CN108588284B - Method for detecting HTLV-II DNA based on enzyme catalysis controllable self-assembly biological bar code - Google Patents
Method for detecting HTLV-II DNA based on enzyme catalysis controllable self-assembly biological bar code Download PDFInfo
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Abstract
The invention provides a terminal-basedThe deoxynucleotide transferase catalyzes the biological bar code to self-assemble the chemical luminescence method for carrying out dendritic amplification detection on human T lymphocyte leukemia virus II (HTLV-II) DNA. The technical scheme comprises two continuous reaction steps: (1) HTLV-II DNA-induced terminal deoxynucleotidyl transferase catalyzed first-step enzymatic extension with dendritic self-assembly of biological barcodes, and (2) terminal deoxynucleotidyl transferase catalyzed second-step enzymatic extension with chemiluminescent detection in the presence of heme. The technical scheme of the invention has the detection lower limit reaching 0.5 multiplied by 10‑ 18mol/L, greatly improved sensitivity compared with the prior art, good specificity and simple and convenient operation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a chemiluminescence method for performing dendritic amplification detection on human T lymphocyte leukemia virus II (HTLV-II) DNA based on terminal deoxynucleotidyl transferase (TdT) catalytic biological bar code self-assembly.
Background
In the prior art, the technical means commonly used for detecting nucleotides include Polymerase Chain Reaction (PCR), rolling circle amplification Reaction (RCA), loop-mediated isothermal amplification reaction (LAMP), Hybrid Chain Reaction (HCR), Catalytic Hairpin Assembly (CHA) amplification, Ligase Chain Reaction (LCR), exonuclease/endonuclease assisted signal amplification reaction (EASA). PCR is a thermal cycling based DNA amplification technique involving stringent primer/template design and precise thermal cycling. RCA and LAMP are isothermal amplification technologies, and complicated thermal cycling steps of PCR are avoided. However, RCA involves complicated steps of preparation and isolation of circular templates, whereas LAMP requires the design of complex DNA hairpin probes. In addition, PCR, RCA and LAMP all rely on DNA template amplification, which necessarily involves non-specific amplification, thereby causing cross-contamination. In addition, HCR is based on a chain hybridization reaction between two sets of DNA hairpin probes, and CHA amplification reaction is based on the reaction in which the target DNA catalyzes the hybridization between the two DNA hairpin probes. Both HCR and CHA amplification reactions are nucleic acid signal amplification reactions that provide enzyme-free participation, but their reactions require precise design of DNA hairpin probes. LCR uses DNA ligase with good thermal stability to connect adjacent hybridized DNA probes for detecting target DNA. However, the reaction conditions of LCR, including appropriate ligation temperature, cycle number and ligase concentration, are relatively complicated and also require gel electrophoresis to participate in the separation of the ligation products. EASA uses cyclic digestion or cyclic cleavage of specific nucleotide sequences with exonucleases (e.g., exonuclease III and lambda exonuclease) and endonucleases (e.g., nicking endonucleases (nt. alwi and nt. bbvci)) to amplify the signal.
The biological barcode amplification (BCA) is a novel amplification technique, and short oligonucleotides are used as recognition strands and substitute for amplification units to amplify signals. It is noteworthy that multiple oligonucleotide chains can be assembled on a single nanoparticle, and subsequent magnetic separation provides a very clean reaction environment for BCA, can be used to detect multiple proteins and nucleotides, and has high sensitivity and specificity. However, there have been reports on that in BCA, the nanoparticles and target chains are 1: 1, the preparation process of the oligonucleotide/protein modified nanoparticle is complex, the procedure for distinguishing the signal probe from the nanoparticle is very complicated, and at present, the realization of high sensitivity and wide dynamic range of detection is still a huge challenge, so that a new technology is urgently needed to be introduced into the BCA.
Human T lymphocyte virus is a member of the human C type RNA tumor virus family, is an oncogenic RNA virus, can be divided into HTLV-I type, HTLV-II type and HTLV-III type, and has a close relation with various human T cell malignant tumor diseases and Acquired Immune Deficiency Syndrome (AIDS). Among them, HTLV-II has strong correlation with nerve diseases, respiratory diseases and inflammatory reactions. Also, injection drug addicts infected with HTLV-II may introduce viruses into the general population and blood donors by secondary transmission, thereby inducing global neurological disability and neuropathy. Given the low abundance, small size, high sequence homology of HTLV family members and its critical role in biomedical research, it is therefore highly desirable to develop an efficient method for detecting HTLV-II with high sensitivity and specificity.
