CN105713966B - Method for rapidly detecting zearalenone - Google Patents

Method for rapidly detecting zearalenone Download PDF

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CN105713966B
CN105713966B CN201610045201.2A CN201610045201A CN105713966B CN 105713966 B CN105713966 B CN 105713966B CN 201610045201 A CN201610045201 A CN 201610045201A CN 105713966 B CN105713966 B CN 105713966B
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zearalenone
ssdna
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aptamer
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CN105713966A (en
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易守军
曾云龙
任婕凤
刘婷
黄昊文
张起豪
邓克勤
唐春然
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Hunan University of Science and Technology
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    • C12N2310/16—Aptamers

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Abstract

The invention relates to a method for rapidly detecting zearalenone, which comprises the following steps: (A) hybridizing a zearalenone aptamer (Apt) and a single-stranded signal probe ssDNA that can hybridize with the zearalenone aptamer to form a hybrid chain; (B) the hybrid chain is contacted with a sample to be detected, and when zearalenone exists in the sample to be detected, the hybrid chain reacts with the zearalenone to release a single-chain signal probe ssDNA; (C) using DNA amplification to make the hybrid chain into double-stranded DNA, then selectively catalyzing the double-stranded DNA to be hydrolyzed into mononucleotide by using exonuclease, and retaining the single-stranded signal probe ssDNA without hydrolysis; (D) and detecting the fluorescence intensity of the system in a copper ion reduction detection system, thereby determining the content of the mycotoxin zearalenone in the sample to be detected. The method can eliminate interference and improve the detection sensitivity and precision of the zearalenone.

