Detection method of TNT explosive

Abstract

The invention discloses a TNT explosive detection method, and belongs to the technical field of explosive detection. The method comprises the following three parts: a first part of primer is reserved, a probe DNA 1 containing a TNT nucleic acid aptamer can be prevented from being degraded by exonuclease I after being combined with TNT, and the probe DNA 1 which is not combined with TNT is degraded by exonuclease I; the second part is circular amplification, the probe DNA 1 reserved in the previous step opens the DNA of the hairpin stem-loop structure, and the generation of circular amplification reaction with the DNA of the hairpin stem-loop structure as a template is promoted; in the third part, naked eye visual detection and / or ultraviolet spectrophotometry detection are / is carried out according to the G-quadruplex contained in the cyclic amplification product. The detection method provided by the invention is sensitive and high in visualization degree, in-situ detection of the TNT explosive can be realized, and the detection limit reaches the concentration level of pmol / L.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of explosive detection, and particularly relates to a detection method of TNT explosive. BACKGROUND

[0002] Explosive detection is one of the most important and urgent problems in anti-terrorism and homeland security. Nitro explosives in explosives have great power, and TNT (2,4,6-trinitrotoluene) is called the king of explosives due to its stable properties and strong explosive power, and is still widely used to date; meanwhile, TNT can pollute the environment by remaining in the soil, can seriously poison plants, and can cause irreversible harm to human body after being taken in. Therefore, developing a detection technology of TNT has important significance for sustainable development and safety of people's life and property.

[0003] In the past few decades, people have developed a multifunctional analysis platform for TNT detection and quantification, including microbial biological reporting, surface-enhanced Raman spectroscopy, infrared hyperspectral imaging, optical spectroscopy, ion mobility spectroscopy, etc. However, these detection methods not only need large, precise and expensive instruments, but also need professional detection personnel trained by the system to operate, and are not suitable for use in complex and real on-site detection. Therefore, it is necessary to actively explore a sensitive and convenient method that can meet different on-site detection of TNT. SUMMARY

[0004] The purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a detection method of TNT explosive, which uses aptamer to specifically and visually detect TNT explosive.

[0005] The purpose of the present application is achieved by the following technical scheme: a detection method of TNT explosive, comprising three parts:

[0006] 1) The primer is reserved, the probe DNA 1 is combined with the target TNT explosive in the sample to be detected, and after the target TNT explosive is combined with the probe DNA 1, the probe DNA 1 can be prevented from being degraded by exonuclease I, and the probe DNA 1 not combined with the target TNT explosive is degraded by exonuclease I;

[0007] 2) Cycle amplification: the reserved probe DNA 1 in the previous step is opened to develop the hairpin stem loop structure DNA, which promotes the occurrence of cycle amplification reaction under the action of DNA polymerase, and makes the probe DNA 2 and the hairpin stem loop structure DNA partially complementary and paired to produce G-quadruplex;

[0008] 3) Visual detection: naked eye visual detection and / or ultraviolet spectrophotometry detection are carried out according to the G-quadruplex contained in the cycle amplification product.

[0009] The probe DNA 1 is a single-stranded DNA containing two parts, N1 and N2, with a structure of 5'-N1-N2-3'; N1 is 22-26 bases in length, and N2 is the aptamer sequence that specifically binds to the target TNT explosive: CTC GGT TGT GGT GTT AGTTGG TAC TGC GCC TCC GTC GTC CTG TCA CTC TGC TGC AGC GCG CGA GCC ATA CAC TATACA CCT TAA AAT ATA GTT GCC TAG G;

[0010] The hairpin stem-loop structure DNA contains four parts: A, B, C, and D, with a structure of 5'-ABCD-3'. A can pair complementaryly with C to form a stem; the AB sequence can pair complementaryly with the N1 sequence; and D is a single-stranded sequence with a length of 11-14 bases, rich in guanine but unable to form a G-quadruplex.

[0011] The probe DNA 2 is a single-stranded DNA containing two parts, E and F, with a structure of 5'-EF-3'. F and the C part of the hairpin stem-loop structure DNA can be complementary. E is a short single-stranded sequence containing 3-4 guanines, which can form a G-quadruplex when close to the D sequence of the hairpin stem-loop structure DNA.