Disclosure of Invention
The invention provides a chemiluminescence method for performing dendritic amplification detection on human T lymphocyte leukemia virus II (HTLV-II) DNA based on terminal deoxynucleotidyl transferase catalytic biological bar code self-assembly, and the method has the advantages of high sensitivity and easiness in operation. In order to achieve the technical purpose, the invention provides the following technical scheme:
the invention aims to provide a kit for carrying out dendritic amplification detection on HTLV-II DNA (human immunodeficiency virus-II) based on self-assembly of a terminal deoxynucleotidyl transferase catalyzed biological bar code, wherein the kit comprises terminal deoxynucleotidyl transferase, a magnetic microsphere functionalized by a capture probe 1, a capture probe 2, a nanogold particle functionalized by a report probe, a luminol reagent, a heme reagent and an incubation reagent; wherein the sequence of the capture probe 1 is: 5'-ATG GGG TCC CAG GTG AG-3' (3-terminal modified with a biotin); the sequence of capture probe 2 was: 5'-AAA AAA AAA AAA AAA AAA TCT TAT CTT-3' (3-terminal modified with a biotin); the sequence of the reporter probe is 5'-ACA TGC TTG GAC TGC-3' (5-terminal modified with a biotin).
The invention also aims to provide a nano sensor for performing dendritic amplification detection on HTLV-II DNA based on terminal deoxynucleotide transferase catalytic biological barcode self-assembly, which is characterized by comprising the following components in parts by weight: terminal deoxynucleotidyl transferase, magnetic microspheres functionalized by capture probes 1, gold nanoparticles functionalized by capture probes 2 and reporter probes, luminol reagent, heme reagent and incubation reagent; the sequence of the capture probe 1 is as follows: 5'-ATG GGG TCC CAG GTG AG-3' (3-terminal modified with a biotin); the sequence of the capture probe 2 is as follows: 5'-AAA AAA AAA AAA AAA AAA TCT TAT CTT-3' (3-terminal modified with a biotin); the sequence of the reporter probe is 5'-ACA TGC TTG GAC TGC-3' (5-terminal modified with a biotin).
The invention also provides a detection method of the kit or the nano sensor, which comprises the following steps:
(1) the target HTLV-II DNA hybridizes to the modified capture probe 1 portion on the magnetic microsphere to form a stable dsDNA duplex with the 3' terminal sequence protruding from the target HTLV-II DNA; adding terminal deoxynucleotidyl transferase, taking a 3' terminal DNA sequence as a primer, and initiating a first-step polymerization extension reaction to obtain a polymerization product rich in thymine;
(2) adding nano-gold particles modified with a capture probe 2 and a report probe, polymerizing a large amount of the nano-gold particles through complementary combination of the capture probe 2 and the thymine-rich polymerization product, and initiating a second polymerization extension reaction by using the report probe on the nano-gold particles as a primer and using deoxyribonucleic acid terminal transferase to generate a large amount of guanine-rich products;
(3) adding heme, folding the guanine-rich product into a spatial G-tetrad structure, catalyzing the oxidation reaction of luminol mediated by hydrogen peroxide, and detecting a chemiluminescence signal to determine the content of HTLV-II DNA.
Preferably, the preparation method of the magnetic microspheres in the step (1) comprises the following steps:
dissolving the capture probe 1 in a 1 xTris-EDTA buffer solution to prepare a stock solution; adding 10 mu mol/L of capture probe 1 and 10mg/mL of magnetic microspheres wrapped with streptavidin into ultrapure water, and incubating for 10min at room temperature to enable the capture probe 1 to be combined with the magnetic microspheres wrapped with streptavidin; and removing excessive capture probes 1 in the solution by using magnetic separation to obtain the streptavidin-coated magnetic microspheres functionalized by the capture probes 1.
Further, capture probe 1: magnetic microspheres: the proportion of the ultrapure water solution is 1 mu L: 1 μ L: 10 μ L.
Preferably, the method for preparing gold nanoparticles in step (2) above includes the following steps: dissolving a capture probe 2 and a report probe in a 1 xTris-EDTA buffer solution to prepare a stock solution; 0.5mg/mL of the gold nanoparticles, 1. mu. mol/L of capture probe 2 and 1. mu. mol/L of the reporter probe were added to ultrapure water and incubated at room temperature for 10min to form capture probe 2 and reporter probe functionalized gold nanoparticles by biotin-streptavidin interaction.
Further, wherein the ratio of the gold nanoparticles: capture probe 2: the reporter probe: the proportion of ultrapure water is 1 mu L: 0.45. mu.L: 4.05 μ L: 1200. mu.L.
Further, the above-mentioned greenhouse incubation needs to be performed in an incubation buffer, and the formulation of the incubation buffer is as follows: 40mmol/L hydroxyethylpiperazine ethanethiosulfonic acid, 300mmol/L sodium chloride, 20mol/L potassium chloride, and pH 8.0.
