CN115521970A - Analysis method for activity of reverse transcription DNA synthetase of organism with high responsiveness - Google Patents

Analysis method for activity of reverse transcription DNA synthetase of organism with high responsiveness Download PDF

Info

Publication number
CN115521970A
CN115521970A CN202210132169.7A CN202210132169A CN115521970A CN 115521970 A CN115521970 A CN 115521970A CN 202210132169 A CN202210132169 A CN 202210132169A CN 115521970 A CN115521970 A CN 115521970A
Authority
CN
China
Prior art keywords
reverse transcription
product
detected
activity
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
CN202210132169.7A
Other languages
Chinese (zh)
Inventor
朱灏
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Huaxiayuan Cell Engineering Group Co ltd
Original Assignee
Huaxiayuan Cell Engineering Group Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Huaxiayuan Cell Engineering Group Co ltd filed Critical Huaxiayuan Cell Engineering Group Co ltd
Priority to CN202210132169.7A priority Critical patent/CN115521970A/en
Publication of CN115521970A publication Critical patent/CN115521970A/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/91245—Nucleotidyltransferases (2.7.7)
    • G01N2333/9125—Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)
    • G01N2333/9128—RNA-directed DNA polymerases, e.g. RT (2.7.7.49)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the technical field of biology, in particular to an analysis method for activity of reverse transcription DNA synthetase of a high-responsiveness organism and application thereof. The method comprises the following specific steps: s1, cracking cells to be detected to obtain a sample; s2, reverse transcription of the sample to obtain a product to be detected; s3, carrying out reverse transcription on the positive control substance to obtain a control product; s4, measuring fluorescence signals of the product to be measured and the reference product; and S5, evaluating the telomerase activity of the cells to be detected according to the determination result. The invention provides an analysis method for the activity of a high-responsiveness organism reverse transcription DNA synthetase, solves the problems of low sensitivity and poor result reliability of a telomerase activity detection method in the prior art, realizes effective detection of low-activity (less than 3%) human mesenchymal stem cell telomerase, has high reliability of a detection result, is economic and time-saving, and is beneficial to clinical popularization.

