CN105606574A - Method and kit for detecting T-2 toxins - Google Patents

Method and kit for detecting T-2 toxins Download PDF

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Publication number
CN105606574A
CN105606574A CN201610041769.7A CN201610041769A CN105606574A CN 105606574 A CN105606574 A CN 105606574A CN 201610041769 A CN201610041769 A CN 201610041769A CN 105606574 A CN105606574 A CN 105606574A
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toxin
chain
ssdna
kit
signal probe
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CN105606574B (en
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夏晓东
冉海宁
杨阳
唐春然
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Hunan University of Science and Technology
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Hunan University of Science and Technology
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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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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to a method and kit for detecting T-2 toxins. The method includes the steps of A, hybridizing T-2 toxin aptamer with single-chain signal probe ssDNA to form a hybridized chain; B, allowing the hybridized chain to contact with a to-be-detected sample, wherein the hybridized chain reacts with the T-2 toxins to release the single-chain signal probe ssDNA when the T-2 toxins exist in the to-be-detected sample; C, using DNA amplification to allow the hybridized chain to form double-chain DNA, and using excision enzyme to hydrolyze the double-chain DNA into mononucleotide to remove the double-chain DNA so as to leave ssDNA in the system; D, under induction of the ssDNA, using silver ion reduction to generate near infrared fluorescence silver nano clusters; detecting system fluorescence intensity to determine the T-2 toxin content in the to-be-detected sample. The method is high in sensitivity, simple to operate, low in cost, and the like.