Preferably, the specific operation of the step (1) is as follows: adding HTLV-II DNA into a hybridization solution of 20 mu L of capture probe 1 functionalized magnetic microspheres, 750mmol/L sodium chloride and 75mmol/L sodium citrate, and incubating for 10min at room temperature to form double-stranded DNA with a protruding 3' -hydroxyl end; after magnetic separation, 0.4 unit of terminal deoxynucleotidyl transferase was added to 20. mu.L of a polymerization reaction solution consisting of the separated precipitated product, which included 2. mu.L of 100. mu. mol/L dTTP, 2. mu.L of 10 Xterminal deoxynucleotidyl transferase reaction buffer, and 2. mu.L of 2.5mmol/L CoCl 2The first extension reaction was performed by incubation at 37 ℃ for 30min with HTLV-II DNA at the overhanging 3' end as primer to give a thymine-rich polymerization product.
Preferably, the specific operation of step (2) is: adding 0.15 mu L of nano-gold particles with the capture probe 2 and the report probe functionalized into a hybridization solution of the thymine polymerization product, wherein the hybridization solution comprises 750mmol/L NaCl and 75mmol/L sodium citrate, and incubating for 10min at room temperature to ensure that the nano-gold particles with the capture probe 2 and the report probe functionalized are hybridized to the thymine polymerization product-containing long chain; removing redundant terminal deoxynucleotidyl transferase, dTTP and gold nanoparticles with capture probes 2 and reporter probes through a magnetic separation step; then 0.4 units of DNAse were added to 20. mu.L of a mixture containing 0.8. mu.L of 100. mu. mol/L dATP, 1.2. mu.L of 100. mu. mol/L dGTP, 2. mu.L of 10 XDNAse buffer, 2. mu.L of 2.5mmol/L CoCl2(ii) a And (3) incubating for 20min at 37 ℃, adding 63.1 mu L of deionized water to suspend the precipitated product after a magnetic separation step, incubating for 3min at 90 ℃, and quickly transferring the supernatant to a clean tube by utilizing magnetic separation for chemiluminescence measurement.
Preferably, the specific operation of step (3) is: adding 0.5mmol/L luminol solution and 750nmol/L heme solution to 63.1. mu.L of the trans-product of step (2)Adding 30 mu L of incubation buffer solution into the reaction product, and incubating for 30min at room temperature to obtain a G-quadruplex structure; to the mixture was added 15. mu.L of 100mmol/L H2O2Thereafter, the chemiluminescent signal was measured by a 96-microplate luminometer at 1.5 second intervals.
The detection method can be applied to basic research such as drug effect evaluation of antiviral drugs, and is not applied to diagnosis and treatment of diseases.
The invention has the advantages of
1. High sensitivity: the technical scheme utilizes two-step high-efficiency polymerization extension reaction catalyzed by terminal deoxynucleotidyl transferase, high-efficiency amplification of BCA technology, high sensitivity of guanine DNA enzyme chemiluminescence and zero background caused by magnetic separation, and the detection limit can reach 0.5 multiplied by 10-18mol/L, therefore, the scheme can realize high-sensitivity detection of HTLV-II DNA.
2. The specificity is good: in this embodiment, since the DNA-terminal transferase can perform the polymerization extension of a single deoxynucleotide without a DNA template, the polymerization reaction is highly accurate. And the self-assembly of the biological barcode is based on a specific hybridization reaction between probes on the nanoparticle. Therefore, based on the two points, the scheme has good specificity.
3. The operation is simple: the reaction in the scheme is carried out under the constant temperature condition, and the temperature is not required to be controlled; the whole reaction process only relates to the chemiluminescent signal amplification mediated by two-step template-free polymerization extension catalyzed by DNA terminal transferase, and does not relate to polymerase, endonuclease or exonuclease; the detection is carried out by the guanine-rich DNA enzyme mediated chemiluminescence, and a signal probe modified by a synthetic dye in a common detection means is not needed, so the operation is very simple.
4. The BCA and terminal deoxynucleotidyl transferase polymerization extension guanine DNA enzyme-mediated chemiluminescence technology in the prior art is fused, a better technical effect is achieved, and the detection precision is greatly improved.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application.
FIG. 1: schematic diagram for dendritic amplification detection of HTLV-II DNA based on controllable self-assembly biological bar code.
The detection method comprises two continuous reaction steps: (1) HTLV-II DNA induced first step enzymatic extension reaction and biological bar code dendritic self assembly; (2) a second step of chemiluminescent detection in the presence of heme induced by enzymatic extension.
