CN103575713A

CN103575713A - Method for detecting ochracin A based on fluorescence anisotropy of nucleic acid aptamer

Info

Publication number: CN103575713A
Application number: CN201310533285.0A
Authority: CN
Inventors: 赵强; 吕琴
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2013-11-01
Filing date: 2013-11-01
Publication date: 2014-02-12
Anticipated expiration: 2033-11-01
Also published as: CN103575713B

Abstract

The invention provides a method for detecting ochracin A based on the fluorescence anisotropy (fluorescence polarization) of a nucleic acid aptamer marked by a fluorescent dye tetramethylrhodamine (TMR for short). According to the method, the TMR is modified to a specific position of the aptamer of the ochracin A, the fluorescence anisotropy (fluorescence polarization) value of the TMR-marked nucleic acid aptamer is obviously changed due to the combination of the ochracin A, and the fluorescence anisotropy (fluorescence polarization) value is detected, so as to realize the quantitative detection of the ochracin A. The method has the advantages of simplicity and rapidness in operation, easiness in high throughput test, good specificity, high sensitivity and low cost.

Description

Based on aptamer fluorescence anisotropy, detect the method for Ochratoxin A

Technical field

The present invention relates to mycotoxin detection technique, specifically belong to a kind of detection technique of fluorescence that detects Ochratoxin A based on aptamer.

Background technology

Ochratoxin A, English name is Ochratoxin A, is a kind of impact mycotoxin widely, and some of Eurotium and Penicillium planted the secondary metabolism product producing, and toxigenic bacterium comprises Aspergillus ochraceus, penicillium verruculosum and carbon black aspergillus etc.Ochratoxin A can directly pollute crops and the numerous food such as cereal, feed, fruit, dry fruit, the plant food that humans and animals pollutes by absorption absorbs and enters in body, also can by taking in animal food, enter in human body because of Ochratoxin A cumulative effect in animal body simultaneously.Ochratoxin A has very strong Toxicity of Kidney, immunosupress toxicity, neurotoxicity, carcinogenicity, teratogenesis.Highly sensitive, high specific detection ochracin is having great importance aspect food security and Environmental Health.Detection method mainly comprises the methods such as thin-layer chromatography, high performance liquid chromatography, enzyme-linked immuno assay, LC-MS at present.These methods often need expensive instrument and complicated operation steps.The analytical approach of employing immune antiboidy need to be utilized the antibody of specific recognition Ochratoxin A, and the preparation of antibody often cost is high, preparation poor reproducibility, and good stability, the conditional request of antibody storage and transport is high.

Aptamer (Aptamer) be the class that goes out by SELEX technology screening can with the oligonucleotide fragment of target molecule specific binding, target molecule is had to high affinity and narrow spectrum recognition capability (Ellington, A.D.and Szostak, J.W.Nature1990,346,818 – 822.Tuerk, C.and Gold, L.Science1990,249,505 – 510.).Aptamer, as affinity ligand, is compared with immune antiboidy, has significant advantage: have high affinity and high specific; Screen easy to prepare; Stability is high; Be easy to chemical modification.Therefore aptamer exhibits a variety of advantages in analyzing and testing and sensory field, can overcome some limitations in stability and preparation etc. of conventional immune antiboidy, demonstrates application prospect (Liu, J.; Cao, Z.; Lu, Y.Chem.Rev.2009,109,1948 – 1998; Cho, E.J.; Lee, J.-W.; Ellington, A.D.Annu.Rev.Anal.Chem.2009,2,241 – 264.).Utilizing of the having reported method that aptamer detects Ochratoxin A is more complicated often, needs to adopt reasonably design with separated etc., as (Yang, X.H. such as electrochemical sensing, affinity chromatographies; Kong, W.J.; Yang, M.H.; Zhao, M.; Ouyang, Z.Chin.J.Anal.Chem.2013,41,297 306; Hayat, A.; Paniel, N.; Rhouati, A.; Marty, J.-L.; Barthelmebs, L.Food Control2012,26,401 – 415; Chapuis-Hugon, F.; Du Boisbaudry, A.; Madru, B.; Pichon, V.Anal.Bioanal.Chem.2011,400,1199 – 1207.).