[0012] Preferably, the method for detecting the TNT explosive includes the following steps:

[0013] 1) Add probe DNA 1, exonuclease I buffer, test sample containing target TNT explosive and water to the system in sequence. After pre-incubation, add exonuclease I to carry out the reaction. After the reaction is completed, heat to inactivate exonuclease I.

[0014] 2) Add Klenow DNA polymerase buffer, hairpin stem-loop structure DNA, probe DNA 2, Klenow DNA polymerase, dNTPs and water to the reaction system of step 1) and react. Inactivate after the reaction is complete.

[0015] 3) Add G-quadruplex buffer to the reaction system from step 2), heat to denature, slowly cool to room temperature, then add chloroheme for incubation, and finally add ABTS. 2- The system was tested with H2O2, and the color change and ultraviolet absorption value at a detection wavelength of 414 nm were observed. In the system with the target TNT explosive present, the color changed from colorless to a visible green, and the ultraviolet absorption value increased with the increase of TNT explosive concentration.

[0016] In the step 1) system, the concentration of the probe DNA 1 is 10 nmol / L, the concentration of the target TNT in the sample to be detected is 0-10 nmol / L, the exonuclease I buffer is 1X, the sample to be detected and the probe DNA 1 are pre-incubated at 25-37℃ for 0.5-1 hour, the concentration of the exonuclease I is 1 U / μL, the reaction is carried out at 37℃ for 1-2 hours, the exonuclease I is inactivated by heating at 80±5℃ for 20 minutes, and then slowly cooled to room temperature.

[0017] In the step 2) system, the Klenow DNA polymerase buffer is 1X, the concentration of dNTPs is 200 μmol / L, the concentration of the hairpin stem-loop structure DNA is 1 μmol / L, the concentration of the probe DNA 2 is 1 μmol / L, the concentration of the Klenow DNA polymerase is 1 U / 10 μL, and the cyclic amplification reaction is incubated at 37℃ for 2±0.5 hours, and then inactivated by incubation at 75℃±5℃ for 15±5 minutes.

[0018] In the step 3) system, the concentration of the G-quadruplex buffer is 1X, heated at 95℃ for 5 minutes, and then slowly cooled to room temperature, the concentration of the chlorocyanin is 125 μmol / L, incubated at room temperature for 15±5 minutes, the concentration of ABTS is 2 mmol / L, and the concentration of H2O2 is 2 mmol / L. 2-

[0019] The components of the G-quadruplex buffer are 25 mmol / L HEPES, NH4OH (pH 8.0), 20 mmol / L KCl, and 200 mmol / L NaCl.

[0020] The stem portion of the hairpin stem-loop structure DNA has a length of 12-15 base pairs.

[0021] The B portion of the hairpin stem-loop structure DNA has a length of 15-20 bases.

[0022] The D portion of the hairpin stem-loop structure DNA has a length of 11-14 bases, and contains 9-10 guanine.

[0023] ​The application provides a method for sensitively, simply, economically and in-situ visual detection of TNT explosives, and has the advantages and beneficial effects that (1) the detection method is sensitive and has high visual degree, can realize in-situ detection of TNT explosives, and the detection limit reaches the pmol / L concentration level; (2) based on the specificity of aptamers, the signal is amplified through cyclic amplification, and the selectivity and sensitivity of the method are improved; (3) the DNA used in the application is ordinary DNA without fluorescent labeling, is cheap and has no background signal interference; (4) no large and precise instrument is needed and the operation is simple, and the activity of G-quadruplex peroxidase is used to realize in-situ visual detection; (5) by screening corresponding aptamers, the purpose of detecting different target explosives can be achieved. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is a schematic diagram of a method for specific visual detection of TNT explosives by using nucleic acid aptamers.

[0025] Figure 2 It is an ultraviolet kinetics curve for detecting TNT.

[0026] Figure 3 It is a visual result graph for detecting TNT.

[0027] Figure 4 It is an ultraviolet kinetics curve for detecting TNT explosives with different concentrations.

[0028] Figure 5 It is a visual result graph for detecting TNT explosives with different concentrations.

[0029] Figure 6 It is a detection result graph of different explosives by the application. DETAILED DESCRIPTION

[0030] The test method or test method described in the following examples is a conventional method unless otherwise specified, and the reagents and materials used in the following examples can be obtained from a conventional commercial channel or prepared by a conventional method unless otherwise specified.