FIG. 2 is a schematic diagram: electrophoretic analysis of the products of the enzymatic extension reaction.
Among them, FIG. (A) is an agarose gel electrophoresis analysis of the deoxynucleotidyl transferase catalyzed first-step extension reaction products at different polymerization time terminals. Reaction conditions are as follows: the terminal deoxynucleotidyl transferase concentration was 0.4 units and the dTTP concentration was 10. mu. mol/L. Lane M is marker; lane 1 is the reaction product in the absence of terminal deoxynucleotidyl transferase; lanes 2-6 are the products in the presence of terminal deoxynucleotidyl transferase for 5, 10, 30, 60 and 120 minutes, respectively.
The graph (B) shows the change in the chemiluminescence intensity and the reaction time for each sample in (a).
FIG. C is an agarose gel electrophoresis analysis of the deoxynucleotidyl transferase catalyzed second-step extension reaction products at different polymerization times. Reaction conditions are as follows: the terminal deoxynucleotidyl transferase concentration was 0.4 units and the dNTP concentration was 10. mu. mol/L. Wherein lane M is marker; lane 1 is the reaction product in the absence of terminal deoxynucleotidyl transferase; lanes 2-6 are the reaction products at 60, 30, 20, 10 and 5 minutes, respectively.
The graph (D) shows the change in the chemiluminescence intensity and the reaction time for each sample in (C). Error bars represent standard deviations of three independent experiments.
FIG. 3: chemiluminescence intensity changes under different concentrations of HTLV-II DNA and linear analysis chart thereof.
Error bars represent standard deviations of three independent experiments.
FIG. 4: chemiluminescent detection of DNA of different mismatched bases.
Single base mismatch DNA (one mismatch), three base mismatch DNA (three mismatches), non-complementary DNA (noncomplementary DNA), and HTLV-II DNA. The concentrations of DNA were all 1 nmol/L. Error bars represent standard deviations of three independent experiments.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As introduced in the background art, the detection means of nucleotides in the prior art has the technical defects of complex operation, low specificity and poor sensitivity, and in order to solve the technical problems, the invention provides a chemiluminescence method for performing dendritic amplification detection on HTLV-II DNA based on terminal deoxynucleotidyl transferase catalytic biological barcode self-assembly.
In a specific embodiment of the invention, a kit for dendritic amplification detection of HTLV-II DNA based on terminal deoxynucleotidyl transferase catalyzed biological barcode self-assembly is provided, wherein the kit comprises terminal deoxynucleotidyl transferase, a magnetic microsphere functionalized by a capture probe 1, a gold nanoparticle functionalized by a capture probe 2 and a report probe, a luminol reagent, a heme reagent and an incubation reagent; wherein the sequence of the capture probe 1 is 5'-ATG GGG TCC CAG GTG AG-3' (the 3 end is modified by biotin); the sequence of the capture probe 2 is 5'-AAA AAAAAA AAA AAA AAATCT TAT CTT-3' (the 3 end is modified by biotin); the sequence of the reporter probe is 5'-ACATGC TTG GAC TGC-3' (5-terminal modified with a biotin).
In another embodiment of the present invention, there is provided a nanosensor for dendritic amplified detection of HTLV-II DNA based on self-assembly of a terminal deoxynucleotidyl transferase-catalyzed biological barcode, comprising: terminal deoxynucleotidyl transferase, magnetic microspheres functionalized by capture probes 1, gold nanoparticles functionalized by capture probes 2 and reporter probes, luminol reagent, heme reagent and incubation reagent; the sequence of the capture probe 1 is as follows: 5'-ATG GGG TCC CAG GTG AG-3' (3-terminal modified with a biotin); the sequence of the capture probe 2 is as follows: 5'-AAAAAAAAAAAAAAA AAA TCT TAT CTT-3' (3-terminal modified with a biotin); the sequence of the reporter probe is 5'-ACA TGC TTG GAC TGC-3' (5-terminal modified with a biotin).
In still another embodiment of the present invention, there is provided a method for detecting the above-mentioned detection kit or nanosensor, the method comprising the steps of:
(1) hybridizing the target HTLV-II DNA to the modified capture probe 1 portion on the magnetic microsphere to form a stable dsDNA duplex with a 3' terminal sequence overhanging the target HTLV-II DNA; adding terminal deoxynucleotidyl transferase, taking a 3' terminal DNA sequence as a primer, and initiating a first-step polymerization extension reaction to obtain a polymerization product rich in thymine;
(2) adding AuNPs modified with capture probe 2 and reporter probe, which will polymerize in large amount by complementary binding of capture probe 2 and thymine-rich polymerization product, taking the reporter probe on AuNPs as primer, deoxynucleic acid terminal transferase will initiate the second polymerization extension reaction and generate large amount of guanine-rich product;
(3) adding heme, folding the guanine-rich product into a spatial G-tetrad structure, catalyzing the oxidation reaction of luminol mediated by hydrogen peroxide, and detecting a chemiluminescence signal to determine the content of HTLV-II DNA.
