CN103808701B - Based on the ochratoxin A homogeneous phase method for quick of nucleic acid interca-lating dyes fluorescent quenching - Google Patents
Based on the ochratoxin A homogeneous phase method for quick of nucleic acid interca-lating dyes fluorescent quenching Download PDFInfo
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Abstract
The invention discloses a kind of ochratoxin A homogeneous phase method for quick based on the fluorescent quenching of nucleic acid interca-lating dyes, comprise the following steps: the formation of (1) ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition; (2) ochratoxin A aptamer is to the specific recognition of ochratoxin A.The present invention adopts ochratoxin A aptamer as molecular recognition elements, the OTA detection method that nucleic acid interca-lating dyes builds as reporter molecules, greatly reduce the cost detecting OTA mycotoxin, favorable reproducibility, specificity is high, simple to operation and can realize the Parallel testing of a large amount of sample; The present invention is based on the ochratoxin A homogeneous phase method for quick of nucleic acid interca-lating dyes fluorescent quenching, after ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition are pre-formed, only need the detection that 5-10min can realize OTA.
Description
Technical field
The present invention relates to and utilize the fluorescent quenching of nucleic acid interca-lating dyes to detect ochratoxin A, feature is the application of ochratoxin A aptamer and nucleic acid interca-lating dyes, and the detection speed of the method is fast, detection flux is high and strong operability, belongs to technical field of fluorescence detection.
Background technology
Ochratoxin A (OchratoxinA, OTA) primarily of aspergillus He Celadon mould wait generation, be widespread in nature, wherein with in the majority on the crops such as grain (wheat, corn, barley, oat, rye, rice and broomcorn millet class etc.), peanut, vegetables (beans), there is many Poisonings such as liver renal toxicity and very strong teratogenesis, carcinogenic and mutagenesis, to animal and human's body health, there is very large potential hazard.Given this, the detection and control to OTA is all paid attention in countries in the world, has made OTA in Fodder and food one after another and to be correlated with limit standard, as European Union regulation cereal materials OTA maximum limitation 5 μ g/kg, cereal fabricated product maximum limitation 3 μ g/kg; OTA maximum limitation 5 μ g/kg in China's regulation cereal, beans, imports and exports grain to protect people ' s health and regulation and control.
The main method of current detection OTA is thin-layered chromatography (TLC), high performance liquid chromatography (HPLC), enzyme linked immunosorbent assay (ELISA) etc.TLC method is simple, and testing cost is low, but sensitivity is poor, reappearance is bad and cannot realize robotization; HPLC method is highly sensitive, but required expensive equipment, complex pretreatment, testing cost are high, is not suitable for the rapid screening of a large amount of sample.ELISA method is highly sensitive, specificity good, but the manufacturing cycle of the high-quality antibody relied on is very long and there is the problem of cross reaction.
Aptamer is as a class single stranded oligonucleotide, to target molecule generation specificity interact before and after space conformation can there is corresponding change, have compared with antibody can in-vitro screening acquisition, Heat stability is good, be easy to the advantage such as chemosynthesis and modification, even can distinguish the target molecule of the single substituting group difference that monoclonal antibody cannot realize; Nucleic acid interca-lating dyes has following features: unstressed configuration or have very weak fluorescence under free state, be fitted in DNA double chain and Fluorescence Increasing occurs, the homogeneous fluorescent detection method therefore based on nucleic acid interca-lating dyes dyestuff has highly sensitive, simple to operate and can carry out the features such as the Parallel testing of a large amount of sample.So, by the homogeneous fluorescent detection method that the high specific of aptamer, high-affinity combine with nucleic acid interca-lating dyes, the shortcoming of the main detection method of current OTA can be overcome well, be expected to become a kind of potential quick screening method.
Summary of the invention
In order to the scheme solving current OTA detection method is complicated, operation is locked, high cost, detection time long shortcoming, the invention provides a kind of ochratoxin A homogeneous phase method for quick based on the fluorescent quenching of nucleic acid interca-lating dyes.
Technical scheme of the present invention is, based on the ochratoxin A homogeneous phase method for quick of nucleic acid interca-lating dyes fluorescent quenching, comprises the following steps:
(1) formation of ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition;
(2) ochratoxin A aptamer is to the specific recognition of ochratoxin A.
