CN103773847A - Lateral flow test strip detection kit for detection of bovine-derived components in foods and feeds and application of kit - Google Patents
Lateral flow test strip detection kit for detection of bovine-derived components in foods and feeds and application of kit Download PDFInfo
- Publication number
- CN103773847A CN103773847A CN201310670600.4A CN201310670600A CN103773847A CN 103773847 A CN103773847 A CN 103773847A CN 201310670600 A CN201310670600 A CN 201310670600A CN 103773847 A CN103773847 A CN 103773847A
- Authority
- CN
- China
- Prior art keywords
- detection
- nucleic acid
- primer
- test strip
- pcr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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
- C12Q1/6804—Nucleic acid analysis using immunogens
Abstract
The invention discloses a preparation method and an application method of a nucleic acid rapid detection kit for Bostaurus components in feeds and belongs to the field of molecular biology and immunology. According to the preparation method and the application method, a high-sensitivity and high-specificity method of a polymerase chain reaction in nucleic acid detection is combined with an immuno gold staining rapid detection technology in immunology detection; a unique primer is designed and the primer is labeled; an extracted target DNA is subjected to specific amplification and an amplified product is combined with a labeled antibody immobilized on a test strip in a developing solution to form a stable and visible detection strip and a quality control strip, so as to realize rapid and accurate detection on the bovine-derived components in food and feed.
Description
Technical field
The invention belongs to molecular biology and field of immunology, the methods for making and using same of the PCR nucleic acid amplification of ox source DNA and lateral flow immunity colloidal gold test paper strip test kit in food and feeds.
Background technology
For preventing mad cow disease (bovine spongiform encephalopathy, BSE) beef in being added on animal-feed, ox bone are propagated, European Union in 1994 and the U.S. in 1997 are formulating the rules of adding ruminating animal endogenous binding protein in forbidding the feed ruminating animal for 1994 and 1997 respectively, and within 2000, European Union expands this ban to forbid to the finished Mammals of the detoxification for edible, birds and fish protein composition to.The Ministry of Agriculture of China provides against and in ruminant feed, uses feed products from animal sources (except milk and milk products) in " the feed products from animal sources safety and sanitation management method " of within 2004, promulgating, but people is that the phenomenon of mixing ruminating animal (being mainly ox, sheep) meat and bone ware happens occasionally in animal-feed.
The taking place frequently of various food safety affairs such as in recent years, both at home and abroad on food products market, the phenomenon of mixing the spurious with the genuine such as false mutton, false beef emerges in an endless stream, and animal derived food doping is adulterated more and more allow the frightened courage of common people's heart quiver.At the beginning of 2013, " the horseflesh disturbance " in Europe grows in intensity, and makes human consumer's " Wen Mase change ".American-European " flesh of fish disturbance " sounded again the alarm bell of food safety again afterwards.Guarantee the safety of food and feeds, needs are sensitive, convenient, detection method provides basis accurately.
In traditional feed, animality Components identification, mainly by microscope inspection, the immunological method based on protein detection and all kinds of PCR method based on detection of nucleic acids, has also developed near infrared detection method (NIRS) in recent years.The various animal source compositions that mix in forage component are easy to be damaged in pyroprocessing and the course of processing, between the protein sequence of different genera animal, difference is little, be difficult to difference, patent of invention " SDS-PAGE method is differentiated animal derived composition in meat and meat product " (publication number: CN 102213720 A) provides a kind of SDS-PAGE electrophoretic with protein to identify pig, ox, sheep, chicken, the method of the animal proteinum of fish, but the protein electrophoresis bands of a spectrum of different sources and processing mode are different, the obstacle that brings result to differentiate, more be difficult to carry out for the animal component in feed.
Because the degeneracy feature of gene coded sequence and the existence of non-coding sequence, nucleic acid becomes the target molecule that more easily detects difference compared with albumen, particularly Mitochondrial DNA, copy number is high, and existence difference between species is main authentication method to the analysis of its differential fragment, these class methods are because of highly sensitive, specificity is good, and rapidly, main method comprises common/fluorescence PCR method and biochip technology for less consuming time and development.Carry out with mtdna sequence species kind differentiate time, conventional region comprise Codocyte pigment B Cytochrome b gene (
cytB), small subunit ribosome 12S RNA encoding sequence (12S ribosomal RNA), cytochrome C subunit I(cytochrome C oxidase subunit I) encoding gene
coxI, coding ATP8 and ATP6
atp8and
atp6gene and control region D-loop fragment.The sequence variations in these regions is moderate, in planting, has certain conservative property, embodies again certain middle difference.
