CN107179404B - A kind of furans metabolite derivatization reagent and its rapid detection card - Google Patents
A kind of furans metabolite derivatization reagent and its rapid detection card Download PDFInfo
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- CN107179404B CN107179404B CN201710406572.3A CN201710406572A CN107179404B CN 107179404 B CN107179404 B CN 107179404B CN 201710406572 A CN201710406572 A CN 201710406572A CN 107179404 B CN107179404 B CN 107179404B
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- furans
- metabolite
- nucleic acid
- acid sequence
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- G—PHYSICS
- 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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- 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
- G01N33/54326—Magnetic particles
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Abstract
The invention discloses a kind of furans metabolite derivatization reagent and its rapid detection cards.Derivatization reagent of the present invention is the compound containing aldehyde radical, and one end modifying and decorating has one section of nucleic acid sequence；The aldehyde radical can be combined with furans metabolite, form furans metabolite-derivatization reagent-nucleic acid sequence compound；There are a nicking enzyme site and a primer binding site in the nucleic acid sequence, a large amount of complementary single strand nucleic acid sequence can be generated under the action of archaeal dna polymerase and nicking enzyme.Furans metabolite derivatization reagent of the present invention can realize nitrofurans metabolin in on-site test complex system, have the advantages that quickly, it is easy, at low cost, detection process is not necessarily to large scale equipment, and testing result can pass through naked eye interpretation.Furans metabolite derivatization reagent of the present invention and its rapid detection card can be used for the fields such as food safety, have a good application prospect.
Description
Technical field
The invention belongs to small molecule rapid detection technical fields, more specifically to a kind of nitrofurans metabolin
The quick quick detection of height used in derivatization reagent and its rapid detection card, with for the scene such as food, water quality to furans medicine
The quick identification of object.
Background technique
Nitrofurans are a kind of efficient broad-spectrum antibiotics, have effect to Gram-positive and negative bacteria, wide
General therapeutic agent and the feed addictive of being used as is for controlling disease in fowl, poultry, aquaculture or epidemic situation, the drug include: furans
XiLin, four class of furazolidone, furaltadone and furantoin.Since nitrofurans are there are certain carcinogenicity, European Union, Australia are big
Leah, the U.S. and China have put into effect relevant law, use of the limitation nitrofurans in aquaculture in succession.
Nitrofurans are easily degraded in animal body as smaller molecule, such small molecule metabolites easily with protein knot
Close, and combine product stabilization be not easily decomposed, can retain in animal body several weeks, common foods cooking method for example boiling, frying,
Baking and microwave heating etc. can not make metabolin degradable.Therefore it is former to be often used as detection nitrofurans for the metabolite
The marker of medicine.
Liquid-liquid extraction high performance liquid chromatography (HPLC) method is mainly used to the method for detection furans drug metabolite at present, is consolidated
Phase extracting efficient liquid-phase chromatograph method, high performance liquid chromatography tandem mass spectrum (LC-MS-MS) method, enzyme-linked immunization, capillary chromatography point
Analysis method etc..The main deficiency of these methods is to require large-scale instrument, and derivative abstraction purification process is complicated, and detection time is long
Deng.And based on there are derivative extractions, recycling effect if immunologic detection method such as enzyme linked immunological and quick colloidal-gold detecting-card
The problems such as rate is low, organic extraction reagent destroys antibody activity, stability and precision to detection will cause larger impact, easily produce
Raw biggish difference between batch, reduces product stability.Therefore, there is an urgent need in the art to develop a kind of Gao Min, quickly, it is easy, stablize
Detection method.
Summary of the invention
It is an object of the invention to: solve the problems, such as the detection of existing nitrofurans, a kind of Gao Min, fast is provided
Speed, easy, stable furans metabolite derivatization reagent and its rapid detection card.
In order to achieve the above-mentioned object of the invention, the present invention provides a kind of furans metabolite derivatization reagent, the derivatization
Reagent is the compound containing aldehyde radical, and one end modifying and decorating has one section of nucleic acid sequence；The aldehyde radical can be metabolized with furans
Object combines, and forms furans metabolite-derivatization reagent-nucleic acid sequence compound；There is one to cut in the nucleic acid sequence
Enzyme site and a primer binding site are carved, a large amount of complementary single strand core can be generated under the action of archaeal dna polymerase and nicking enzyme
Acid sequence.
As a kind of improvement of furans metabolite derivatization reagent of the present invention, the furans metabolite-derivatization examination
Agent-nucleic acid sequence compound can resist with the specific recognition furans metabolite-derivatization reagent for being marked on magnetic bead surfaces
Body combines, and is enriched with and cleans through Magnetic Isolation, and it is compound to form magnetic bead-furans metabolite-derivatization reagent-nucleic acid sequence
Object.
