CN105506128A - Method for detecting aflatoxin B1 - Google Patents

Method for detecting aflatoxin B1 Download PDF

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CN105506128A
CN105506128A CN201610020877.6A CN201610020877A CN105506128A CN 105506128 A CN105506128 A CN 105506128A CN 201610020877 A CN201610020877 A CN 201610020877A CN 105506128 A CN105506128 A CN 105506128A
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dna
aflatoxin
solution
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nucleotide sequence
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CN105506128B (en
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张晶珠
毛瑜
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Shenzhen Kun Jian Original New Drug Research Institute
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions

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Abstract

The invention provides a method for detecting aflatoxin B1. The method includes the following steps that 1, a sample which is to be detected and contains the aflatoxin B1 is mixed with an aflatoxin B1 extracting solution, and liquid supernatant is centrifuged and extracted; 2, the liquid supernatant is mixed with a DNA sensor solution and DNA polymerase and subjected to incubation; 3, a heme chloride solution is added, and ABTS reduction state colourless substrate and hydrogen peroxide are added after room temperature incubation is conducted. When the method is utilized to detect the aflatoxin B1, no large instrument and equipment are needed, cost is low, a thermostatic reaction is conducted, qualitative detection conducted through naked eyes can be achieved, and quantitative detection can be further achieved; besides, fluorochrome is not needed, and cost is greatly lowered.

