CN105606574B - The detection method and detection kit of T-2 toxin - Google Patents
The detection method and detection kit of T-2 toxin Download PDFInfo
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- CN105606574B CN105606574B CN201610041769.7A CN201610041769A CN105606574B CN 105606574 B CN105606574 B CN 105606574B CN 201610041769 A CN201610041769 A CN 201610041769A CN 105606574 B CN105606574 B CN 105606574B
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Abstract
The present invention relates to a kind of methods and kit of detection 2 toxin of T, and this method comprises the following steps：（A）So that 2 toxin aptamers of T is hybridized with single-stranded signal probe ssDNA, forms hybridization chain；（B）The hybridization chain is set to be contacted with sample to be tested, in the presence of having 2 toxin of T in sample to be tested, hybridization chain is reacted with 2 toxin of T releases single-stranded signal probe ssDNA；（C）Using DNA cloning, make hybridization chain at double-stranded DNA, then uses excision enzyme, double-stranded DNA is hydrolyzed into mononucleotide and removes double-stranded DNA, leaves ssDNA in system at this time；（D）Under ssDNA inductions, silver ion reduction generates near-infrared fluorescent silver nanoclusters；Detection architecture fluorescence intensity, to measure the content of 2 toxin of T in sample to be tested.This method have high sensitivity, it is easy to operate, it is inexpensive the features such as.
Description
Technical field
The invention belongs to nano-biosensing and technical field of biological, specifically, being to provide a kind of T-2 toxin
Detection method and detection kit.
Background technology
T-2 toxin (trichothecenes, TS) is mainly mould by a variety of mycetogenetic single-ended spores such as fusarium tricinctum
One of aliphatic compound.FAO (Food and Agriculture Organization of the United Nation) in 1973（FAQ）And the World Health Organization（WHO）It is held in Geneva joint
In meeting, using this toxoid with aflatoxins equally as the most dangerous food pollution source of naturally occurring, it is distributed widely in
Nature is the primary toxins of common pollution field crops and inventory's cereal, endangers people, animal larger.T-2 toxin is in room
Highly stable under the conditions of temperature, its toxicity cannot be destroyed in 1 hour by placing 6~7 years or being heated to 100~120 DEG C.There is presently no right
The specific prevention and treatment method of T-2 toxin poisonings, the only effective prevention method are to avoid contact with or reduce contact.Therefore, to T-
The detection of 2 toxin is very necessary.
Currently, the method for detection T-2 toxin mainly has：The side such as chromatography, immunochromatographic method, chromatograph-mass spectrometer coupling method
Method.Recently, 105021593 A of Chinese patent CN disclose a kind of based on foot point domain and hybridization chain reaction measurement T-2 toxin
Method.
But these methods respectively has its disadvantage：Such as chromatograph-mass spectrometer coupling method, not only instrument price is expensive, and needs medicine special
Gate technique personnel could be competent at, it is difficult to universal；Immunization reagent is expensive and is easy to inactivate；It is surveyed based on foot point domain and hybridization chain reaction
The method for determining T-2 toxin, the deficiencies of needing using expensive the DNA by label.
Invention content
The purpose of the present invention is being directed to problems of the prior art, a kind of method of inspection T-2 toxin is provided.The present invention
The technical solution of use：A method of inspection T-2 toxin includes the following steps：
（A）T-2 toxin aptamers（Apt）Hybridize with single-stranded signal probe ssDNA, forms hybridization chain；
（B）Testing sample solution is added to the hybridization chain solution, when there is T-2 toxin in sample to be tested, hybridization chain choosing
It is reacted with T-2 toxin to selecting property and releases single-stranded signal probe ssDNA；
（C）Eliminate the interference of hybridization chain makes hybridization chain at double-stranded DNA using DNA cloning, then uses exonuclease, will
Double-stranded DNA is hydrolyzed into mononucleotide and removes double-stranded DNA, leaves single-stranded signal probe ssDNA；
（D）Silver ion reduction can not can induce near-infrared fluorescent using T-2 toxin aptamer-T-2 toxin combinations
Silver nanoclusters, only single-stranded signal probe ssDNA can induce silver ion reduction into near-infrared fluorescent silver nanoclusters, silver ion also
Detection architecture fluorescence intensity in former detection architecture, to measure the content of the T-2 toxin in sample to be tested.
The silver ion reduction detection architecture includes the silver ion successively added and makes silver ion reduction at near-infrared fluorescent
The borohydride reduction agent of silver nanoclusters, such as sodium borohydride.Step（D）Detection, such as including successively first in backward system
Silver ion solution and sodium borohydride solution is added, near-infrared fluorescent silver nanoclusters are generated after reaction.