In a preferred embodiment, the method for preparing the magnetic microspheres in the step (1) comprises the following steps:
Dissolving the capture probe 1 in a 1 xTris-EDTA buffer solution to prepare a stock solution; adding 10 mu mol/L of capture probe 1 and 10mg/mL of magnetic microspheres wrapped with streptavidin into ultrapure water, and incubating for 10min at room temperature to enable the capture probe 1 to be combined with the magnetic microspheres wrapped with streptavidin; and removing excessive capture probes 1 in the solution by using magnetic separation to obtain the streptavidin-coated magnetic microspheres functionalized by the capture probes 1.
The capture probe 1: magnetic microspheres: the proportion of the ultrapure water solution is 1 mu L: 1 μ L: 10 μ L.
Preferably, the method for preparing AuNPs in the step (2) comprises the following steps: dissolving a capture probe 2 and a report probe in a 1 xTris-EDTA buffer solution to prepare a stock solution; AuNPs functionalized with capture probes 2 and reporter probes were formed by adding 0.5mg/mL AuNPs, 1. mu. mol/L capture probes 2, and 1. mu. mol/L reporter probes to ultrapure water and incubating for 10min at room temperature via biotin-streptavidin interaction.
Further, wherein AuNPs: capture probe 2: the reporter probe: the proportion of ultrapure water is 1 mu L: 0.45. mu.L: 4.05 μ L: 1200. mu.L.
Further, the above-mentioned greenhouse incubation needs to be performed in an incubation buffer, and the formulation of the incubation buffer is as follows: 40mmol/L hydroxyethylpiperazine ethanethiosulfonic acid, 300mmol/L sodium chloride, 20mol/L potassium chloride, and pH 8.0.
Preferably, the specific operation of step (1) is: adding HTLV-II DNA into 20 mu L of a hybridization solution of capture probe 1 functionalized magnetic microspheres, 750mmol/L sodium chloride and 75mmol/L sodium citrate, and incubating for 10min at room temperature to form double-stranded DNA with a protruding 3' -hydroxyl end; after magnetic separation, 0.4 unit of terminal deoxynucleotidyl transferase was added to 20. mu.L of a polymerization reaction solution consisting of the separated precipitated product, which included 2. mu.L of 100. mu. mol/L dTTP, 2. mu.L of 10 Xterminal deoxynucleotidyl transferase reaction buffer, and 2. mu.L of 2.5mmol/L CoCl2The first extension reaction was performed by incubation at 37 ℃ for 30min with HTLV-II DNA at the overhanging 3' end as primer to give a thymine-rich polymerization product.
Preferably, the specific operation of step (2) is: adding 0.15 mu L of AuNPs with the capture probe 2 and the report probe functionalized into a hybridization solution of the thymine polymerization product, wherein the hybridization solution comprises 750mmol/L NaCl and 75mmol/L sodium citrate, and incubating for 10min at room temperature to ensure that the AuNPs with the capture probe 2 and the report probe functionalized are hybridized to the thymine polymerization product long chain; removing redundant terminal deoxynucleotidyl transferase, dTTP and AuNPs with capture probe 2 and reporter probe functionalization through a magnetic separation step; then 0.4 units of DNAse were added to 20. mu.L of a mixture containing 0.8. mu.L of 100. mu. mol/L dATP, 1.2. mu.L of 100. mu. mol/L dGTP, 2. mu.L of 10 XDNAse buffer, 2. mu.L of 2.5mmol/L CoCl 2(ii) a Incubating at 37 ℃ for 20min, adding 63.1 mu L of deionized water to suspend the precipitated product after a magnetic separation step, incubating at 90 ℃ for 3min, and quickly transferring the supernatant into a clean tube by using magnetic separation for chemiluminescence determination.
Preferably, the specific operation of step (3) is: adding 0.5mmol/L luminol solution and 750nmol/L heme solution into 63.1. mu.L of the reaction product in the step (2), adding 30. mu.L incubation buffer, and incubating at room temperature for 30min to obtain a G-quadruplex structure; to the mixture was added 15. mu.L of 100mmol/L H2O2Thereafter, the chemiluminescent signal was measured by a 96-microplate luminometer at 1.5 second intervals.