Description

Analysis method for activity of reverse transcription DNA synthetase of organism with high responsiveness
Technical Field
The invention relates to IPC 12Q1/48, in particular to an analysis method for activity of reverse transcription DNA synthetase of a high-responsiveness organism and application thereof.
Background
A basic detection method of telomerase activity, a telomere repetitive sequence extension method, was originally proposed by Greider in 1985, by heat preservation of cell extract and oligonucleotide primers, active telomerase adds a DNA sequence at the 3' end of the primers by using the provided raw materials and taking the RNA of the active telomerase as a template, and results are displayed by polyacrylamide gel electrophoresis and autoradiography. However, the method has lower sensitivity and large required sample amount, and has great limitation on the application of telomerase to clinical early diagnosis and treatment. Currently, improved methods for detecting telomerase activity include a TRAP method, a TRAP silver staining method, a TRAP-ethidium bromide method, a TRAP-hybridization protection method, a TRAP-enzyme-linked immunosorbent assay method and the like, but the detection methods often have the problems of limited detection sample range, relatively long detection time, relatively high detection cost and the like, and based on the problems, the detection method for detecting telomerase activity, which is high in reliability and sensitivity, economical and time-saving and beneficial to clinical popularization, becomes a problem to be solved urgently in the field.
Disclosure of Invention
The invention provides a method for analyzing the activity of a high-responsiveness organism reverse transcription DNA synthetase, solves the problems of low sensitivity and poor result reliability of a telomerase activity detection method in the prior art, and realizes a telomerase activity detection method which has high reliability and sensitivity, is economical and time-saving and is beneficial to clinical popularization.
In order to solve the above problems, the present invention provides, in a first aspect, a method for analyzing reverse transcription DNA synthetase activity of a highly responsive organism, comprising the steps of:
s1, cracking cells to be detected to obtain a sample;
s2, reverse transcription of the sample to obtain a product to be detected;
s3, carrying out reverse transcription on the positive control substance to obtain a control product;
s4, measuring fluorescence signals of the product to be measured and the reference product;
and S5, evaluating the telomerase activity of the cells to be detected according to the determination result.
In some preferred embodiments, the step S1 is specifically to mix, disperse, and perform a lysis reaction on the cell to be detected and the lysis solution to obtain the sample.
In some preferred embodiments, the temperature of the pyrolysis is-10 to 0 ℃ and the pyrolysis time is 15 to 40min.
In some preferred embodiments, the amount of lysis solution added is 15-25. Mu.L/10 6 And (4) detecting the cells to be detected.
In some preferred embodiments, the starting materials for the lysing solution include, by volume parts, 0.5 to 1.5 parts of sulfonyl fluoride, 0.1 to 2 parts of antioxidant, and 160 to 200 parts of solvent.
Further preferably, the solute of the enzyme inhibitor is phenylmethylsulfonyl fluoride, and the solvent is lysis buffer; the molar concentration of PMSF is 0.01-0.03mol/L.
Further preferably, the solute of the antioxidant is beta-mercaptoethanol, and the solvent is lysis buffer; the mol concentration of the beta-mercaptoethanol is 0.001-0.01mol/L.
Further preferably, the solvent is a lysis buffer; the lysis buffer may be commercially available, for example.
In some preferred embodiments, the step S2 is specifically to add the sample into the reaction solution a to perform a reverse transcription reaction, wherein the reverse transcription temperature is 35 to 40 ℃; the reverse transcription time is 2.6-3.2h.
In some preferred embodiments, the volume ratio of the sample to the reaction solution a is 1:35-45.
More preferably, the reaction solution A is 5 Xtelomerase reaction buffer solution and nuclease H-free reaction solution 2 Mixture of O, 5 Xtelomerase reaction buffer and nuclease-free H 2 The volume ratio of O is 1: (3-5).
In some preferred embodiments, the step S4 is specifically to add the reaction solution B to the product to be detected and the control product, respectively, and then to place the reaction solution B in a PCR instrument (specifically, ABI Stepone) to perform the detection of the fluorescent signal by an upper machine.
Further preferably, the volume ratio of the product to be measured to the reaction solution B is 1: (17-22).
In some preferred embodiments, the raw materials of the reaction solution B include, by volume parts, 1 to 4 parts of the primer, 6 to 12 parts of the premix, and 6 to 9 parts of water.
In some preferred embodiments, the program for the computer to detect is configured to:
Figure BDA0003503150170000021
further preferably, the channel for on-machine detection is a SYBR channel.
