CN107164384A - Detect aflatoxin M1Aptamer, sensor, kit and application - Google Patents

Detect aflatoxin M1Aptamer, sensor, kit and application Download PDF

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CN107164384A
CN107164384A CN201710383877.7A CN201710383877A CN107164384A CN 107164384 A CN107164384 A CN 107164384A CN 201710383877 A CN201710383877 A CN 201710383877A CN 107164384 A CN107164384 A CN 107164384A
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solution
aflatoxin
sensor
dna
aptamer
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CN107164384B (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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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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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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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    • G01—MEASURING; TESTING
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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 M1Aptamer, sensor, kit and application.The present invention discloses nucleotides for SEQ ID No first:Detection aflatoxin M shown in 11Aptamer, the detection kit the invention also discloses the sensor prepared with the aptamer and containing the sensor solution.The present invention uses round pcr as signal transduction and carries out signal amplification, test limit greatly reduces, test limit is up to 0.03ng/L.The present invention uses aptamer as recognition unit, and the aflatoxin M in sample is detected with its sensor prepared1Content, determine aflatoxin M1Sensitivity is high, the range of linearity is wide, can realize accurately quantitative detection.

Description

Detect aflatoxin M1Aptamer, sensor, kit and application
Technical field
The present invention relates to detection aflatoxin M1Aptamer, the invention further relates to contain the nucleic acid adaptation 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 technology
Aflatoxin M1(AFM1) it is one of dairy products Poisoning most strong mycotoxin, huge is brought to human health Harm.AFM11 class carcinogenic substance is appointed as by the World Health Organization (WHO) international cancer research institution.Contain when cow feeding There is AFB1Moldy feed after, metabolin AFM will be produced in milk1.In order to prevent due to the feed of pollution and calling together for food The food security fear and economic loss come are rewinded, many countries are to AFM1Limited the quantity.Different food products AFM1Limitation scope For 0.05-0.5 μ g kg-1.Therefore, to ensure food security, exploitation is simple, sensitive and specific AFM1Detection method is non- It is often important.
Current existing Determination Methods of Aflatoxins mainly has：
Quantitatively detect 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 epidemiologies method such as ELISA, immunosensor method is also widely used.But in transport The application for limiting this kind of method is difficult to ensure that with the stability of antibody in storage process.
There is the aptamer of similar functions with antibody, with low price, high stability easily modifies and be easily-synthesized etc. excellent Point, may be reused and preserve for a long time.The aptamer obtained through in-vitro screening can be with target substance with high specificity With reference to, therefore it is widely used in biology sensor detection field.
The content 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 be to provide it is a kind of containing the aptamer sensor be used for detect aspergillus flavus poison Plain M1Kit；
The fourth object of the present invention is to be used to described aptamer, aptamer sensor and kit examine Survey aflatoxin M1, realize the detection aflatoxin M that sensitivity is high, the range of linearity is wide, simple to operate1Effect.
The above-mentioned purpose of the present invention is achieved through the following technical solutions：
