CN108588284B - A method for detection of HTLV-II DNA based on enzymatically controllable self-assembled biological barcodes - Google Patents

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^{‑ 18}mol/L, greatly improved sensitivity compared with the prior art, good specificity and simple and convenient operation.

Description

A method for detection of HTLV-II DNA based on enzyme-catalyzed controllable self-assembled biological barcodes

technical field

The invention relates to the field of biotechnology, in particular to a chemiluminescence method for dendritic amplification detection of human T lymphocytic leukemia virus II (HTLV-II) DNA based on the self-assembly of biological barcodes catalyzed by terminal deoxynucleotidyl transferase (TdT). method.

Background technique

In the prior art, technical means commonly used to detect nucleotides include polymerase chain reaction (PCR), rolling circle amplification reaction (RCA), loop-mediated isothermal amplification reaction (LAMP), and hybridization chain reaction (HCR). ), catalytic hairpin assembly (CHA) amplification, ligase chain reaction (LCR), exonuclease/endonuclease assisted signal amplification (EASA). PCR is a thermocycling-based DNA amplification technique that involves stringent primer/template design and precise thermocycling. RCA and LAMP are isothermal amplification techniques that avoid the tedious thermal cycling steps of PCR. However, RCA involves complex circular template preparation and isolation steps, while LAMP requires the design of complex DNA hairpin probes. In addition, PCR, RCA, and LAMP all rely on DNA template amplification, which inevitably involves non-specific amplification, thereby causing cross-contamination. In addition, HCR is based on the chain hybridization reaction between two sets of DNA hairpin probes, and the CHA amplification reaction is based on the target DNA catalyzing the hybridization between two DNA hairpin probes. Both HCR and CHA amplification reactions can provide nucleic acid signal amplification reactions without the participation of enzymes, 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 the detection of target DNA. However, the reaction conditions of LCR, including suitable ligation temperature, cycle times and ligase concentration, are relatively complicated, and gel electrophoresis is also required for the separation of ligation products. EASA cyclically digests or cyclically cleaves specific nucleosides using exonucleases (eg, Exonuclease III and Lambda Exonuclease) and endonucleases (eg, nickases (Nt.AlwI and Nt.BbvCI) acid sequences, which amplify the signal. However, exonuclease III and lambda exonuclease induce non-specific digestion, making the reaction less sensitive and specific.

Biological Barcode Amplification (BCA) is a new type of amplification technology in which short oligonucleotides act as identification strands and substitute amplification units to amplify the signal. Notably, multiple oligonucleotide chains can be assembled on a single nanoparticle, and the subsequent magnetic separation provides a very clean reaction environment for BCA, which can be used to detect a variety of proteins and nucleotides with high sensitivity and specificity. However, the reported hybridization ratio of nanoparticles and target strands in BCA is 1:1, the preparation process of oligonucleotide/protein-modified nanoparticles is complicated, and the procedure for distinguishing signaling probes from nanoparticles is also very complicated. It is cumbersome. At present, it is still a huge challenge to achieve high sensitivity and wide dynamic range of detection, which urgently requires the introduction of new technologies into BCA.

Human T-lymphocyte virus is a member of the human C-type RNA tumor virus family and is an oncogenic RNA virus that can be divided into HTLV-I, HTLV-II and HTLV-III. Cell malignancy diseases and acquired immunodeficiency syndrome (AIDS) are closely linked. Among them, HTLV-II has a strong correlation with neurological diseases, respiratory diseases, and inflammatory reactions. Furthermore, HTLV-II-infected injecting drug users may introduce the virus into the general population and blood donors through secondary transmission, thereby inducing neurological disability and neuropathy globally. Considering the low abundance, small size, high sequence homology of HTLV family members and its key role in biomedical research, it is very urgent to develop an effective method for the detection of HTLV-II with high sensitivity and specificity. .