Fluorescent quantitative PCR technique is that a kind of highly sensitive nucleic acid detects and quantivative approach, ultimate principle is in real-time fluorescence quantitative PCR reaction, has introduced a kind of fluorescence chemical material, along with the carrying out of PCR reaction, PCR reaction product constantly accumulates, and fluorescence signal intensity also equal proportion increases.Fluorescent quantitative PCR technique steps qualitative detection and has gone up the step that can quantize, and there is highly sensitive, specificity and reliability is stronger, can realize multiple reaction, level of automation is high, nonstaining property, there is the feature such as real-time and accuracy, particularly rely on the TaqMan method of probe combination, be widely used at present the field such as molecular biology research and medical research.Identify application aspect at species, the domestic national standard of having issued or industry standard have contained Mammals or the hydrobionts such as ox, sheep, deer, camel, horse, donkey, pig, rabbit, dog and fish.In view of the importance of ruminating animal, patent of invention " is differentiated fluorescent PCR detection reagent and the preparation method and application of ruminating animal derived component ", and (publication number: CN 101659996A) described one and can have been differentiated ox simultaneously, the fluorescence PCR method of sheep and goat, carry out animal-origin examination with three probes, publication number is that the patent of invention " donkey or donkey derived component real-time fluorescence PCR detection method and detection primer and probe " of CN 102311999 A provides the fluorescence PCR method that carrys out derived components with particular probe discriminating donkey, publication number is that the patent of invention " horse or horse derived component real-time fluorescence PCR detection method and detection primer and probe " of CN 102311998 A provides a kind of fluorescence PCR method of identifying Malaysia derived components.Publication number is that the patent of invention " a kind of discrimination method of animal derived materials " of CN101196463 provides one group can differentiate ox, sheep, pig, horse, the vertebrate primer sequence such as chicken and conventional PCR-gel electrophoresis method, similarly, aspect the evaluation of poultry source, patent of invention " a pair of goose derived component PCR detection primer " (publication number: CN 102337337 A) has been described a kind of method of identifying in conjunction with agarose gel electrophoresis with conventional PCR, publication number is that CN 101270392A patent of invention " PCR-mtDNA detects amplimer and detection kit and the using method of total avian composition " further provides the method that detects multiple poultry derived Mitochondrial DNA with pair of primers, in aforesaid method, real-time quantitative PCR detects strong to device dependence, be difficult to realize Site Detection, conventional pcr amplification detects need to be to carrying out IMAQ and analysis after product gel electrophoresis, expend time in longer, and be difficult to guarantee the sensitivity of detection.