As a kind of improvement of furans metabolite derivatization reagent of the present invention, the primer binding site is located at the core
3 ' ends of acid sequence, nicking enzyme site is adjacent with the primer binding site, and the length of the nucleic acid sequence is 30~200
A base.
As a kind of improvement of furans metabolite derivatization reagent of the present invention, the length of the nucleic acid sequence is 40~60
A base.Furans metabolite derivatization reagent of the present invention can be used in nitrofurans metabolism analyte detection.
In order to achieve the above-mentioned object of the invention, the present invention also provides a kind of rapid detection card of nitrofurans metabolin, by
Bottom plate, sample pad, nitrocellulose filter and blotting paper assemble；
The bottom plate has viscosity, and sample pad, nitrocellulose filter and blotting paper are pasted on bottom plate；
The sample pad is the glass fibre after the buffer containing pH7~8 is handled and dried, and in the middle part of the sample pad
It is provided with loading hole；The buffer includes phosphate buffer, borate buffer, HEPES buffer solution or Tris buffer；
Have that 2~3mm's is overlapping between the sample pad and the nitrocellulose filter；
It is secured on the nitrocellulose filter with the nucleic acid sequence of product single strand nucleotide sequence partial complementarity as detection
Line also secures the nucleic acid sequence with the nucleic acid array complementation of colloid gold label as nature controlling line；
The blotting paper is pasted onto the other end of the nitrocellulose filter, and has the overlapping of 2~3mm between the two.
A kind of improvement of rapid detection card as nitrofurans metabolin of the present invention, the nucleotide of the partial complementarity
Number is 8~30.
A kind of improvement of rapid detection card as nitrofurans metabolin of the present invention, the rapid detection card also can be used
In the concentration of detection nitrofurans metabolin.
In order to achieve the above-mentioned object of the invention, the present invention also provides a kind of detection method of nitrofurans metabolin, packets
Include following steps:
(1) magnetic bead solution is prepared；
(2) sample to be tested is mixed with furans metabolite derivatization reagent, adds magnetic bead solution obtained by step (1) simultaneously
Then mixing separates magnetic bead and cleans resuspension, obtains suspension；
(3) suspension obtained by step (2) is added in equal volume in isothermal strand displacement reaction system and is reacted, the SDA is anti-
The system is answered to include:
Buffer2 (New England biolabEB) 20 μ L, archaeal dna polymerase 5U, nicking enzyme 5U, dNTP 5mM, primer 1
μM, 50 μ L of glycerol adds water to 100 μ L；
(4) reaction product obtained by step (3) is added dropwise to the loading of the rapid detection card of the nitrofurans metabolin
It is detected in hole, when the detection line in the rapid detection card of nitrofurans metabolin takes on a red color, prompts to deposit in sample to be tested
In nitrofurans metabolin；When the detection line in the rapid detection card of nitrofurans metabolin does not take on a red color, and nature controlling line
When taking on a red color, prompt that nitrofurans metabolin is not present in sample to be tested.
It is understood that thinking and method of the invention is not limited to the detection of nitrofurans metabolin, also can be used
In the molecule for detecting other types, as long as derivatization reagent, which is changed into, to fit with the substance of target specific binding, such as antibody
Ligand, receptor etc., while target nucleic acids being marked to meet mentality of designing of the invention.
Equally possible understanding, nucleic acid of the present invention also could alternatively be fluorophor, passes through detection after enrichment
Fluorescence intensity to judge indirectly the concentration of target molecules；The nucleic acid can also be one section of arbitrarily sequence, can lead to after being enriched with
Quantitative fluorescent PCR is crossed to detect its content, determines the concentration of target molecules indirectly；In addition, magnetic bead used in the present invention can also
Think the nano particle that colloid of iron oxide particle, colloid gold particle, granules of polystyrene or other compounds generate, enrichment process
It can be realized by being centrifuged.
Compared with prior art, derivatization reagent of the present invention is metabolized by marking specific nucleic acid sequence, and with furans
Object and surface markers have the magnetic bead combination of antibody to form compound, compound massive amplification nucleic acid sequence in enzyme reaction system
Column are detected using nucleic acid colloidal gold strip sandwich method, and testing result is determined by naked eye interpretation.Furans metabolism of the present invention
Object derivatization reagent can realize nitrofurans metabolin in on-site test complex system, have quickly, it is easy, at low cost excellent
Point, detection process are not necessarily to large scale equipment.Furans metabolite derivatization reagent of the present invention and its rapid detection card can be used for food
The fields such as safety, have a good application prospect.
Detailed description of the invention
With reference to the accompanying drawings and detailed description, the detection to furans metabolite derivatization reagent of the present invention and its quickly
Card and beneficial effect are described in detail.
Fig. 1 is the schematic diagram of furans metabolite detection method of the present invention.
