Colorimetric method for detecting tobramycin based on double-heavy-chain replacement and three-dimensional DNA structure
阅读说明：本技术 基于双重链置换和三向dna结构检测妥布霉素的比色方法 (Colorimetric method for detecting tobramycin based on double-heavy-chain replacement and three-dimensional DNA structure ) 是由 周楠迪 欧莹 田亚平 于 2019-09-20 设计创作，主要内容包括：基于双重链置换和三向DNA结构检测妥布霉素的比色方法,属于食品安全、医药分析及环境污染检测领域。本发明首先设计了双链T1/T2；当存在妥布霉素时,Bsm DNA聚合酶通过强链置换反应合成完全互补的双链,Nt.BstNBI切刻内切酶切割双链上的识别位点；三向DNA结构捕捉报告探针,再生并置换出大量含有G-四链体形成序列的S1链。此后,G-四链体/血红素催化ABTS<Sup>2-</Sup>/H<Sub>2</Sub>O<Sub>2</Sub>显色反应,利用吸光值与妥布霉素浓度间的线性关系可测定妥布霉素含量。本发明通过适配体捕获妥布霉素触发Nt.BstNBI切刻内切酶和Bsm DNA聚合酶介导的双重链置换反应产生大量的报告探针,同时,报告探针触发λ核酸外切酶辅助的环路扩增,实现了比色信号的多重放大,拓宽了检测范围,提高了检测灵敏度。(A colorimetric method for detecting tobramycin based on double-heavy chain replacement and three-dimensional DNA structure belongs to the fields of food safety, medical analysis and environmental pollution detection. The invention firstly designs double chains T1/T2; when tobramycin exists, Bsm DNA polymerase synthesizes a complete complementary double strand through a strong strand displacement reaction, and the recognition site on the double strand is cut by an Nt.BstNBI nicking endonuclease; the three-way DNA structure captures the reporter probe, regenerates and replaces a large number of S1 strands containing the G-quadruplex forming sequence. Thereafter, G-quadruplexes/heme catalyze ABTS 2‑ /H 2 O 2 And (3) performing color reaction, and determining the content of the tobramycin by utilizing a linear relation between a light absorption value and the concentration of the tobramycin. The invention generates a large amount of report probes by the double heavy chain replacement reaction mediated by the aptamer capture tobramycin trigger Nt.BstNBI nicking endonuclease and Bsm DNA polymerase, and simultaneously, the report probes trigger lambda exonuclease assisted loop amplification, thereby realizing the multiple amplification of colorimetric signals, widening the detection range and improving the detection sensitivity.)
1. A colorimetric method for detecting tobramycin based on double heavy chain replacement and three-dimensional DNA structure is characterized in that: firstly, sequences T1 and T2 are mixed at the same concentration, are denatured at high temperature and are annealed to form double chains, in the presence of tobramycin, aptamer part of T1 in the double chain sequences recognizes and is combined with tobramycin, the 3' end of T2 is exposed, then primer 1, primer 2, Nt.BstNBI nicking endonuclease, Bsm DNA polymerase and free deoxyribonucleoside triphosphate are added into a reaction system, the Bsm DNA polymerase synthesizes completely complementary double chains through strong chain displacement reaction, and the Nt.BstNBI nicking endonuclease cuts recognition sites on the double chains to generate a large number of report probes;
secondly, hybridizing a three-way DNA structure formed by high-temperature denaturation and annealing of the three single-stranded DNAs S1, S2 and S3 with a reporter probe, triggering lambda exonuclease-assisted loop amplification, and regenerating the reporter probe and replacing a large number of S1 strands containing a G-quadruplex forming sequence;
g-quadruplexes/heme catalyzed ABTS after heme addition2-/H2O2And (3) carrying out a color development reaction, measuring the absorbance by using a UV-vis spectrophotometer, and calculating the tobramycin content in the sample by using the linear relation between the absorbance and the concentration of the tobramycin.