The present invention adopts the aptamer of fluorescent dye tetramethyl rhodamine (tetramethylrhodamine is called for short TMR) mark to set up the detection technique that a kind of fluorescence anisotropy simply and easily (fluorescence polarization) method detects Ochratoxin A.

Summary of the invention

The object of this invention is to provide a kind of method and corresponding reagent that detects Ochratoxin A based on aptamer fluorescence anisotropy, that the method has advantages of is simple to operate, quick, specificity is good, highly sensitive.

The present invention utilizes Ochratoxin A and tetramethyl rhodamine (tetramethylrhodamine, the selective binding of the aptamer of abbreviation TMR) modifying, the fluorescence anisotropy (fluorescence polarization) that causes the aptamer that TMR modifies changes, by measuring the variation of fluorescence anisotropy (fluorescence polarization), realize the quantitative detection to Ochratoxin A.(see figure 1).

A kind of fluorescent dye tetramethyl rhodamine (tetramethylrhodamine provided by the invention, abbreviation TMR) aptamer of mark, the nucleotides sequence of its aptamer is classified SEQ ID NO:1(5 '-GAT CGG GTG TGG GTG GCGTAA AGG GAG CAT CGG ACA-3 ' as); TMR mark is held in the 10th the T base starting to 5 ' of nucleotide sequence SEQ ID NO:1, or in the 8th T base, or in the 19th T base.

A kind of method that detects Ochratoxin A based on aptamer fluorescence anisotropy provided by the invention, step comprises: by the aptamer of TMR mark, in binding buffer solution, mix with Ochratoxin A, incubation, the fluorescence anisotropy of measuring the aptamer of TMR mark, excitation wavelength is 560 nanometers, emission wavelength is 578 nanometers, according to the variation of the fluorescence anisotropy of aptamer, can try to achieve the concentration of Ochratoxin A in sample.

Described binding buffer solution is: 10mM Tris-HCl, 120mM NaCl, 20mM CaCl ₂, 0.1%Tween20, pH8.5.

Compared with prior art the present invention has following advantage and effect:

The present invention for the specific recognition to Ochratoxin A, makes full use of the high stability of aptamer, easily preparation by the aptamer of fluorochrome label, easily introduces the advantage of the aspects such as functional group.That fluorescence anisotropy (fluorescence polarization) detection technique has is simple to operate, sensitive, favorable reproducibility, without separated, easily realize the advantages such as high throughput analysis.The present invention only need to adopt a kind of aptamers and corresponding binding buffer solution of fluorochrome label, and whole testing process is simple, easily operation, and do not require complicated instrument and equipment.The present invention adopts aptamer can overcome and use immune antiboidy in the limitation of the aspects such as preparation and stability as identification agent.Be convenient to fast processing batch samples, be easy to commercialization and practical application, there is the advantage of high flux, automated analysis.

Accompanying drawing explanation:

Fig. 1 adopts the aptamer (T10-TMR-O36) of TMR mark to detect the Ochratoxin A of variable concentrations

Fig. 2 adopts the aptamer (T10-TMR-O36) of TMR mark to detect the selectivity of Ochratoxin A.

Fig. 3 adopts the aptamer (T10-TMR-O36) of TMR mark to detect the Ochratoxin A of variable concentrations in the grape wine that adopts 50 times of dilutions of buffer solution

Embodiment:

Embodiment 1: the Ochratoxin A (Fig. 1) that adopts the aptamer detection variable concentrations of mark TMR in the 10th T base.

The aptamer of TMR mark has following sequence: 5 '-GAT CGG GTG TGG GTG GCG TAA AGGGAG CAT CGG ACA-3, and TMR is tagged in the 10th T base, and the aptamer of corresponding TMR mark is referred to as T10-TMR-O36.