SUMMARY OF THE INVENTION

The invention provides a chemiluminescence method for dendritic amplification detection of human T lymphocytic leukemia virus II (HTLV-II) DNA based on the self-assembly of biological barcodes catalyzed by terminal deoxynucleotidyl transferase. The method has the advantages of high sensitivity and easy operation. Operational advantages. In order to realize the above technical purpose, the present invention provides the following technical solutions:

One of the objectives of the present invention is to provide a kit for dendritic amplification detection of HTLV-II DNA based on the self-assembly of biological barcodes catalyzed by terminal deoxynucleotidyl transferase, the kit includes terminal deoxynucleotidyl transferase, Magnetic microspheres functionalized with capture probe 1, gold nanoparticles functionalized with capture probe 2 and reporter probe, luminol reagent, heme reagent, and incubation reagent; the sequence of capture probe 1 is: 5' -ATG GGG TCC CAG GTG AG-3' (modified with a biotin at the 3 end); the sequence of capture probe 2 is: 5'-AAA AAA AAA AAA AAA AAA TCT TAT CTT-3' (modified with a biotin at the 3 end) ; The sequence of the reporter probe is 5'-ACA TGC TTG GAC TGC-3' (5-terminal modified with a biotin).

The second objective of the present invention is to provide a nanosensor for dendritic amplification detection of HTLV-II DNA based on the self-assembly of biological barcodes catalyzed by terminal deoxynucleotidyl transferase, characterized in that the nanosensor comprises: a terminal deoxynucleotide nucleus Glycosyltransferase, magnetic microspheres functionalized with capture probe 1, gold nanoparticles functionalized with capture probe 2 and reporter probe, luminol reagent, heme reagent, incubation reagent; the capture probe 1 The sequence is: 5'-ATG GGG TCCCAG GTG AG-3' (3 ends are modified with a biotin); the sequence of the capture probe 2 is: 5'-AAA AAA AAA AAA AAAAAA TCT TAT CTT-3' (3 The sequence of the reporter probe is 5'-ACA TGC TTG GAC TGC-3' (one biotin is modified at the 5 end).

The third object of the present invention is to provide the detection method of the above-mentioned test kit or nanosensor, and the steps of the method are as follows:

(1) The target HTLV-II DNA is partially hybridized with the modified capture probe 1 on the magnetic microspheres to form a stable dsDNA duplex with a sequence protruding from the 3' end of the target HTLV-II DNA; terminal deoxynucleotidyl transferase is added , using the 3'-end DNA sequence as a primer to initiate the first step of polymerization and extension reaction to obtain a thymine-rich polymer product;

(2) Adding the gold nanoparticles modified with the capture probe 2 and the reporter probe, which will polymerize in a large amount through the complementary binding of the capture probe 2 and the thymine-rich polymer product to the reporter probe on the gold nanoparticles As a primer, deoxynucleic acid terminal transferase will initiate the second-step polymerization and extension reaction, and generate a large number of guanine-rich products;

(3) Adding heme, the guanine-rich product folds into a space G-quadruplex structure, and catalyzes the oxidation reaction of luminol mediated by hydrogen peroxide, and determines the content of HTLV-II DNA by detecting chemiluminescence signal detection .

Preferably, the preparation method of magnetic microspheres in the above step (1) includes the following steps:

Dissolve capture probe 1 in 1×Tris-EDTA buffer to prepare a stock solution; add 10 μmol/L capture probe 1 and 10 mg/mL streptavidin-coated magnetic microspheres to ultrapure water , and incubated at room temperature for 10 min to bind the capture probe 1 to the streptavidin-coated magnetic microspheres; use magnetic separation to remove excess capture probe 1 in the solution to obtain functionalized capture probe 1 of streptavidin-coated magnetic microspheres.

Further, the ratio of capture probe 1: magnetic microsphere: ultrapure aqueous solution is 1 μL: 1 μL: 10 μL.

Preferably, the preparation method of gold nanoparticles in the above step (2) includes the following steps: dissolving the capture probe 2 and the reporter probe in 1×Tris-EDTA buffer to prepare a stock solution; Gold nanoparticles, 1 μmol/L capture probe 2 and 1 μmol/L reporter probe were added to ultrapure water and incubated at room temperature for 10 min to form capture probes through biotin-streptavidin interaction 2. Gold nanoparticles functionalized with reporter probes.