2. The colorimetric method for detecting tobramycin based on double heavy chain replacement and three-dimensional DNA structure as claimed in claim 1, wherein: the sequences of the T1, the T2, the primer 1 and the primer 2 are specifically as follows:
T1: 5’-CTG CCG TGA CTA GGC ACT AGT CTC AAC GAG TCG CGT-3’；
T2: 5’-TTT TTT TTT TTT AGT CAT GCT TGA TGA CTC GTT GAC TTA TCC CAA TTG TCA CGG CAG-3’；
primer 1: 5 '-CTG CCG-3';
primer 2: 5'-TTT TTT TTT TTT AGT CAT GCT ACG CGA CTC G-3';
the sequences of S1, S2 and S3 are specifically as follows:
S1: 5’-P-TTT TTT TTT TTT AGT CAT GCT TCT CGG TGT GAC AGG CAA CTC CGG GTT GGG CGG GAT GG-3’；
S2: 5’-TTA ATT ATA ATA ACC AGT TGC CTG GAT GAT CGA GA-3’；
S3: 5’-P-TTT TTT TTT TTT AGT CAT GCT CCC ATC CCG CCC AAC CCC CTT ATT ATA ATT AA-3’。
3. the colorimetric method for detecting tobramycin based on double heavy chain replacement and three-dimensional DNA structure as claimed in claim 2, wherein: the S1 and S3 are DNA sequences of 5' end modified phosphate groups.
4. The colorimetric method for detecting tobramycin based on double heavy chain replacement and three-dimensional DNA structure as claimed in claim 1, which is characterized by comprising the following steps:
(1) target recognition, wherein T1 comprises a tobramycin aptamer sequence and a sequence complementary with T2 and a primer 2, T1 and T2 are uniformly mixed at the same concentration, and renaturation is carried out at 37 ℃ after denaturation at 95 ℃ to form a partially complementary double strand, 10 ~ 50 nM double strand and 4 mu L of tobramycin solution with different concentrations are uniformly mixed, and incubation is carried out for 30 min at 37 ℃;
(2) an isothermal amplification system comprising adding 10 ~ 60 nM primer 1,10 ~ 60 nM primer 2, 1 ~ 6U Nt.BstNBI nicking endonuclease, 1.6U 1.6 ~ 9.6.6U Bsm DNA polymerase, 1 Xbuffer and 2. mu.L, 10 mmol. L to the mixture obtained in step (1)-1Free deoxyribonucleoside triphosphates are evenly mixed and incubated for 30 ~ 150 min at 55 ℃ to generate a large amount of report probes;
(3) three-dimensional DNA structures were prepared by first preparing 0.5 ~ 4. mu. mol. L of each-110. mu.L of S1, 10. mu.L of S2 and 10. mu.L of S3, heating at 95 ℃ for 5 min, and then gradually cooling to room temperature; thereafter, 30. mu.L of the mixed DNA mixture was incubated at 25 ℃ for 60 min to construct a three-dimensional DNA structure;
(4) forming G-quadruplex/heme by mixing the mixed solution obtained in the step (2) with the mixed solution obtained in the step (3), incubating at 37 ℃ for 120 min, adding 1 ~ 6U lambda exonuclease and 1 x lambda exonuclease reaction buffer solution, mixing uniformly, performing enzymatic reaction at 37 ℃ for 30 ~ 150 min to generate a large amount of single-chain S1, adding 400 mu L of working buffer solution and 10 mu L of hemin, and incubating the mixture at 25 ℃ for 60 min to form a G-quadruplex/heme complex;
(5) absorbance detection and standard curve plotting: 20. mu.L of 50 mmol. multidot.L was added to the reaction solution of the step (4)-1ABTS and 10. mu.L H at a volume concentration of 0.3%2O2Incubating at 37 ℃ for 10 min, and reading a blank and an absorbance value of the tobramycin-containing solution at 420nm by using a UV-vis spectrophotometer;
drawing a corresponding linear relation curve according to the relation between the measured light absorption value and the concentration of the tobramycin;
(6) and (3) actual sample detection, namely, measuring corresponding absorbance values of a tobramycin-containing water sample by the operation of the step (1) ~ (5), and calculating corresponding tobramycin concentration from a standard curve.
