CN107164384B - Detect aflatoxin M1Aptamer, sensor, kit and application - Google Patents

Detect aflatoxin M1Aptamer, sensor, kit and application Download PDF

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Publication number
CN107164384B
CN107164384B CN201710383877.7A CN201710383877A CN107164384B CN 107164384 B CN107164384 B CN 107164384B CN 201710383877 A CN201710383877 A CN 201710383877A CN 107164384 B CN107164384 B CN 107164384B
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solution
aflatoxin
sensor
dna
aptamer
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CN107164384A (en
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郑楠
文芳
郭晓东
李松励
张养东
赵圣国
王加启
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Institute of Animal Science of CAAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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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
    • C12Q1/6851—Quantitative amplification
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers

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Abstract

The invention discloses detection aflatoxin Ms1Aptamer, sensor, kit and application.It is to detect aflatoxin M shown in SEQ ID No:1 that the present invention discloses nucleotide first1Aptamer, the invention also discloses the sensor prepared with the aptamer and contain the detection kit of the sensor solution.The present invention uses round pcr to carry out signal amplification as signal transduction, and detection limit greatly reduces, and detection is limited up to 0.03ng/L.The present invention uses aptamer as recognition unit, with the aflatoxin M in the sensor test sample of its preparation1Content, measure aflatoxin M1High sensitivity, the range of linearity are wide, are able to achieve accurate quantitative detection.

