CN106526168B - A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant and its application - Google Patents

A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant and its application Download PDF

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CN106526168B
CN106526168B CN201610989401.3A CN201610989401A CN106526168B CN 106526168 B CN106526168 B CN 106526168B CN 201610989401 A CN201610989401 A CN 201610989401A CN 106526168 B CN106526168 B CN 106526168B
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doxycycline
remnant
aptamer
reagent box
quick detection
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CN106526168A (en
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乐涛
张磊
孙琦
张志浩
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Chongqing Normal University
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    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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    • G01—MEASURING; TESTING
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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    • G01N2446/20—Magnetic particle immunoreagent carriers the magnetic material being present in the particle core

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Abstract

The nucleotide aptamers probe reagent box of quick detection doxycycline remnant provided by the invention, for the Doxycycline aptamer nucleic acid probe using nanoscale magnetic bead as kernel, outer surface is bonded with Doxycycline aptamer chain；Wherein, Doxycycline aptamer chain（Doxycycline aptamer artificial sequence）For：5’‑GTA CGG AAT TCG CTA GCC GGG CGG CAG GCC ACG GCT GGG GTT GGT CCC ACT GCG GAT CCG AGC TCC ACG TG‑3’；The kit aptamers probe is the novel recognition component of Doxycycline, have have good stability, high sensitivity, it is at low cost, it is easy prepare, easily modify and label high specific advantage.

Description

A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant and its Using
Technical field
The present invention relates to Doxycycline specific recognition DNA aptamers screening technique and its application, and in particular to it is raw Object technology and food antibiotic detection field.
Background technology
Doxycycline (Doxycycline) is tetracycline antibiotics (Tetracyclines), dynamic commonly used to treatment Object infectious disease, is also act as feed addictive, to prevent disease and promote growth of animal.In actual operation, due to arbitrarily adding Big dosage and not in accordance with the off-drug period, causes drug to be remained in animal tissue.Tetracycline medication extended residual is in animal In tissue, drug-fast bacteria is caused to be propagated between the mankind, seriously damages health.Many countries and international organization are dynamic by establishing Effective detection method of physical property food veterinary drug residue, to control medicament residue problem.Such as, China and European Union to Dox in animal Maximum residue limit (MRL) is remained in tissue to make explicit provisions：The MRL of muscle, liver and kidney be respectively 100 μ g/kg, 300 μ g/kg and 600 μ g/kg.In order to control residue of veterinary drug, human health is ensured, it is necessary to establish a kind of fast and effectively residual It stays detection method to remain it to be monitored.The method for the detection Doxycycline (OTC) reported recently includes mainly efficient liquid phase Chromatography, fluorescence method, mass spectrography and other methods etc..But these methods usually relatively time-consuming and price it is relatively expensive, Also there is higher requirement to experimenter, it is higher to the pre-processing requirements of sample and be not easy to promote.Immunoassay is using anti- Original antibody specifically bind the various substances of reaction detection, food antibiotics leftover detection field it is relatively broad apply.Enzyme Linked immunosorbent assay (ELISA), which reacts the efficient catalytic of the immune response of antigen and antibody and enzyme, to be organically combined, and is effectively protected It is highly selective and highly sensitive in card analysis, report that most is immunoassay method.It is American-European at present to have the life of some companies Production is directed to the ELISA kit product of antibiotics leftover detection, but expensive, and the type of enzyme labelled antibody is limited, Zhi Nengjian Survey the common antibiotic residue of a few species.Since Doxycycline is micromolecular compound, itself do not have immunogenicity, need by It as the antigenic determinant stimulation animal of carrier could generate specific antibody after being connect with large biological molecule such as protein, raw There are more limitations with application for production, including：Antigen coupling synthesis, the screening of high-titer antibody；Result from the animals such as mouse, rabbit Heterologous antibody is possible to generate nonspecific reaction in use；The manufacturing cost of antibody is high, time-consuming and laborious, monoclonal cell strain It is not easy to maintain；Detection performance is unstable between being criticized in batch.These all greatly limit immunoassay method in how western ring Application in element detection.