Detect the detection efficiency that can effectively improve nucleic acid product by the method for immunology detection being transplanted in nucleic acid product.Can in its amplified production, introduce by the specific protein of primer mark or compound that pcr amplification is used the nucleic acid complexes that simultaneously comprises two kinds of compound/protein labelings, the product of this mark can detect in the mode of similar double-antibody sandwich by antibody or part, the immuno-gold labeling detection technique of the similar routine of this process, this method is used in the method for single nucleotide polymorphism and constant-temperature amplification target gene, the particularly evaluation to pathogenic micro-organism or disease association gene, publication number is that a kind of patent of invention-" method based on nucleic acid transverse flow ELISA test strip single nucleotide polymorphism " of CN 102134596 A carried out the detection of single nucleotide polymorphism with this method.And the patent of invention " mono-increasing listeria spp nucleic acid chromatography detection kit and detection method and application " that publication number is CN 102520172 A has been described a kind of cow genome group detection method that adopts respectively vitamin H and digoxigenin labeled primer.Therefore class methods only judge having or not of amplified production, the molecular weight of product is not confirmed, therefore the interfering factors in amplified reaction is more more, the common false-positive reason that causes comprises: the abnormal renaturation between non-specific amplification, primer dimer, amplified production, the interference that solution primer dimer, abnormal annealing cause can be carried out from the several aspects of control of the selection of PCR polysaccharase, primer sequence optimization, dNTP substrate, crossover process.The archaeal dna polymerase that selection has warm start (Hot-start) function can obviously reduce the formation of primer dimer and non-specific amplification product.Aspect primer optimization, publication number be patent of invention-" a pair of part reduces its dimeric method with order primer " of CN 102146432 A described a kind of at primer 5 ' end the primer design method with short palindromic sequence, recirculation under this primer normal temperature, avoid forming heterodimer, publication number is that patent of invention-" detecting the real-time fluorescence quantitative PCR reagent of HER2 gene expression dose " of CN 102719547 A also adopted the amplification of similar method for real-time quantitative PCR, publication number is that patent of invention-" using chimeric primers to reduce heterodimer forms " of CN 101842494 A described a kind of method that uses chimeric primers to increase, in to the optimization of reaction substrate, the patent of invention " 3 ' modified oligonucleotide that contains false iso-cytosine nucleobase derivative and the application as primer or probe thereof " of publication number CN 101171343A provides a kind of method that uses the Nucleotide of special modification to form to reduce primer dimer as substrate, use probe or introduce internal control probe the interference that also can reduce non-specific amplification, publication number is that a kind of patent of invention-" method that adds internal control nucleic acid pathogen nucleic acid to be carried out to half-quantitative detection " of CN 101957373 A adopts internal control probe to reduce interference, in aforesaid method, using warm start technology and cyclisation primer is current method, all the other methods or adopted special synthetic substrate and substrate, or need to introduce crossover process for the second time by probe, capital increases the complexity of testing process, particularly probe hybridization method, because needing insulating process to lose the convenience of test strip method.
In design of primers of the present invention, keeping on specificity basis, to in primer pair and primer 5 ' and the free energy of 3 ' end annealing is assessed rear selection, and dimeric formation is avoided in suitable region, for the dimeric disturbed condition of ELISA test strip method with suitable amplicon length, in cooperation expansion damping fluid, the concentration optimization of stain remover and denaturing agent, can realize effective removing of primer dimer and non-specific amplification product.
Summary of the invention
The nucleic acid molecule of biological micromolecule or compound mark can be identified by the antibodies specific of this small molecules or compound, thereby detect by immunological method.The common molecule that can be used for labeled nucleic acid molecule comprises hapten molecule vitamin H (Biotin), digoxin (Digoxin), luminescent dye molecule rhodamine (RBITC), fluorescein isothiocyanate (fluorescein isothiocyanate, FITC), Cy3, Cy5 etc.In above-mentioned tagged molecule, digoxin, fluorescein isothiocyanate are all easy to obtain special antibody, and biotin molecule and its part-streptavidin have the binding characteristic of high special, therefore these several molecules can be selected mark and the immunology detection for nucleic acid molecule.The present invention is intended to using few to antigen or haptens small molecules mark strand nucleic acid as primer, to target nucleic acid specific amplification to be checked, the complex molecule forming possesses two kinds of antigens or hapten-marked simultaneously, immunogenic, this mixture is to be availablely combined with specific antibody or sepcific ligands, and the immune colloid gold of realizing nucleic acid amplification product detects.