Telomerase is a reverse transcribed DNA synthetase, widely distributed in eukaryotic cells. According to the invention, an SYBR channel is selected, and a SYBR Green dye and a specific on-computer detection program are combined to jointly act on a product to be detected and a control product, so that the reliable detection of the activity of the human mesenchymal stem cell telomerase is realized; further controlling the composition and the dosage of the lysate and a reverse transcription reaction medium, and protecting target cells from being interfered by external environment in a stable physiological activity state; the conditions such as temperature, reaction time and the like of the cleavage reaction and the reverse transcription reaction are controlled, the response of the instrument to a low-activity target product is promoted, the effective detection of the telomerase activity of the human umbilical cord mesenchymal stem cells with the telomerase activity less than 3 percent is realized, and the sensitivity and the practical operation feasibility of the detection method of the telomerase activity of the human umbilical cord mesenchymal stem cells are effectively promoted.
The second aspect of the invention provides an application of an analysis method of reverse transcription DNA synthetase activity of a high-responsiveness organism, which is applied to the research of a new way for diagnosing and treating cancers and delaying senescence.
Has the advantages that:
the invention provides a method for analyzing the activity of a high-responsiveness organism reverse transcription DNA synthetase, solves the problems of low sensitivity and poor result reliability of a telomerase activity detection method in the prior art, realizes effective detection of low-activity (less than 3%) human mesenchymal stem cell telomerase, has high reliability of a detection result, is economic and time-saving, and is favorable for clinical popularization.
Detailed Description
Examples
Example 1.
The embodiment provides an analysis method for activity of reverse transcription DNA synthetase of a high-responsiveness organism, which comprises the following specific steps:
s1, cracking cells to be detected to obtain a sample;
s2, reverse transcription of the sample to obtain a product to be detected;
s3, carrying out reverse transcription on the positive control substance to obtain a control product;
s4, measuring fluorescence signals of the product to be measured and the reference product;
and S5, evaluating the telomerase activity of the cell to be detected according to the determination result.
And the step S1 is specifically to mix, disperse and crack the cell to be detected and the lysate, perform a cracking reaction, centrifuge for 20min at 4 ℃, and collect the supernatant, namely the sample.
The cell to be detected is a human umbilical cord mesenchymal stem cell and is purchased from China-derived cell engineering group, inc.
Specifically, the dispersion operation was performed by pipetting 20 times a mixture of the cell to be detected and the solution a.
The cracking operation was performed on ice, setting the ambient temperature at 0 ℃ and the cracking time at 30min.
The addition amount of the lysis solution is 20 mu L/10 6 And (4) detecting the cells to be detected.
The raw materials of the cracking liquid comprise, by volume, 1 part of sulfonyl fluoride, 1.2 parts of antioxidant and 200 parts of solvent.
The solute of the enzyme inhibitor is phenylmethylsulfonyl fluoride, and the solvent is a lysis buffer solution; the molar concentration of PMSF was 0.02mol/L.
The solute of the antioxidant is beta-sulfenyl ethanol, and the solvent is lysis buffer solution; the molar concentration of the beta-mercaptoethanol is 0.005mol/L.
The solvent is a lysis buffer solution; lysis buffer was purchased from the Sciencell laboratory, model 8928a.
The S2 step is specifically that a sample is added into the reaction liquid A for reverse transcription reaction, and the reverse transcription temperature is 37 ℃; the reverse transcription time was 3h, after which time the temperature was raised to 85 ℃ for 10min, and the reaction was terminated.
The volume ratio of the sample to the reaction solution A is 1:39.
the reaction solution A is 5 Xtelomerase reaction buffer solution and nuclease H-free 2 Mixture of O, 5 Xtelomerase reaction buffer and nuclease-free H 2 The volume ratio of O is 1:4.
the 5 × telomerase reaction buffer was purchased from Sciencell laboratories, model 8928b; the nuclease-free H 2 O was purchased from Sciencell laboratories, model 8928d.
And the step S4 is specifically that the reaction solution B is respectively added into the product to be detected and the control product, and then the reaction solution B is placed in a PCR instrument (specifically ABI Stepone) to detect a fluorescence signal by an upper machine.
The volume ratio of the product to be detected to the reaction liquid B is 1:19.
the raw materials of the reaction solution B comprise, by volume, 2 parts of primers, 10 parts of premix and 7 parts of water.
The primers were Telomere Primer Set (TPS) purchased from Sciencell laboratories, model 8928c; the premix was a 2x qPCR master mix purchased from roche under No. 06402712001; the water was nuclease-free water purchased from the Sciencell laboratory, model 8928d.
The program of the on-computer detection is set as follows:
Figure BDA0003503150170000041
and the channel for the on-machine detection is an SYBR channel.
The positive control was telomerase positive cell lysate purchased from Sciencell laboratory, model 8928e. The operations of reverse transcription and fluorescence signal determination of the positive control are the same as those of the product to be tested.
Example 2.