Description

Detect aflatoxin M1Aptamer, sensor, kit and application
Technical field
The present invention relates to detection aflatoxin Ms1Aptamer, the invention further relates to containing the nucleic acid be adapted to The sensor of body and the detection aflatoxin M prepared with it1Kit, the invention further relates to they detection aspergillus flavus poison Plain M1In application, belong to aflatoxin M1Detection field.
Background technique
Aflatoxin M1(AFM1) it is one of strongest mycotoxin of dairy products Poisoning, human health is brought huge Harm.AFM11 class carcinogenic substance is appointed as by the World Health Organization (WHO) international cancer research institution.When cow feeding contains There is AFB1Moldy feed after, metabolin AFM will be generated in milk1.In order to prevent due to the feed of pollution and calling together for food Bring food safety fear and economic loss are returned, many countries are to AFM1It is limited the quantity.Different food products AFM1Limitation range For 0.05-0.5 μ g kg-1.Therefore, to ensure food safety, exploitation is simple, sensitive and specific AFM1Detection method is non- It is often important.
Current existing Determination Methods of Aflatoxins mainly has:
Quantitative detection AFM1Liquid chromatogram combination fluorescence detector and liquid chromatogram combination mass-spectrometric technique, these instruments inspection Survey method needs professional operator and expensive instrument and equipment.
Some rapid free epidemiology method such as ELISA, immunosensor method are also widely used.But it is transporting It is difficult to ensure with the stability of antibody in storage process and limits the application of such methods.
There is the aptamer of similar functions with antibody, there is low price, high stability is easily modified and is easily-synthesized etc. and is excellent Point may be reused and save for a long time.The aptamer obtained through in-vitro screening can be with target substance with high specificity In conjunction with being therefore widely used in biosensor detection field.
Summary of the invention
An object of the present invention is to provide a kind of for detecting aflatoxin M1Aptamer；
The second object of the present invention is to provide a kind of detection aflatoxin M1Aptamer sensor；
The third object of the present invention is to provide a kind of malicious for detecting aspergillus flavus containing the aptamer sensor Plain M1Kit；
The fourth object of the present invention is to be used to examine by the aptamer, aptamer sensor and kit Survey aflatoxin M1, realize high sensitivity, the detection aflatoxin M that the range of linearity is wide, easy to operate1Effect.
Above-mentioned purpose of the invention is achieved through the following technical solutions:
Present invention firstly provides a kind of aflatoxin Ms1Aptamer is as follows nucleotide sequence:
5′-ATCCGTCACACCTGCTCTGACGCTGGGGTCGACCCG-3′(SEQ ID No:1)。
The present invention also provides a kind of detection aflatoxin Ms1Aptamer sensor, the aptamer sensor It is made of following four part:
(1) DNA sequence dna A: the DNA profiling being made of following DNA sequence dna: 5 '-GGTGTGACGGATXaXb-3 '；Wherein, Xa and Xb respectively represents a and b any bases, a=b=20-30；
Preferably, the DNA sequence dna A is following nucleotide sequence:
5′-GGTGTGACGGATAATCTGGTTTAGCTACGCCTTCCCCGTGG CGATGTTTCTTAGCGCCTTAC-3′ (SEQ ID No:2)。
(2) aflatoxin M described in DNA sequence dna B:SEQ ID No:11Aptamer, wherein in 3 ' terminal modified lifes Object element；
(3) length of PCR primer 1, the PCR primer 1 is 20-30nt, and sequence has the feature that 5 '-Xc-3 ', The 20-30 base at 5 ' ends and the Xb section complementary pairing of DNA sequence dna A；Wherein, X is any base, and c or b are base quantity, c= B=20-30；
Preferably, the nucleotide sequence of the PCR primer 1 is as follows:
5′-GTAAGGCGCTAAGAAACATCG-3′(SEQ ID No:3)。
(4) length of PCR primer 2, the PCR primer 2 is 20-30nt, and sequence has the feature that 5 '-Xd-3 ', The 20-30 base at 5 ' ends is identical as the Xa section of DNA sequence dna A；Wherein, X represents any base, and a or d represent base quantity, d =a=20-30.
Preferably, the nucleotide sequence of the PCR primer 2 is as follows:
5′-AATCTGGTTTAGCTACGCCTTC-3′(SEQ ID No:4)。
Aflatoxin M of the present invention1Aptamer sensor is assembled i.e. by mixing above-mentioned four kinds of components It can get.
The present invention also provides a kind of detection aflatoxin Ms1Kit, which includes following components:
(1) PCR pipe modifies solution；
(2) sensor solution；
(3) PCR system solution；
(4) aflatoxin M1Extract solution；
Wherein, the PCR pipe modification solution includes following components: glutaraldehyde solution, carbonate buffer solution, strepto- are affine Element；Wherein, the glutaraldehyde solution, carbonate buffer solution concentration be respectively preferably 0.8%, 0.01M, Streptavidin it is dense Degree is preferably 2.5-5ng mL-1, further preferably 2.5ng mL-1。
The sensor solution includes following each component:
The aflatoxin M1Aptamer sensor, PBST buffer, hybridization buffer, Tris buffer；Its In, the concentration of DNA sequence dna A is preferably 1 × 10 in the sensor solution-3- 10 nM, further preferably 10nM；The sensing The concentration of DNA sequence dna B is preferably 5-40nM in device solution, further preferably 10nM；PCR primer 1 in the sensor solution Concentration with PCR primer 2 is preferably 5-20 μM, and further preferably 10 μM.
The PCR system solution can be the reaction solution of commercially available archaeal dna polymerase when institute band, or according to reaction The reaction solution that principle is formulated, including following components: SYBR Green dyestuff, Taq archaeal dna polymerase, PCR buffering Liquid, dNTP, water.
The aflatoxin M1Extracting solution can dissolve aflatoxin M to be any1Solution, preferably methanol is molten The concentration of liquid, the methanol solution can according to need determination, for example, can be 70% methanol solution.
Further, the present invention provides the kits to detect aflatoxin M1Method, comprising the following steps:
(1) aflatoxin M will be contained1Sample to be tested and aflatoxin M1Extracting solution mixing, be vortexed concussion, centrifugation And take supernatant；