Description

The detection method of T-2 toxin and detection kit
Technical field
The invention belongs to nano-biosensing and technical field of biological, specifically, be to provide a kind of T-2 toxinDetection method and detection kit.
Background technology
T-2 toxin (trichothecenes, TS) is mainly mould by multiple mycetogenetic single-ended spores such as fusarium tricinctumsOne of aliphatic compound. It is joint that FAO (Food and Agriculture Organization of the United Nation) (FAQ) in 1973 and the World Health Organization (WHO) hold in GenevaIn meeting, using this toxoid, with the same the most dangerous food pollution source as naturally existing of aflatoxins, it is distributed widely inNature, is common pollution field crops and the main toxin of stock's cereal, larger to people, animal harm. T-2 toxin is in chamberHighly stable under temperature condition, place 6～7 years or be heated to 100～120 DEG C and can not destroy its toxicity in 1 hour. Also not right at presentThe specificity prevention and treatment method of T-2 toxin poisoning, unique effective prevention method is to avoid contact or reduce contact. Therefore, to T-The detection of 2 toxin is very necessary.
At present, the method for detection T-2 toxin mainly contains: the side such as chromatography, immunochromatographic method, chromatograph-mass spectrometer coupling methodMethod. Recently, Chinese patent CN105021593A discloses a kind of based on foot point territory and hybridization chain reaction mensuration T-2 toxinMethod.
But, these methods respectively have its shortcoming: as chromatograph-mass spectrometer coupling method, not only instrument is expensive, and needs medicine specialGate technique personnel could be competent at, and are difficult to popularize; Immunization reagent is expensive and be easy to inactivation; Survey based on foot point territory and hybridization chain reactionDetermine the method for T-2 toxin, 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 the T-2 of inspection toxin is provided. The present inventionThe technical scheme adopting: a kind of method of examining T-2 toxin, comprises the steps:
(A) T-2 toxin aptamer (Apt) and strand signal probe ssDNA hybridization, form hybridization chain;
(B) testing sample solution is joined to this hybridization chain solution, in the time having T-2 toxin in testing sample, hybridization chain is selectiveGround reacts with T-2 toxin and discharges strand signal probe ssDNA;
(C) eliminate the interference of hybridizing chain, utilize DNA cloning, make to hybridize chain and become double-stranded DNA, then use exonuclease, by two strandsDNA is hydrolyzed into mononucleotide and removes double-stranded DNA, leaves strand signal probe ssDNA;
(D) utilize T-2 toxin aptamer-T-2 toxin combination can not induce silver ion reduction to become the Yin Na of near-infrared fluorescentRice bunch, only has strand signal probe ssDNA can induce silver ion reduction to become near-infrared fluorescent silver nanoclusters, examines at silver ion reductionDetection architecture fluorescence intensity in survey system, thereby the content of the T-2 toxin in mensuration testing sample.
Described silver ion reduction detection architecture comprises the silver ion that successively adds and makes silver ion reduction become near-infrared fluorescentThe borohydride reduction agent of silver nanoclusters, for example sodium borohydride. The detection of step (D), for example, comprise successively first in backward systemAdd silver ion solution and sodium borohydride solution, after reaction, generate near-infrared fluorescent silver nanoclusters.
Described T-2 toxin aptamer is
5’-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCGC-3’。
Describedly can be 5 ' with the strand signal probe ssDNA of T-2 toxin aptamer hybridization-CCCCCCACACCCGATCCCCCCCGCGAGTCTTCACC-3’。
Detection principle of the present invention is as follows: first allow Apt and ssDNA hybridize (underscore part); When there being T-2 toxin to existTime, hybridization chain reacts and discharges ssDNA with T-2 toxin; In system, there is Apt-ssDNA (remaining), Apt-T-2 toxinAnd ssDNA. Apt-T-2 toxin can not induce silver ion reduction to become near-infrared fluorescent silver nanoclusters, and Apt-T-2 toxin exists surveyingFixed noiseless; Hybridization chain may have interference; In order to eliminate the interference of hybridization chain: a. utilizes DNA cloning, make to hybridize chain and become double-strandedDNA, b. exonuclease, is hydrolyzed into mononucleotide by double-stranded DNA, thus the double-stranded DNA in removing system. Now systemIn only stay and can induce the ssDNA that generates near-infrared fluorescent silver nanoclusters, by the fluorescence intensity of detection architecture, can measure T-The content of 2 toxin. Due to the interference of background fluorescence in elimination system, the sensitivity and the precision that detect are improved.
The present invention provides a kind of kit of the T-2 of detection toxin in addition, and it at least comprises: T-2 toxin aptamer, canStrand signal probe ssDNA, DNA cloning system, excision enzyme, silver ion reduction detection body with the hybridization of T-2 toxin aptamerSystem.
Described T-2 toxin aptamer is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCGC-3’。
Described strand signal probe ssDNA is 5 '-CCCCCCACACCCGATCCCCCCCGCGAGTCTTCACC-3 '.
Described DNA cloning system comprises cushioning liquid, dNTP and Phi29DNA polymerase; Described cushioning liquid byTris-HCl、MgCl2、(NH4)2SO4Composition.
Described excision enzyme is ExoIII exonuclease.
In described silver ion reduction detection architecture, reducing agent is boron hydride.
Described boron hydride is sodium borohydride.
The present invention also provides a kind of T-2 toxin aptamer that can be used for detecting T-2 toxin, and its base sequence is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACAC
ATGCGAGAGGTGAAGACTCGC-3’。
Advantage of the present invention
According to detection method of the present invention and kit, eliminate the interference of background fluorescence, the detection that has improved T-2 toxin is sensitiveDegree and precision.
Brief description of the drawings
Fig. 1 is the concentration relationship of ssDNA-Ag nano-cluster fluorescence intensity and T-2 toxin.
Detailed description of the invention
Embodiment 1
A kind of kit of the T-2 of detection toxin at least comprises: T-2 toxin aptamer, strand signal probe ssDNA, DNA cloningSystem, exonuclease, silver ion reduction detection architecture. T-2 toxin aptamer is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCGC-3 '. Strand letterNumber probe ssDNA is 5 '-CCCCCCACACCCGATCCCCCCCGCGAGTCTTCACC-3 '. DNA cloning system comprises that buffering is moltenLiquid, dNTP and Phi29DNA polymerase; Described cushioning liquid is by Tris-HCl, MgCl2、(NH4)2SO4Composition. Nucleic acid is circumscribedEnzyme is ExoIII exonuclease. Reducing agent in described silver ion reduction detection architecture is boron hydride, as hydroborationSodium.
Embodiment 2
A method that detects T-2 toxin, specific operation process is as follows:
By each DNA storing solution at 95 DEG C through heat treated 5 minutes, before use, and at room temperature place 30 minutes. Then, divideDo not get hybridization cushioning liquid 40 μ L and 3.0 μ mol signal probe ssDNA containing 3.0 μ molT-2 toxin aptamers (Apt)Hybridization cushioning liquid 40 μ L be placed in 2ml centrifuge tube, at 37 DEG C, hybridize 1 hour, generate T-2 toxin aptamer-letterNumber Probe Hybridization thing (Apt-ssDNA).
At 37 DEG C, the T-2 toxin that is 0 ~ 1.0ng/mL by concentration successively respectively adds Apt-ssDNA solution toIn, T-2 toxin reacts with T-2 toxin aptamer, generates aptamer-T-2 toxin, discharges ssDNA. At this moment, in systemThere are the materials such as ssDNA, residue (unreacted) APT-ssDNA and aptamer-T-2 toxin.
(cushioning liquid consists of 50mMTris-HCl, 10mMMgCl to add 10 μ L cushioning liquid2，10mM(NH4)2SO4, pH7.5), then add dNTP (10mM) 18 μ L. In system, add 2 μ LPhi29DNA polymerizations again(10u/ μ l), reacts 15 minutes enzyme at 37 DEG C, makes with T-2 toxin aptamer-signal probe hybridization sequences (Ap-SsDNA) increase into double-stranded DNA for touching plate. At 65 DEG C, keep making for 10 minutes Phi29DNA deactivation.
In this reaction system, add 2 μ LExoIII exonucleases (20u/ μ L) again, at 37 DEG C, react 30 pointsClock, makes optionally double-stranded DNA to be hydrolyzed into mononucleotide and removes double-stranded DNA, and single-stranded probe ssDNA is not hydrolyzed and retainsCome.
In reactant liquor, add 25 μ L1mmol silver nitrates and 180 μ L sodium citrate buffer solutions (10mM, pH7.0).Then, mixture is at room temperature placed after 10 minutes in lucifuge or darkroom, and under rapid stirring, adding 100 μ L concentration is 200 μ MFreshly prepd sodium borohydride solution. Then at 45 DEG C, react 5 ~ 10min. Solution is transferred to micro-cuvette, measuresThe fluorescence intensity of system, carries out the quantitative assay of T-2 toxin, and result as shown in Figure 1.
The range of linearity 0.008 – 0.20ng/mL, detectability 5pg/mL, the rate of recovery is 95.5 ~ 105.7%. Other fungi poisonThe detection of the biological micromolecule T-2 toxin such as element, vitamin C, glucose is noiseless.
<110>University Of Science and Technology Of Hunan
<120>detection method of T-2 toxin and detection kit
<160>2
<210>1
<211>67
<212>DNA
<213>T-2 toxin aptamer
<400>1
5’CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCGC-3’
<210>2
<211>35
<212>DNA
<213>strand signal probe
<400>2
5’-CCCCCCACACCCGATCCCCCCCGCGAGTCTTCACC-3’