Further, the ratio of gold nanoparticles: capture probe 2: reporter probe: ultrapure water is 1 μL: 0.45 μL: 4.05 μL: 1200 μL.

Further, the incubation under the above-mentioned greenhouse needs to be carried out in an incubation buffer, and the formula of the incubation buffer is as follows: 40mmol/L hydroxyethylpiperazine ethanethiosulfonic acid, 300mmol/L sodium chloride, 20mol/L potassium chloride , pH8.0.

Preferably, the specific operation of the above step (1) is: adding HTLV-II DNA to a hybridization solution of 20 μL of capture probe 1 functionalized magnetic microspheres, 750 mmol/L sodium chloride and 75 mmol/L sodium citrate, Incubate for 10 min at room temperature to form double-stranded DNA with a 3'-hydroxyl end overhang; after magnetic separation, 0.4 units of terminal deoxynucleotidyl transferase were added to 20 μL of the polymerization reaction solution consisting of the separated precipitated products, and the polymerization reaction The solution consisted of 2 μL of 100 μmol/L dTTP, 2 μL of 10× terminal deoxynucleotidyl transferase reaction buffer, 2 μL of 2.5 mmol/L CoCl ₂ , and incubated at 37 degrees Celsius with the overhanging 3′-end HTLV-II DNA as a primer. The first extension reaction was carried out for 30 min to obtain a thymine-rich polymer product.

Preferably, the specific operation of the above step (2) is: adding 0.15 μL of gold nanoparticles functionalized with the capture probe 2 and the reporter probe into the hybridization solution of the thymidine polymerization product, the hybridization solution comprising 750mmol/L L of NaCl and 75 mmol/L of sodium citrate, and incubated at room temperature for 10 min to hybridize the gold nanoparticles functionalized with capture probe 2 and reporter probe to the long chain of thymine-containing polymer products; Separation step to remove excess terminal deoxynuclease, dTTP and gold nanoparticles functionalized with capture probe 2 and reporter probe; then add 0.4 units of deoxynuclease to 20 μL The mixed solution includes 0.8 μL of 100 μmol/L dATP, 1.2 μL of 100 μmol/L dGTP, 2 μL of 10× deoxynucleoterminal transferase buffer, 2 μL of 2.5 mmol/L CoCl ₂ ; incubate at 37°C for 20 min, and go through a magnetic separation step After that, 63.1 μL of deionized water was added to suspend the precipitated product, and after incubation at 90 degrees Celsius for 3 min, the supernatant was quickly transferred to a clean tube by magnetic separation for chemiluminescence assay.

Preferably, the specific operation of the above step (3) is: adding 0.5 mmol/L luminol solution and 750 nmol/L heme solution to 63.1 μL of the reaction product of the step (2), adding 30 μL warm Incubation buffer, incubate for 30 min at room temperature to obtain the G-quadruplex structure; after adding 15 μL of 100 mmol/L H ₂ O ₂ to the mixture, measure the chemiluminescence signal at 1.5 s time intervals through a 96-well plate photometer .

The above detection methods can be applied to basic research such as the evaluation of the efficacy of antiviral drugs, but not to the diagnosis and treatment of diseases.

The beneficial effects of the present invention

1. High sensitivity: This technical solution utilizes the two-step high-efficiency polymerization extension reaction catalyzed by terminal deoxynucleic acid transferase, the high-efficiency amplification of BCA technology, the high sensitivity of guanine-rich DNase chemiluminescence, and the zero background caused by magnetic separation. , the detection limit can reach 0.5×10 ^-18 mol/L, so this protocol can achieve highly sensitive detection of HTLV-II DNA.

2. Good specificity: In this design scheme, since deoxynucleic acid terminal transferase can carry out the polymerization and extension reaction of a single deoxynucleotide without a DNA template, the precision of the polymerization reaction is very high. The self-assembly of biological barcodes is based on specific hybridization reactions between probes on nanoparticles. Therefore, based on the above two points, this scheme will have good specificity.