Aptamer is a kind of single stranded oligonucleotide filtered out through external artificial synthesized developed in recent years, Can efficiently, specifically to combine various biological target molecules, the appearance of aptamer be chemical-biological educational circles and biomedicine Boundary provides a kind of new research platform.Aptamer is good with self stability, it is relatively easy, quick, easy to prepare synthesis The advantages that acquisition, easy functional modification are with label, therefore, using flexibly extensive in biosensor design.In recent years, it was based on The biosensor research of aptamer is greatly paid close attention to by people.
It is existing at present to utilize the report that nucleic acid aptamer is the antibiotic detection in food of identification molecular application, detection method Mostly based on electrochemica biological sensor.The present invention is directed to traditional large-scale instrument, and to can not achieve field quick detection, pre-treatment numerous The shortcomings that trivial and immunoassay method needs to prepare antibody establishes the fluoroscopic examination based on Beads enrichment-aptamers identification Method can be used for quick, the highly sensitive detection of doxycycline remnant in food.
Invention content
The present invention screens the single stranded DNA aptamers sequence that can be combined with Doxycycline high-affinity, establishes and is based on The fluorescence detection method of Beads enrichment-aptamers identification, can be used for quick, the Gao Ling of doxycycline remnant in animal derived food Sensitivity detects.
The object of the invention：It is to provide a kind of construction method for the aptamers measuring Doxycycline, in view of Doxycycline point The design feature of son, the method for devising the preferred Doxycycline of magnetic bead SELEX technologies of improvement, by the combination based on SELEX Chemical technology, screening obtain to specifically bind the DNA aptamers of Doxycycline.Aptamers are passed through into biotin-avidin system System is fixed on nanometer magnetic bead surface, and the aptamers complementary strand thereof with FAM labels detects under different excitations and launch wavelength Fluorescent marker on complementary strand.When target molecule is added, aptamer is specifically bound with target molecule, causes to be complementary to miscellaneous The dissociation of the fluorescent label DNA short chain of friendship, causes the variation of fluorescence intensity to carry out Doxycycline in quantitative analysis sample.
The nucleotide aptamers probe reagent box of quick detection doxycycline remnant provided by the invention, including following reagent For Doxycycline aptamer nucleic acid probe, PBS buffer solution, dissociation solution；It is characterized in that：The Doxycycline nucleic acid adaptation For body nucleic acid probe using nanoscale magnetic bead as kernel, outer surface is bonded with Doxycycline aptamer chain；Wherein, Doxycycline nucleic acid Aptamers chain (Doxycycline aptamer artificial sequence) is：5’-GTA CGG AAT TCG CTA GCC GGG CGG CAG GCC ACG GCT GGG GTT GGT CCC ACT GCG GAT CCG AGC TCC ACG TG-3 ' are referring specifically to attachment：《Core Nucleotide sequence table》.
The dissociation solution is 8g NaCl, 0.37g KCl, 0.135g Na2HPO4.2H2O, 1g glucose in 900mL water, 5g HEPES, dissolving, NaOH tune PH to 7.05 are settled to 1000ml.
The preparation method of the aptamers nucleic acid probe is：Fe is prepared using chemical coprecipitation3O4Nano particle, then Amidized nano material and Avidin are coupled using glutaraldehyde method；Doxycycline aptamers are modified in nanometer magnetic bead table Face, the hybridize chain complementary with Doxycycline aptamers are marked fluorophor at 5 ' ends, pass through complementary, the composition with aptamers respectively The aptamers nucleic acid probe of fluorescent marker.
The nucleotide aptamers probe reagent box of the quick detection doxycycline remnant is more in detecting poultry meat Application in western ring element residual.
Magnetic nano-particle used in the present invention and aptamers are Chongqing biological medicine and instrument research center laboratory Synthesis.
Wherein, above-mentioned probe is by the modification of Doxycycline aptamers in nanometer magnetic bead surface, the adaptation of difference fluorescent marker Body complementary strand thereof, after target molecule is added, aptamer is specifically bound with target molecule, causes to be complementary to the glimmering of hybridization The dissociation of signal DNA short chains causes fluorescence intensity change, establishing criteria curve to acquire the content of Doxycycline in sample.
