CN105548119A - Method for rapidly detecting T-2 toxin - Google Patents
Method for rapidly detecting T-2 toxin Download PDFInfo
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- CN105548119A CN105548119A CN201610043520.XA CN201610043520A CN105548119A CN 105548119 A CN105548119 A CN 105548119A CN 201610043520 A CN201610043520 A CN 201610043520A CN 105548119 A CN105548119 A CN 105548119A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention relates to a method for rapidly detecting a T-2 toxin. The method comprises the following steps that A, a T-2 toxin aptamer and single-strand signal probe DNA are hybridized to form a hybrid strand; B, the hybrid strand makes contact with a sample to be detected, and the hybrid strand reacts with the T-2 toxin to release out the single-strand signal probe DNA when T-2 toxin exists in the sample to be detected; C, the hybrid strand is made to form two-strand DNA by means of DNA amplification, then excision enzyme is used for hydrolyzing the two-strand DNA into mononucleotide, and at the moment, the single-strand signal probe DNA is left in the system; D, under the induction of the single-strand DNA, copper ions are restored to generate near infrared fluorescent copper nano particles; the system fluorescence intensity is detected, and therefore the content of the T-a toxin in the sample to be detected is detected. The method has the advantages of being high in sensitivity, easy and fast to operate, low in cost and the like.
Description
Technical field
The invention belongs to nano-biosensing and technical field of biological, specifically, be to provide a kind of method of quick detection T-2 toxin.
Background technology
T-2 toxin is primarily of one of mycetogenetic trichothecenes such as fusarium tricinctum, and it is distributed widely in nature, is common pollution field crops and the primary toxins of stock's cereal, large to people, animal harm.FAO (Food and Agriculture Organization of the United Nation) (FAQ) and the World Health Organization (WHO) are using the most dangerous food pollution source of this toxoid as existence naturally.T-2 toxin is highly stable at ambient temperature, places 6 ~ 7 years or is heated to 100 ~ 120 DEG C and can not destroy its toxicity in 1 hour.Up to now, there is no the specificity prevention and treatment method of T-2 toxin poisoning, unique effective prevention method avoids contact or reduces contact.Therefore, very necessary to the detection of T-2 toxin.
At present, the method detecting T-2 toxin mainly contains: the methods such as chromatography, immunochromatographic method, chromatograph-mass spectrometer coupling method.Recently, Chinese patent CN105021593A discloses a kind of method measuring T-2 toxin based on foot point territory and hybridization chain reaction.
But, these methods respectively have its shortcoming: as chromatograph-mass spectrometer coupling method, not only instrument price is expensive, and needs medicine dedicated technician to be competent at, and is difficult to popularize; Immunization reagent is expensive and be easy to inactivation; Measure the method for T-2 toxin based on foot point territory and hybridization chain reaction, need to use the deficiencies such as the expensive DNA by mark.
Summary of the invention
The object of the invention is for problems of the prior art, a kind of method of quick detection T-2 toxin is provided.
The technical solution used in the present invention: a kind of method of quick detection T-2 toxin, comprises the steps:
(A) T-2 toxin aptamer Apt and single-stranded signal probe ssDNA is hybridized, and forms hybridization chain;
(B) testing sample solution is joined this hybridization chain solution, when there being T-2 toxin in testing sample, hybridization chain optionally reacts with T-2 toxin and discharges single-stranded signal probe ssDNA;
(C) eliminate the interference of hybridization chain, utilize DNA cloning, make hybridization chain become double-stranded DNA, then use exonuclease, double-stranded DNA is hydrolyzed into mononucleotide, leave single-stranded signal probe ssDNA;
(D) utilize T-2 toxin aptamer-T-2 toxin combination that copper ion can not be induced to be reduced into the copper nano-particle of near-infrared fluorescent, only have single-stranded signal probe ssDNA that copper ion can be induced to be reduced into near-infrared fluorescent copper nano-particle, detection system fluorescence intensity in copper ion reduction detection system, thus measure the content of the T-2 toxin in testing sample.
Described copper ion reduction detection system comprises the copper ion of successively interpolation and makes copper ion be reduced into the reductive agent vitamin C of near-infrared fluorescent copper nano-particle.The detection of step (D), such as, comprise successively and first add copper ion solution and vitamin c solution in backward system, generate near-infrared fluorescent copper nano-particle after reaction.
Described T-2 toxin aptamer is
5’-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGA
GGTGAAGACTCGCA-3’。
The described signal probe ssDNA can hybridized with T-2 toxin aptamer is 5 '-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
tGCGAGTCTTCACC-3 '.