3. Simple operation: The reaction in this scheme is carried out under constant temperature conditions, and does not require temperature control; the entire reaction process only involves the amplification of chemiluminescence signal mediated by two-step template-free polymerization extension catalyzed by deoxynucleic acid terminal transferase, and does not involve Polymerase, endonuclease or exonuclease; detection by guanine-rich DNase-mediated chemiluminescence does not require synthetic dye-modified signal probes in general detection methods, so the operation is very simple.

4. The chemiluminescence technology mediated by BCA and terminal deoxynucleic acid transferase polymerization extension guanine DNase in the prior art is integrated, and better technical effects are obtained, and the detection accuracy is greatly improved.

Description of drawings

The accompanying drawings that form a part of the present application are used to provide further understanding of the present application, and the schematic embodiments and descriptions of the present application are used to explain the present application and do not constitute improper limitations on the present application.

Figure 1: Schematic diagram of dendrimer-based detection of HTLV-II DNA based on controllable self-assembled biological barcodes.

The detection method includes two consecutive reaction steps: (1) the first step of enzymatic extension reaction induced by HTLV-II DNA and the dendritic self-assembly of the biological barcode; (2) the second step of enzymatic extension reaction induced heme Chemiluminescence detection in the presence of.

Figure 2: Electrophoretic analysis of the product of the enzymatic extension reaction.

Among them, Figure (A) agarose gel electrophoresis analysis of the first-step extension reaction products catalyzed by terminal deoxynucleotidyl transferase at different polymerization times. Reaction conditions: terminal deoxynucleotidyl transferase concentration was 0.4 units, and dTTP concentration was 10 μmol/L. Lane M is the marker; lane 1 is the reaction product in the absence of terminal deoxynucleotidyl transferase; lanes 2-6 are in the presence of terminal deoxynucleotidyl transferase, respectively, and the reaction time is 5, 10, 30, 60 and 120 minute products.

Figure (B) shows the changes in the chemiluminescence intensity and reaction time of the corresponding samples in (A).

Panel (C) agarose gel electrophoresis analysis of the second-step extension reaction products catalyzed by terminal deoxynucleotidyl transferase at different polymerization times. Reaction conditions: terminal deoxynucleotidyl transferase concentration was 0.4 units, and dNTP concentration was 10 μmol/L. Lane M is the 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.

Figure (D) shows the changes in the chemiluminescence intensity and reaction time of the corresponding samples in (C). Error bars represent the standard deviation of three independent experiments.

Figure 3: Changes of chemiluminescence intensity under different concentrations of HTLV-II DNA and its linear analysis.

Error bars represent the standard deviation of three independent experiments.

Figure 4: Chemiluminescence detection of DNA with different base mismatches.

One mismatch, Three mismatches, Noncomplementary DNA and HTLV-II DNA. The concentration of DNA was 1 nmol/L. Error bars represent the standard deviation of three independent experiments.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the application. Unless otherwise defined, 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 should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof.

As described in the background art, the detection methods of nucleotides in the prior art have technical defects such as complicated operation, low specificity and poor sensitivity. In order to solve the above technical problems, the present invention proposes a terminal deoxynucleoside-based detection method. A chemiluminescent method for dendritic amplification of HTLV-II DNA by acid transferase-catalyzed self-assembly of biological barcodes.

In a specific embodiment of the present invention, a kit for dendritic amplification detection of HTLV-II DNA based on terminal deoxynucleotidyl transferase-catalyzed self-assembly of biological barcodes is provided, and the kit includes a terminal deoxynucleotide nucleus Glycosyltransferase, magnetic microspheres functionalized with capture probe 1, gold nanoparticles functionalized with capture probe 2 and reporter probe, luminol reagent, heme reagent, and incubation reagent; The sequence is 5'-ATG GGG TCC CAG GTG AG-3' (modified with a biotin at the 3 end); the sequence of capture probe 2 is 5'-AAA AAAAAA AAA AAA AAATCT TAT CTT-3' (modified with a biotin at the 3 end) ); the sequence of the reporter probe is 5'-ACATGC TTG GAC TGC-3' (5-terminal modified with a biotin).