Example one
The terminal deoxynucleotide transferase catalyzes the self-assembly of biological barcodes to realize the amplification of dendritic chemiluminescent signals: the terminal deoxynucleotidyl transferase is required to perform two polymerization extension reactions, in the first polymerization extension reaction, different concentrations of the target HTLV-II DNA are added to 20. mu.L of a hybridization solution containing freshly prepared capture probe 1 functionalized magnetic microspheres, 750mmol/L NaCl and 75mmol/L sodium citrate, followed by incubation at room temperature for 10 minutes to form 3' -hydroxyl-end protruding double-stranded DNA. After magnetic separation, 0.4 unit of terminal deoxynucleotidyl transferase was added to 20. mu.L of the product group precipitated by separation The resulting polymerization reaction solution contained 2. mu.L of 100. mu. mol/L dTTP, 2. mu.L of 10X terminal deoxynucleotidyl transferase reaction buffer, and 2. mu.L of 2.5mmol/L CoCl2And the first extension reaction was performed by incubating with the highlighted 3' end of HTLV-II DNA as a primer at 37 ℃ for 30 min.
After the first extension reaction is finished, the HTLV-II DNA primer at the 3' end extends out of a polymerization product containing thymine. Then 0.15. mu.L of freshly prepared AuNPs with capture probe 2 and reporter probe functionalized were added to the hybridization solution of the polymerization product of thymine. The hybridization solution comprised 750mmol/L NaCl and 75mmol/L sodium citrate and was incubated at room temperature for 10min to allow hybridization of AuNPs with capture probes 2 and reporter probes functionalized to long chains of thymine-containing polymerizate. Excess terminal deoxynucleotidyl transferase, dTTP and AuNPs with capture probe 2 and reporter probe functionalization were removed by a magnetic separation step. Then, the second-step extension reaction of DNAse was performed by adding 0.4 unit of DNAse to a mixture containing 20. mu.L of the product of the first-step extension polymerization reaction, which mixture contained 0.8. mu.L of 100. mu. mol/L dATP, 1.2. mu.L of 100. mu. mol/L dGTP, 2. mu.L of 10 XDNAse buffer, 2. mu.L of 2.5mmol/L CoCl 2And incubated at 37 deg.C for 20 min. After the magnetic separation step, 63.1 mul of deionized water is added to suspend the precipitated product, the product is incubated for 3min at 90 ℃, and then the supernatant is quickly transferred to a clean tube by using magnetic separation for chemiluminescence determination.
And (3) chemiluminescence detection: freshly prepared 0.5mmol/L luminol solution and 750nmol/L heme solution were added to the reaction product containing 63.1. mu.L, followed by 30. mu.L incubation buffer and incubation at room temperature for 30min to fold the complex polymerization product into a G-quadruplex structure. To the mixture was added 15. mu.L of 100mmol/L H2O2Thereafter, the chemiluminescent signal was measured by a 96-microplate luminometer at 1.5 second intervals.
The first test example: experimental verification of the principle
In order to verify the feasibility of the experiment, the present invention performed agarose gel electrophoresis and corresponding chemiluminescence detection analysis on the two-step extension reaction product of terminal deoxynucleotidyl transferase, and the results are shown in FIG. 2, (A) agarose gel electrophoresis analysis of the first-step extension reaction product catalyzed by terminal deoxynucleotidyl transferase at different polymerization times. The reaction conditions are as follows: the terminal deoxynucleotidyl transferase concentration was 0.4 units and the dTTP concentration was 10. mu. mol/L. Wherein lane M is DNA marker; lane 1 is the reaction product in the absence of terminal deoxynucleotidyl transferase; lanes 2-6 are the products in the presence of terminal deoxynucleotidyl transferase for 5, 10, 30, 60 and 120min, respectively. (B) Change in chemiluminescence intensity and reaction time of each sample corresponding to (a). (C) The products of the second step extension reaction catalyzed by terminal deoxynucleotidyl transferase (TdT) were analyzed by agarose gel electrophoresis at different polymerization times. The reaction conditions are as follows: the terminal deoxynucleotidyl transferase concentration was 0.4 units and the dNTP concentration was 10. mu. mol/L. Wherein lane M is DNA marker; lane 1 is the reaction product in the absence of terminal deoxynucleotidyl transferase; lanes 2-6 are the reaction products at 60, 30, 20, 10 and 5min, respectively. (D) Change in chemiluminescence intensity and reaction time of each sample corresponding to (C). The above results show that both template-free polymerization extension reaction catalyzed by terminal deoxynucleotidyl transferase and detection of chemiluminescence can be performed.