[0031] The detection method of the TNT explosives provided by the application mainly consists of three parts: the first part is that the primer is reserved, and the DNA probe 1 nucleic acid and the target TNT explosives are assembled to avoid degradation by exonuclease I; the second part is that the displacement function of Klenow DNA polymerase is used to occur a cyclic amplification reaction, so that the signal is further amplified; and the third part is visual detection of the cyclic amplification product.

[0032] The detection principle of the application is shown in Figure 1 .

[0033] The N2 sequence of the probe DNA 1 combined with the target TNT explosive is specifically combined, so as to protect the probe DNA 1 from being degraded by exonuclease. The N1 sequence part of the probe DNA 1 reserved in the above step can be paired with the A-B part of the hairpin stem loop structure DNA to open the hairpin stem loop structure, and then the F part of the probe DNA 2 is paired with the C part of the hairpin stem loop structure DNA. Under the action of Klenow DNA polymerase, the probe DNA 2 can be used as a primer, and the hairpin stem loop structure DNA can be used as a template to carry out an amplification reaction; the amplification reaction can also displace the probe DNA 1 combined with the hairpin stem loop structure DNA, and the displaced probe DNA 1 opens more hairpin stem loop structure DNA to make more probe DNA 2 and the hairpin stem loop structure DNA complementary and polymerize, so that a cyclic amplification reaction occurs, the signal is further amplified, and the detection limit is reduced; the designed probe DNA 2 can form a G-quadruplex after being paired with the hairpin stem loop structure DNA, and therefore the cyclic amplification product obtained contains a large amount of G-quadruplex sequence. The G-quadruplex can have peroxidase activity with chlorocruorin product, and in the presence of H2O2, the ABTS 2- (2,2'-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt) is oxidized to form ABTS .- The ultraviolet absorption at 414 nm is enhanced, and the color of the system changes from colorless to green, so that the content of TNT explosive can be quantitatively detected.

[0034] The above probe DNA 1, hairpin stem loop structure DNA and probe DNA 2 are all single-stranded DNA, and the design principles are as follows:

[0035] The probe DNA 1 sequence is divided into two parts, N1 and N2. N1 is 22-26 bases in length, and N2 is an aptamer sequence CTC GGT TGT GGT GTT AGT TGG TAC TGC GCC TCC GTC GTC CTG TCA CT CTG C TGC AGC GCG CGA GCC ATA CAC TAT ACA CCT TAA AAT ATA GTT GCC TAG G which specifically binds to TNT.

[0036] The hairpin stem loop structure DNA comprises four parts, and the structure is A-B-C-D. The sequences of A and C are complementary to each other to form a stem region, B is a loop region, and D is rich in guanine G. The stem end regions A and C are complementary to each other, and the complementary pairing length is 12-15 base pairs, so as to ensure the stability of the hairpin structure. N1 is complementary to the A-B part with 22-26 bases.

[0037] Probe DNA 2 contains two parts E and F, and its structure is E-F. E is a short sequence containing three consecutive guanine G, and E and D are close to K + F can be complementary paired with C with 10-14 bases.

[0038] Example 1

[0039] In this embodiment, TNT is used as the detection target.

[0040] 1. Design and synthesis of probe DNA

[0041] (1) The sequence of probe DNA 1 is:

[0042]

[0043] Among them, the underlined sequence of N1 can be complementary paired with the hairpin stem-loop structure DNA to open the hairpin structure of the hairpin stem-loop structure DNA; N2 can specifically bind to TNT, and after N2 binds to TNT, it can prevent exonuclease I from degrading probe DNA 1 from the 3'-5' direction.

[0044] (2) The sequence of the hairpin stem-loop structure DNA is:

[0045]

[0046] Among them, the sequence under the dashed line can be complementary paired to form a "stem", and the B part forms a loop ring at the head and tail. The double-underlined sequence is rich in G but cannot form a G-quadruplex structure.

[0047] (3) The sequence of probe DNA 2 is:

[0048] Among them, the sequence under the wavy line can be complementary paired with the hairpin stem-loop structure DNA but cannot open the hairpin structure of the hairpin stem-loop structure DNA.