In another specific embodiment of the present invention, a nanosensor for dendritic amplification detection of HTLV-II DNA based on the self-assembly of biological barcodes catalyzed by terminal deoxynucleotidyl transferase is provided, characterized in that the nanosensor The sensor includes: terminal deoxynucleotidyl transferase, magnetic microspheres functionalized with capture probe 1, gold nanoparticles functionalized with capture probe 2 and reporter probe, luminol reagent, heme reagent, and incubation reagent; The sequence of the capture probe 1 is: 5'-ATGGGG TCC CAG GTG AG-3' (modified with a biotin at the 3 end); the sequence of the capture probe 2 is: 5'-AAAAAAAAAAAAAAA AAA TCT TAT CTT-3 ' (one biotin modified at the 3 end); the sequence of the reporter probe is 5'-ACATGC TTG GAC TGC-3' (one biotin modified at the 5 end).

In yet another specific embodiment of the present invention, the above-mentioned detection kit or the detection method of the nanosensor is provided, and the steps of the method are as follows:

(1) The target HTLV-II DNA is partially hybridized with the modified capture probe 1 on the magnetic microspheres to form a stable dsDNA duplex with a sequence protruding from the 3' end of the target HTLV-II DNA; terminal deoxynucleotidyl transferase is added , using the 3'-end DNA sequence as a primer to initiate the first step of polymerization and extension reaction to obtain a thymine-rich polymer product;

(2) AuNPs modified with the capture probe 2 and the reporter probe are added, which will polymerize in a large amount through the complementary binding of the capture probe 2 and the thymine-rich polymer product. Using the reporter probe on the AuNPs as a primer, deoxygenation Nucleic acid terminal transferase will initiate the second-step polymerization and extension reaction and generate a large number of guanine-rich products;

(3) Adding heme, the guanine-rich product folds into a space G-quadruplex structure, and catalyzes the oxidation reaction of luminol mediated by hydrogen peroxide, and determines the content of HTLV-II DNA by detecting chemiluminescence signal detection .

In a preferred embodiment, the preparation method of magnetic microspheres in the above-mentioned step (1) comprises the following steps:

Dissolve capture probe 1 in 1×Tris-EDTA buffer to prepare a stock solution; add 10 μmol/L capture probe 1 and 10 mg/mL streptavidin-coated magnetic microspheres to ultrapure water , and incubated at room temperature for 10 min to bind the capture probe 1 to the streptavidin-coated magnetic microspheres; use magnetic separation to remove excess capture probe 1 in the solution to obtain functionalized capture probe 1 of streptavidin-coated magnetic microspheres.

The above capture probe 1: magnetic microspheres: ultrapure aqueous solution in a ratio of 1 μL: 1 μL: 10 μL.

Preferably, the preparation method of AuNPs in the above step (2) includes the following steps: dissolving the capture probe 2 and the reporter probe in 1×Tris-EDTA buffer to prepare a stock solution; dissolving 0.5 mg/mL AuNPs, 1 μmol/L of the capture probe 2 and 1 μmol/L of the reporter probe were added to ultrapure water and incubated at room temperature for 10 min to form the capture probe 2 and the reporter probe through the interaction of biotin-streptavidin. Needle functionalized AuNPs.

Further, the ratio of AuNPs: capture probe 2: reporter probe: ultrapure water is 1 μL: 0.45 μL: 4.05 μL: 1200 μL.

Further, the incubation under the above-mentioned greenhouse needs to be carried out in an incubation buffer, and the formula of the incubation buffer is as follows: 40mmol/L hydroxyethylpiperazine ethanethiosulfonic acid, 300mmol/L sodium chloride, 20mol/L potassium chloride , pH8.0.

Preferably, the specific operation of the above step (1) is: adding HTLV-II DNA to a hybridization solution of 20 μL of capture probe 1 functionalized magnetic microspheres, 750 mmol/L sodium chloride and 75 mmol/L sodium citrate, Incubate for 10 min at room temperature to form double-stranded DNA with a 3'-hydroxyl end overhang; after magnetic separation, 0.4 units of terminal deoxynucleotidyl transferase were added to 20 μL of the polymerization reaction solution consisting of the separated precipitated products, and the polymerization reaction The solution consisted of 2 μL of 100 μmol/L dTTP, 2 μL of 10× terminal deoxynucleotidyl transferase reaction buffer, 2 μL of 2.5 mmol/L CoCl ₂ , and the overhanging 3′-end HTLV-II DNA was used as a primer at 37° C. Incubate for 30 min for the first extension reaction to obtain a thymine-rich polymer product.