Above-mentioned nanometer magnetic bead is to prepare Fe using chemical coprecipitation3O4Then nano particle utilizes glutaraldehyde method by ammonia The nano material of base is coupled with Avidin.
Biotin has been modified at the 5 ' ends of the Doxycycline aptamer apt 1, passes through biotin-avidin system System, while being fixed on the nanometer magnetic bead surface of Avidin modification.
Fluorophor is marked at 5 ' ends in the chain that hybridizes complementary with Doxycycline aptamers respectively, by mutual with aptamers It mends, constitutes fluorescent marker nucleic acid probe.
A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant, including following reagent：Above-mentioned institute The Doxycycline aptamer nucleic acid probe stated, PBS buffer solution, dissociation solution.
The nucleotide aptamers probe reagent box of quick detection doxycycline remnant described above, the dissociation solution are In 900mL water, 8g NaCl, 0.37g KCl, 0.135g Na2HPO4.2H2O, 1g glucose, 5g HEPES, dissolving, NaOH tune PH to 7.05, is settled to 1000ml.
The preparation method of the nucleotide aptamers probe reagent box of quick detection doxycycline remnant described above, uses Chemical coprecipitation prepares Fe3O4Then nano particle utilizes glutaraldehyde method to be coupled amidized nano material and Avidin. By the modification of Doxycycline aptamers in nanometer magnetic bead surface, the hybridize chain complementary with Doxycycline aptamers marks respectively at 5 ' ends Fluorophor, by with aptamers complementation, constitute the aptamers nucleic acid probe of fluorescent marker.
Specially：1) 1, the 6- hexamethylene diamines of 13g are added in 60mL ethylene glycol, 4.0g anhydrous sodium acetates and 2.0g six are hydrated Ferric trichloride (FeCl36H2O), 50 DEG C of stirrings, obtains colloidal solution, transfers the solution into 100mL with polytetrafluoroethyllining lining In reaction kettle, 198 DEG C of reaction 6h；Room temperature cools down, and abandons supernatant liquid, and deionized water goes out lower layer's solid matter, and Magneto separate is collected； It is washed 2 times with deionized water and ethyl alcohol respectively, 50 DEG C of dry 5-10h；
2) it quantitatively weighs nano material to be suspended in 10mM PBS, ultrasonic disperse 15min, glutaraldehyde solution is added, makes it Final concentration of volume ratio 5%；Then shaken at room temperature 2h, Magneto separate abandon supernatant, and 10mM PBS washings three times, remove extra penta Dialdehyde；Then 10mM PBS are added, a certain amount of Avidin, shaken at room temperature 12h is added in ultrasonic disperse, and Magneto separate abandons supernatant, 10mM PBS cleanings are three times；
3) nano material that Avidin is modified is suspended in ultrasonic disperse in 10mM PBS, it is suitable that certain density nucleic acid is added Ligand solution, shaken at room temperature 4h, Magneto separate abandon supernatant, and 10mM PBS cleanings are resuspended in three times to remove unbonded aptamers In PBS buffer solution.
Kit described above is in the application in detecting doxycycline remnant；After target molecule is added, nucleic acid is suitable Ligand is specifically bound with target molecule, leads to the dissociation for being complementary to the fluorescent label DNA short chain of hybridization, fluorescence intensity is caused to become Change, establishing criteria curve acquires the content of Doxycycline in sample.It is residual that this method can detect Doxycycline in animal derived food It stays.
The method have the benefit that：The kit aptamers probe is the novel recognition component of Doxycycline, And have good stability, high sensitivity, it is at low cost, it is easy prepare, easily modify and label high specific advantage.
Specific implementation mode
Embodiment 1 quickly detects the preparation of the nucleotide aptamers probe reagent box of doxycycline remnant
1. the preparation of amination nanometer magnetic bead
1, the 6- hexamethylene diamines of 13g, six water of 4.0g anhydrous sodium acetates (CH3COONa) and 2.0g are added in 60mL ethylene glycol Ferric trichloride (FeCl36H2O) is closed, 50 DEG C of stirrings obtain colloidal solution, transfer the solution into 100mL band polytetrafluoroethyllining linings Reaction kettle in, 198 DEG C reaction 6h.Room temperature cools down, and abandons supernatant liquid, and deionized water goes out lower layer's solid matter, and Magneto separate is received Collection.It is washed 2 times with deionized water and ethyl alcohol respectively, 50 DEG C of dry 5-10h.