Present invention firstly provides a kind of aflatoxin M1Aptamer, it is as follows nucleotide sequence：
5′-ATCCGTCACACCTGCTCTGACGCTGGGGTCGACCCG-3′(SEQ ID No:1)。
The present invention also provides a kind of detection aflatoxin M1Aptamer sensor, the aptamer sensor It is made up of following four part：
(1) DNA sequence dna A：The DNA profiling being made up of following DNA sequence dna： 5′-GGTGTGACGGATXaXb-3′；Wherein, Xa and Xb represent a and b any bases, a=b=20-30 respectively；
It is preferred that, the DNA sequence dna A is following nucleotide sequence：
5′-GGTGTGACGGATAATCTGGTTTAGCTACGCCTTCCCCGTGG CGATGTTTCTTAGCGCCTTAC-3′ (SEQ ID No:2)。
(2) DNA sequence dna B：SEQ ID No:Aflatoxin M described in 11Aptamer, wherein, in 3 ' terminal modified lifes Thing element；
(3) PCR primer 1, the length of the PCR primer 1 is 20-30nt, and sequence has following feature：5 '-Xc-3 ', The 20-30 base and DNA sequence dna A Xb section complementary pairings at 5 ' ends；Wherein, X is any base, and c or b are base quantity, c= B=20-30；
It is preferred that, the nucleotide sequence of the PCR primer 1 is as follows：
5′-GTAAGGCGCTAAGAAACATCG-3′(SEQ ID No:3)。
(4) PCR primer 2, the length of the PCR primer 2 is 20-30nt, and sequence has following feature：5 '-Xd-3 ', The 20-30 base at 5 ' ends is identical with DNA sequence dna A Xa sections；Wherein, X represents any base, and a or d represent base quantity, d =a=20-30.
It is preferred that, 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 obtain.
The present invention also provides a kind of detection aflatoxin M1Kit, the kit include following components：
(1) PCR pipe modification 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, the concentration of carbonate buffer solution are respectively preferably 0.8%, 0.01M, Streptavidin it is dense Degree is preferably 2.5-5ng mL-1, more preferably 2.5ng mL-1。
The sensor solution includes following each component：
The aflatoxin M1Aptamer sensor, PBST buffer solutions, hybridization buffer, Tris buffer solutions；Its In, DNA sequence dna A concentration is preferably 1 × 10 in the sensor solution-3- 10 nM, more preferably 10nM；The sensing DNA sequence dna B concentration is preferably 5-40nM in device solution, more preferably 10nM；PCR primer 1 in the sensor solution Concentration with PCR primer 2 is preferably 5-20 μM, more preferably 10 μM.
Described PCR system solution, can for commercially available archaeal dna polymerase when institute band reaction solution, or according to reaction The reaction solution that principle is formulated, including following components：SYBR Green dyestuffs, Taq archaeal dna polymerases, PCR bufferings Liquid, dNTP, water.
The aflatoxin M1Extract solution can dissolve aflatoxin M to be any1Solution, preferably methanol is molten Liquid, the concentration of the methanol solution can be determined as needed, for example, can be 70% methanol solution.
Further, aflatoxin M is detected the invention provides the kit1Method, comprise the following steps：
(1) aflatoxin M will be contained1Testing sample and aflatoxin M1Extract solution is mixed, and be vortexed concussion, centrifugation And take supernatant；
(2) supernatant is mixed and is incubated with sensor solution；
(3) step (2) described mixed liquor is discarded, and is eluted with Tris buffer solutions；
(4) PCR system solution is added, through quantitative PCR detection fluorescence signal.
It is preferred that, testing sample and aflatoxin M described in step (1)1The weight ratio of extract solution is 1:3—1:100.
The temperature being incubated described in step (2) is preferably 45 DEG C, and incubation time is preferably 1h.
The PCR system solution can be determined according to operation instruction during commercially available polymerase.