Description

Method for rapidly detecting zearalenone
Technical Field
The invention belongs to the technical field of nano biosensing and biological detection, and relates to a method and a kit for detecting zearalenone.
Background
Zearalenone is mainly produced by fusarium graminearum, and various fusarium such as fusarium roseum, fusarium scurrorum and fusarium trilinear can also produce the toxin. In 1980, the study of plum quarten revealed that zearalenone also exists in plants of many crops such as wheat, soybean and the like. Zearalenone mainly pollutes corn, wheat, rice, barley, millet, oat and other grains. Wherein the positive detection rate of zearalenone of the corn is 45 percent, and the highest toxic content can reach 2909 mg/kg; the detection rate of wheat is 20%, and the toxin content is 0.364-11.05 mg/kg. The zearalenone has strong heat resistance, and can be completely destroyed after being treated at 110 ℃ for 1 hour. Zearalenone has estrogen-like effect, can cause acute and chronic poisoning of animals, causes abnormal and even death of animal reproductive function, and can cause huge economic loss to animal farms. Therefore, the method is very necessary for the detection of zearalenone.
The existing analysis methods comprise a chromatographic method and an immunological method, wherein the former method has insufficient sensitivity, and the latter method has the defects of expensive reagents, easy activation and the like. Chinese patent CN102443585A discloses a zearalenone aptamer and application thereof, which can be used for detecting zearalenone, but has the problem that the price of DNA of a biomarker is high.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting zearalenone, aiming at the problems in the prior art.
The technical scheme adopted by the invention is as follows: a method for rapidly detecting zearalenone comprises the following steps:
(A) hybridizing a zearalenone aptamer (Apt) and a single-stranded signal probe ssDNA that can hybridize with the zearalenone aptamer to form a hybrid chain;
(B) enabling the hybrid chain to act with a sample to be detected, and selectively reacting the hybrid chain with zearalenone to release a single-chain signal probe ssDNA when the zearalenone exists in the sample to be detected;
(C) in order to eliminate the interference of the hybrid strands, DNA amplification is utilized to make the hybrid strands into double-stranded DNA, then exonuclease is used to selectively catalyze the hydrolysis of the double-stranded DNA into mononucleotide, and a single-stranded signal probe ssDNA in the system is not hydrolyzed and is reserved;
(D) the method comprises the steps of detecting the fluorescence intensity of a system in a copper ion reduction detection system by utilizing the principle that copper ions cannot be reduced to generate copper nanoparticles with fluorescence emission peaks around 620nm in a zearalenone aptamer-zearalenone combination, and the copper ions are reduced to generate ssDNA-modified copper nanoparticles with strong fluorescence emission at around 620nm (emission peak positions) only in the presence of a single-chain signal probe ssDNA, so that the content of mycotoxin zearalenone in a sample to be detected is determined.
The copper ion reduction detection system comprises copper ions and a reducing agent vitamin C, wherein the copper ions are added in sequence, and the reducing agent vitamin C is used for reducing the copper ions into copper nano particles. The detection in the step (D) comprises, for example, sequentially adding a copper ion solution and a vitamin C solution into the system, and generating copper nanoparticles with a fluorescence emission peak of about 620nm after reaction.
The zearalenone aptamer is 5' -GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’。
The ssDNA of the single-stranded signal probe is 5' -TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCACTTCACACGATT-3’。
The detection principle of the invention is as follows: apt was first hybridized to ssDNA (underlined); when zearalenone exists, the hybrid chain reacts with zearalenone to release ssDNA; Apt-ssDNA (residual), Apt-zearalenone and ssDNA are present in the system. Copper ions can not be reduced in Apt-zearalenone to generate copper nanoparticles with a fluorescence emission peak of about 620nm, and the existence of Apt-zearalenone causes no interference to the determination; the hybrid strand may interfere; in order to eliminate the interference of impurity cross-linking: a. using DNA amplification to make the hybrid chain into double-stranded DNA, b, selectively catalyzing the double-stranded DNA to be hydrolyzed into mononucleotide by using exonuclease, and retaining the single-stranded signal probe ssDNA in the system without hydrolysis; only ssDNA inducing reduction of copper ions into near-infrared copper nanoparticles is left in the system, the copper ions are reduced in the presence of the ssDNA to generate copper nanoparticles with a fluorescence emission peak about 620nm modified by the ssDNA, 340 nm light is used as excitation light, the fluorescence intensity of a fluorescence emission (520 nm-660 nm) spectrum is measured, and the content of the mycotoxin zearalenone can be measured according to the relation between the intensity of the fluorescence emission spectrum of the system and the amount of zearalenone. Because the interference of background fluorescence in the system is eliminated, the sensitivity and the precision of detection can be improved.
The invention also provides a kit for detecting zearalenone, which at least comprises: zearalenone aptamer, single-stranded signal probe DNA, a DNA amplification system, exonuclease and copper ion reduction detection system.
The zearalenone aptamer is 5'-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3'.
The possible single-stranded signaling probe DNA is 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCACTTCACACGATT-3'.
The DNA amplification system comprises a buffer solution (the buffer solution is Tris-HCl and MgCl)2、 (NH4)2SO4Composition), deoxymononucleotide triphosphate mixed solution (dNTP) and Phi29DNA polymerase.
The exonuclease is Exo III exonuclease.
The invention also provides a zearalenone nucleic acid aptamer for detecting zearalenone, wherein the base sequence of the zearalenone nucleic acid aptamer is 5' -GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTT
GTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’。
THE ADVANTAGES OF THE PRESENT INVENTION
According to the detection method and the kit, the interference can be eliminated, and the detection sensitivity and precision of the zearalenone can be improved.
Drawings
FIG. 1 shows fluorescence excitation and emission spectra of ssDNA-copper nanoparticles.
Detailed Description
Example 1
A kit for detecting zearalenone at least comprises: single-stranded signal probe ssDNA capable of hybridizing zearalenone aptamer, DNA amplification system, exonuclease and copper ion reduction detection bodyIs described. The zearalenone aptamer is 5'-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3'. The single-stranded signal probe DNA is 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCACTTCACACGATT-3'. The DNA comprises a buffer solution (the buffer solution is Tris-HCl and MgCl)2、 (NH4)2SO4Composition), deoxymononucleotide triphosphate mixed solution dNTP and Phi29DNA polymerase. The exonuclease is Exo III exonuclease. The reducing agent used by the copper ion reduction detection system is vitamin C.
Example 2
A method for rapidly detecting zearalenone comprises the following specific operation processes:
each DNA stock was heat-treated at 95 ℃ for 5 minutes and allowed to stand at room temperature for 30 minutes before use. Then, 50. mu.L of a hybridization buffer solution containing 2.5. mu. mol of zearalenone aptamer Apt and 50. mu.L of a hybridization buffer solution containing 2.5. mu. mol of signal probe ssDNA were placed in a 2ml centrifuge tube, and hybridization was carried out at 37 ℃ for 1 hour to produce a zearalenone aptamer-signal probe hybrid (Apt-ssDNA).
At 37 ℃, zearalenone with the concentration of 0-50 ng/mL is added into the Apt-ssDNA solution in sequence, and the zearalenone reacts with a zearalenone aptamer segment in the hybrid chain to generate the aptamer-zearalenone, and ssDNA is released. In this case, ssDNA, the remaining (unreacted) Apt-ssDNA, and the aptamer, zearalenone, are present in the system.
10 μ L of buffer (buffer composition 50mM Tris-HCl, 10 mM MgCl) was added2，10 mM(NH4)2SO4pH 7.5), and then 20. mu.L of dNTP (10 mM) was added. Then 2 μ L Phi29DNA polymerase (10 u/μ L) is added into the system and reacted for 15 minutes at 37 ℃, so that the zearalenone aptamer-signal probe hybridization sequence (Apt-ssDNA) is used as a template for amplification into double-stranded DNA. The Phi29DNA was inactivated by holding at 65 ℃ for 10 minutes.
Adding 2 mu L of Exo III exonuclease (20 u/mu L) into the reaction system, reacting for 30 minutes at 37 ℃, wherein the Exo III exonuclease selectively catalyzes double-stranded DNA to hydrolyze into mononucleotide, and a single-stranded signal probe ssDNA in the system is not hydrolyzed but remains; the solution was heated to 95 ℃ for 5 minutes to inactivate the exonuclease Exo III and the solution was rapidly cooled to room temperature in an ice bath.
To the reaction solution were added 25. mu.L of 1 mmol copper nitrate and 200. mu.L of PBS buffer (10 mM, pH 7.8). Then, after the mixture was left at room temperature in the dark or dark for 10 minutes, 100. mu.L of a freshly prepared vitamin C solution with a concentration of 1mM was added under rapid stirring. Then reacted at 45 ℃ for 5 min. The solution was transferred to a microcuvette, and the fluorescence intensity of the fluorescence emission (520-660 nm) spectrum of the system was measured using light with a wavelength of 340 nm as excitation light, and the results of quantitative measurement of zearalenone were shown in FIG. 1.
Linear range 0.008-0.50 ng/mL, linear equation y = 260.87 + 99.37 lgCThe linear correlation coefficient r = 0.9978, the detection limit is 1pg/mL, and the recovery rate is 96.5-106.2%. Other biological micromolecules such as mycotoxin and the like do not interfere with the detection of the zearalenone.
<110> Hunan university of science and technology
<120> method for rapidly detecting zearalenone
<160>2
<210>1
<211>73
<212>DNA
<213> zearalenone nucleic acid aptamer
<400>1
5’-GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’
<210>2
<211>55
<212>DNA
<213> Single-stranded Signal Probe
<400>2
5’-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGCACTTCACACGATT-3’