The concrete operation step that the present invention is based on the ochratoxin A homogeneous phase method for quick that the fluorescent quenching of nucleic acid interca-lating dyes realizes is as follows:
The formation of ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition
First ochratoxin A aptamer is carried out pre-service: 95 DEG C of sex change 5min, 4 DEG C of cooling 5min.Then 50 μ L10mMTris-HCl reaction buffers (pH8.5) are got, wherein the concentration of ochratoxin A aptamer is 50nM, the dilution ratio of GeneFinder nucleic acid interca-lating dyes is 8:10000, room temperature reaction 30min, to make nucleic acid interca-lating dyes fully be combined with ochratoxin A aptamer, form ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition; Ochratoxin A aptamer is ssDNA probe, and sequence is 5 '-CTTCGGGTGTGGGTGGCATTCCGGTAGGCTCTG-3 '.
Ochratoxin A aptamer is to the specific recognition of ochratoxin A
Appropriate ochratoxin A solution is added in above-mentioned steps (1) reacted solution, adjusting liquor capacity with 10mMTris-HCl reaction buffer (pH8.5) is 100 μ L, wherein the concentration of ochratoxin A aptamer is 25nM, the dilution ratio of GeneFinder nucleic acid interca-lating dyes is 4:10000, room temperature reaction 5-10min, fully be combined with ochratoxin A to make ochratoxin A aptamer, form ochratoxin A aptamer and ochratoxin A compound, thus cause dissociating of ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition, produce the change of fluorescence intensity.Reacted solution is transferred in particle fluorescence cuvette and puts into fluorescence spectrophotometer, or put into the fluorescence spectrophotometer with microwell plate read module by reacted solution transfer microwell plate, take 490nm as excitation wavelength, 525nm is emission wavelength, measure its fluorescence signal intensity and record the change of fluorescence intensity: the fluorescence intensity (I of blank
0) maximum, along with the increase fluorescence intensity (I) of OTA concentration weakens gradually.
Detect by above-mentioned (1), the variable concentrations ochratoxin A titer of (2) step to preparation, record changing value (the Δ I=I of the fluorescence intensity that each standard solution sample produces
0-I).Obtain typical curve with Δ I to ochratoxin A concentration mapping in standard solution, in unknown sample, the changing value of the fluorescence intensity that ochratoxin A produces determines its concentration with mark curve control.
Beneficial effect imbody of the present invention is in the following areas in sum:
1) the present invention adopts ochratoxin A aptamer as molecular recognition elements, the OTA detection method that nucleic acid interca-lating dyes builds as reporter molecules, greatly reduce the cost detecting OTA mycotoxin, favorable reproducibility, specificity is high, simple to operation and can realize the Parallel testing of a large amount of sample;
2) the present invention is based on the ochratoxin A homogeneous phase method for quick of nucleic acid interca-lating dyes fluorescent quenching, after ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition are pre-formed, only need the detection that 5-10min can realize OTA.
Accompanying drawing explanation
Fig. 1: the phenomenon figure based on the ochratoxin A homogeneous phase method for quick of nucleic acid interca-lating dyes fluorescent quenching: wherein (a) is GeneFinder+Aptamer+AFB1, b () is GeneFinder+Aptamer, c () is GeneFinder+Aptamer+OTA, d () is GeneFinder, (e) is OTAaptamer.[OTAaptamer]=25nM,[OTA]=[AFB1]=50nM，DilutionratioofGeneFinder=4:10000。AFB1 is aflatoxin B1.
Fig. 2: the working curve diagram that ochratoxin A standard solution is detected.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.
(1) formation of ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition
First ochratoxin A aptamer is carried out pre-service: 95 DEG C of sex change 5min, 4 DEG C of cooling 5min.Then 50 μ L10mMTris-HCl reaction buffers (pH8.5) are got, wherein the concentration of ochratoxin A aptamer is 50nM, the dilution ratio of GeneFinder nucleic acid interca-lating dyes is 8:10000, room temperature reaction 30min, to make nucleic acid interca-lating dyes fully be combined with ochratoxin A aptamer, form ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition; Ochratoxin A aptamer is ssDNA probe, and sequence is 5 '-CTTCGGGTGTGGGTGGCATTCCGGTAGGCTCTG-3 '.