Description

A kind of method detecting aflatoxin B1
Technical field
The present invention relates to field of biological detection, more specifically, relate to a kind of method detecting aflatoxin B1.
Background technology
Aflatoxin is the secondary metabolite produced primarily of flavus, Aspergillus parasiticus, occurs that the probability of aflatoxin is the highest in the food and feed of damp-heat area.They are present in soil, animals and plants, various nut, particularly easily pollute the grain oil products such as peanut, corn, rice, soybean, wheat, are mycotoxicosis class mycotoxins that is maximum, that very give prominence to human health risk.The most common with aflatoxin B1 in natural food, hazardness is also the strongest.
Current existing Determination Methods of Aflatoxins mainly contains:
(1) thin layer chromatography
Thin-layer chromatography is isolation technique most widely used in aflatoxin research.From nineteen ninety, it is listed in AOAC (AssociationofOfficialAgriculturalChemists) standard method, and the method has the function of qualitative and quantitative analysis aflatoxin simultaneously.
(2) liquid phase chromatography
Liquid chromatography and thin-layer chromatography have similarity in many aspects, and the two complements each other.Usually carry out the condition setting in early stage with TLC, after selecting suitable separation condition, carry out the quantitative assay of aflatoxin again with LC.
(3) immunochemical analyses method
The immune analysis method utilizing the aflatoxin of monoclonal antibody or the polyclonal antibody design with high specificity is also conventional Determination Methods of Aflatoxins.These class methods generally include radioimmunoassay method, euzymelinked immunosorbent assay (ELISA) and immunochromatographic method, and they all can carry out quantitative assay to aflatoxin.
But these method costs are high, and need the instrument using Large expensive, complex operation.
Aptamer is section of DNA (thymus nucleic acid), RNA (Yeast Nucleic Acid) or XNA (nucleic acid analog) sequence.Normally utilize in-vitro screening technology---the Fas lignand system evolution technology (Systematicevolutionofligandsbyexponentialenrichment, SELEX) of index concentration, the oligonucleotide fragment obtained from nucleic acid molecule libraries.Aptamer can be combined with target substance high specific, highly selective, is therefore widely used in field of biosensors.
DNA sensor take DNA as sensor, the biological signals acted on is changed into the physical signallings such as detectable optical, electrical, sound wave by transverter between DNA and other materials.In recent years, DNA sensor is in widespread attention in the applied research in the fields such as gene diagnosis, environmental monitoring, drug research.
Summary of the invention
The object of the invention is the above-mentioned defect overcoming prior art, a kind of method detecting aflatoxin B1 is provided, the method is simple to operate, highly sensitive, cost is low.
To achieve these goals, the invention provides a kind of method detecting aflatoxin B1, the method comprises the following steps:
(1) testing sample containing aflatoxin B1 is mixed with aflatoxin B1 extracting solution, centrifugal and extracting supernatant liquor;
(2) described supernatant liquor mixed with DNA sensor solution, archaeal dna polymerase and hatch;
(3) add protohemine solution, HEPES buffered soln, after incubated at room, add ABTS reduction-state colorless substrate and hydrogen peroxide;
Wherein, described DNA sensor solution comprises DNA sensor, RCA reaction solution and dNTP solution,
Described DNA sensor is made up of following three parts:
(1) DNA (A): hold by following DNA sequence dna 5 ' end and 3 ' circular DNA template be formed by connecting: 5 '-XaACCCAACCCGCCCTACCCXb-3 ' (SEQIDNO:2); Wherein, Xa and Xb represents a and b base arbitrarily respectively, and a+b=40-80;
(2) DNA (B): aflatoxin B1 aptamer, this aptamer has following nucleotide sequence:
5 '-GTTmmmmmmmTGTTGTCTCTCTGTGTCTnnnnnnnTTCGCTAGGCCCACA-3 ' (SEQIDNO:1); Wherein, each m and n is A, T, C or G and m segment and n segment complementary pairing independently of one another;
(3) DNA (C): RCA primer, the length of described DNA (C) is 25-50nt and sequence has following characteristics: 5 ' flank complementary pairing of the 5 ' 15-30 a held base and DNA (A) fixed sequence program, and 5 ' of the 3 ' 10-20 a held base and DNA (B) holds complementary pairing.
Wherein, described DNA (A) fixed sequence program is the sequence between Xa and Xb, because DNA (A) is circular DNA template, therefore, 5 ' flank of described DNA (A) fixed sequence program is not limited to the part in Xa, also can to comprise in Xb at least partially.The complementary sequence of described fixed sequence program, can the oxidation of catalysis ABTS under the existence of protohemine.
In the present invention, described RCA refers to rolling circle amplification (rollingcircleamplification, RCA), and implication and the technological step of this term are known to the skilled person.
Preferably, described DNA (A) has following nucleotide sequence:
5’-ACTGATATTAACCCAACCCGCCCTACCCATTTCTTGTTTCGTATCATTGCGAATACCGTG-3’(SEQIDNO：3)。
Preferably, described DNA (C) has following nucleotide sequence:
5’-TAATATCAGTCACGGTATTCTTTTCCAGCACGGGAAC-3’(SEQIDNO：4)。
Or preferably, described DNA (C) has following nucleotide sequence:
5’-TAATATCAGTCACGGTATTCTTTTCACAACAGCACGGGAAC-3’(SEQIDNO：5)。
Again or preferably, described DNA (C) has following nucleotide sequence:
5’-TAATATCAGTCACGGTATTCTTTTCAGAGACAACAGCACGGGAAC-3’(SEQIDNO：6)。
DNA sensor of the present invention is carried out self-assembly by the above-mentioned three kinds of components of mixing and can be obtained.
Wherein, in described DNA sensor solution, the concentration of DNA (A), DNA (B) and DNA (C) is all preferably 10-1000nM, more preferably 20-100nM.
Wherein, described aflatoxin B1 extracting solution can be any solution that can dissolve aflatoxin B1, and be preferably methanol solution, the concentration of described methanol solution can be determined as required, such as, can be 70% methanol solution.