Claims (10)

1. a detection method for T-2 toxin, is characterized in that, the method comprises the steps:
(A) T-2 toxin aptamer and strand signal probe DNA(ssDNA) hybridization, form hybridization chain;
(B) make this hybridization chain and testing sample effect, in the time having T-2 toxin to exist in testing sample, hybridization chain optionally withThe reaction of T-2 toxin discharges strand signal probe ssDNA;
(C) eliminate the interference of hybridizing chain, utilize DNA cloning, make to hybridize chain and become double-stranded DNA, then use excision enzyme, by double-stranded DNABe hydrolyzed into mononucleotide and remove double-stranded DNA, leave strand signal probe ssDNA;
(D) utilizing T-2 toxin aptamer-T-2 toxin combination can not induce silver ion reduction to generate near-infrared fluorescent silver receivesRice bunch, only has strand signal probe ssDNA can induce silver ion reduction to become near-infrared fluorescent silver nanoclusters, detection architecture glimmeringLuminous intensity, thereby the content of the T-2 toxin in mensuration testing sample.
2. method according to claim 1, is characterized in that, described T-2 toxin aptamer is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCGC-3’。
3. method according to claim 1 and 2, is characterized in that, described strand signal probe ssDNA is 5 '-CCCCCCACACCCGATCCCCCCCGCGAGTCTTCACC-3’。
4. a kit that detects T-2 toxin, is characterized in that, it at least comprises: T-2 toxin aptamer, can with T-2 poisonStrand signal probe ssDNA, DNA cloning system, exonuclease, the silver ion reduction detection architecture of the hybridization of element aptamer.
5. kit according to claim 4, is characterized in that, described T-2 toxin aptamer is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCGC-3’。
6. kit according to claim 4, is characterized in that, described strand signal probe DNA is 5 '-CCCCCCACACCCGATCCCCCCCGCGAGTCTTCACC-3’。
7. according to the kit of the detection T-2 toxin described in claim 4, it is characterized in that, described DNA cloning system comprisesCushioning liquid, dNTP and Phi29DNA polymerase; Described cushioning liquid is by Tris-HCl, MgCl2、(NH4)2SO4) composition.
8. according to the kit of the detection T-2 toxin described in claim 4, it is characterized in that, described exonuclease is ExoIII exonuclease.
9. according to the kit of the detection T-2 toxin described in claim 4, it is characterized in that, described silver ion reduction detectsIn system, reducing agent is boron hydride.
10. according to the kit of the detection T-2 toxin described in claim 9, it is characterized in that, described boron hydride is hydroborationSodium.
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CN108219783A (en) * 2017-12-27 2018-06-29 温州大学 Fluorescent silver nanocluster, preparation method and its application in Mycotoxin identification of nucleic acid stability
CN113376120A (en) * 2021-05-27 2021-09-10 江苏科技大学 Optical fiber LSPR aptamer biosensor and preparation method and application thereof

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CN108219783A (en) * 2017-12-27 2018-06-29 温州大学 Fluorescent silver nanocluster, preparation method and its application in Mycotoxin identification of nucleic acid stability
CN113376120A (en) * 2021-05-27 2021-09-10 江苏科技大学 Optical fiber LSPR aptamer biosensor and preparation method and application thereof

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