A kind of screening preparation of ochratoxin A aptamer: ochratoxin A molecule is coupled on the magnetic bead of carboxyl modified by coupling agent EDC and NHS, reacts with the reaction buffer solution containing initial libraries and initial libraries is fixed on magnetic bead.Be anti-Screening target with the magnetic bead of carboxyl modified respectively; making (warfarin) and ochratoxin B molecule for negative Screening target with N-acetyl group-L-Phe (N-acetyl-L-phenylalanine), method China, screening for just screening molecule with ochratoxin A.Deposit in case at ochratoxin A, there is the oligonucleotide sequence of binding ability to disintegrate down from magnetic bead with it, after pcr amplification, streptavidin magnesphere partition method is adopted to prepare secondary library, repeat screening 15 to take turns, then carry out cloning and sequencing, finally carry out sequential analysis, Activity determination and sequence truncation and optimization, thus obtain the specific nucleic acid aptamers of ochratoxin A.
(2) ochratoxin A aptamer is to the specific recognition of ochratoxin A
Appropriate ochratoxin A solution is added in above-mentioned steps (1) reacted solution, adjusting liquor capacity with 10mMTris-HCl reaction buffer (pH8.5) is 100 μ L, wherein the concentration of ochratoxin A aptamer is 25nM, the dilution ratio of GeneFinder nucleic acid interca-lating dyes is 4:10000, room temperature reaction 5-10min, fully be combined with ochratoxin A to make ochratoxin A aptamer, form ochratoxin A aptamer and ochratoxin A compound, thus cause dissociating of ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition, produce the change of fluorescence intensity.Reacted solution is transferred in particle fluorescence cuvette and puts into fluorescence spectrophotometer, or put into the fluorescence spectrophotometer with microwell plate read module by reacted solution transfer microwell plate, take 490nm as excitation wavelength, 525nm is emission wavelength, slit width is excited to be set as 10nm, launch slit width and be set as 5nm, measure its fluorescence signal intensity and record the change of fluorescence intensity: the fluorescence intensity (I of blank
0) maximum, along with the increase fluorescence intensity (I) of OTA concentration weakens gradually.
As shown in Figure 1, nucleic acid interca-lating dyes (GeneFinder) autofluorescence is very weak, negligible; OTA aptamer (aptamer) self does not have fluorescence; The two is deposited in case jointly, forms aptamer/GeneFinder fluorescent composition, has very strong fluorescence, Fluorescence Increasing more than 10 times.Deposit in case at OTA, aptamer and OTA fully combines, and forms aptamer/OTA compound, thus causes dissociating of aptamer/GeneFinder fluorescent composition, produces fluorescent quenching, causes fluorescence intensity to reduce; And depositing in case in aflatoxin B1 (AFB1), aptamer nonrecognition AFB1, therefore aptamer/GeneFinder fluorescent composition can not dissociate, so can not produce fluorescent quenching, fluorescence intensity can not reduce.
By above-mentioned (1), (2) step to variable concentrations ochratoxin A titer (0nM, 1nM, 2.5nM, 5.0nM, the 7.5nM of preparation, 10nM, 15nM, 20nM, 25nM, 35nM, 50nM, 75nM) detect, and record changing value (the Δ I=I of the fluorescence intensity that each standard solution sample produces
0-I).Obtain working curve (Fig. 2) with Δ I to ochratoxin A concentration mapping in standard solution, in unknown sample, the changing value of the fluorescence intensity that ochratoxin A produces contrasts with working curve and determines its concentration.
Should be understood that, for those of ordinary skills, can be improved according to the above description or convert, and all these improve and convert the protection domain that all should belong to claims of the present invention.