2. Avidin is coupled amidized nanometer magnetic bead
It quantitatively weighs nano material to be suspended in PBS (10mM), ultrasonic disperse 15min, glutaraldehyde solution is added, makes its end A concentration of 5% (v/v).Then shaken at room temperature 2h, Magneto separate abandon supernatant, and 10mM PBS washings three times, remove extra penta 2 Aldehyde.Then the PBS of 10mM is added, a certain amount of Avidin, shaken at room temperature 12h is added in ultrasonic disperse, and Magneto separate abandons supernatant, 10mM PBS cleanings are three times.Suction before and after surveying Avidin combination magnetic nanoparticle at 280nm using ultraviolet specrophotometer Light value.
3. the nanometer magnetic bead of the aptamers connection Avidin modification of biotin modification
The nano material that Avidin is modified is suspended in ultrasonic disperse in 10mM PBS, certain density nucleic acid adaptation is added Body (middle Doxycycline aptamer artificial sequence described in nucleotides sequence list) solution, shaken at room temperature 4h, Magneto separate are abandoned Clearly, 10mM PBS cleanings are resuspended in three times to remove unbonded aptamers in PBS buffer solution.
4. aptamers and complementary strand thereof
The complementary strand that certain density FAM is marked is added in above-mentioned suspension, and 37 DEG C are protected from light incubation 1h, and Magneto separate is abandoned Supernatant, PBS washings to remove free complementary strand, are resuspended in PBS buffer solution three times, using sepectrophotofluorometer, respectively at Under 495nm, 588nm excitation wavelength and 520nm, 608nm launch wavelength, its fluorescent value is surveyed.
5. sample-adding and detection
The nano material compound of step 3 is dissolved in dissociation solution (in 900mL water, 8g NaCl, 0.37g KCl, 0.135g Na2HPO4.2H2O, 1g glucose, 5g HEPES, dissolving, NaOH tune PH to 7.05 are settled to 1000mL), it is added Sample to be tested or mark product solution, 45 DEG C of incubations are protected from light 40-60min, and Magneto separate abandons supernatant, and PBS washings remove dissociation three times Under complementary strand, be resuspended in PBS, fluorescent value detected under similarity condition.
6. the drafting of standard curve
Doxycycline standard items are detected by aforesaid operations step, establish standard curve.Standard concentration is followed successively by 0,2.5,5,10,20,40,80 μ g/L, are placed in quartz colorimetric utensil, long in Ex495nm excitation wavelengths and hair Em520nm ejected waves Under, each experiment is repeated 5 times, and surveys its fluorescent value.Standard concentration is that fluorescent value corresponding to 0ng/mL is B0, various concentration standard The corresponding fluorescent value of product is B, the average value divided by B of the standard items fluorescent value obtained0The light absorption value (inhibiting rate) of standard.With not It is abscissa with standard concentration, with inhibiting rate B/B0It maps for ordinate, draws standard curve.
7. eating medicine prison sample to measure
Any healthy animal tissue without antibacterials is chosen, the blank tissue after screening is shredded, refiner is used 4000r/min homogeneous 5min weigh tissue homogeneous object 1.0g in 50mL centrifuge tubes, and 3% trichloroacetic acid (meat 4mL, liver is added 9mL), vortex mixing overturns oscillation 20min, room temperature 10000r/min and centrifuges 10min.200 μ L of supernatant are taken to be centrifuged in 1.5mL 20 μ L of 1mol/L sodium hydroxide solutions are added in Guan Zhong, and sample buffer (180 μ L of meat, 380 μ L of liver) mixing is added in vortex mixing, 5min is centrifuged with 10000r/min, supernatant is as sample solution.Muscle dilution gfactor is 10, and liver dilution gfactor is 30.It is accurate The appropriate standard items storing solution of Doxycycline is really measured, Doxycycline standard items storing solution is dissolved in PBS, pig muscle, pork liver 3 respectively In kind matrix, final concentration of 0,2.5,5,10,20,40,80 μ g/L draw curve according to the operating method of step 6, are tested As a result comparative analysis.