5. The colorimetric method for detecting tobramycin based on double heavy chain replacement and three-dimensional DNA structure as claimed in claim 4, wherein: the buffer solution in the step (2) specifically contains 100 mmol. L-1 NaCl，50 mmol·L-1 Tris-HCl，10 mmol·L-1 MgCl2，0.1 mg·mL-1A mixed solution of BSA.
6. The colorimetric method for detecting tobramycin based on double heavy chain replacement and three-dimensional DNA structure as claimed in claim 4, wherein: the lambda exonuclease reaction buffer solution in the step (4) specifically contains 67 mmol.L-1 Glycine-KOH，2.5 mmol·L-1 MgCl2，50 mg·mL-1A mixed solution of BSA;
the working buffer solution is specifically 50 mmol.L-1 Tris-HCl，150 mmol·L-1 NH4Cl，20 mmol·L-1KCl, the volume concentration of which is 0.03 percent Triton-X-100, and the pH value is 7.5;
dissolving stock solution of chlorhematin in dimethyl sulfoxide, and diluting stock solution of chlorhematin with the above working buffer solution to 20 μmol.L-1。
7. The colorimetric method for detecting tobramycin based on double heavy chain replacement and three-dimensional DNA structure as claimed in claim 4, wherein: in step (5), the ABTS stock solution is dissolved in dimethyl sulfoxide.
8. The colorimetric method for detecting tobramycin based on double heavy chain replacement and three-dimensional DNA structure as claimed in claim 4, wherein the concentration of tobramycin in the step (6) is 20 ~ 800 nmol.L-1。
Technical Field
The invention relates to a colorimetric method for detecting tobramycin based on double-heavy chain replacement and three-dimensional DNA structure, belonging to the fields of food safety, medical analysis and environmental pollution detection.
Background
Aminoglycoside antibiotics are antibiotics consisting of two or more aminosugars linked to the hexose ring via glycosidic bonds and have good therapeutic effects on common bacterial diseases. Tobramycin is a broad-spectrum aminoglycoside antibiotic used mainly for the treatment of infections caused by certain gram-positive and aerobic gram-negative microorganisms. The mechanism is binding to ribosomes, which destroy the synthetic proteins, resulting in cell membrane damage and cell death. Tobramycin is widely used in animal husbandry due to its good water solubility, low cost and broad antimicrobial spectrum. However, excessive and erroneous use of tobramycin results in a large amount of residues in animal derived foods (such as milk, eggs and meat) and the environment, which residues can cause serious side effects to human health, such as allergy, nephrotoxicity and neurotoxicity.
To date, a variety of conventional and reliable methods have been used for the determination of aminoglycoside antibiotics (including tobramycin), including High Performance Liquid Chromatography (HPLC), capillary zone electrophoresis, enzyme-linked immunosorbent assays (ELISA), and surface plasmon resonance for transfer localization, among others. However, these methods have some disadvantages, such as high detection limits, expensive equipment, long test periods, personnel training and complex sample preparation. Therefore, there is a need to establish a simple, rapid and accurate detection method for detecting tobramycin residues in food and environment.
The G-quadruplex is a special DNA configuration formed by folding guanine-rich nucleic acid sequences under specific ionic conditions and is hydrogen-stabilized with monovalent cations and hoogsteen. When the G-quadruplex is bound to heme, the G-quadruplex/heme complex formed has horseradish peroxidase activity, which is capable of catalyzing H2O2A redox reaction mediated to produce an electrochemical signal and a colorimetric signal. Meanwhile, compared with the traditional biological enzyme, the G-quadruplex/heme has low cost and stabilityThe method has the advantages of good qualitative property and easy preparation, and is widely applied to the development of various biosensors.
Disclosure of Invention
The invention aims to provide a colorimetric method for detecting tobramycin based on double-heavy chain replacement and three-dimensional DNA structure, which has the advantages of high sensitivity, high specificity, low cost, visual result and the like.