(2) supernatant is mixed and is incubated for sensor solution；
(3) step (2) described mixed liquor is discarded, and is eluted with Tris buffer；
(4) PCR system solution is added, through quantitative PCR detection fluorescence signal.
Preferably, sample to be tested and aflatoxin M described in step (1)1The weight ratio of extracting solution is 1:3-1:100.
The temperature of incubation described in step (2) is preferably 45 DEG C, and incubation time is preferably 1h.
Operation instruction when the PCR system solution can be according to commercially available polymerase determines.
Kit of the invention uses quantitative PCR method, and testing principle is as follows:
Solution is modified by Streptavidin in PCR pipe surface modification first with PCR pipe；By the aspergillus flavus of biotin modification Toxin M1Aptamer is in conjunction with the Streptavidin of PCR pipe surface modification, thus by aflatoxin M1Aptamer is solid Surely PCR pipe surface is arrived；By DNA sequence dna A and aflatoxin M1Aptamer DNA (B) is assembled into sensor by complementation.Sample The aflatoxin M to dissociate in product1With aflatoxin M1Aptamer DNA (B) specific binding so that DNA sequence dna A from PCR pipe surface falls off, and is discharged into solution, so that trigger sensor, expands to obtain the amplification of signal by round pcr.If The aflatoxin M to dissociate in sample1Amount is more, then the DNA sequence dna A split away off is more, the DNA sequence dna A of PCR pipe surface residual It is few, then need more recurring numbers to can be only achieved fluorescence threshold.Conversely, then few.The aflatoxin M of various concentration gradient is set1 Standard items detect signal by the above method, will test the aflatoxin M of various concentration1Obtained signal is made into standard Curve, the aflatoxin M in actual sample1Concentration can be obtained by contrast standard curve.
Aflatoxin M of the present invention1Aptamer, the aflatoxin M1Aptamer sensor and The kit can be applied to detection aflatoxin M1。
The present invention uses aptamer as recognition unit, has the characteristics that selectivity is good.Using nucleic acid of the invention The sensor of aptamers preparation measures aflatoxin M1High sensitivity uses round pcr to carry out signal as signal transduction and puts Greatly, detection limit greatly reduces, up to 0.03ng/L.
In the present invention, the PCR refers to polymerase chain reaction (Polymerase Chain Reaction), the term Meaning and technical step are known to the skilled person.
Detailed description of the invention
Fig. 1 aptamer sensor of the present invention detects aflatoxin M1Schematic diagram.
Fig. 2 quantitative PCR detection aflatoxin M1Standard curve.
Fig. 3 quantitative PCR detection aflatoxin M1Amplification curve.
Specific embodiment
Embodiments of the present invention will be described in more by the following example, it should be understood that the example is only example Property, any restrictions are not constituted to the scope of the present invention.It will be understood by those skilled in the art that without departing from of the invention Can be with the details and forms of the technical scheme of the invention are modified or replaced under spirit and scope, but these modifications or substitutions are equal Fall into protection scope of the present invention.
In PCR system solution in the present inventionPremix ExWith ROX Reference Dye II (50 ×) reaction solution is commercially available.
The preparation of 1 sensor solution of embodiment
DNA sequence dna A and DNA sequence dna B reaction solution are mixed.Wherein, the concentration of DNA sequence dna A, DNA sequence B is 10nM.
Wherein, the sequence of DNA sequence dna A are as follows:
5′-GGTGTGACGGATAATCTGGTTTAGCTACGCCTTCCCCGTGG CGATGTTTCTTAGCGCCTTAC- 3′。
The sequence of DNA sequence dna B are as follows:
5′-ATCCGTCACACCTGCTCTGACGCTGGGGTCGACCCG-Biotin -3′。
Prepare PCR system solution:
By PCR primer 1, PCR primer 2,Premix ExWith ROX Reference Dye II The mixing of (50 ×) reaction solution；Wherein, PCR primer 1, the concentration of PCR primer 2 are 10 μM.
Wherein, the sequence of PCR primer 1 are as follows:
5′-GTAAGGCGCTAAGAAACATCG-3′。
The sequence of PCR primer 2 are as follows:
5′-AATCTGGTTTAGCTACGCCTTC-3′。
Embodiment 2 makes standard curve
50 μ L, 0.8% glutaraldehyde solution handles 37 DEG C of 5h of PCR pipe；After ultrapure washing three times, 0.01M carbonate is added The 50 μ L of Streptavidin of buffer solution is incubated for 2h (37 DEG C)；
After phosphate buffer is washed twice, aptamers and complementary DNA 1:1 (v/v) are sufficiently mixed, the mixture of 50 μ L It is added to incubation 1h (37 DEG C) in each pipe；
Hybridization buffer is washed three times, 50 μ l various concentration gradients (concentration be respectively 0.1ng/L, 1ng/L, 0.01 μ g/L, 0.1 μ g/L, 1 μ g/L) aflatoxin M1Standard items；
Tris buffer adds PCR system solution after washing three times, and through quantitative PCR detection fluorescence signal, it is bent to make standard Line, made standard curve are shown in Fig. 2.
Aflatoxin M in 3 test sample of embodiment1Content
It weighs 0.5g and is detected sample, 2.5mL 0.05ng mL is added-1Aflatoxin M1Standard solution is sufficiently mixed with it It is even, 2.5mL70% methanol is then added.Mixture is vortexed after concussion 5min and is centrifuged 10min in 10000g.Collect supernatant liquid nitrogen It blows and is concentrated into 0.5mL.5% methanol of 2mL is added to redissolve；Quantitative PCR detection fluorescence signal.
Quantitative PCR detection aflatoxin M1Amplification curve see Fig. 3, according to the standard curve that embodiment 2 is measured, determine Aflatoxin M in test sample1Content be 0.045ng mL-1。
SEQUENCE LISTING
<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
<120>aptamer, sensor, kit and the application of Aflatoxins M1 are detected
<130> BJ-2001-160540A-H
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 36
<212> DNA
<213> artifical sequence
<400> 1
atccgtcaca cctgctctga cgctggggtc gacccg 36
<210> 2
<211> 62
<212> DNA
<213> artifical sequence
<400> 2
ggtgtgacgg ataatctggt ttagctacgc cttccccgtg gcgatgtttc ttagcgcctt 60
ac 62
<210> 3
<211> 21
<212> DNA
<213> artifical sequence
<400> 3
gtaaggcgct aagaaacatc g 21
<210> 4
<211> 22
<212> DNA
<213> artifical sequence
<400> 4
aatctggttt agctacgcct tc 22

