CN105603070A - Detection method and kit of aflatoxin B1 - Google Patents

Detection method and kit of aflatoxin B1 Download PDF

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Publication number
CN105603070A
CN105603070A CN201610041767.8A CN201610041767A CN105603070A CN 105603070 A CN105603070 A CN 105603070A CN 201610041767 A CN201610041767 A CN 201610041767A CN 105603070 A CN105603070 A CN 105603070A
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aflatoxin
afb1
ssdna
signal probe
strand
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CN105603070B (en
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夏晓东
陈佳鑫
田彩霞
黄昊文
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Hunan University of Science and Technology
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means

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Abstract

The invention relates to a detection method and kit of aflatoxin B1. The detection method includes following steps: (A), enabling aptamer Apt of aflatoxin B1 to hybridize with ssDNA of a single strand signal probe to form a hybrid strand; (B), enabling the hybrid strand to contact with a to-be-detected sample, wherein when the to-be-detected sample contains aflatoxin B1, the hybrid strand reacts with aflatoxin B1 to release ssDNA; (C), utilizing DNA amplification to enable the hybrid strand to have double strand DNA, using an excision enzyme to hydrolyze the double strand DNA to be mononucleotide to remove the double strand DNA, wherein the ssDNA is left in a system at that time; (D), induced by the ssDNA, reducing silver ions to generate near infrared fluorescent silver nano cluster, detecting fluorescent intensity of the system so as to determine content of aflatoxin B1 in the to-be-detected sample. The detection method has the advantages of high sensitivity, simplicity in operation and low cost.