The technical scheme of the invention specifically comprises the design of a non-complete complementary double strand and the sequence design of a three-way DNA connection structure; bsm DNA polymerase synthesizes a complete complementary double strand through a strong strand displacement reaction, and Nt.BstNBI nicking endonuclease cuts recognition sites on the double strand to generate a large number of report probes; hybridizing the three-way DNA structure with a reporter probe, triggering lambda exonuclease-assisted loop amplification, stimulating the reporter probe to regenerate and replace a large amount of S1 (containing a G-quadruplex forming sequence); g-quadruplex/heme complex catalyzed ABTS2- / H2O2The system generates a color reaction and the absorbance is measured by a UV-vis spectrophotometer as shown in fig. 1.
The method comprises the following steps: the sequences T1 and T2 were first mixed at the same concentration, T1 and T2 were denatured at high temperature and annealed to form a double strand, and the 3' end of T2 was blocked, ensuring that there was no non-specific amplification in the absence of target. When tobramycin was present, the target bound to the aptamer region on T1, exposing the 3' end of T2. Primer 1, primer 2, nt.bstnbi nicking endonuclease, Bsm DNA polymerase and free deoxyribonucleoside triphosphates are then added to the reaction system, and then the Bsm DNA polymerase synthesizes a DNA duplex containing the nt.bstnbi recognition site by a strong strand displacement reaction and can cleave it to release the reporter probe. Meanwhile, as primer 2 is extended, tobramycin can dissociate from the T1-tobramycin complex due to the strong strand displacement activity of the polymerase, and the released tobramycin can recognize a new T1-T2 duplex to start the next round of isothermal amplification, resulting in more reporter probe. Thus, tobramycin-triggered polymerase and endonuclease assisted isothermal amplification systems have a multi-cycle signal amplification mechanism and produce large quantities of reporter probes.
Meanwhile, a three-dimensional DNA structure compound is designed. The three-way DNA structure formed by high-temperature denaturation and annealing of S1, S2 and S3 is subjected to high-temperature denaturation, the three-way DNA connecting structure is hybridized with a reporter probe, lambda exonuclease-assisted loop amplification is triggered, the regeneration of the reporter probe is stimulated, and a large amount of S1 (containing a G-quadruplex forming sequence) is replaced. After addition of heme, the G-quadruplex/heme complex catalyzes ABTS2-/H2O2The system generates a color reaction and the absorbance is measured by a UV-vis spectrophotometer. And calculating the tobramycin content in the sample by utilizing the linear relation between the light absorption value and the concentration of the tobramycin.
Further, the sequences of the T1, the T2, the primer 1 and the primer 2 are specifically as follows:
T1: 5’-CTG CCG TGA CTA GGC ACT AGT CTC AAC GAG TCG CGT-3’；
T2: 5’-TTT TTT TTT TTT AGT CAT GCT TGA TGA CTC GTT GAC TTA TCC CAA TTG TCA CGG CAG-3’；
primer 1: 5 '-CTG CCG-3';
primer 2: 5'-TTT TTT TTT TTT AGT CAT GCT ACG CGA CTC G-3'.
The sequences of S1, S2 and S3 are specifically as follows:
S1: 5’-P-TTT TTT TTT TTT AGT CAT GCT TCT CGG TGT GAC AGG CAA CTC CGG GTT GGG CGG GAT GG-3’；
S2: 5’-TTA ATT ATA ATA ACC AGT TGC CTG GAT GAT CGA GA-3’；
S3: 5’-P-TTT TTT TTT TTT AGT CAT GCT CCC ATC CCG CCC AAC CCC CTT ATT ATA ATT AA-3’。
further, the S1 and S3 are DNA sequences of 5' modified phosphate groups.