The kit of the present invention uses quantitative PCR method, and its Cleaning 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 combined with the Streptavidin of PCR pipe surface modification, so that by aflatoxin M1Aptamer is consolidated 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 dissociated in product1With aflatoxin M1Aptamer DNA (B) specifically bind so that DNA sequence dna A from PCR pipe surface comes off, and is discharged into solution, so that trigger sensor, the amplification for obtaining signal is expanded by round pcr.If The aflatoxin M dissociated 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 periods to can be only achieved fluorescence threshold.Conversely, then few.The aflatoxin M of various concentrations gradient is set1 Standard items, signal is detected by the above method, will detect the aflatoxin M of various concentrations1Obtained signal makes standard Aflatoxin M in curve, 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 detect aflatoxin M1。
The present invention use aptamer as recognition unit, with it is selectively good the characteristics of.Using the nucleic acid of the present invention Sensor prepared by aptamers determines aflatoxin M1Sensitivity is high, and signal is carried out using round pcr as signal transduction and is put Greatly, test limit greatly reduces, up to 0.03ng/L.
In the present invention, the PCR refers to polymerase chain reaction (Polymerase Chain Reaction), the term Implication and technical step are known to the skilled person.
Brief description of the drawings
Fig. 1 aptamer sensor detection aflatoxin Ms of the present invention1Schematic diagram.
Fig. 2 quantitative PCR detection aflatoxin Ms1Standard curve.
Fig. 3 quantitative PCR detection aflatoxin Ms1Amplification curve.
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 limitation is not constituted to the scope of the present invention.It will be understood by those skilled in the art that without departing from the present invention's The details and form of technical solution of the present invention can be 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 the sensor solution of embodiment 1
DNA sequence dna A and DNA sequence dna B reaction solutions are mixed.Wherein, the concentration of DNA sequence dna A, DNA sequence Bs is 10nM.
Wherein, DNA sequence dna A sequence is：
5′-GGTGTGACGGATAATCTGGTTTAGCTACGCCTTCCCCGTGG CGATGTTTCTTAGCGCCTTAC- 3′。
DNA sequence dna B sequence is：
5′-ATCCGTCACACCTGCTCTGACGCTGGGGTCGACCCG-Biotin -3′。
Prepare PCR system solution：
By PCR primer 1, PCR primer 2,Premix ExWith ROX Reference Dye II (50 ×) reaction solution is mixed；Wherein, PCR primer 1, the concentration of PCR primer 2 are 10 μM.
Wherein, the sequence of PCR primer 1 is：
5′-GTAAGGCGCTAAGAAACATCG-3′。
The sequence of PCR primer 2 is：
5′-AATCTGGTTTAGCTACGCCTTC-3′。
Embodiment 2 makes standard curve
37 DEG C of 5h of the glutaraldehyde solutions of 50 μ L 0.8% processing PCR pipe；It is ultrapure washing three times after, add 0.01M carbonate The μ L of Streptavidin 50 of buffer solution are incubated 2h (37 DEG C)；
After phosphate buffer is washed twice, aptamers and complementary DNA 1:1 (v/v) is sufficiently mixed, 50 μ L mixture It is added in each pipe and is incubated 1h (37 DEG C)；
Hybridization buffer is washed three times, 50 μ l various concentrations 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；
Addition PCR system solution after Tris buffer solutions are washed three times, through quantitative PCR detection fluorescence signal, makes standard bent Line, made standard curve is shown in Fig. 2.
Aflatoxin M in the detection sample of embodiment 31Content
Weigh 0.5g and be detected sample, add 2.5mL 0.05ng mL-1Aflatoxin M1Standard liquid is fully mixed with it It is even, then add 2.5mL70% methanol.Mixture is vortexed concussion 5min after 10000g centrifugations 10min.Collect supernatant liquid nitrogen Blow and be concentrated into 0.5mL.The methanol of 2mL 5% is added to redissolve；Quantitative PCR detection fluorescence signal.
Quantitative PCR detection aflatoxin M1Amplification curve see Fig. 3, the standard curve measured according to embodiment 2, it is determined that Detect aflatoxin M in sample1Content be 0.045ng mL-1。
SEQUENCE LISTING
<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
<120>Detect aptamer, sensor, kit and the application of Aflatoxins M1
<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 (10)