The embodiment provides an analysis method for activity of reverse transcription DNA synthetase of a high-responsiveness organism, which comprises the following specific steps:
s1, cracking cells to be detected to obtain a sample;
s2, carrying out reverse transcription on the sample to obtain a product to be detected;
s3, carrying out reverse transcription on the positive control substance to obtain a control product;
s4, measuring fluorescence signals of the product to be measured and the reference product;
and S5, evaluating the telomerase activity of the cells to be detected according to the determination result.
And the step S1 is specifically to mix, disperse and crack the cell to be detected and the lysate, perform a cracking reaction, centrifuge for 20min at 4 ℃, and collect the supernatant, namely the sample.
The cell to be detected is a human umbilical cord mesenchymal stem cell and is purchased from China-derived cell engineering group, inc.
Specifically, the dispersion operation was performed by pipetting 20 times a mixture of the cell to be detected and the solution a.
The cracking operation was carried out on ice, setting the ambient temperature at 0 ℃ and the cracking time at 30min.
The addition amount of the lysis solution is 20 mu L/10 6 And (4) detecting the cells to be detected.
The raw materials of the cracking liquid comprise, by volume, 1 part of sulfonyl fluoride, 1.2 parts of antioxidant and 200 parts of solvent.
The solute of the enzyme inhibitor is phenylmethylsulfonyl fluoride, and the solvent is a lysis buffer solution; the molar concentration of PMSF was 0.02mol/L.
The solute of the antioxidant is beta-sulfenyl ethanol, and the solvent is lysis buffer solution; the molar concentration of the beta-mercaptoethanol is 0.005mol/L.
The solvent is a lysis buffer solution; lysis buffer was purchased from Sciencell laboratories, model 8928a.
The S2 step is specifically that a sample is added into the reaction liquid A for reverse transcription reaction, and the reverse transcription temperature is 37 ℃; the reverse transcription time was 3h, after which time the temperature was raised to 85 ℃ for 10min, and the reaction was terminated.
The volume ratio of the sample to the reaction solution A is 1:39.
the reaction solution A is 5 Xtelomerase reaction buffer solution and nuclease H-free 2 Mixture of O, 5 Xtelomerase reaction buffer and nuclease-free H 2 The volume ratio of O is 1:4.
the 5 × telomerase reaction buffer was purchased from Sciencell laboratories, model 8928b; the nuclease-free H 2 O was purchased from Sciencell laboratories, model 8928d.
And the step S4 is specifically that the reaction solution B is respectively added into the product to be detected and the control product, and then the reaction solution B is placed in a PCR instrument (specifically ABI Stepone) to detect a fluorescence signal by an upper machine.
The volume ratio of the product to be detected to the reaction liquid B is 1:19.
the raw materials of the reaction solution B comprise, by volume, 2 parts of primers, 10 parts of premix and 7 parts of water.
The primers were Telomere Primer Set (TPS) purchased from Sciencell laboratories, model 8928c; the premix was a 2x qPCR master mix purchased from roche under No. 06402712001; the water was nuclease-free water purchased from Sciencell laboratories, model 8928d.
The program of the on-machine detection is set as follows:
Figure BDA0003503150170000061
and the channel for the on-machine detection is an SYBR channel.
The positive control was telomerase positive cell lysate purchased from Sciencell laboratory model 8928e. The operations of reverse transcription and fluorescence signal determination of the positive control are the same as those of the product to be tested.
Example 3.
This example provides a method for analyzing the activity of a reverse transcription DNA synthetase in a highly responsive organism, comprising the steps of:
s1, cracking cells to be detected to obtain a sample;
s2, carrying out reverse transcription on the sample to obtain a product to be detected;
s3, carrying out reverse transcription on the positive control substance to obtain a control product;
s4, measuring fluorescence signals of the product to be measured and the reference product;
and S5, evaluating the telomerase activity of the cells to be detected according to the determination result.
And the step S1 specifically comprises the steps of mixing the cells to be detected with the lysate, dispersing, carrying out a lysis reaction, centrifuging for 20min at 4 ℃, and collecting the supernatant, namely the sample.
The cell to be detected is a human umbilical cord mesenchymal stem cell and is purchased from China-derived cell engineering group, inc.
Specifically, the dispersion operation was performed by pipetting 20 times a mixture of the cell to be detected and the solution a.
The cracking operation was carried out on ice, setting the ambient temperature at 0 ℃ and the cracking time at 30min.
The addition amount of the lysis solution is 20 mu L/10 6 And (4) detecting the cells to be detected.
The raw materials of the cracking liquid comprise, by volume, 1 part of sulfonyl fluoride, 1.2 parts of antioxidant and 200 parts of solvent.
The solute of the enzyme inhibitor is phenylmethylsulfonyl fluoride, and the solvent is a lysis buffer solution; the molar concentration of PMSF was 0.02mol/L.
The solute of the antioxidant is beta-sulfenyl ethanol, and the solvent is lysis buffer solution; the molar concentration of the beta-mercaptoethanol is 0.005mol/L.