Test example two: evaluation of sensitivity
To evaluate the sensitivity of the present protocol for detecting HTLV-II DNA, various concentrations of the assay were performed and the results are shown in FIG. 3. In order to evaluate the quantitative analysis capability of the HTLV-II DNA, the invention logarithms the concentration of the HTLV-II DNA, and observes that the chemiluminescence intensity and the concentration logarithm value thereof show good linear relation in a certain concentration range, and the detection limit can reach 0.5 multiplied by 10-18mol/L, therefore, the technical scheme has ultrahigh detection sensitivity.
Test example three: evaluation of specificity
In order to evaluate the specificity of the scheme, the invention designs three types of mismatched base DNA sequences for specificity verification experiments, such as single base mismatched DNA, three base mismatched DNA, non-complementary DNA and HTLV-II DNA, as shown in FIG. 4. The technical scheme has good specificity according to judgment of a chemiluminescence signal.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> university of Shandong Master
<120> method for detecting HTLV-II DNA based on enzyme catalysis controllable self-assembly biological bar code
<130> 2010
<160> 3
<170> PatentIn version 3.3
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aaaaaaaaaa aaaaaaaatc ttatctt 27
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Claims (7)
1. A kit for carrying out dendritic amplification detection on HTLV-II DNA based on terminal deoxynucleotidyl transferase catalytic biological barcode self-assembly comprises terminal deoxynucleotidyl transferase, a magnetic microsphere with capture probe 1 functionalized, a gold nanoparticle with capture probe 2 and reporter probe functionalized, a luminol reagent, a heme reagent and an incubation reagent; the sequence of the capture probe 1 is as follows: 5'-ATG GGG TCC CAG GTG AG-3', modifying one biotin at the 3' end; the sequence of the capture probe 2 is as follows: 5'-AAA AAA AAA AAA AAA AAA TCT TAT CTT-3', modifying one biotin at the 3' end; the sequence of the reporter probe is 5'-ACA TGC TTG GAC TGC-3', and the 5' end is modified by biotin;
the preparation method of the modified capture probe 1 magnetic microsphere comprises the following steps: dissolving the capture probe 1 in a 1 xTris-EDTA buffer solution to prepare a stock solution; adding 10 mu mol/L of capture probe 1 and 10 mg/mL of magnetic microspheres wrapped with streptavidin into ultrapure water, and incubating for 10 min at room temperature to enable the capture probe 1 to be combined with the magnetic microspheres wrapped with streptavidin; removing excessive capture probes 1 in the solution by magnetic separation to obtain streptavidin-coated magnetic microspheres functionalized by the capture probes 1; capture probe 1: magnetic microspheres: the proportion of the ultrapure water solution is 1 mu L: 1 μ L: 10 mu L of the solution;
The preparation method of the gold nanoparticles comprises the following steps: dissolving a capture probe 2 and a report probe in a 1 xTris-EDTA buffer solution to prepare a stock solution; adding 0.5 mg/mL of nano-gold particles, 1 mu mol/L of capture probe 2 and 1 mu mol/L of report probe into ultrapure water, incubating for 10 min at room temperature, and forming capture probe 2 and report probe functionalized nano-gold particles through biotin-streptavidin interaction; nano gold particles: capture probe 2: the reporter probe: the proportion of ultrapure water is 1 mu L: 0.45. mu.L: 4.05 μ L: 1200. mu.L.
2. A nanosensor for dendritic amplified detection of HTLV-II DNA based on self-assembly of a terminal deoxynucleotidyl transferase catalyzed biological barcode, comprising: terminal deoxynucleotidyl transferase, magnetic microspheres functionalized by capture probes 1, gold nanoparticles functionalized by capture probes 2 and reporter probes, luminol reagent, heme reagent and incubation reagent; the sequence of the capture probe 1 is as follows: 5'-ATG GGG TCC CAG GTG AG-3', modifying one biotin at the 3' end; the sequence of the capture probe 2 is as follows: 5'-AAA AAA AAA AAA AAA AAA TCT TAT CTT-3', modifying one biotin at the 3' end; the sequence of the reporter probe is 5'-ACA TGC TTG GAC TGC-3', and the 5' end is modified by biotin;
The preparation method of the modified capture probe 1 magnetic microsphere comprises the following steps: dissolving the capture probe 1 in a 1 xTris-EDTA buffer solution to prepare a stock solution; adding 10 mu mol/L of capture probe 1 and 10 mg/mL of magnetic microspheres wrapped with streptavidin into ultrapure water, and incubating for 10 min at room temperature to enable the capture probe 1 to be combined with the magnetic microspheres wrapped with streptavidin; removing excessive capture probes 1 in the solution by magnetic separation to obtain streptavidin-coated magnetic microspheres functionalized by the capture probes 1; capture probe 1: magnetic microspheres: the proportion of the ultrapure water solution is 1 mul: 1 μ L: 10 mu L of the solution;
the preparation method of the gold nanoparticles comprises the following steps: dissolving a capture probe 2 and a report probe in a 1 xTris-EDTA buffer solution to prepare a stock solution; adding 0.5 mg/mL of nano-gold particles, 1 mu mol/L of capture probe 2 and 1 mu mol/L of report probe into ultrapure water, incubating for 10 min at room temperature, and forming capture probe 2 and report probe functionalized nano-gold particles through biotin-streptavidin interaction; nano gold particles: capture probe 2: the reporter probe: the proportion of ultrapure water is 1 mu L: 0.45. mu.L: 4.05 μ L: 1200. mu.L.