The colorimetric method for detecting tobramycin based on double-heavy chain replacement and three-dimensional DNA structure comprises the following specific steps:
(1) target recognition, wherein T1 comprises a tobramycin aptamer sequence and a sequence complementary with T2 and a primer 2, T1 and T2 are uniformly mixed at the same concentration, and renaturation is carried out at 37 ℃ after denaturation at 95 ℃ to form a partially complementary double strand, 10 ~ 50 nM double strand and 4 mu L of tobramycin solution with different concentrations are uniformly mixed, and incubation is carried out for 30 min at 37 ℃;
(2) an isothermal amplification system comprising adding 10 ~ 60 nM primer 1,10 ~ 60 nM primer 2, 1 ~ 6U Nt.BstNBI nicking endonuclease, 1.6U 1.6 ~ 9.6.6U Bsm DNA polymerase, 1 Xbuffer and 2. mu.L, 10 mmol. L to the mixture obtained in step (1)-1Free deoxyribonucleoside triphosphates are evenly mixed and incubated for 30 ~ 150 min at 55 ℃ to generate a large amount of report probes;
(3) three-dimensional DNA structures were prepared by first preparing 0.5 ~ 4. mu. mol. L of each-110. mu.L of S1, 10. mu.L of S2 and 10. mu.L of S3 were mixed, heated at 95 ℃ for 5 min, and then gradually cooled to room temperature. Thereafter, 30. mu.L of the mixed DNA mixture was incubated at 25 ℃ for 60 min to construct a three-dimensional DNA structure;
(4) forming G-quadruplex/heme by uniformly mixing the mixed solution obtained in the step (2) with the mixed solution obtained in the step (3), incubating at 37 ℃ for 120 min, adding 1 ~ 6U lambda exonuclease and 1 x lambda exonuclease reaction buffer solution, performing enzymatic reaction at 37 ℃ for 30 ~ 150 min after uniformly mixing to generate a large amount of single-chain S1, then adding 400 mu L of working buffer solution and 10 mu L of hemin, and incubating the mixture at 25 ℃ for 60 min to form a G-quadruplex/heme complex;
(5) absorbance detection and standard curve plotting: 20. mu.L of 50 mmol. multidot.L was added to the reaction solution of the step (4)-1ABTS and 10. mu.L H at a volume concentration of 0.3%2O2Incubating at 37 ℃ for 10 min, and reading a blank and an absorbance value of the tobramycin-containing solution at 420nm by using a UV-vis spectrophotometer;
drawing a corresponding linear relation curve according to the relation between the measured light absorption value and the concentration of the tobramycin;
(6) and (3) actual sample detection, namely, measuring corresponding absorbance values of a tobramycin-containing water sample by the operation of the step (1) ~ (5), and calculating corresponding tobramycin concentration from a standard curve.
Further, the buffer solution in the step (2) specifically contains 100 mmol. L-1 NaCl，50 mmol·L-1Tris-HCl，10 mmol·L-1 MgCl2，0.1 mg·mL-1A mixed solution of BSA.
Further, the lambda exonuclease reaction buffer in the step (4) specifically contains 67 mmol.L-1 Glycine-KOH，2.5 mmol·L-1 MgCl2，50 mg·mL-1A mixed solution of BSA;
the working buffer solution is specifically 50 mmol.L-1 Tris-HCl，150 mmol·L-1 NH4Cl，20 mmol·L-1KCl, the volume concentration of which is 0.03 percent Triton-X-100, and the pH value is 7.5;
dissolving stock solution of chlorhematin in dimethyl sulfoxide, and diluting stock solution of chlorhematin with the above working buffer solution to 20 μmol.L-1。
Further, the ABTS stock solution in step (5) is dissolved in dimethyl sulfoxide.
Further, the concentration of tobramycin in the step (6) is specifically 20 ~ 800 nmol.L-1。
The invention has the beneficial effects that: the invention uses aptamer to capture tobramycin to trigger double heavy chain replacement reaction mediated by Nt.BstNBI nicking endonuclease and Bsm DNA polymerase to generate a large amount of report probes, and simultaneously, the report probes trigger lambda exonuclease assisted loop amplification to realize multiple amplification of colorimetric signals. The detection range of the method is expanded and the detection sensitivity is improved by the multiple amplification of colorimetric signals. Compared with the traditional method for detecting tobramycin, the method has the advantages of strong specificity, high sensitivity and simple operation.
Drawings
FIG. 1 is a schematic diagram of a colorimetric method for detecting tobramycin based on double heavy chain displacement and three-dimensional DNA structure.
FIG. 2 Tobramycin colorimetric assay standard curve.
Detailed Description