SEQUENCELISTING
<110> applicant Henan Academy of Agricultural Sciences
<120> is based on the ochratoxin A homogeneous phase method for quick of nucleic acid interca-lating dyes fluorescent quenching
<130>
<160>1
<170>PatentInversion3.5
<210>1
<211>33
<212>DNA
<213> ochratoxin A aptamer
<400>1
cttcgggtgtgggtggcattccggtaggctctg
33
Claims (2)
1., based on an ochratoxin A homogeneous phase method for quick for nucleic acid interca-lating dyes fluorescent quenching, it is characterized in that, comprise the following steps:
(1) formation of ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition;
(2) ochratoxin A aptamer is to the specific recognition of ochratoxin A,
The concrete operation step of described method is as follows:
(1) formation of ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition
First ochratoxin A aptamer is carried out pre-service: 95 DEG C of sex change 5min, 4 DEG C of cooling 5min, then get 50 μ L10mMTris-HCl and react buffer solution, pH8.5, wherein the concentration of ochratoxin A aptamer is the dilution ratio of 50nM, GeneFinder nucleic acid interca-lating dyes is 8:10000, room temperature reaction 30min, to make nucleic acid interca-lating dyes fully be combined with ochratoxin A aptamer, form ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition;
(2) ochratoxin A aptamer is to the specific recognition of ochratoxin A
Appropriate ochratoxin A solution is added in above-mentioned steps (1) reacted solution, use 10mMTris-HCl reaction buffer, pH8.5, adjustment liquor capacity is 100 μ L, wherein the concentration of ochratoxin A aptamer is 25nM, the dilution ratio of GeneFinder nucleic acid interca-lating dyes is 4:10000, room temperature reaction 5-10min, fully be combined with ochratoxin A to make ochratoxin A aptamer, form ochratoxin A aptamer and ochratoxin A compound, thus cause dissociating of ochratoxin A aptamer and nucleic acid interca-lating dyes fluorescent composition, produce the change of fluorescence intensity, reacted solution is put into fluorescence spectrophotometer, take 490nm as excitation wavelength, 525nm is emission wavelength, measure its fluorescence signal intensity and record the change of fluorescence intensity: the fluorescence intensity I of blank
0maximum, along with the increase fluorescence intensity (I) of OTA concentration weakens gradually,
Detect by above-mentioned (1), the variable concentrations ochratoxin A titer of (2) step to preparation, record changing value (the Δ I=I of the fluorescence intensity that each standard solution sample produces
0-I); Obtain typical curve with Δ I to ochratoxin A concentration mapping in standard solution, in unknown sample, the changing value of the fluorescence intensity that ochratoxin A produces determines its concentration with mark curve control.
2. the ochratoxin A homogeneous phase method for quick based on the fluorescent quenching of nucleic acid interca-lating dyes according to claim 1, is characterized in that: ochratoxin A aptamer is ssDNA probe, and sequence is
5’-CTTCGGGTGTGGGTGGCATTCCGGTAGGCTCTG-3’。
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| CN104178568B (en) * | 2014-07-25 | 2016-03-30 | 清华大学 | A kind of method based on the target substance in nucleic acid aptamer probe fluorescence sense analyzing and testing sample to be tested |
| CN104818279A (en) * | 2015-04-23 | 2015-08-05 | 河南省农业科学院 | Ochratoxin A nucleic acid adapter and application thereof to sample magnetic separation preprocessing |
| CN106546724A (en) * | 2015-09-23 | 2017-03-29 | 中国医学科学院药用植物研究所 | A kind of new method of molecular beacon probe quick detection ochratoxin A |
| CN106124781B (en) * | 2016-07-07 | 2018-01-02 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of method based on the sandwich fluorescent quenching technology detection demethyl testosterone of aptamers |
| CN106706737B (en) * | 2016-12-10 | 2019-03-26 | 武汉市农业科学技术研究院农业环境安全检测研究所（武汉市农业科学技术研究院中心实验室） | A kind of ochratoxin A rapid detection method |
| CN110823846A (en) * | 2018-08-09 | 2020-02-21 | 河北农业大学 | Application of novel fluorescent dye AccuBlue in detection of aptamer |
| CN109444101B (en) * | 2018-12-13 | 2021-03-02 | 四川大学 | Proportional aptamer fluorescent probe and method for detecting ochratoxin A by using same |
| CN109668864A (en) * | 2018-12-14 | 2019-04-23 | 福建中医药大学 | Azotized carbon nano piece couples the ochratoxin A fluorescence detection method of aptamers sensing |
| CN111896508A (en) * | 2020-07-08 | 2020-11-06 | 西南大学 | Novel method for rapidly detecting ochratoxin A based on nucleic acid dye GeneFinder |
| CN113866405A (en) * | 2021-10-15 | 2021-12-31 | 河南工业大学 | Preparation method of fluorescent aptamer sensor for simultaneously detecting ochratoxin A and aflatoxin B1 |
| CN114121154A (en) * | 2021-10-18 | 2022-03-01 | 江南大学 | Method for improving aptamer specificity and affinity by utilizing molecular design guidance |
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