The T-2 toxin aptamer is
5’-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCG C-3’。
The single-stranded signal probe ssDNA that can hybridize with T-2 toxin aptamers is 5 '-CCCCCCACACCCGATCCC
CCCCGCGAGTCTTCACC-3’。
The testing principle of the present invention is as follows：First Apt is allowed to hybridize with ssDNA（Underscore part）；When with the presence of T-2 toxin
When, hybridization chain is reacted with T-2 toxin releases ssDNA；There are Apt- ssDNA (remaining), Apt-T-2 toxin in system
And ssDNA.Apt-T-2 toxin not can induce silver ion reduction into near-infrared fluorescent silver nanoclusters, and Apt-T-2 toxin exists to surveying
It is fixed noiseless；Hybridization chain may have interference；In order to eliminate the interference of hybridization chain：A. DNA cloning is utilized, makes hybridization chain at double-strand
Double-stranded DNA is hydrolyzed into mononucleotide by DNA, b. exonuclease, to the double-stranded DNA in removing system.System at this time
In leave behind can induce generate near-infrared fluorescent silver nanoclusters ssDNA T- can be measured by the fluorescence intensity of detection architecture
The content of 2 toxin.Due to the interference of background fluorescence in elimination system, sensitivity and the precision of detection are improved.
Invention additionally provides a kind of kits of detection T-2 toxin, include at least：T-2 toxin aptamer, can
Single-stranded signal probe ssDNA, DNA cloning system, excision enzyme, the silver ion reduction detection body hybridized with T-2 toxin aptamers
System.
The T-2 toxin aptamer is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACA
CATGCGAGAGGTGAAGACTCGC-3’。
The single-stranded signal probe ssDNA is 5 '-CCCCCCACACCCGATCCCCCCCGCGAGTCTTCACC-3 '.
The DNA cloning system includes buffer solution, dNTP and Phi29 DNA polymerases；The buffer solution by
Tris-HCl、MgCl2、 (NH4)2SO4Composition.
The excision enzyme is Exo III exonucleases.
Reducing agent is boron hydride in the silver ion reduction detection architecture.
The boron hydride is sodium borohydride.
The present invention also provides a kind of T-2 toxin aptamers can be used for detecting T-2 toxin, base sequences 5 '-
CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACAC
ATGCGAGAGGTGAAGACTCGC-3’。
Advantages of the present invention
Detection method and kit according to the present invention, eliminate the interference of background fluorescence, improve the detection of T-2 toxin
Sensitivity and precision.
Description of the drawings
Fig. 1 is the concentration relationship of ssDNA-Ag nanocluster fluorescences intensity and T-2 toxin.
Specific implementation mode
Embodiment 1
It is a kind of detection T-2 toxin kit include at least：T-2 toxin aptamer, single-stranded signal probe ssDNA, DNA
Amplification system, exonuclease, silver ion reduction detection architecture.T-2 toxin aptamers are 5 '-CAGCTCAGAAGCTTGATC
CTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCGC-3’.Single-stranded signal probe ssDNA is 5 '-
CCCCCCACACCCGATCCCCCCCGCGAGTCTTCACC-3’.DNA cloning system includes buffer solution, dNTP and Phi29
DNA polymerases；The buffer solution is by Tris-HCl, MgCl2、 (NH4)2SO4Composition.Exonuclease is Exo III nucleic acid
Excision enzyme.Reducing agent in the silver ion reduction detection architecture is boron hydride, such as sodium borohydride.
Embodiment 2
A method of detection T-2 toxin, specific operation process are as follows：
By each DNA storing solutions heat-treated 5 minutes at 95 DEG C, before use, simultaneously placing 30 minutes at room temperature.So
Afterwards, it takes respectively containing 3.0 μm of ol T-2 toxin aptamers（Apt）40 μ L of hybridization buffer and 3.0 μm of ol signal probes
The 40 μ L of hybridization buffer of ssDNA are placed in 2ml centrifuge tubes, are hybridized 1 hour at 37 DEG C, and it is suitable to generate T-2 toxin nucleic acid
Body-signal probe hybrid（Apt - ssDNA）.
At 37 DEG C, the T-2 toxin of a concentration of 0 ~ 1.0 ng/mL is added to Apt- ssDNA solution by difference successively
In, T-2 toxin is reacted with T-2 toxin aptamers, is generated aptamer-T-2 toxin, is released ssDNA.At this moment, in system
There are ssDNA, residue（It is unreacted）The substances such as APT- ssDNA and aptamer-T-2 toxin.
10 μ L buffer solutions are added（Buffer solution group becomes 50 mM Tris-HCl, 10 mM MgCl2, 10 mM
(NH4)2SO4,pH 7.5 ）, dNTP (10 mM) 18 μ L are then added.2 μ L Phi29 DNA polymerizations are added into system again
Enzyme (10 u/ μ l), reacts 15 minutes at 37 DEG C so that with T-2 toxin aptamer-signal probe hybridization sequences（Ap-
ssDNA）It is expanded into double-stranded DNA for template.It keeps that Phi29 DNA is made within 10 minutes to deactivate at 65 DEG C.