The solvent is a lysis buffer solution; lysis buffer was purchased from the Sciencell laboratory, model 8928a.
The S2 step is specifically that a sample is added into the reaction liquid A for reverse transcription reaction, and the reverse transcription temperature is 37 ℃; the reverse transcription time was 3h, after which time the temperature was raised to 85 ℃ for 10min, and the reaction was terminated.
The volume ratio of the sample to the reaction solution A is 1:39.
the reaction solution A is 5 Xtelomerase reaction buffer solution and nuclease H 2 Mixture of O, 5 Xtelomerase reaction buffer and nuclease-free H 2 The volume ratio of O is 1:4.
the 5 × telomerase reaction buffer was purchased from Sciencell laboratories, model 8928b; the nuclease-free H 2 O was purchased from Sciencell laboratories, model 8928d.
And the step S4 is specifically that the reaction solution B is respectively added into the product to be detected and the control product, and then the reaction solution B is placed in a PCR instrument (specifically ABI Stepone) to detect a fluorescence signal by an upper machine.
The volume ratio of the product to be detected to the reaction solution B is 1:19.
the raw materials of the reaction solution B comprise, by volume, 2 parts of primers, 10 parts of premix and 7 parts of water.
The primers were Telomere Primer Set (TPS) purchased from Sciencell laboratories, model 8928c; the premix was 2x qPCR master mix, purchased from the roche group, cat # 06402712001; the water was nuclease-free water purchased from the Sciencell laboratory, model 8928d.
The program of the on-machine detection is set as follows:
Figure BDA0003503150170000071
Figure BDA0003503150170000081
and the channel for the on-machine detection is an SYBR channel.
The positive control was telomerase positive cell lysate purchased from Sciencell laboratory model 8928e. The operations of reverse transcription and fluorescence signal determination of the positive control are the same as those of the product to be detected.
Example 4.
This example provides a method for analyzing the activity of a reverse transcription DNA synthetase in a highly responsive organism, comprising the steps of:
s1, cracking cells to be detected to obtain a sample;
s2, reverse transcription of the sample to obtain a product to be detected;
s3, carrying out reverse transcription on the positive control substance to obtain a control product;
s4, measuring fluorescence signals of the product to be measured and the reference product;
and S5, evaluating the telomerase activity of the cells to be detected according to the determination result.
And the step S1 specifically comprises the steps of mixing the cells to be detected with the lysate, dispersing, carrying out a lysis reaction, centrifuging for 20min at 4 ℃, and collecting the supernatant, namely the sample.
The cell to be detected is a human umbilical cord mesenchymal stem cell and is purchased from China-derived cell engineering group, inc.
Specifically, the dispersion operation is to blow the mixture of the cell to be detected and the solution a by a pipette 20 times.
The cracking operation was carried out on ice, setting the ambient temperature at 0 ℃ and the cracking time at 30min.
The addition amount of the lysis solution is 20 mu L/10 6 And (4) detecting the cells to be detected.
The raw materials of the cracking liquid comprise, by volume, 1 part of sulfonyl fluoride, 1.2 parts of antioxidant and 200 parts of solvent.
The solute of the enzyme inhibitor is phenylmethylsulfonyl fluoride, and the solvent is a lysis buffer solution; the molar concentration of PMSF was 0.02mol/L.
The solute of the antioxidant is beta-sulfenyl ethanol, and the solvent is lysis buffer solution; the molar concentration of the beta-mercaptoethanol is 0.005mol/L.
The solvent is a lysis buffer solution; lysis buffer was purchased from Sciencell laboratories, model 8928a.
The S2 step is specifically that a sample is added into the reaction liquid A for reverse transcription reaction, and the reverse transcription temperature is 37 ℃; the reverse transcription time was 3h, after which time the temperature was raised to 85 ℃ for 10min, and the reaction was terminated.
The volume ratio of the sample to the reaction solution A is 1:39.
the reaction solution A is 5 Xtelomerase reaction buffer solution and nuclease H-free 2 Mixture of O, 5 Xtelomerase reaction buffer and nuclease-free H 2 The volume ratio of O is 1:4.
the 5 × telomerase reaction buffer was purchased from Sciencell laboratories, model 8928b; the nuclease-free H 2 O was purchased from Sciencell laboratories, model 8928d.
And the step S4 is specifically that the reaction solution B is respectively added into the product to be detected and the control product, and then the reaction solution B is placed in a PCR instrument (specifically ABI Stepone) to detect a fluorescence signal by an upper machine.
The volume ratio of the product to be detected to the reaction solution B is 1:19.
the raw materials of the reaction solution B comprise, by volume, 2 parts of primers, 10 parts of premix and 7 parts of water.
The primers were Telomere Primer Sets (TPS) purchased from Sciencell laboratories, model 8928c; the premix was a 2x qPCR master mix purchased from roche under No. 06402712001; the water was nuclease-free water purchased from Sciencell laboratories, model 8928d.
The program of the on-computer detection is set as follows:
Figure BDA0003503150170000091
and the channel for the on-machine detection is an SYBR channel.