Fig. 2 is the specificity experiments result figure of furans metabolite detection method of the present invention, and specially furans metabolism produces
The testing result of object AOZ, AMOZ, AHD and SEM.
Fig. 3 is the detection sensitivity result figure of furans metabolite rapid detection card of the present invention.
Specific embodiment
In order to be more clear goal of the invention of the invention, technical solution and advantageous effects, with reference to embodiments,
The present invention will be described in further detail.It should be understood that embodiment described in this specification is just for the sake of explanation
The present invention, be not intended to limit the present invention, formula, ratio of embodiment etc. can adaptation to local conditions make a choice and reality had no to result
Matter influences.
Embodiment
By taking the o-nitrobenzaldehyde detection AOZ of nucleic acid sequence modification as an example:
(1) prepared by magnetic bead
It takes in 0.5 μm of diameter of the carboxyl magnetic bead to 1.5mL centrifuge tube of 0.5mL (10mg), and is placed on Magneto separate frame
After supernatant change is completely thorough, is carefully moved with suction pipe and abandon supernatant.The MES buffer of 1mL is added to mix well washing.It will centrifugation
Pipe is placed on Magneto separate frame after supernatant becomes clear, is carefully moved with suction pipe and is abandoned supernatant.Repeated washing is primary.Finally, magnetic is resuspended
Pearl is in the MES buffer of 1mL.EDC is placed in room temperature 30 minutes from refrigeration place taking-up.Weigh 10mg 1- (3- dimethylamino third
Base) -3- ethyl-carbodiimide hydrochloride (EDC) addition is equipped in the centrifuge tube of magnetic bead, and acutely oscillation shakes up.It at room temperature, will be from
Heart pipe is placed on rotation blending instrument priming reaction 30 minutes.In reaction process, pay attention to that magnetic bead precipitating is not allowed to be built up together.It will be from
Heart pipe, which is placed on Magneto separate frame carefully to be moved after supernatant becomes clear with suction pipe, abandons supernatant.Repeated washing is twice.Take 200mg 2-
NP-AOZ antibody is added in the solution, is cleaned 3 times after reacting 1h, is then closed 4 degree of preservations with the PBS containing 1%BSA.
(2) nucleic acid sequence information
Nucleic acid sequence needed for the present embodiment is as follows:
O-nitrobenzaldehyde molecular labeling nucleic acid sequence is as shown in SEQ ID NO:1, wherein and 5 ' ends are conjugation sites,
GCTGAGG is nicking enzyme site.
Amplimer nucleic acid sequence is as shown in SEQ ID NO:2.
Colloid gold label nucleic acid sequence is as shown in SEQ ID NO:3, wherein 5 ' terminal modified sulfydryls.
Detection line (T line) crosses nucleic acid sequence as shown in SEQ ID NO:4.
Nature controlling line (C line) crosses nucleic acid sequence as shown in SEQ ID NO:5.
(3) reaction MIX system is prepared
Every 100 μ L 2 × SDA MIX system is prepared:
(4) detection line and nature controlling line
Detection line and the scribing line concentration of nature controlling line are 10 μM, and scribing line amount is 0.6 μ L/cm.Ultraviolet irradiation 15 divides after scribing line
Clock is fixed.
(5) prepared by sample pad
Add 0.5%triton X-100 mixed liquid dipping glass fibre with the borate buffer of the pH 50mM for being 8.0, dries
It is spare afterwards.
(6) test strips assemble
Sample pad, nitrocellulose membrane, blotting paper are successively pasted on bottom liner according to 2-3mm overlapping, and are cut to cutting machine
3mm strip, dry room temperature preservation after packaging.
(7) animal tissue's sample pre-treatments
It will be separately added into not through Mass Spectrometer Method without the broken homogenate of the remaining structure of fish muscle tissue homogenizer of nitrofurans
It is mixed with the AOZ of concentration and 10 μ g/L difference furans drug class metabolite SEM, AMOZ, AHD homogeneous.Accurately weigh 1g homogeneous
Tissue samples afterwards are diluted with 4mL ultrapure water, the HCl of 300 μ L 1M are added after mixing, and the modification of 100 μ L nucleic acid is added after mixing
37 DEG C concussion 1-2 hours after aqueous solution (20g/L) mixing for the o-nitrobenzaldehyde crossed.Thereafter supernatant is taken after 5000rpm centrifugation
100-150 μ L is added in the PBS solution of 20 μ L magnetic beads, separates magnetic bead with magnet stand after mixing 20min, with 100 μ L weight after cleaning 3 times
It is outstanding.