Wherein, described hydrogen peroxide can be conventional various concentration, such as, be the hydrogen peroxide of 30%.
In the present invention, described RCA reaction solution can for being purchased archaeal dna polymerase time with reaction solution, also can for the reaction solution according to the configuration of this reaction principle.(330mMTris-acetic acid (at 37 DEG C, pH is 7.9), 100mM magnesium acetate, 660mM Potassium ethanoate, 1% (v/v) polysorbas20,10mMDTT)).
According to the present invention, the weight ratio of described testing sample and aflatoxin B1 extracting solution can be 1:1-1000.
The condition of hatching in step (2) can be the incubation conditions of conventional DNA polymerase reaction, such as, be 30 DEG C, hatch 60min, 90 DEG C of heating 5min termination reactions.Described archaeal dna polymerase can be the various archaeal dna polymerases of this area routine, is preferably Phi29DNA polysaccharase.The consumption of described archaeal dna polymerase and dNTP can be determined according to operation instruction when being purchased this enzyme.
According to principle of the present invention, those skilled in the art can the consumption of each component in determining step (3), and such as, the final concentration of described protohemine is 0.1-1 μM; The final concentration of described ABTS reduction-state colorless substrate is 1-5mM; The final concentration of described hydrogen peroxide is 0.05-0.2mM; Described HEPES damping fluid is 2 × HEPES damping fluid.Meet on the basis of above-mentioned final concentration, those skilled in the art can select suitable mother liquid concentration as required.
The present invention adopts a step competition law, first the DNA sensor containing aflatoxin aptamer is assembled, aflatoxin B1 free in sample and aflatoxin aptamer DNA (B) specific binding, it is made to come off from DNA sensor, thus triggering DNA sensor, the amplification of signal is obtained by RCA technology.RCA product chain there is n section can be oxidized by catalytic substrate ABTS, become green DNA sequence dna from colourless.Thus realize naked eyes detection.According to competitive principle, if the free aflatoxin B1 amount in sample is many, then the aflatoxin aptamer split away off is many, and the DNA sensor simultaneously triggered is many, and then has many signals.Otherwise, then few.The aflatoxin B1 standard substance of different concns gradient are set, its signal is detected by aforesaid method, the signal that the aflatoxin B1 detecting different concns gradient obtains is drawn as typical curve, and after this in actual sample, the concentration of aflatoxin B1 is obtained by contrast standard curve.
Measure aflatoxin B1 without the need to large-scale instrument and equipment according to method of the present invention, cost is low.Isothermal reaction, can realize the qualitative detection of naked eyes, also can realize detection by quantitative, and, without the need to fluorescence dye, greatly reduce cost.Use aptamer as signal receiver, there is the feature of highly selective.Amplify while using RCA technology and DNA catalytic sequence to carry out signal, greatly reduce detectability (2ppb).This test kit is easy to carry, simple to operate.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Embodiment
The present invention is further illustrated by following examples.Wherein, hemin, ABTS reduction-state colorless substrate, Phi29 polysaccharase are and are purchased.RCA reaction solution is Phi29 polymeric enzyme reaction damping fluid.
Preparation example 1: preparation DNA sensor solution.
By DNA (A), DNA (B), DNA (C), the mixing of dNTP and RCA reaction solution.Wherein, the concentration of DNA (A), DNA (B), DNA (C), dNTP is respectively 40nM, 60nM, 40nM and 0.5nM.
Wherein, the sequence of DNA (A) is:
5’-ACTGATATTAACCCAACCCGCCCTACCCATTTCTTGTTTCGTATCATTGCGAATACCGTG-3’。
The sequence of DNA (B) is:
5’-GTTCCCGTGCTGTTGTCTCTCTGTGTCTGCACGGGTTCGCTAGGCCCACA-3’(SEQIDNO：7)。
The sequence of DNA (C) is:
5’-TAATATCAGTCACGGTATTCTTTTCCAGCACGGGAAC-3’。
Embodiment 1
(1) with the aflatoxin B1 standard substance of 70% methyl alcohol configuration different concns gradient (concentration is respectively 500 μ g/L, 50 μ g/L, 5 μ g/L, 500 μ g/L, 50 μ g/L);
(2) add 50 μ lDNA sensor solution, 10UPhi29 polysaccharase wherein respectively, mixing, hatches 60min for 30 DEG C, 90 DEG C of heating 5min termination reactions;
Add hemin solution and 2 × HEPES buffered soln, add ABTS reduction-state colorless substrate and 30% hydrogen peroxide after incubated at room 30min, the final concentration of described hemin is 0.5 μM; The final concentration of described ABTS reduction-state colorless substrate is 2mM; The final concentration of described hydrogen peroxide is 0.1mM; Color signal is read after 5min.
Production standard curve.
Embodiment 2
Get 10g and be detected sample (content of known wherein aflatoxin B1 is 1 μ g/L), add 500 μ l aflatoxin extracting solutions (70% methyl alcohol) and fully mix with it, centrifugal, extract 50 μ l supernatant liquors;
In supernatant liquor, add 50 μ lDNA sensor solution, 10UPhi29 polysaccharase, mixing, hatches 60min for 30 DEG C, 90 DEG C of heating 5min termination reactions;
Add hemin solution and HEPES buffered soln, after incubated at room 30min, add ABTS reduction-state colorless substrate and 30% hydrogen peroxide; The final concentration of described hemin is 0.5 μM; The final concentration of described ABTS reduction-state colorless substrate is 2mM; The final concentration of described hydrogen peroxide is 0.1mM.After 5min, naked eyes can find out colour-change.Read color signal, determine that the content of the aflatoxin B1 of detected sample is 1 μ g/L according to the typical curve that embodiment 1 is measured.
Be described above various embodiments of the present invention, above-mentioned explanation is exemplary, and non-exclusive, and be also not limited to disclosed each embodiment.When not departing from the scope and spirit of illustrated each embodiment, many modifications and changes are all apparent for those skilled in the art.