Claims (2)

1. A method for rapidly detecting zearalenone is characterized by comprising the following steps:
(A) hybridizing the zearalenone aptamer Apt with a single-stranded signal probe ssDNA to form a hybrid chain;
(B) enabling the hybrid chain to act with a sample to be detected, and selectively reacting the hybrid chain with zearalenone to release a single-chain signal probe ssDNA when the zearalenone exists in the sample to be detected;
(C) in order to eliminate the interference of the hybridization, the hybridized strand is made into double-stranded DNA by using DNA amplification, then the double-stranded DNA is selectively catalyzed by exonuclease ExoIII to be hydrolyzed into mononucleotide, and the single-stranded signal probe ssDNA is not hydrolyzed but remains in the system;
(D) detecting the fluorescence intensity of a system in a copper ion reduction detection system by utilizing the principle that copper ions cannot be reduced in a zearalenone aptamer-zearalenone combination to generate copper nanoparticles with a fluorescence emission peak at about 620nm and only reduced in a single-stranded signal probe ssDNA to generate copper nanoparticles modified by the ssDNA and having fluorescence emission at about 620nm, so as to determine the content of mycotoxin zearalenone in a sample to be detected; the copper ion reduction detection system is a reduction reaction system formed by adding a PBS buffer solution of copper nitrate and a vitamin C solution into a reaction solution;
the zearalenone aptamer is 5' -GCATCACTACAGTCATTACGCATCGTGGGGATGG
GAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’；
The ssDNA of the single-stranded signal probe is 5' -TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
TTTTTTGCACTTCACACGATT-3’。
2. A kit for rapidly detecting zearalenone is characterized by comprising: zearalenone aptamer, single-stranded signal probe ssDNA, a DNA amplification system, exonuclease ExoIII and a copper ion reduction detection system; the copper ion reduction detection system is a reduction reaction system formed by adding a PBS buffer solution of copper nitrate and a vitamin C solution into a reaction solution;
the zearalenone aptamer is 5' -GCATCACTACAGTCATTACGCATCGTGGGGATGG
GAGGTTGTTTACGCAGGAGATGTTAATCGTGTGAAGTGC-3’；
The ssDNA of the single-stranded signal probe is 5' -TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
TTTTTTGCACTTCACACGATT-3’；
The DNA amplification system comprises a buffer solution, a deoxymononucleotide triphosphate mixed solution dNTP and Phi29DNA polymerase; the buffer solution is prepared from Tris-HCl and MgCl2、 (NH4)2SO4And (4) forming.
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CN109676128B (en) * 2019-02-28 2021-05-04 湖南科技大学 Preparation method and application of molybdenum trisulfide coated gold nanorods
CN110330974B (en) * 2019-07-11 2022-09-09 南京工业大学 Preparation and application of zearalenone ratiometric fluorescent probe

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102443585A (en) * 2011-11-25 2012-05-09 国家纳米技术与工程研究院 Zearalenone nucleic acid aptamer and application thereof
CN102830113A (en) * 2012-06-14 2012-12-19 青岛科技大学 Signal amplification technology establishment based on target induced chain release and restriction enzyme digestion circulation and detection of ochracin A
CN105543345A (en) * 2015-12-17 2016-05-04 湖南科技大学 Method and kit for detecting zearalenone

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102443585A (en) * 2011-11-25 2012-05-09 国家纳米技术与工程研究院 Zearalenone nucleic acid aptamer and application thereof
CN102830113A (en) * 2012-06-14 2012-12-19 青岛科技大学 Signal amplification technology establishment based on target induced chain release and restriction enzyme digestion circulation and detection of ochracin A
CN105543345A (en) * 2015-12-17 2016-05-04 湖南科技大学 Method and kit for detecting zearalenone

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新型光学核酸探针的制备及其在生化分析中的应用研究;卿志和;《中国博士学位论文全文数据库，工程科技I辑》;20150915(第9期);B014-106 *

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