The positive control was telomerase positive cell lysate purchased from Sciencell laboratory, model 8928e. The operations of reverse transcription and fluorescence signal determination of the positive control are the same as those of the product to be detected.
Example 5.
The embodiment provides an analysis method for activity of reverse transcription DNA synthetase of a high-responsiveness organism, which comprises the following specific steps:
s1, cracking cells to be detected to obtain a sample;
s2, carrying out reverse transcription on the sample to obtain a product to be detected;
s3, carrying out reverse transcription on the positive control substance to obtain a control product;
s4, measuring fluorescence signals of the product to be measured and the reference product;
and S5, evaluating the telomerase activity of the cells to be detected according to the determination result.
And the step S1 specifically comprises the steps of mixing the cells to be detected with the lysate, dispersing, carrying out a lysis reaction, centrifuging for 20min at 4 ℃, and collecting the supernatant, namely the sample.
The cell to be detected is a human umbilical cord mesenchymal stem cell and is purchased from China-derived cell engineering group, inc.
Specifically, the dispersion operation is to blow the mixture of the cell to be detected and the solution a by a pipette 20 times.
The cracking operation was carried out on ice, setting the ambient temperature at 0 ℃ and the cracking time at 30min.
The addition amount of the lysis solution is 20 mu L/10 6 And (4) detecting the cells to be detected.
The raw materials of the cracking liquid comprise, by volume, 1 part of sulfonyl fluoride, 1.2 parts of antioxidant and 100 parts of solvent.
The solute of the enzyme inhibitor is phenylmethylsulfonyl fluoride, and the solvent is a lysis buffer solution; the molar concentration of PMSF was 0.02mol/L.
The solute of the antioxidant is beta-sulfenyl ethanol, and the solvent is lysis buffer solution; the molar concentration of the beta-mercaptoethanol is 0.005mol/L.
The solvent is a lysis buffer solution; lysis buffer was purchased from the Sciencell laboratory, model 8928a.
The S2 step is specifically that a sample is added into the reaction liquid A for reverse transcription reaction, and the reverse transcription temperature is 37 ℃; the reverse transcription time is 3h, after 3h, the temperature is raised to 85 ℃ and the reaction is stopped after 10min of storage.
The volume ratio of the sample to the reaction solution A is 1:39.
the reaction solution A is 5 Xtelomerase reaction buffer solution and nuclease H-free 2 Mixture of O, 5 Xtelomerase reaction buffer and nuclease-free H 2 The volume ratio of O is 1:4.
the 5 × telomerase reaction buffer was purchased from Sciencell laboratories, model 8928b; the nuclease-free H 2 O was purchased from Sciencell laboratories, model 8928d.
And the step S4 is specifically to add the reaction solution B into the product to be detected and the control product respectively, and then place the reaction solution B in a PCR instrument (specifically ABI Stebone) for detecting a fluorescence signal by an upper computer.
The volume ratio of the product to be detected to the reaction solution B is 1:19.
the raw materials of the reaction solution B comprise, by volume, 2 parts of primers, 10 parts of premix and 7 parts of water.
The primers were Telomere Primer Sets (TPS) purchased from Sciencell laboratories, model 8928c; the premix was a 2x qPCR master mix purchased from roche under No. 06402712001; the water was nuclease-free water purchased from Sciencell laboratories, model 8928d.
The program of the on-computer detection is set as follows:
Figure BDA0003503150170000111
and the channel for the on-machine detection is an SYBR channel.
The positive control was telomerase positive cell lysate purchased from Sciencell laboratory model 8928e. The operations of reverse transcription and fluorescence signal determination of the positive control are the same as those of the product to be detected.
Performance test method
The method simultaneously determines the telomerase activity of the negative control, and the operations of reverse transcription and fluorescence signal determination of the negative control are the same as the operations of reverse transcription and fluorescence signal determination of the product to be detected in the embodiment 1. The negative controls were: heating 5.5 μ L of positive control at 85 deg.C for 10min to obtain negative control; the fluorescence value after reverse transcription reaction of the negative control was > 33.
Human umbilical cord mesenchymal stem cell telomerase activity:
defining the fluorescence signal of the product to be tested as Cq1, the fluorescence signal of the control product as Cq2, and the telomerase activity delta =2 of the cells to be tested (Cq1-Cq2) . Each set of examples was run in parallel 5 times and the delta average was calculated.
Reproducibility:
RSD% of the above parallel detection results were calculated. The result reproducibility is defined as that RSD% is less than 10%, the result reproducibility is good when RSD% is more than or equal to 10% and less than 15%, and the result reproducibility is poor when RSD% is more than or equal to 15%.
Performance test data
TABLE 1 Performance test results
δ Reproducibility of
Example 1 1.16％ Good taste
Example 2 0.76％ Difference (D)
Example 3 0.81％ In
Example 4 0.84％ In (1)
Example 5 0.71％ In