[0049] The underlined sequence of N1 of probe DNA 1 will have two reactions after opening the hairpin stem-loop structure DNA:

[0050] (1) The wavy line sequence of probe DNA 2 can be complementary paired with the hairpin stem-loop structure DNA, and E and D can be close to form a G-quadruplex structure;

[0051] (2) With the hairpin stem-loop structure DNA as the template, the probe DNA 2 as the primer, under the action of Klenow DNA polymerase, the amplification reaction is initiated; the amplification reaction can also displace the probe DNA 1 combined with the hairpin stem-loop structure DNA, the displaced probe DNA 1 opens more hairpin stem-loop structure DNA, so that more probe DNA 2 and hairpin stem-loop structure DNA complementary pairing and polymerization reaction occur, thereby the cyclic amplification reaction occurs, so that the E and D close to each other become more and more, and the detection limit is reduced;

[0052] 2. Visual detection of TNT explosives

[0053] (1) Exonuclease reaction

[0054] In a 10 μL exonuclease reaction system, 1 μL of 100 nmol / L probe DNA 1, 1 μL of 1 nmol / L TNT, 1 μL of 10× exonuclease I buffer, and ddH2O were added to make up the volume to 9 μL. After pre-incubation at 37℃ for 1 hour, 10 U of exonuclease I (1 μL) was added, and incubated at 37℃ for 1 hour. The exonuclease I was inactivated at 80℃ for 20 minutes, and then slowly cooled to 30℃.

[0055] (2) Amplification reaction

[0056] 3 μL of 10× Klenow DNA polymerase buffer, 0.6 μL of 10 mmol / L dNTPs, 3 μL of 10 μmol / L hairpin stem-loop structure DNA, 3 μL of 10 μmol / L probe DNA 2, 3 U of Klenow DNA polymerase (0.6 μL) were directly added to the exonuclease reaction system in the previous step, and ddH2O was added to make up the volume to 30 μL. After incubation at 37℃ for 2 hours, inactivation was performed at 70℃ for 10 minutes.

[0057] (3) Result detection

[0058] 20 μL of 10× G-quadruplex buffer (250 mmol / L HEPES, NH4OH (pH 8.0), 200 mmol / L KCl, 2000 mmol / L NaCl) and ddH2O were directly added to the product obtained by the above cyclic amplification to a total volume of 200 μL, heated at 95℃ for 5 minutes, and then placed at room temperature for 0.5 hours. After incubation at room temperature for 15 minutes after adding 1 μL of 250 mmol / L chlorohaemin, 2 μL of ABTS (200 mmol / L) and 2 μL of H2O2 (200 mmol / L) were added, and the 414 nm ultraviolet absorption kinetics was detected. 2- (200 mmol / L), 2 μL of H2O2 (200 mmol / L), and the 414 nm ultraviolet absorption kinetics was detected. Figure 2 、 3As shown, the UV dynamics of the reaction system containing TNT increased significantly, while the UV dynamics of the blank control corresponding to TNT was basically unchanged, without enhancement.

[0059] 3. Quantitative detection of TNT explosives

[0060] In the quantitative detection reaction system, a series of different concentration gradients of TNT explosives were detected, and the concentrations of TNT explosives were 0 pmol / L, 1 pmol / L, 10 pmol / L, 100 pmol / L, and 1 nmol / L, respectively.

[0061] The probe DNA 1, the hairpin stem-loop structure DNA, and the probe DNA 2 were used in the detection of TNT explosives, and the methods were the same. Figure 4 、 5 As shown, with the increase of TNT concentration, the UV absorption value at 414 nm increased and the green color of the system gradually deepened.

[0062] 4. Specific detection

[0063] Other explosives used for specific visual detection include RDX, HMX, and DNT.

[0064] The probe DNA 1, the hairpin stem-loop structure DNA, and the probe DNA 2 were used in the detection of TNT explosives, and the methods were the same. Figure 6 Only the system corresponding to TNT turned green.