It takes 25 μ 2 × SDA of L MIX into 200 μ L test tubes, the resuspension product of 25 μ L is added, after drifting along or through, be placed in 37 DEG C of hot plates
It after reaction 15 minutes, takes 5 μ L products to be added dropwise in the loading hole of test strips, adds the developping solution of 60 μ L, after five minutes observation knot
Fruit, in as a result 30 minutes effectively.
Fig. 2~3 are referred to, as seen from the figure: in Fig. 2, the detection card of AOZ, only CT line is same when detecting AOZ standard items
Shi Xianse, interpretation are the positive.Other three kinds of metabolism analyte detections only have the colour developing of C line, and interpretation is feminine gender, thus judge the detection card
There is preferable specificity.In Fig. 3, the concentration of examination criteria product 2-NP-AOZ is successively increased, the T line when concentration reaches 0.005ppb
Colour developing, as concentration increases, T line color is deepened, and thus the interpretation detection card reaches the detection sensitivity of 2-NP-AOZ
0.005ppb。
According to the disclosure and teachings of the above specification, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula carries out change and modification appropriate.Therefore, the invention is not limited to the specific embodiments disclosed and described above, to this
Some modifications and changes of invention should also be as falling into the scope of the claims of the present invention.In addition, although this specification
In use some specific terms, these terms are merely for convenience of description, does not limit the present invention in any way.
SEQ ID NO:1
5’-AGGTACATCTACAGTACAGACATCTACAGCTGAGGTCACTAGCCAG-3’
SEQ ID NO:2
5’-CTGGCTAGTGAC-3’
SEQ ID NO:3
5’SH-AGGTACATCTACAGTA-3’
SEQ ID NO:4
5’CAGACATCTACAGCT-3’
SEQ ID NO:5
5’-TACTGTAGATGTACCT-3’
Claims (3)
1. a kind of reagent combination, which is characterized in that including a kind of furans metabolite derivatization reagent and a kind of magnetic bead；
The furans metabolite derivatization reagent is the o-nitrobenzaldehyde that one end modifying and decorating has one section of nucleic acid sequence；It is described
The aldehyde radical in o-nitrobenzaldehyde in furans metabolite derivatization reagent forms furan for combining with furans metabolite
It mutters metabolite-o-nitrobenzaldehyde-nucleic acid sequence compound；There are a nicking enzyme site and one in the nucleic acid sequence
Primer binding site, for generating a large amount of complementary single strand nucleic acid sequence under the action of archaeal dna polymerase and nicking enzyme；
The surface markers of the magnetic bead have specific recognition furans metabolite-o-nitrobenzaldehyde antibody；It is marked on magnetic bead
The antibody of the specific furans metabolite-o-nitrobenzaldehyde on surface is used for and the furans metabolite-ortho-nitrophenyl first
Aldehyde-nucleic acid sequence compound combines, and is enriched with and cleans through Magnetic Isolation, forms magnetic bead-furans metabolite-ortho-nitrophenyl first
Aldehyde-nucleic acid sequence compound；
The primer binding site is located at 3 ' ends of the nucleic acid sequence, nicking enzyme site and the primer binding site phase
Neighbour, the length of the nucleic acid sequence are 30~200 bases.
2. reagent combination according to claim 1, which is characterized in that the length of the nucleic acid sequence is 40~60 alkali
Base.
3. application of the reagent combination of any of claims 1 or 2 in nitrofurans metabolism analyte detection.
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| CN110618259A (en) * | 2019-09-17 | 2019-12-27 | 华南农业大学 | Colloidal gold test strip for detecting grouper iridovirus and preparation and detection methods thereof |
| CN110579591A (en) * | 2019-09-17 | 2019-12-17 | 华南农业大学 | Colloidal gold test strip for detecting nervous necrosis virus of grouper and preparation and detection methods thereof |
| CN111207991A (en) * | 2020-02-26 | 2020-05-29 | 杭州南开日新生物技术有限公司 | Pretreatment method and detection method of nitrofuran metabolite |
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| CN101561439A (en) * | 2009-03-31 | 2009-10-21 | 深圳出入境检验检疫局食品检验检疫技术中心 | Nitrofurantoin residue enzyme-linked immunoassay kit |
| CN103436608B (en) * | 2013-08-08 | 2015-02-25 | 中国科学院广州生物医药与健康研究院 | Rapid detection method based on nucleic acid aptamers and kit |
| CN104374910A (en) * | 2014-11-19 | 2015-02-25 | 无锡中德伯尔生物技术有限公司 | Kit and method applied to detection of nitrofurans drug metabolite |
| GB2552427A (en) * | 2015-01-29 | 2018-01-24 | Neogen Corp | Methods for immuno chromatographic assay desensitization |
| CN106093379A (en) * | 2016-06-08 | 2016-11-09 | 中国科学院广州生物医药与健康研究院 | A kind of semicarbazides derivatization reagent and rapid detection card thereof |
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