Claims (13)

1. one kind is for detecting aflatoxin M1Aptamer, it is characterised in that: its nucleotide is SEQ ID No:1 institute Show.
2. a kind of detection aflatoxin M1Aptamer sensor, it is characterised in that: the aptamer sensor by Four parts form below:
(1) DNA sequence dna A: the DNA profiling being made of following DNA sequence dna: 5 '-GGTGTGACGGATXaXb-3 '；Wherein, Xa and Xb Respectively represent a and b any bases, a=b=20-30；
(2) aflatoxin M described in DNA sequence dna B:SEQ ID No:11Aptamer, wherein in 3 ' terminal modified biotins；
(3) PCR primer 1, the length of the PCR primer 1 is 20-30nt, and sequence has the feature that 5 '-Xc-3 ', 5 ' ends 20-30 base and DNA sequence dna A Xb section complementary pairing；Wherein, X is any base, and c or b are base quantity, c=b= 20-30；
(4) PCR primer 2, the length of the PCR primer 2 is 20-30nt, and sequence has the feature that 5 '-Xd-3 ', 5 ' ends 20-30 base it is identical as the Xa section of DNA sequence dna A；Wherein, X represents any base, and a or d represent base quantity, d=a= 20-30。
3. aptamer sensor according to claim 2, it is characterised in that: the nucleotide sequence of the DNA sequence dna A For shown in SEQ ID No:2.
4. aptamer sensor according to claim 2, it is characterised in that: the nucleotide sequence of the PCR primer 1 For shown in SEQ ID No:3.
5. aptamer sensor according to claim 2, it is characterised in that: the nucleotide sequence of the PCR primer 2 For shown in SEQ ID No:4.
6. a kind of detection aflatoxin M1Kit, it is characterised in that: the kit includes following components:
(1) PCR pipe modifies solution；
(2) sensor solution；
(3) PCR system solution；
(4) aflatoxin M1Extract solution；
The sensor solution includes following each component:
Aptamer sensor, PBST buffer, hybridization buffer and Tris buffer as claimed in claim 2.
7. kit according to claim 6, it is characterised in that: the PCR pipe modification solution includes following components: penta 2 Aldehyde solution, carbonate buffer solution, Streptavidin；The concentration of DNA sequence dna A is 1 × 10 in the sensor solution-3-10nM；Institute The concentration for stating DNA sequence dna B in sensor solution is 5-40nM；The concentration of PCR primer 1 and PCR primer 2 in the sensor solution It is 5-20 μM；
The PCR system solution includes following components: SYBR Green dyestuff, Taq archaeal dna polymerase, PCR buffer, dNTP And water；
The aflatoxin M1Extracting solution is methanol solution.
8. kit according to claim 7, it is characterised in that: wherein, the concentration of the glutaraldehyde solution is 0.8%, The concentration of carbonate buffer solution is 0.01M；The concentration of Streptavidin is 2.5-5ngmL-1；
The concentration of DNA sequence dna A is 10nM in the sensor solution；
The concentration of DNA sequence dna B is 10nM in the sensor solution；
The concentration of PCR primer 1 and PCR primer 2 is 10 μM in the sensor solution；
The methanol solution is 70% methanol solution.
9. kit according to claim 7, it is characterised in that: the concentration of the Streptavidin is 2.5ngmL-1。
10. application claim 6 or 7 kit detects aflatoxin M1Method, comprising the following steps:
(1) aflatoxin M will be contained1Sample to be tested and aflatoxin M1Extracting solution mixing, be vortexed concussion, is centrifuged and takes Supernatant；
(2) supernatant is mixed and is incubated for sensor solution；
(3) step (2) described mixed liquor is discarded, and is eluted with Tris buffer；
(4) PCR system solution is added, through quantitative PCR detection fluorescence signal；
The method belongs to non-diagnostic and non-treatment purpose method.
11. according to the method for claim 10, it is characterised in that: sample to be tested and aflatoxin M described in step (1)1 The weight ratio of extracting solution is 1:3-1:100；
The temperature of incubation described in step (2) is 45 DEG C, incubation time 1h.
12. aptamer described in claim 1 detects aflatoxin M in preparation1Application in the reagent of content.
13. aptamer sensor as claimed in claim 2 detects aflatoxin M in preparation1Answering in the reagent of content With.
CN201710383877.7A 2016-05-26 2017-05-26 Detect aflatoxin M1Aptamer, sensor, kit and application Active CN107164384B (en)

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CN110095442B (en) * 2019-04-24 2020-03-03 中国科学院生态环境研究中心 Method for analyzing aflatoxin B1 through fluorescence anisotropy of sensitive aptamer
CN111999502B (en) * 2020-08-24 2023-08-04 湖南农业大学 Aflatoxin B1 detection kit and method based on PBNPs in-situ growth regulation multimode signal output

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