Claims (10)

1. A method for analyzing the activity of a reverse transcription DNA synthetase of a high-responsiveness organism is characterized by comprising the following specific steps:
s1, cracking cells to be detected to obtain a sample;
s2, reverse transcription of the sample to obtain a product to be detected;
s3, carrying out reverse transcription on the positive control substance to obtain a control product;
s4, measuring fluorescence signals of the product to be measured and the reference product;
and S5, evaluating the telomerase activity of the cell to be detected according to the determination result.
2. The method according to claim 1, wherein the step S1 comprises mixing the cell to be assayed and the lysate, dispersing the mixture, and performing a lysis reaction to obtain the sample.
3. The method according to claim 2, wherein the temperature of the lysis is-10 to 0 ℃ and the time of the lysis is 15 to 40min.
4. The method according to claim 2, wherein the starting materials for the lysis buffer comprise, by volume, 0.5 to 1.5 parts of an enzyme inhibitor, 0.1 to 2 parts of an antioxidant, and 160 to 200 parts of a solvent.
5. The method according to claim 1, wherein the step S2 is carried out by adding the sample into the reaction solution A to carry out the reverse transcription reaction at a temperature of 35-40 ℃; the reverse transcription time is 2.6-3.2h.
6. The method according to claim 5, wherein the volume ratio of the sample to the reaction solution A is 1:35-45.
7. The method according to claim 1, wherein the step S4 comprises adding the reaction solution B to the test product and the control product, respectively, and then placing the reaction solution B in a PCR instrument for detecting the fluorescent signal by an up-converter.
8. The method according to claim 7, wherein the raw materials of the reaction solution B comprise, by volume, 1 to 4 parts of the primer, 6 to 12 parts of the premix, and 6 to 9 parts of water.
9. The method according to claim 7, wherein the program for performing the on-machine detection is configured to:
Figure FDA0003503150160000011
Figure FDA0003503150160000021
10. use of the method according to any one of claims 1 to 9 for analyzing reverse transcriptase DNA synthetase activity of a highly responsive organism for exploring a novel mechanism for cancer diagnosis and treatment and aging delay.
CN202210132169.7A 2022-02-14 2022-02-14 Analysis method for activity of reverse transcription DNA synthetase of organism with high responsiveness Withdrawn CN115521970A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210132169.7A CN115521970A (en) 2022-02-14 2022-02-14 Analysis method for activity of reverse transcription DNA synthetase of organism with high responsiveness