Description

The detection method of AFB1 and detection kit
Technical field
The present invention relates to nano-biosensing and field of biological detection, specifically, be to provide a kind of aflatoxinThe detection method of B1 and detection kit.
Background technology
AFB1 has strong toxicity to comprising people and some animals, is carcinogenicity in known chemical substanceThe strongest one, very harmful to people and animals. Aflatoxin is the metabolite that aspergillus flavus, aspergillus parasiticus etc. produce. Aspergillus flavusToxin is extensively present in soil, soybean, paddy, corn, macaroni, flavouring, milk and goods thereof, edible oil, meat (fish)In the medium animals and plants of goods, peanut and walnut and various nut, particularly in peanut and walnut. When people takes in containing aflatoxin, can there is the acute poisoning such as oxyhepatitis, hemorrhagic necrosis in the food that B1 pollutes, even dead; When trace continues to take in, can makeBecome slow poisoning, growth disorder, carcinogenic, teratogenesis etc. Aflatoxin almost cannot be avoided in agricultural product, in natural foodThe most common with AFB1, harmfulness is the strongest, and State General Administration for Quality Supervision's regulation AFB1 is most of foodOne of essential items for inspection. In China's food hygienic standard, specify: in corn, peanut oil, peanut and goods thereof aflatoxin must not >20 μ g/kg; Rice, other edible oil must not > 10 μ g/kg; Other grain, beans, fermented food must not > 5 μ g/kg;Baby's milk powder substitute must not detect aflatoxin. The states such as European Union in 2002 to grain, the aspergillus flavus in peanut and products thereofToxin B1 content regulation, the Aflatoxin in Peanut byHigh B1 content that the mankind directly use needs≤2 μ g/kg, enters as raw-food materialThe peanut aflatoxin B1 content of mouth needs≤8 μ g/kg. If in people's food, aflatoxin content exceeds certain standard, justCan directly threaten people's health. Therefore the detection method of, setting up high selectivity, high-sensitive AFB1 has valency very muchValue.
Research mainly contains thin-layered chromatography (TLC), high performance liquid chromatography (HPLC), immune affinity column method both at home and abroad at presentAnd ELISA etc. The specificity of TLC method is poor, and sensitivity is also relatively poor, and required standard items concentration is higher, potentialContaminative higher. The personnel that HPLC method need to have specialized operations technology just can detect, and technical requirement is high; Decontaminating columnConsume morely, testing cost is higher, and immune affinity column specificity is better, but still needs know-how personnel could be competent at detectionWork, detection technique requires high. That immunization has is easy and simple to handle, quick, less pollution, also more high many advantages of sensitivity,But conventionally need to use the biological reagents such as expensive enzyme marking reagent, enzyme, antigen, antibody, and these biological reagents also veryEasily inactivation.
Summary of the invention
The invention provides a kind of method of examining AFB1, the method comprises the steps:
(A) nucleic acid aptamer of aflatoxin B 1 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 AFB1 in testing sample, the choosing of hybridization chainReact with AFB1 and discharge strand signal probe ssDNA to selecting property;
(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 nucleic acid aptamer of aflatoxin B 1-aflatoxin combination can not induce silver ion reduction to become near-infrared glimmeringThe silver nanoclusters of light, only has strand signal probe ssDNA can induce silver ion reduction to become near-infrared fluorescent silver nanoclusters, silver fromDetection architecture fluorescence intensity in son reduction detection architecture, thereby the content of the AFB1 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 nucleic acid aptamer of aflatoxin B 1 is 5 '-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’。
Described strand signal probe ssDNA is 5 '-CCCCCCACACCCGATCCCCCCTGTGGGCCTAGCG-3’。
Detection principle of the present invention is as follows: first allow Apt and ssDNA hybridize (underscore part); When there being AFB1While existence, hybridization chain reacts and discharges ssDNA with AFB1; In system, there is Apt-ssDNA (remaining),Apt-AFB1 and ssDNA. Apt-AFB1 can not induce silver ion reduction to become near-infrared fluorescent silver nanoparticleBunch, Apt-AFB1 exists noiseless to measuring; Hybridization chain may have interference; In order to eliminate the interference of hybridization chain: a.Utilize DNA cloning, make to hybridize chain and become double-stranded DNA, b. exonuclease, is hydrolyzed into mononucleotide by double-stranded DNA and removes twoChain DNA. Now in system, only stay and can induce the ssDNA that generates near-infrared fluorescent silver nanoclusters, by the fluorescence of detection architectureIntensity, can measure the content of AFB1. Due to the interference of background fluorescence in elimination system, improve detect sensitiveDegree and precision.
The present invention also provides a kind of kit that detects AFB1, and it at least comprises: AFB1 coreFit, the strand signal probe ssDNA of acid, DNA cloning system, excision enzyme, silver ion reduction detection architecture.
Described nucleic acid aptamer of aflatoxin B 1 is 5 '-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’。
Described strand signal probe ssDNA is 5 '-CCCCCCACACCCGATCCCCCCTGTGGGCCTAGCG-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 nucleic acid aptamer of aflatoxin B 1 that can be used for detecting AFB1, its baseSequence is 5 '-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3 '.
Advantage of the present invention
According to detection method of the present invention and kit, eliminate the interference of background fluorescence, improve the inspection of AFB1Survey sensitivity and precision.
Brief description of the drawings
Fig. 1 is the concentration relationship of ssDNA-Ag nano-cluster fluorescence intensity and AFB1.
Detailed description of the invention
Embodiment 1
A kind of kit that detects AFB1 at least comprises: nucleic acid aptamer of aflatoxin B 1, strand signal probeSsDNA, DNA cloning system, exonuclease, silver ion reduction detection architecture. Described nucleic acid aptamer of aflatoxin B 1 is5’-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3 '. Described strand signalProbe ssDNA is 5 '-CCCCCCACACCCGATCCCCCCTGTGGGCCTAGCG-3 '. Described DNA cloning system comprises slowDissolved liquid (Tris-HCl, MgCl2、(NH4)2SO4), dNTP and Phi29DNA polymerase. The exonuclease of stating be ExoIII exonuclease. Reducing agent in described silver ion reduction detection architecture is boron hydride, as sodium borohydride.
Embodiment 2
A method that detects AFB1, 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 probes containing 3.0 μ mol nucleic acid aptamer of aflatoxin B 1 AptThe hybridization cushioning liquid 40 μ L of ssDNA are placed in 2ml centrifuge tube, hybridize 1 hour at 37 DEG C, generate AFB1Aptamer-signal probe hybrid (Apt-ssDNA).
At 37 DEG C, it is molten that the AFB1 that is 0 ~ 2ng/mL by concentration successively respectively adds Apt-ssDNA toIn liquid, AFB1 reacts with nucleic acid aptamer of aflatoxin B 1, generates aptamer-AFB1, dischargesSsDNA. At this moment, in system, there are the things such as ssDNA, residue (unreacted) APT-ssDNA and aptamer-AFB1Matter.
(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 nucleic acid aptamer of aflatoxin B 1-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 be hydrolyzed into mononucleotide and remove, and single-stranded probe ssDNA is not extremely hydrolyzed slowly because of reaction speedAnd remain.
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 μThe freshly prepd sodium borohydride solution of M. Then at 45 DEG C, react 5 ~ 10min. Solution is transferred to micro-cuvette, surveysDetermine the fluorescence intensity of system, carry out the quantitative assay of AFB1, result as shown in Figure 1.
The range of linearity 0.008 – 0.20ng/mL, detectability 0.9pg/mL, the rate of recovery is 95.8 ~ 106.6%. Other fungiThe detection of the biological micromolecule AFB1s such as toxin, vitamin C, glucose is noiseless.
<110>University Of Science and Technology Of Hunan
<120>detection method of AFB1 and detection kit
<160>2
<210>1
<211>55
<212>DNA
<213>nucleic acid aptamer of aflatoxin B 1
<400>1
5’-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’
<210>2
<211>35
<212>DNA
<213>strand signal probe
<400>2
5’-CCCCCCACACCCGATCCCCCCCTGTGGGCCTAGCG-3’

