system-signal enzyme complex without the corresponding second oligonucleotide-signal enzyme connector;
(4) separating the telomere repeat-second oligonucleotide-signaling enzyme linker hybrid from the magnetic bead system-signaling enzyme complex with the corresponding second oligonucleotide-signaling enzyme linker removed to form a separated telomere repeat-second oligonucleotide-signaling enzyme linker hybrid;
(5) enzymatically reacting said isolated telomeric repeat-second oligonucleotide-signaling enzyme linker hybrid with a substrate for said signaling enzyme, and determining a signal generated by said enzymatic reaction, wherein the magnitude of said signal reflects the magnitude of said telomerase activity.
10. The method for detecting telomerase activity of claim 9, wherein the signaling enzyme is a glycosidic bond hydrolase, the substrate is a non-fluorescent fluorescein derivative comprising a glycosidic bond, and the signal is a change in fluorescence; alternatively, the signaling enzyme is a phosphoester bond hydrolase, and the signal is a change in color when the substrate is p-nitrophenyl phosphate, or a decrease in adenosine triphosphate content when the substrate is adenosine triphosphate.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10215900A (en) * | 1997-02-04 | 1998-08-18 | Dai Ichi Seiyaku Co Ltd | Measurement of enzyme activity |
| US5863726A (en) * | 1993-11-12 | 1999-01-26 | Geron Corporation | Telomerase activity assays |
| CN101115847A (en) * | 2004-11-16 | 2008-01-30 | 希艾娜癌症诊疗有限公司 | Methods of detecting an analyte in a sample |
| CN102876792A (en) * | 2012-09-27 | 2013-01-16 | 浙江今复康生物科技有限公司 | AETCA (anchored-extension and telomeric complements amplification) detection reagent kit for telomerase and detection method |
| CN103497997A (en) * | 2013-09-29 | 2014-01-08 | 天津理工大学 | Quantum dot-riboflavin molecular beacon for detecting telomerase activity |
| CN106811524A (en) * | 2017-01-19 | 2017-06-09 | 陕西师范大学 | A kind of telomerase activation colorimetric detection method |
-
2019
- 2019-08-22 CN CN201910776813.2A patent/CN112410400A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5863726A (en) * | 1993-11-12 | 1999-01-26 | Geron Corporation | Telomerase activity assays |
| JPH10215900A (en) * | 1997-02-04 | 1998-08-18 | Dai Ichi Seiyaku Co Ltd | Measurement of enzyme activity |
| CN101115847A (en) * | 2004-11-16 | 2008-01-30 | 希艾娜癌症诊疗有限公司 | Methods of detecting an analyte in a sample |
| CN102876792A (en) * | 2012-09-27 | 2013-01-16 | 浙江今复康生物科技有限公司 | AETCA (anchored-extension and telomeric complements amplification) detection reagent kit for telomerase and detection method |
| CN103497997A (en) * | 2013-09-29 | 2014-01-08 | 天津理工大学 | Quantum dot-riboflavin molecular beacon for detecting telomerase activity |
| CN106811524A (en) * | 2017-01-19 | 2017-06-09 | 陕西师范大学 | A kind of telomerase activation colorimetric detection method |
Non-Patent Citations (3)
| Title |
|---|
| BING YANG ET AL.: "Advances in optical assays for detecting telomerase activity", 《LUMINESCENCE》, vol. 34, no. 2, pages 136 - 152 * |
| YAOCAI WANG ET AL.: "An ultra-sensitive colorimetric assay for reliable visual detection of telomerase activity", 《ANALYST》, vol. 142, no. 17, pages 3235 - 3240 * |
| 郑婷婷 等: "端粒酶活性检测研究进展", 《应用技术学报》, vol. 18, no. 1, pages 1 - 13 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113481279A (en) * | 2021-07-01 | 2021-10-08 | 广州博徕斯生物科技股份有限公司 | Method for detecting telomerase activity |
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