Cleaning Principle of the present invention is as follows: first allow Apt and ssDNA hybridize (underscore part); When there being T-2 toxin to exist, hybridization chain and T-2 toxin react and discharge ssDNA; Apt-ssDNA (remaining) is there is, Apt-T-2 toxin and ssDNA in system.Apt-T-2 toxin can not induce copper ion to be reduced into near-infrared fluorescent copper nano-particle, and Apt-T-2 toxin exists mensuration noiseless; Hybridization chain may have interference; In order to eliminate the interference of hybridization chain: a. utilizes DNA cloning, and make hybridization chain become double-stranded DNA, b. exonuclease, is hydrolyzed into mononucleotide by double-stranded DNA, eliminates double-stranded DNA.Now only staying in system can the ssDNA of inductive formation near-infrared fluorescent copper nano-particle, by the fluorescence intensity of detection system, can measure the content of T-2 toxin.Due to the interference of background fluorescence in elimination system, improve sensitivity and the precision of detection.
Invention additionally provides a kind of kit detecting T-2 toxin, it at least comprises: T-2 toxin aptamer, the single-stranded signal probe ssDNA can hybridized with T-2 toxin aptamer, DNA cloning system, excision enzyme, copper ion reduce detection system.
Described T-2 toxin aptamer is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGG TGAAGACTCGCA-3 '.
Described single-stranded signal probe ssDNA is 5 '-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCGAGTCTTCA CC-3 '.
Described DNA cloning system comprises buffer solution, triphosphoric acid deoxymononucleotide mixed solution (dNTP) and Phi29DNA polymerase.Described buffer solution is by Tris-HCl, MgCl
2(NH
4)
2sO
4composition.
Described excision enzyme is ExoIII exonuclease.
In described copper ion reduction detection system, reductive agent is vitamin C.
Present invention also offers a kind of T-2 toxin aptamer that can be used for detecting T-2 toxin, its base sequence is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGG TGAAGACTCGCA-3 '.
Advantage of the present invention
According to detection method of the present invention and kit, eliminate the interference of background fluorescence, improve detection sensitivity and the precision of T-2 toxin.
Accompanying drawing explanation
Fig. 1 is the concentration relationship of ssDNA – copper nano-particle fluorescence intensity and T-2 toxin.
Embodiment
Embodiment 1
The kit of detection T-2 toxin of the present invention at least comprises: T-2 toxin aptamer, single-stranded signal probe ssDNA, DNA cloning system, exonuclease, copper ion reduction detection system.Described T-2 toxin aptamer is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGG TGAAGACTCGCA-3 '.Described single-stranded signal probe ssDNA is 5 '-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCGAGTCTTC ACC-3 '.Described DNA cloning system comprises buffer solution (Tris-HCl, MgCl
2, (NH
4)
2sO
4), dNTP and Phi29DNA polymerase.Described exonuclease is ExoIII exonuclease.
Embodiment 2
A method for quick detection T-2 toxin, specific operation process is as follows:
By each DNA storing solution at 95 DEG C through heating 5 minutes, before using, and at room temperature place 30 minutes.Then, the hybridization buffer 40 μ L got respectively containing hybridization buffer 40 μ L and the 3.0 μm of ol signal probe ssDNA of 3.0 μm of olT-2 toxin aptamer Apt is placed in 2ml centrifuge tube, hybridize 1 hour at 37 DEG C, generate T-2 toxin aptamer-signal probe hybrid (Apt-ssDNA).
At 37 DEG C, be that the T-2 toxin of 0 ~ 50ng/mL adds in Apt-ssDNA solution respectively successively by concentration, T-2 toxin and T-2 toxin aptamer react, and generate aptamer-T-2 toxin, discharge ssDNA.At this moment, the materials such as ssDNA, residue (unreacted) Apt-ssDNA and aptamer-T-2 toxin are had in system.
(buffer solution consists of 50mMTris-HCl, 10mMMgCl to add 10 μ L buffer solution
2, 10mM (NH
4)
2sO
4, pH7.8), then add dNTP (10mM) 18 μ L.In system, add 2 μ LPhi29DNA polymerases (10u/ μ l) again, reacting 15 minutes at 37 DEG C, making to increase into double-stranded DNA with T-2 toxin aptamer-signal probe hybridization sequences (Apt-ssDNA) for touching plate.Keep Phi29DNA being deactivated in 10 minutes at 65 DEG C.