3. The kit of claim 1 or the nanosensor of claim 2, wherein the detection method comprises the steps of:
(1) the target HTLV-II DNA hybridizes to the modified capture probe 1 portion on the magnetic microsphere to form a stable dsDNA duplex with the 3' terminal sequence protruding from the target HTLV-II DNA; adding terminal deoxynucleotidyl transferase, taking a 3' terminal DNA sequence as a primer, and initiating a first-step polymerization extension reaction to obtain a polymerization product rich in thymine;
(2) adding nano-gold particles modified with capture probes 2 and reporter probes, wherein the nano-gold particles are subjected to mass polymerization through complementary combination of the capture probes 2 and the polymerization product rich in thymine, and the reporter probes on the nano-gold particles are used as primers, and the deoxyribonucleic acid terminal transferase initiates a second-step polymerization extension reaction to generate a product rich in guanine;
(3) adding heme, folding the guanine-rich product into a spatial G-tetrad structure, catalyzing oxidation reaction of luminol mediated by hydrogen peroxide, and detecting a chemiluminescence signal to determine the content of HTLV-II DNA.
4. The kit or nanosensor of claim 3, wherein said incubation at room temperature is performed in an incubation buffer having a formulation of: 40 mmol/L hydroxyethylpiperazine ethanethiosulfonic acid, 300 mmol/L sodium chloride, 20 mol/L potassium chloride, pH 8.0.
5. The kit or nanosensor of claim 3, wherein the specific operation of step (1) is: adding HTLV-II DNA into a hybridization solution of 20 mu L of capture probe 1 functionalized magnetic microspheres, 750 mmol/L sodium chloride and 75 mmol/L sodium citrate, and incubating for 10 min at room temperature to form double-stranded DNA with a protruding 3' -hydroxyl end; after magnetic separation, 0.4 unit of terminal deoxynucleotidyl transferase was added to 20. mu.L of a polymerization reaction solution consisting of the separated precipitated product, which included 2. mu.L of 100. mu. mol/L dTTP, 2. mu.L of 10 Xterminal deoxynucleotidyl transferase reaction buffer, and 2. mu.L of 2.5 mmol/L CoCl2The first extension reaction was performed by incubation at 37 ℃ for 30 min with HTLV-II DNA at the overhanging 3' end as primer to give a thymine-rich polymerization product.
6. The kit or nanosensor of claim 3, wherein the specific operation of step (2) is: adding 0.15 mu L of the gold nanoparticles with the capture probe 2 and the reporter probe functionalized into a hybridization solution of the thymine polymerization product, wherein the hybridization solution comprises 750 mmol/L NaCl and 75 mmol/L sodium citrate, and incubating at room temperature for 10 min to allow the gold nanoparticles with the capture probe 2 and the reporter probe functionalized to hybridize to the thymine-containing polymerization product On the object chain; removing redundant terminal deoxynucleotidyl transferase, dTTP and gold nanoparticles with capture probes 2 and reporter probes through a magnetic separation step; then 0.4 units of DNAse were added to 20. mu.L of a mixture containing 0.8. mu.L of 100. mu. mol/L dATP, 1.2. mu.L of 100. mu. mol/L dGTP, 2. mu.L of 10 XDNAse buffer, 2. mu.L of 2.5 mmol/L CoCl2(ii) a Incubating at 37 ℃ for 20 min, adding 63.1 mu L of deionized water to suspend the precipitated product after a magnetic separation step, incubating at 90 ℃ for 3 min, and quickly transferring the supernatant into a clean tube by using magnetic separation for chemiluminescence determination.
7. The kit or nanosensor of claim 3, wherein the specific operation of step (3) is: adding 0.5 mmol/L luminol solution and 750 nmol/L heme solution into 63.1. mu.L of the reaction product in the step (2), adding 30. mu.L incubation buffer, and incubating at room temperature for 30 min to obtain a G-quadruplex structure; to the mixture was added 15. mu.L of 100mmol/L H2O2Thereafter, the chemiluminescent signal was measured by a 96-microplate luminometer at 1.5 second intervals.
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