2 μ L Exo III exonucleases are added into the reaction system again（20 u/μL）, 30 points are reacted at 37 DEG C
Clock makes that selectively double-stranded DNA is hydrolyzed into mononucleotide and removes double-stranded DNA, and single-stranded probe ssDNA is not hydrolyzed and retained
Come.
25 μ L, 1 mmol silver nitrates and 180 μ L sodium citrate buffer solutions are added into reaction solution（10 mM, pH7.0）.
Then, mixture be protected from light at room temperature or darkroom in place after ten minutes, under fast stirring, be added a concentration of 200 μM of 100 μ L
Freshly prepd sodium borohydride solution.Then 5 ~ 10 min are reacted at 45 DEG C.Solution is transferred to microcolorimetric ware, is measured
The fluorescence intensity of system carries out the quantitative determination of T-2 toxin, and the results are shown in Figure 1.
0.008-0.20 ng/mL of the range of linearity, detection limit 5pg/mL, the rate of recovery is 95.5 ~ 105.7%.Other fungi poison
The detection of the biological micromolecules T-2 toxin such as element, vitamin C, glucose is noiseless.
<110>University Of Science and Technology Of Hunan
<120>The detection method and detection kit of T-2 toxin
<160> 2
<210> 1
<211> 67
<212> DNA
<213>T-2 toxin aptamers
<400> 1
5’CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCGC-3’
<210> 2
<211> 35
<212> DNA
<213>Single-stranded signal probe
<400> 2
5’-CCCCCCACACCCGATCCCCCCCGCGAGTCTTCACC-3’
Claims (8)
1. a kind of detection method of T-2 toxin, characterized in that this method comprises the following steps：
（A）T-2 toxin aptamer and single-stranded signal DNA probe（ssDNA）Hybridization forms hybridization chain；
（B）So that the hybridization chain and sample to be tested is acted on, in the presence of having T-2 toxin in sample to be tested, hybridization chain selectively with
The reaction of T-2 toxin releases single-stranded signal probe ssDNA；
（C）Eliminate the interference of hybridization chain makes hybridization chain at double-stranded DNA using DNA cloning, excision enzyme is then used, by double-stranded DNA
It is hydrolyzed into mononucleotide and removes double-stranded DNA, leave single-stranded signal probe ssDNA；
（D）Silver ion reduction generation near-infrared fluorescent silver is not can induce using T-2 toxin aptamer-T-2 toxin combinations to receive
Rice cluster, only single-stranded signal probe ssDNA can induce silver ion reduction at near-infrared fluorescent silver nanoclusters, detection architecture it is glimmering
Luminous intensity, to measure the content of the T-2 toxin in sample to be tested；
The T-2 toxin aptamer is 5 '-CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATG
CGAGAGGTGAAGACTCGC-3’；The single-stranded signal probe ssDNA is 5 '-CCCCCCACACCCGATCCCCCCCGCGAG
TCTTCACC-3’。
2. a kind of kit of detection T-2 toxin, characterized in that it is included at least：T-2 toxin aptamer, can with T-2 poison
The single-stranded signal probe ssDNA of plain aptamer hybridization, DNA cloning system, exonuclease, silver ion reduction detection architecture.
3. kit according to claim 2, characterized in that the T-2 toxin aptamer is 5 '-
CAGCTCAGAAGCTTGATCCTGTATATCAAGCATCGCGTGTTTACACATGCGAGAGGTGAAGACTCGC-3’。
4. kit according to claim 2, characterized in that the single-stranded signal DNA probe is 5 '-
CCCCCCACACCCGATCCCCCCCGCGAGTCTTCACC-3’。
5. according to the kit of the detection T-2 toxin described in claim 2, characterized in that the DNA cloning system includes slow
Rush solution, dNTP and Phi29 DNA polymerases；The buffer solution is by Tris-HCl, MgCl2、 (NH4)2SO4Composition.
6. according to the kit of the detection T-2 toxin described in claim 2, characterized in that the exonuclease is Exo
III exonucleases.
7. according to the kit of the detection T-2 toxin described in claim 2, characterized in that the silver ion reduction detection
Reducing agent is boron hydride in system.
8. according to the kit of the detection T-2 toxin described in claim 7, characterized in that the boron hydride is hydroboration
Sodium.
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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 |
| CN113376120B (en) * | 2021-05-27 | 2022-07-15 | 江苏科技大学 | Optical fiber LSPR aptamer biosensor and preparation method and application thereof |
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