In this reaction system, add 2 μ LExoIII exonucleases (20u/ μ L) again, react 30 minutes, make optionally double-stranded DNA to be hydrolyzed into mononucleotide at 37 DEG C, single-stranded probe ssDNA is not cut and remain.
25 μ L1mmol copper nitrates and 180 μ LPBS damping fluid (10mM, pH7.8) are added in reactant liquor.Then, potpourri is at room temperature placed after 10 minutes in lucifuge or darkroom, under fast stirring, adds the freshly prepd vitamin c solution that 100 μ L concentration are 1mM.Then at 45 DEG C, 5 ~ 10min is reacted.Solution is transferred to microcolorimetric ware, measure the fluorescence intensity of system, carry out the quantitative measurement of T-2 toxin, result as shown in Figure 1.
The range of linearity 0.08 – 25ng/mL, detectability 0.05ng/mL, the recovery is 94.5 ~ 112.2%.The detection of the biological micromolecule T-2 toxin such as other mycotoxin is noiseless.
<110> University Of Science and Technology Of Hunan
<120> mono-kind detects the method for T-2 toxin fast
<160>2
<210>1
<211>68
<212>DNA
<213>T-2 toxin aptamer
<400>1
5’-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCGCA-3’
<210>2
<211>58
<212>DNA
<213> single-stranded signal probe
<400>2
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCGAGTCTTCACC-3’
Claims (10)
1. detect a method for T-2 toxin fast, it is characterized in that, the method comprises the steps:
(A) T-2 toxin aptamer Apt and single-stranded signal probe ssDNA is hybridized, and forms hybridization chain;
(B) make this hybridization chain and testing sample effect, when there being T-2 toxin to exist in testing sample, hybridization chain optionally reacts with T-2 toxin and discharges single-stranded signal probe ssDNA;
(C) in order to eliminate the interference of hybridization chain, utilizing DNA cloning, making hybridization chain become double-stranded DNA, then use excision enzyme, optionally double-stranded DNA is hydrolyzed into mononucleotide, leaving single-stranded signal probe ssDNA;
(D) utilize T-2 toxin aptamer-T-2 toxin combination that copper ion reduction can not be induced to generate the copper nano-particle of near-infrared fluorescent, only have single-stranded signal probe ssDNA that copper ion can be induced to be reduced into near-infrared fluorescent copper nano-particle, the fluorescence intensity of detection system, thus the content measuring the T-2 toxin in testing sample.
2. the method for quick detection T-2 toxin according to claim 1, is characterized in that, described T-2 toxin aptamer is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGG TGAAGACTCGC-3 '.
3. the method for quick detection T-2 toxin according to claim 1 and 2, is characterized in that, described signal probe ssDNA is 5 '-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCGAGTCTTCA CC-3 '.
4. the method for quick detection T-2 toxin according to claim 1, comprise the kit detecting T-2 toxin, it at least comprises: T-2 toxin aptamer, signal probe ssDNA, DNA cloning system, exonuclease, copper ion reduction detection system.
5. kit according to claim 4, is characterized in that, described T-2 toxin aptamer is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGG TGAAGACTCGC-3 '.
6. kit according to claim 4, is characterized in that, described signal probe DNA is 5 '-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCGAGTCTTCA CC-3 '.
7. kit according to claim 4, is characterized in that, described DNA cloning system comprises buffer solution, dNTP and Phi29DNA polymerase; Described buffer solution is by Tris-HCl, MgCl
2, (NH
4)
2sO
4composition.
8. kit according to claim 4, is characterized in that, described exonuclease is ExoIII exonuclease.
9. kit according to claim 4, is characterized in that, in described copper ion reduction detection system, reductive agent is vitamin C.
10. can be used for the T-2 toxin aptamer detecting T-2 toxin, it is characterized in that, its base sequence is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGG TGAAGACTCGC-3 '.
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Cited By (2)
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| CN111118181A (en) * | 2018-10-31 | 2020-05-08 | 华东师范大学 | Bacterium detection method |
| CN112763472A (en) * | 2020-12-29 | 2021-05-07 | 南京师范大学 | Detection system for detecting T-2 toxin residue and preparation method and application thereof |
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Cited By (4)
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| CN111118181A (en) * | 2018-10-31 | 2020-05-08 | 华东师范大学 | Bacterium detection method |
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| CN112763472A (en) * | 2020-12-29 | 2021-05-07 | 南京师范大学 | Detection system for detecting T-2 toxin residue and preparation method and application thereof |
| WO2022141653A1 (en) * | 2020-12-29 | 2022-07-07 | 南京师范大学 | Detection system for detecting t-2 toxin residue, and preparation method therefor and use thereof |
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