Claims (10)

1. a detection method for AFB1, is characterized in that, the method comprises the steps:
(A) nucleic acid aptamer of aflatoxin B 1 Apt and strand signal probe ssDNA hybridization, form hybridization chain;
(B) make this hybridization chain and testing sample effect, in the time having AFB1 to exist in testing sample, hybridization chain is selectiveGround reacts with AFB1 and discharges strand signal probe ssDNA;
(C) in order to eliminate the interference of hybridization chain, utilize DNA cloning, make to hybridize chain and become double-stranded DNA, then use excision enzyme, by two strandsDNA is hydrolyzed into mononucleotide and removes double-stranded DNA, leaves strand signal probe ssDNA;
(D) utilize nucleic acid aptamer of aflatoxin B 1-AFB1 combination can not induce silver ion reduction to generate closely redThe silver nanoclusters of outer fluorescence, only has strand signal probe ssDNA can induce silver ion reduction to become near-infrared fluorescent silver nanoclusters,The fluorescence intensity of detection architecture, thereby the content of the AFB1 in mensuration testing sample.
2. method according to claim 1, is characterized in that, described nucleic acid aptamer of aflatoxin B 1 is 5 '-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’。
3. method according to claim 1 and 2, is characterized in that, described strand signal probe ssDNA is 5 '-CCCCCCACACCCGATCCCCCCTGTGGGCCTAGCG-3’。
4. a kit that detects AFB1, is characterized in that, it at least comprises: AFB1 nucleic acid is suitableBody, strand signal probe ssDNA, DNA cloning system, exonuclease, silver ion reduction detection architecture.
5. kit according to claim 4, is characterized in that, described nucleic acid aptamer of aflatoxin B 1 is 5 '-AAAAAGTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3’。
6. kit according to claim 4, is characterized in that, described strand signal probe DNA is 5 '-CCCCCCACACCCGATCCCCCCTGTGGGCCTAGCG-3’。
7. kit according to claim 4, is characterized in that, described DNA cloning system comprise cushioning liquid, dNTP andPhi29DNA polymerase; Described cushioning liquid is by Tris-HCl, MgCl2、(NH4)2SO4Composition.
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 silver ion reduction detection architecture, reducing agent is boronHydride.
10. kit according to claim 9, is characterized in that, described boron hydride is sodium borohydride.
CN201610041767.8A 2016-01-21 2016-01-21 Detection method and detection kit for aflatoxin B1 Active CN105603070B (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108444992A (en) * 2018-02-09 2018-08-24 河南工业大学 A kind of quantitative aflatoxin detection kit and its detection method
CN109402128A (en) * 2018-12-21 2019-03-01 青岛农业大学 Aflatoxin B1Aptamer, the aflatoxin B containing the aptamer1Detection kit and detection method

Citations (1)

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CN104267192A (en) * 2014-03-06 2015-01-07 上海大学 Bio-electrochemical sensor for detecting thrombin as well as preparation method and application of bio-electrochemical sensor

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CN104267192A (en) * 2014-03-06 2015-01-07 上海大学 Bio-electrochemical sensor for detecting thrombin as well as preparation method and application of bio-electrochemical sensor

Non-Patent Citations (4)

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WON-BO SHIM: "An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1", 《BIOSENSORS AND BIOELECTRONICS》 *
XIAODONG GUO: "Development of an ultrasensitive aptasensor for the detection of aflatoxin B 1", 《BIOSENSORS AND BIOELECTRONICS》 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108444992A (en) * 2018-02-09 2018-08-24 河南工业大学 A kind of quantitative aflatoxin detection kit and its detection method
CN108444992B (en) * 2018-02-09 2020-11-13 河南工业大学 Aflatoxin quantitative detection kit and detection method thereof
CN109402128A (en) * 2018-12-21 2019-03-01 青岛农业大学 Aflatoxin B1Aptamer, the aflatoxin B containing the aptamer1Detection kit and detection method

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