Claims (10)

1. detect a method for aflatoxin B1, it is characterized in that, the method comprises the following steps:
(1) testing sample containing aflatoxin B1 is mixed with aflatoxin B1 extracting solution, centrifugal and extracting supernatant liquor;
(2) described supernatant liquor mixed with DNA sensor solution, archaeal dna polymerase and hatch;
(3) add protohemine solution, HEPES buffered soln, after incubated at room, add ABTS reduction-state colorless substrate and hydrogen peroxide;
Wherein, described DNA sensor solution comprises DNA sensor, RCA reaction solution and dNTP solution,
Described DNA sensor is made up of following three parts:
(1) DNA (A): hold by following DNA sequence dna 5 ' end and 3 ' circular DNA template be formed by connecting: 5 '-XaACCCAACCCGCCCTACCCXb-3 '; Wherein, Xa and Xb represents a and b base arbitrarily respectively, and a+b=40-80;
(2) DNA (B): aflatoxin B1 aptamer, this aptamer has following nucleotide sequence:
5 '-GTTmmmmmmmTGTTGTCTCTCTGTGTCTnnnnnnnTTCGCTAGGCCCACA-3 '; Wherein, each m and n is A, T, C or G and m segment and n segment complementary pairing independently of one another;
(3) DNA (C): RCA primer, the length of described DNA (C) is 25-50nt and sequence has following characteristics: 5 ' flank complementary pairing of the 5 ' 15-30 a held base and DNA (A) fixed sequence program, and 5 ' of the 3 ' 10-20 a held base and DNA (B) holds complementary pairing.
2. method according to claim 1, wherein, described DNA (A) has following nucleotide sequence:
5’-ACTGATATTAACCCAACCCGCCCTACCCATTTCTTGTTTCGTATCATTGCGAATACCGTG-3’。
3. method according to claim 1, wherein, described DNA (C) has following nucleotide sequence:
5’-TAATATCAGTCACGGTATTCTTTTCCAGCACGGGAAC-3’。
4. method according to claim 1, wherein, described DNA (C) has following nucleotide sequence:
5’-TAATATCAGTCACGGTATTCTTTTCACAACAGCACGGGAAC-3’。
5. method according to claim 1, wherein, described DNA (C) has following nucleotide sequence:
5’-TAATATCAGTCACGGTATTCTTTTCAGAGACAACAGCACGGGAAC-3’。
6. according to the method in claim 1-5 described in any one, wherein, in described DNA sensor solution, the concentration of DNA (A), DNA (B) and DNA (C) is 10-1000nM.
7. according to the method in claim 1-5 described in any one, wherein, described aflatoxin B1 extracting solution is methanol solution.
8. according to the method in claim 1-5 described in any one, wherein, described archaeal dna polymerase is Phi29DNA polysaccharase.
9. according to the method in claim 1-5 described in any one, wherein, the weight ratio of described testing sample and aflatoxin B1 extracting solution is 1:1-1000.
10. according to the method in claim 1-5 described in any one, wherein, the final concentration of described protohemine is 0.1-1 μM; The final concentration of described ABTS reduction-state colorless substrate is 1-5mM; The final concentration of described hydrogen peroxide is 0.05-0.2mM.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107164384A (en) * 2016-05-26 2017-09-15 中国农业科学院北京畜牧兽医研究所 Detect aflatoxin M1Aptamer, sensor, kit and application
CN111122847A (en) * 2020-01-22 2020-05-08 福建中医药大学 Method for rapidly detecting aflatoxin B1 on site based on aptamer

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CN102952802A (en) * 2012-09-29 2013-03-06 江南大学 Group of oligonucleotides aptamers capable of specifically recognizing aflatoxin B1
CN104964969A (en) * 2015-05-28 2015-10-07 广东省生态环境与土壤研究所 Label-free visualization detection method and label-free visualization detection kit of aflatoxin B1

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CN102952802A (en) * 2012-09-29 2013-03-06 江南大学 Group of oligonucleotides aptamers capable of specifically recognizing aflatoxin B1
CN104964969A (en) * 2015-05-28 2015-10-07 广东省生态环境与土壤研究所 Label-free visualization detection method and label-free visualization detection kit of aflatoxin B1

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
CN107164384A (en) * 2016-05-26 2017-09-15 中国农业科学院北京畜牧兽医研究所 Detect aflatoxin M1Aptamer, sensor, kit and application
CN107164384B (en) * 2016-05-26 2019-01-04 中国农业科学院北京畜牧兽医研究所 Detect aflatoxin M1Aptamer, sensor, kit and application
CN111122847A (en) * 2020-01-22 2020-05-08 福建中医药大学 Method for rapidly detecting aflatoxin B1 on site based on aptamer
CN111122847B (en) * 2020-01-22 2022-09-20 福建中医药大学 Method for rapidly detecting aflatoxin B1 on site based on aptamer

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