Claims

1. A method for detecting TNT explosives, characterized in that: include: 1) Primer retention: Probe DNA 1 binds to the target TNT explosive in the sample to be tested, and exonuclease I degrades the probe DNA 1 that has not bound to the target TNT explosive; 2) Cyclic amplification: The probe DNA 1 retained in the previous step opens the hairpin stem-loop structure DNA, promoting a cyclic amplification reaction using the hairpin stem-loop structure DNA as a template and probe DNA 2 as a primer; 3) Visual detection: The presence of G-quadruplexes in the cyclic amplification products is detected by naked-eye visual detection and / or ultraviolet spectrophotometry. The probe DNA 1 is a single-stranded DNA containing two parts, N1 and N2, with a structure of 5'-N1-N2-3'; N2 is the aptamer sequence that specifically binds to the target TNT explosive: CTC GGT TGT GGT GTT AGT TGG TAC TGC GCC TCC GTC GTCCTG TCA CTC TGC TGC AGC GCG CGA GCC ATA CAC TAT ACA CCT TAA AAT ATA GTT GCCTAG G; The hairpin stem-loop structure DNA contains four parts: A, B, C, and D, with a structure of 5'-ABCD-3'. A can pair complementaryly with C to form a stem; the AB sequence can pair complementaryly with the N1 sequence; and D is a single-stranded sequence with a length of 11-14 bases, rich in guanine but unable to form a G-quadruplex. The probe DNA 2 is a single-stranded DNA containing two parts, E and F, with a structure of 5'-EF-3'. F and the C part of the hairpin stem-loop structure DNA can be complementary. E is a short single-stranded sequence containing 3-4 guanines, which can form a G-quadruplex when close to the D sequence of the hairpin stem-loop structure DNA.

2. The method for detecting TNT explosives according to claim 1, characterized in that: Includes the following steps: 1) Add probe DNA 1, exonuclease I buffer, test sample and water to the system in sequence, pre-incubate and then add exonuclease I to react. After the reaction is complete, heat to inactivate exonuclease I. 2) Add Klenow DNA polymerase buffer, hairpin stem-loop structure DNA, probe DNA2, Klenow DNA polymerase, dNTPs and water to the reaction system of step 1) and react. Inactivate after the reaction is complete. 3) Add G-quadruplex buffer to the reaction system from step 2), heat to denature, slowly cool to room temperature, then add chloroheme for incubation, and finally add ABTS. 2- The system was tested with H2O2, and the color change and ultraviolet absorption value at a detection wavelength of 414 nm were observed. In the system with the target TNT explosive present, the color changed from colorless to a visible green, and the ultraviolet absorption value increased with the increase of TNT explosive concentration.

3. The method for detecting TNT explosives according to claim 2, characterized in that: In step 1), the concentration of probe DNA 1 is 10 nmol / L, the exonuclease I buffer is 1X, the test sample and probe DNA 1 are pre-incubated at 25-37℃ for 0.5-1 hours, the concentration of exonuclease I is 1U / μL, the reaction is carried out at 37℃ for 1-2 hours, the exonuclease I is inactivated by heating at 80±5℃ for 20 minutes, and then slowly cooled to room temperature.

4. The method for detecting TNT explosives according to claim 2, characterized in that: In step 2), the Klenow DNA polymerase buffer is 1X, the concentration of dNTPs is 200 μmol / L, the concentration of hairpin stem-loop DNA is 1 μmol / L, the concentration of probe DNA 2 is 1 μmol / L, the concentration of Klenow DNA polymerase is 1 U / 10 μL, and the cyclic amplification reaction is incubated at 37℃ for 2 ± 0.5 hours, followed by incubation at 75℃ ± 5℃ for 15 ± 5 minutes for inactivation.

5. The method for detecting TNT explosives according to claim 2, characterized in that: In step 3), the G-quadruplex buffer concentration is 1X, heated at 95°C for 5 minutes, then slowly cooled to room temperature; the chloroheme concentration is 125 μmol / L, and incubated at room temperature for 15 ± 5 minutes. (ABTS) 2- The concentration of H2O2 is 2 mmol / L.

6. The method for detecting TNT explosives according to claim 5, characterized in that: The G-quadruplex buffer solution consists of 25 mmol / L HEPES, NH4OH (pH 8.0), 20 mmol / L KCl, and 200 mmol / L NaCl.

7. The method for detecting TNT explosives according to claim 1, characterized in that: The hairpin stem-loop structure DNA stem portion is 12-15 base pairs in length.

8. The method for detecting TNT explosives according to claim 1, characterized in that: The B portion of the hairpin stem-loop structure DNA is 15-20 bases in length.

9. The method for detecting TNT explosives according to claim 1, characterized in that: The D portion of the hairpin stem-loop structure DNA is 11-14 bases in length and contains 9-10 guanines.

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