Therefore, on the one hand, the invention provides the detection technique of bovine material in a kind of test strip method detection food and feeds, mainly comprise:
1) two unique amplimers of design, wherein forward primer Bos-ATP-F has following sequence: 5 '-GCCATATACTCTCCTTGGTGAC-3 ', with a kind of mark in antigen or hapten molecule vitamin H (Biotin), fluorescein isothiocyanate (FITC) or digoxin (Digoxin); The sequence of downstream primer Cat-T-ATP-R is 5 '-TAGTAGGCTTGGGAATAGTACG-3 ', to be different from a kind of mark of upstream primer; Under suitable amplification condition, bovine mitochondrial gene group can increase
atp8-atp6region one segment length is the DNA fragmentation of 270 bps; When without tested nucleic acid-templated existence, without specific nucleic acid fragment amplification product;
2) by a kind of universal antibody of standard, as crosslinked in anti-FITC mAb and colloid gold particle, form coated antibody at particle surface;
3) by the antibody of another kind of tagged molecule or part, as part-streptavidin molecule of anti digoxin antibody or biotin molecule is fixed on and forms detection line on film with linear;
4), in the time there is specific amplification products to be checked, because of the effect of upstream and downstream primer, in amplified production, simultaneously with two kinds in above-mentioned three kinds of markers, form the mixture of tagged molecule 1-amplified production-tagged molecule 2;
5) by step 4) the mark 1-amplified production-mark 2 forming and the antibodies of the mark 1 of colloid gold particle pan coating, form anti-mark 1 antibody-mark 1-amplified production-mark 2 coloured particle mixtures;
6) by step 5) the coloured particle mixture that obtains travels up to and is coated with on the antibody of mark 2 or the lines of part along tunica fibrosa by capillarity in solution, mixture is because depositing with antibody (part) combination of mark 2, be trapped on detection line, form macroscopic colored line, be judged to be the positive; 7) in the time that specific amplification products does not exist, there is not above-mentioned steps 4)-6), can not form mark 1-amplified production-mark 2 mixtures, also just can not shape be deposited on the antibody (part) of mark 2 on detection line, do not form visible band, be judged to feminine gender.
On the other hand, the present invention also provides the developping solution detecting for nucleotide sequence, and this developping solution can reduce the interference of primer dimer to experimental result;
On the other hand, the invention provides the test strip for the rapid detection of food borne pathogenic microorganism, the liner that it is included in non-setting adhesive, has in order: 1: absorbent filter pad; 2: binding substances pad, with the antibody of the first nucleic acid markers or the particle that adds lustre to of part (Radioactive colloidal gold or latex); 3: lateral chromatography matrix (nitrocellulose filter or nylon membrane); 4: detect band, with the antibody of the second nucleic acid markers; 5: quality control band, having can be in conjunction with the antibody of specific Species origin antibody; 6: substrate; 7: absorbent pad.
On the other hand, the present invention also provides the universal standard test strip for detection of nucleic acid amplification product.
Finally, the present invention also provides above-mentioned detection method and the test strip purposes in the detection of ox derived component in food and feeds.
The explanation of technical terms using in specification sheets of the present invention:
The initiator that primer: DNA is synthetic.Be generally a pair of single stranded oligonucleotide, after hybridizing with template, DNA is synthetic from its 3 ' end.
Mark: the method by detectable signaling molecule (as haptens, fluorescence, radioactivity etc.) with the coupling of single stranded oligonucleotide phase.
Hybridization: refer in particular to complementary DNA single chain and form duplex structure by base pairing.
Launch: under the chromatography effect of expansion damping fluid, start by test strip absorbent pad bottom the process moving to detection line, nature controlling line direction through the PCR of amplified reaction product.
Nucleic acid: the common name of thymus nucleic acid (DNA) and Yeast Nucleic Acid (RNA).
Antigen and haptens: possess immunogenic material.Be generally macro-molecular protein or cellular component.But some small molecules also possesses immunogenicity, be called as haptens ((Hapten).Haptens is often used to label probe.
Antibody: the protein molecule that can be combined with antigen or hapten specificity.
Complex body: the binding substances of being formed by two or more molecular specificity.
Primer dimer: in the process of polymerase chain reaction (PCR), because of between primer pair or wall scroll primer mutually anneal form dimer molecule, the dimer being made up of two different primers is heterodimer, because causing with the combination of kind of an antibody (part) and cause false positive with two kinds of marks, the dimer being formed by single primer annealing is homodimer, only can not cause false positive with a kind of mark, but the meeting of formation of too much dimer reduces amplification efficiency.
Immunity test strip: for the medical tool of rapid detection.Be called again colour generation membrane chromatographic.
Nucleic acid polymerase: the enzyme of nucleic acid long-chain.Be divided into archaeal dna polymerase and the large class of RNA polymerase two.
beneficial effect:
Because the present invention is the gene fragment with the primer amplified suitable length of mark, can guarantee the high degree of specificity of detection, thereby guarantee the accuracy of detected result.Novel part of the present invention has been avoided the flow processs such as amplified production dilution and probe hybridization, the advantages such as immune colloid gold (test strip) technology is directly perceived, quick, convenient, ripe, cheap are kept, possesses again the feature of pcr amplification method height sensitivity, high degree of specificity, therefore, method of the present invention is a kind of effectively ox source animal component detection method.