Preferably, the specific operation of the above step (2) is: adding 0.15 μL of AuNPs functionalized with the capture probe 2 and the reporter probe into the hybridization solution of the thymidine polymerization product, the hybridization solution includes 750mmol/L of NaCl and 75 mmol/L sodium citrate, and incubated at room temperature for 10 min to hybridize the AuNPs functionalized with capture probe 2 and reporter probe to the long chain of the thymine-containing polymer product; after a magnetic separation step, remove Excess terminal deoxynuclease, dTTP, and AuNPs functionalized with capture probe 2 and reporter probe; then add 0.4 units of terminal deoxynuclease to a 20 μL mix containing 0.8 μL of 100 μmol /L of dATP, 1.2 μL of 100 μmol/L dGTP, 2 μL of 10× deoxynucleic acid terminal transferase buffer, 2 μL of 2.5 mmol/L CoCl ₂ ; incubate at 37°C for 20 min, after the magnetic separation step, add 63.1 μL The precipitated product was suspended in deionized water, incubated at 90 degrees Celsius for 3 min, and the supernatant was quickly transferred to a clean tube by magnetic separation for chemiluminescence assay.

Preferably, the specific operation of the above step (3) is: adding 0.5 mmol/L luminol solution and 750 nmol/L heme solution to 63.1 μL of the reaction product of the step (2), adding 30 μL warm Incubation buffer, incubate for 30 min at room temperature to obtain the G-quadruplex structure; after adding 15 μL of 100 mmol/L H ₂ O ₂ to the mixture, measure the chemiluminescence signal at 1.5 s time intervals through a 96-well plate photometer .

Example 1

Terminal deoxynucleotidyl transferase catalyzes the self-assembly of biological barcodes to achieve dendritic chemiluminescence signal amplification: Terminal deoxynucleotidyl transferase requires two polymerization extension reactions. In the first polymerization extension reaction, different concentrations of the target HTLV-II DNA was added to 20 μL of hybridization solution containing freshly prepared capture probe 1 functionalized magnetic microspheres, 750 mmol/L NaCl and 75 mmol/L sodium citrate, followed by incubation at room temperature for 10 min to form 3′- Double-stranded DNA with protruding hydroxyl ends. After magnetic separation, 0.4 units of terminal deoxynucleotidyl transferase were added to 20 μL of the polymerization reaction solution consisting of the separated precipitated product, including 2 μL of 100 μmol/L dTTP, 2 μL of 10× terminal deoxynucleotidyl transferase reaction buffer, 2 μL of 2.5 mmol/L CoCl ₂ was used for the first extension reaction at 37° C. with the overhanging 3′-end HTLV-II DNA as a primer and incubated for 30 min.

After the first-step extension reaction, the HTLV-II DNA primer at the 3' end extends a thymine-containing polymer product. Then 0.15 μL of freshly prepared AuNPs functionalized with capture probe 2 and reporter probe were added to the hybridization solution of the thymine polymerized product. The hybridization solution includes 750 mmol/L NaCl and 75 mmol/L sodium citrate, and is incubated at room temperature for 10 min to hybridize the AuNPs functionalized with the capture probe 2 and the reporter probe to the thymine-containing polymer product long chain. After a magnetic separation step, excess terminal deoxynuclease, dTTP and AuNPs functionalized with capture probe 2 and reporter probe were removed. Then carry out the second-step extension reaction of deoxynucleic acid terminal transferase, and add 0.4 units of deoxynucleic acid terminal transferase to the mixture containing 20 μL of the first-step extension polymerization reaction product, and the mixture includes 0.8 μL of 100 μmol/L of dATP, 1.2 μL of 100 μmol/L dGTP, 2 μL of 10× deoxynuclease terminal transferase buffer, 2 μL of 2.5 mmol/L CoCl ₂ , and incubated at 37 degrees Celsius for 20 min. After the magnetic separation step, 63.1 μL of deionized water was added to suspend the precipitated product, and after incubation at 90 degrees Celsius for 3 min, the supernatant was quickly transferred to a clean tube by magnetic separation for chemiluminescence assay.