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210132169.7A CN115521970A (en) 2022-02-14 2022-02-14 Analysis method for activity of reverse transcription DNA synthetase of organism with high responsiveness

Publications (1)

Publication Number Publication Date
CN115521970A true CN115521970A (en) 2022-12-27

Family

ID=84694025

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210132169.7A Withdrawn CN115521970A (en) 2022-02-14 2022-02-14 Analysis method for activity of reverse transcription DNA synthetase of organism with high responsiveness

Country Status (1)

Country Link
CN (1) CN115521970A (en)

Similar Documents

Publication Publication Date Title
CN104099425B (en) A kind of test kit for detecting B-raf transgenation
CN109750088B (en) TdT-RCA-based sensor and application thereof in DNA methyltransferase detection
Green et al. Developmental validation of VeriFiler™ Plus PCR Amplification Kit: A 6-dye multiplex assay designed for casework samples
CN105624296A (en) Universal fluorescent hairpin primers for detecting gene polymorphism on basis of ARMS (amplification refractory mutation system) fluorescent PCR (polymerase chain reaction) process
CN111334580A (en) PIK3CA gene mutation detection kit
Hanson et al. RNA profiling for the identification of the tissue origin of dried stains in forensic biology
CN112481417A (en) Rapid detection method and kit for new coronavirus typing and mutation sites
CN109251964B (en) Circulating microRNAs detection kit, method for specifically detecting circulating microRNAs and application
EP2233579B1 (en) Method and appartus for analyzing nucleic acid amplification
CN104830992B (en) Detect primer, kit and its PCR method of methylenetetrahydrofolate reductase C677T polymorphic sites
CN111394434B (en) CHO host cell DNA residue detection kit adopting TaqMan probe method and application thereof
Yajima et al. Establishment of quantitative reverse transcription–polymerase chain reaction assays for human telomerase-associated genes
CN116716415A (en) Primer probe for quantitative detection of DNA residual content of insect cells, method and application thereof
CN115521970A (en) Analysis method for activity of reverse transcription DNA synthetase of organism with high responsiveness
CN111500687A (en) Breast cancer 21 gene detection kit
CN102134595B (en) Method for detecting nucleic acid mass of sample
ITMI20101132A1 (en) HIGH SENSITIVITY METHOD TO DETECT NUCLEIC TARGET ACID IN A SAMPLE
CN112592965B (en) E.coli host DNA residue detection kit adopting TaqMan probe method
CN109576350B (en) Kit and method for simultaneously quantifying DNA and RNA and quality control method
CN112029836A (en) Nucleic acid composition and kit for detecting BRAF gene mutation and detection method of BRAF gene mutation
CN112779322A (en) Gene mutation detection kit based on non-fluorescence labeled probe and high-resolution melting curve, detection method and application thereof
CN117106865B (en) High-sensitivity detection method based on double amplification and double LNA probe specific recognition DNA mutation
RU2642273C1 (en) Method of differentiating yersinia pestis strains on basic and nonbasic subtypes by pcr method in real time mode
JPWO2006070667A1 (en) EGFR gene mutation detection method and detection kit
CN117778523B (en) Poly (A) polymerase activity determination method

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
WW01 Invention patent application withdrawn after publication

Application publication date: 20221227

WW01 Invention patent application withdrawn after publication