The current techique platform detecting as a nucleic acid amplification product, method of the present invention and test strip thereof can be widely used in the evaluation of animal source composition and the detection of food borne pathogenic microorganism in food and feeds, all have wide practical use in husbandry, customs inspection quarantine.Though there is the animal source Components identification method of multiple PCR-based at present, but its design of primers and product fragment are all suitable for probe method or gel electrophoresis, lack necessary consideration and remove for the interference that reduces primer dimer and non-specific renaturation in colloidal gold strip method, solution is also also inapplicable.The object of the invention is to provide a kind of PCR primer, amplification and detection method that detects ox source animal component based on immune colloid gold method, and the application of the method.Other common species, as the evaluation of ox, sheep, pig, donkey, rabbit, camel, duck, goose, chicken, fox, ermine, deer etc. can adopt method similarly to complete.
The invention of nucleic acid test strip detection method is by the trace routine of greatly simplifying after nucleic acid amplification.Result interpretation simple and clear, directly perceived (detecting signal result as accompanying drawing 2).
In the food and feeds that this patent is applied for ox derived component Rapid detection test strip technology as a kind of novel nucleic acid amplification after detection technique, have the following advantages:
Simple to operate: only the sample after nucleic acid amplification directly need to be dripped to nucleic acid reagent and detect version above, not need professional to operate, be convenient to promote;
Quick: to detect sentence read result after 5 minutes;
Sensitive: compared with agarose gel electrophoresis, detection sensitivity improves nearly 100～200 times;
Specificity is high: be specificity labeled primers due to what add use in testing process, make result more accurate;
Low price: expense is significantly less than traditional gel electrophoresis and ELISA detects.
Accompanying drawing explanation
Fig. 1 shows the basic structure of the test strip of amplification of nucleic acid sequences product detection of the present invention;
1: absorbent filter pad; 2: binding substances pad, with the antibody of the first nucleic acid markers or the particle that adds lustre to of part (Radioactive colloidal gold or latex); 3: lateral chromatography matrix (nitrocellulose filter or nylon membrane); 4: detect band, with the antibody of the second nucleic acid markers; 5: quality control band, having can be in conjunction with the antibody of specific Species origin antibody; 6: substrate; 7: absorbent pad.
Fig. 2 is the detected result schematic diagram of detection of nucleic acids test strip;
Fig. 3 is the method showing with embodiment 1, adopts specificity labeled primers to detect the detected result of the PCR-test strip of ox;
Fig. 4 is the method showing with embodiment 2, adopts the detected result of the specific PCR-test strip that detects ox specificity labeled primers;
Fig. 5 is the method with embodiment 2, the susceptibility comparative result of test strip and detected through gel electrophoresis method after show nucleic acid amplification.
Embodiment
Following embodiment illustrates detection method of the present invention and detects effect.Embodiment is only used for for example, not forming any restriction to protection domain of the present invention, and protection scope of the present invention illustrates at appending claims.
1 materials and methods
Cow genome group DNA
1.2 design of primers
1.3 pcr amplification systems:
1.3 pcr amplification systems:
| Template DNA: cow genome group DNA | 1.0 μl |
| PrimerF/R | 0.8 μl |
| dNTP | 2.0 μl |
| 10X PCRbuffer | 2.0 μl |
| HS Taq DNA polymerase | 0.2 μl |
| ddH2O | 13.2 μl |
| Cumulative volume | 20 μl |
Reaction conditions:
| 95 |
5 min |
| 94 ℃ | 30 sec |
| 55?℃ | 30 sec |
| 72 ℃ | 30 sec |
| 72 |
5 min |
| Totally 30 circulations | ? |
Get respectively 3 μ l simultaneously and detect and get 3 μ L amplified productions for nucleic acid test strip, join in 97 μ L developping solutions and carry out check point in sample pad, observations after 5 minutes.