Embodiment 2 quickly detects the application of the nucleotide aptamers probe reagent box of doxycycline remnant
1. the foundation of standard curve
Method in application invention content measures Doxycycline standard items, establishes standard curve.Doxycycline is respectively configured A concentration of 0,0,2.5,5,10,20,40,80 μ g/L of series standard, with a concentration of abscissa of various criterion product, with inhibiting rate B/ B0For ordinate, standard curve is drawn, regression equation is established.Standard curve is in typical S types, Regression Equations y=- 43.76x+94.95 R2=0.99, IC50For 10.7 μ g/L, lowest detection line is 1.79 μ g/L, detection method standard curve Detection range is 1.8~80 μ g/L.
2. tissue sample matrix effect is tested
Doxycycline standard working solution is used into 3 kinds of samples such as PBS, pig liver sample treatment liquid, musculature treatment fluid respectively Product matrix carries out series concentration dilution, under optimum reaction condition, effect experiment numerical value of the measured standard items in each matrix. This method tests a series of concentration Doxycyclines in matrix sample, and acquired results draw standard curve, linear relationship Be all higher than 98%, gained matrix PBS, pig muscle, liver IC50Respectively 11.7 μ g/L, 14.5 μ g/kg, 16.3 μ g/kg.Knot Although fruit illustrates that bio-matrix difference has certain influence to the light absorption value of standard items, its IC50Variation is little, illustrates different samples Treatment fluid influences less the sensibility of method and testing result, and the processing method of sample is feasible.
3. detection method minimum detection limit in the tissue
With the detection method, standard curve is made.The fluorescent value of 20 parts of blank tissues (pig muscle and liver) is measured respectively, Inhibiting rate calculating is carried out, regression equation, computation organization's sample detection line, by testing Doxycycline in pig muscle and pork liver are substituted into Detection line in dirty sample is respectively 1.6 μ g/kg, 1.7 μ g/kg.
4. organizing TIANZHU XINGNAO Capsul
Animal tissue is tested, the rate of recovery of liver sample is between 83.5%~98.6%, and average 91.2%；Muscle samples The rate of recovery is between 84.2%~98.7%, and average 93.7%.The average variation within batch coefficient of pig liver and Swine muscle sample Respectively 6.1%, 5.3%；The average interassay coefficient of variation of pig liver and Swine muscle sample is respectively 6.8%, 8.2%.It is flat It criticizes interior interassay coefficient of variation and is respectively less than 9%.It is seen by the result of batch interior interassay coefficient of variation and the rate of recovery, the experimental results showed that should The repeatability of method, accuracy, accuracy are relatively good, can meet food medicine prison on-site verification needs.
SEQUENCE LISTING
<110>Chongqing Normal University
<120>A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant and its application
<130> 2016
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 71
<212> DNA
<213>Doxycycline aptamer artificial sequence
<400> 1
gtacggaatt cgctagccgg gcggcaggcc acggctgggg ttggtcccac tgcggatccg 60
agctccacgt g 71

Claims (3)

1. a kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant, including following reagent：Doxycycline Aptamer nucleic acid probe, PBS buffer solution and dissociation solution；It is characterized in that：The Doxycycline aptamer nucleic acid is visited For needle using nanoscale magnetic bead as kernel, outer surface is bonded with Doxycycline aptamer chain；Wherein, Doxycycline aptamer chain Nucleotides sequence be classified as：5’-GTA CGG AAT TCG CTA GCC GGG CGG CAG GCC ACG GCT GGG GTT GGT CCC ACT GCG GAT CCG AGC TCC ACG TG-3’。
2. the nucleotide aptamers probe reagent box of quick detection doxycycline remnant according to claim 1, feature It is：The dissociation solution is 8g NaCl, 0.37g KCl, 0.135g Na in 900mL water2HPO4.2H2O, 1g glucose, 5g HEPES, dissolving, NaOH tune PH to 7.05 are settled to 1000ml.
3. the nucleotide aptamers probe reagent box of quick detection doxycycline remnant as claimed in claim 1 or 2 is in detection man Application in meat of poultris in doxycycline remnant.
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