Chemiluminescence detection: Add freshly prepared 0.5 mmol/L luminol solution and 750 nmol/L heme solution to the reaction product containing 63.1 μL, then add 30 μL of incubation buffer, and incubate at room temperature For 30 minutes, the complex polymer product was folded into a G-quadruplex structure. After adding 15 μL of 100 mmol/L H ₂ O ₂ to the mixture, the chemiluminescence signal was measured at 1.5 sec intervals by a 96 microplate luminometer.

Test Example 1: Experimental Verification of the Principle

In order to verify the feasibility of the experiment, the present invention carried out agarose gel electrophoresis and corresponding chemiluminescence detection and analysis on the two-step extension reaction product of terminal deoxynucleotidyl transferase. The results are shown in Figure 2, (A) agarose The products of the first extension reaction catalyzed by terminal deoxynucleotidyl transferase were analyzed by gel electrophoresis at different polymerization times. Reaction conditions: terminal deoxynucleotidyl transferase concentration was 0.4 units, and dTTP concentration was 10 μmol/L. Lane M is the DNA marker; lane 1 is the reaction product in the absence of terminal deoxynucleotidyl transferase; lanes 2-6 are in the presence of terminal deoxynucleotidyl transferase, respectively, and the reaction time is 5, 10, 30, 60 and 120 min product. (B) Changes in chemiluminescence intensity and reaction time for each sample corresponding to (A). (C) Agarose gel electrophoresis analysis of the second-step extension reaction products catalyzed by terminal deoxynucleotidyl transferase (TdT) at different polymerization times. Reaction conditions: terminal deoxynucleotidyl transferase concentration was 0.4 units, and dNTP concentration was 10 μmol/L. Lane M is the 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 5 min, respectively. (D) Changes in chemiluminescence intensity and reaction time for each sample corresponding to (C). The above results show that both the template-free polymerization extension reaction catalyzed by terminal deoxynucleotidyl transferase and the detection of chemiluminescence can be carried out.

Test Example 2: Sensitivity Evaluation

In order to evaluate the sensitivity of this protocol to detect HTLV-II DNA, different concentrations were analyzed and determined, and the results are shown in Figure 3. In order to evaluate its quantitative analysis ability, the present invention takes the logarithm of the concentration of HTLV-II DNA, and it is observed that the chemiluminescence intensity and its concentration logarithm show a good linear relationship within a certain concentration range, and the detection limit can reach 0.5×10 ^{− 18} mol/L, so this technical solution has ultra-high detection sensitivity.

Test Example 3: Specificity Evaluation

In order to evaluate the specificity of this scheme, the present invention designed three types of mismatched base DNA sequences for specificity verification experiments, as shown in Figure 4, which are single-base mismatched DNA, three-base mismatched DNA, non-complementary DNA sequence DNA and HTLV-II DNA. Judging by the chemiluminescence signal, the technical solution has good specificity.

The above descriptions are only preferred embodiments of the present application, and are not intended to limit the present application. For those skilled in the art, the present application may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included within the protection scope of this application.

SEQUENCE LISTING

<110> Shandong Normal University

<120> Detection of HTLV-II DNA based on enzyme-catalyzed controllable self-assembled biological barcodes

<130> 2010

<160> 3

<170> PatentIn version 3.3

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<211> 17

<212> DNA

<213> Artificial sequences

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atggggtccc aggtgag 17

<210> 2

<211> 27

<212> DNA

<213> Artificial sequences

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aaaaaaaaaa aaaaaaaatc ttatctt 27

<210> 3

<211> 15

<212> DNA

<213> Artificial sequences

<400> 3

acatgcttgg actgc 15

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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