1. one kind is used to detect aflatoxin M1Aptamer, it is characterised in that：Its nucleotides is SEQ ID No:1 institute Show.
2. one kind detection aflatoxin M1Aptamer sensor, it is characterised in that：The aptamer sensor by Four parts are constituted below：
(1) DNA sequence dna A：The DNA profiling being made up of following DNA sequence dna：5′-GGTGTGACGGATXaXb-3′；Wherein, Xa and Xb A and b any bases, a=b=20-30 are represented respectively；
(2) DNA sequence dna B：SEQ ID No:Aflatoxin M described in 11Aptamer, wherein, in 3 ' terminal modified biotins；
(3) PCR primer 1, the length of the PCR primer 1 is 20-30nt, and sequence has following feature：5 '-Xc-3 ', 5 ' ends 20-30 base and DNA sequence dna A Xb section complementary pairings；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 following feature：5 '-Xd-3 ', 5 ' ends 20-30 base it is identical with DNA sequence dna A Xa sections；Wherein, X represents any base, and a or d represent base quantity, d=a= 20-30。
3. according to the aptamer sensor described in claim 2, it is characterised in that：The nucleotide sequence of the DNA sequence dna A For SEQ ID No:Shown in 2.
4. according to the aptamer sensor described in claim 2, it is characterised in that：The nucleotide sequence of the PCR primer 1 For SEQ ID No:Shown in 3.
5. according to the aptamer sensor described in claim 2, it is characterised in that：The nucleotide sequence of the PCR primer 2 For SEQ ID No:Shown in 4.
6. one kind detection aflatoxin M1Kit, it is characterised in that：The kit includes following components：
(1) PCR pipe modification solution；
(2) sensor solution；
(3) PCR system solution；
(4) aflatoxin M1Extract solution；
The sensor solution includes following each component：
Aptamer sensor, PBST buffer solutions, hybridization buffer and Tris buffer solutions described in claim 2.
7. according to the kit described in claim 6, it is characterised in that：The PCR pipe modification solution includes following components：Penta 2 Aldehyde solution, carbonate buffer solution, Streptavidin；Wherein, the glutaraldehyde solution, the concentration of carbonate buffer solution distinguish preferred For 0.8%, 0.01M；The concentration of Streptavidin is preferably 2.5-5ng mL-1, more preferably 2.5ng mL-1；
DNA sequence dna A concentration is 1 × 10 in the sensor solution-3- 10nM, more preferably 10nM；The sensor is molten DNA sequence dna B concentration is 5-40nM, preferably 10nM in liquid；PCR primer 1 and PCR primer 2 is dense in the sensor solution Spend for 5-20 μM, preferably 10 μM.
Described PCR system solution includes following components：SYBR Green dyestuffs, Taq archaeal dna polymerases, PCR buffer solutions, dNTP And water；
The aflatoxin M1Extract solution is methanol solution, and the methanol solution is preferably 70% methanol solution.
8. kit detection aflatoxin M described in application claim 6 or 71Method, comprise the following steps：
(1) aflatoxin M will be contained1Testing sample and aflatoxin M1Extract solution is mixed, and be vortexed concussion, centrifuges and takes Supernatant；
(2) supernatant is mixed and is incubated with sensor solution；
(3) step (2) described mixed liquor is discarded, and is eluted with Tris buffer solutions；
(4) PCR system solution is added, through quantitative PCR detection fluorescence signal.
9. in accordance with the method for claim 8, it is characterised in that：Testing sample and aflatoxin M described in step (1)1Carry The weight ratio for taking liquid is 1:3-1:100；
The temperature being incubated described in step (2) is 45 DEG C, and incubation time is 1h.
10. the reagent described in the aptamer sensor or claim 6 of the aptamer of claim 1, claim 2 Box is in detection aflatoxin M1Application in content.
CN201710383877.7A 2016-05-26 2017-05-26 Detect aflatoxin M1Aptamer, sensor, kit and application Active CN107164384B (en)

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CN110095442A (en) * 2019-04-24 2019-08-06 中国科学院生态环境研究中心 A kind of method of sensitive aptamers fluorescence anisotropy assay aflatoxin B1
CN111999502A (en) * 2020-08-24 2020-11-27 湖南农业大学 Aflatoxin B1 detection kit and method for regulating multimode signal output based on PBNPs in-situ growth

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110095442A (en) * 2019-04-24 2019-08-06 中国科学院生态环境研究中心 A kind of method of sensitive aptamers fluorescence anisotropy assay aflatoxin B1
CN111999502A (en) * 2020-08-24 2020-11-27 湖南农业大学 Aflatoxin B1 detection kit and method for regulating multimode signal output based on PBNPs in-situ growth
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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