At buffer solution (10mM Tris-HCl, 120mM NaCl, 20mM CaCl ₂, 0.1%Tween20, pH8.5) in by T10-TMR-O36(concentration, be 27nM) mix with the Ochratoxin A of variable concentrations, at room temperature incubation is 40 minutes, then measure the fluorescence anisotropy of T10-TMR-O36, excitation wavelength 560 nanometers, wavelength of transmitted light 578 nanometers.Along with the increase of the concentration of Ochratoxin A (Ochratoxin A), the fluorescence anisotropy value r of T10-TMR-O36 reduces gradually, detects and is limited to 3nM, and sensing range 3nM, to 3 μ M, detects relative standard deviation and is less than 3%.

Embodiment 2: the selectivity (Fig. 2) of investigating aptamer (T10-TMR-O36) the detection Ochratoxin A based on TMR mark.

(10mM Tris-HCl, 120mM NaCl, 20mM CaCl in buffer solution ₂, 0.1%Tween20, pH8.5) by T10-TMR-O36(concentration, be 27nM) mix from different determinands, under room temperature, incubation is 40 minutes, measures the fluorescence anisotropy changes delta r of T10-TMR-O36.Only have Ochratoxin A (620nM) to cause the remarkable reduction of the fluorescence anisotropy value of T10-TMR-O36, other measured matter all can not cause the marked change of the fluorescence anisotropy value of T10-TMR-O36.In Fig. 2, (a) be not for containing the buffer solution of Ochratoxin A, (b) be 620nM Ochratoxin A, by (c) to (j) measured matter, be respectively 10 μ M N-acetyl-L-phenylalanine(NAP) (c), 7 μ M warfarin (d), 9 μ M7-amino-4-methylcoumarin(AMC) (e), 62 μ M arginine (f), 62 μ M phenylalanine (g), 62 μ M aspartic acid (h), 62 μ M serine (i), 62 μ M tyrosine (j).

Embodiment 3: adopt the aptamer (T10-TMR-O36) of TMR mark to detect Ochratoxin A (Fig. 3) in the claret of dilution.

Claret is adopted to buffer solution (10mM Tris-HCl, 120mM NaCl, 20mM CaCl2,0.1%Tween20, pH8.5) dilute 50 times, in the claret solution of dilution, by T10-TMR-O36(concentration, be then 27nM) mix with the Ochratoxin A of variable concentrations, at room temperature incubation is 40 minutes, then measure the fluorescence anisotropy of T10-TMR-O36, excitation wavelength 560 nanometers, wavelength of transmitted light 578 nanometers.Along with the increase of the concentration of Ochratoxin A (Ochratoxin A), the fluorescence anisotropy value r of T10-TMR-O36 reduces gradually.

Claims

1. an aptamer for fluorescent dye tetramethyl rhodamine mark, is characterized in that, the nucleotides sequence of described aptamer is classified SEQ ID NO:1 as; Fluorescent dye tetramethyl rhodamine mark is held in the 10th the T base starting to 5 ' of nucleotide sequence SEQ ID NO:1, or in the 8th T base, or in the 19th T base.

2. a method that detects Ochratoxin A based on aptamer fluorescence anisotropy, step comprises: by the aptamer of fluorescent dye tetramethyl rhodamine mark claimed in claim 1, in binding buffer solution, mix with Ochratoxin A, incubation, measure the fluorescence anisotropy of the aptamer of fluorescent dye tetramethyl rhodamine mark, excitation wavelength is 560 nanometers, emission wavelength is 578 nanometers, according to the variation of the fluorescence anisotropy of aptamer, try to achieve the concentration of Ochratoxin A in sample.

3. a kind of method that detects Ochratoxin A based on aptamer fluorescence anisotropy as claimed in claim 2, described binding buffer solution is: 10mM Tris-HCl, 120mM NaCl, 20mM CaCl ₂, 0.1%Tween20, pH8.5.