1.4 PCR specificity experiments
The PCR reaction system that utilization is set up is to 1: mouse; 2: ox; 3: rabbit; 4: horse; 5: sheep; 6: dog; 7: pig; 8: cat; 9: chicken; 10: duck; 11: goose; 12:NTC (blank: water) verifies its specificity.
2 results
2.1 PCR reaction system and conditions
The HS Taq DNA polymerase that adopts TAKARA company, total reaction system is 20 μ L.Detect with Bio-Rad PCR instrument, reaction parameter is: 94 ℃ of 5 min, and 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30 s increases, and proceeds to 72 ℃ of 5 min, 4 ℃ of preservations after 30 circulations.Get 5 μ L products carrying out electrophoresis containing in 2% agarose gel electrophoresis of ethidium bromide, after electrophoresis, judge and have or not specific band.After nucleic acid amplification, the detected result of test strip and gel electrophoresis is shown in accompanying drawing 4.
2.2 specific test
The PCR method that the present invention sets up has good specificity to cow genome group DNA, to no cross reactions such as other Mammalss and poultry species.
The susceptibility of nucleic acid test strip detection method, by pcr template with 1/10,1/10
2, 1/10
3, 1/10
4, 1/10
5, 1/10
6, 1/10
7, 1/10
8, 1/10
9, 1/10
10dilution proportion after, increase by the PCR system of having set up.
1 materials and methods
Ox Mitochondrial DNA
1.2 design of primers
1.3 pcr amplification systems:
| Template DNA: |
10 μl |
| Upstream primer | 0.8 μl |
| Downstream primer | 0.8 μl |
| dNTP | 2.0 μl |
| 10X PCR?buffer | 2.0 μl |
| HS Taq DNA polymerase | 0.2 μl |
| ddH 2O | 4.2 μl |
| Cumulative volume | 20 μl |
Reaction conditions:
| 95 |
5 min |
| 94 ℃ | 30 sec |
| 56?℃ | 30 sec |
| 72 ℃ | 30 sec |
| 72 ℃ | 5 min |
| Totally 30 circulations | ? |
Get 5 μ l for agarose gel electrophoresis.Gel electrophoresis condition is: 1X tbe buffer liquid, voltage 100V, 30 points of electrophoresis times.Separately get 5 μ l and detect for nucleic acid test strip, get 5 μ l sample spot in sample pad, join in 95 μ L developping solutions and detect, observations after 5 minutes simultaneously.
2 results
2.1 PCR reaction system and conditions
The HS Taq DNA polymerase that adopts TaKaRa company, total reaction system is 20 μ l.Increase with Bio-Rad T-100 PCR instrument, reaction parameter is: 94 ℃ of 5 min, and 94 ℃ of 30s, 56 ℃ of 30s, 72 ℃ of 30 s increases, and proceeds to 72 ℃ of 5 min, 4 ℃ of preservations after 30 circulations.Get 5 μ l products carrying out electrophoresis containing in 2% agarose gel electrophoresis of ethidium bromide, after electrophoresis, judge and have or not specific band.The susceptibility comparative result of test strip and gel electrophoresis after nucleic acid amplification is shown in accompanying drawing 5, can be found out by the result of accompanying drawing 5, and compared with traditional gel electrophoresis, its susceptibility increases by 100～200 times.
GCCATATACTCTCCTTGGTGACATGCCGCAACTAGACACGTCAACATGACTGACAATGATCTTATCAATATTCTTGACCCTTTTTATCATCTTTCAACTAAAAGTTTCAAAACACAACTTTTATCACAATCCAGAACTGACACCAACAAAAATATTAAAACAAAACACCCCTTGAGAAACAAAATGAACGAAAATTTATTTACCTCTTTTATTACCCCTGTAATTTTAGGTCTCCCTCTCGTAACCCTTATCGTACTATTCCCAAGCCTACTA
Claims (7)
1. the invention provides the test strip method for quick of bovine material in a kind of food and feeds.
2. detection method as claimed in claim 1, adopt the primer PCR of two unique design to carry out amplified reaction, wherein the sequence of upstream primer Cat-ATP-F is: 5 '-GTACATAAAATTAATGTATTA GGAC-3 ', the sequence of downstream primer Cat-ATP-R is 5 '-ATTAAATAGCTACCCCCA CGATTC-3 ', and 5 of this primer pair ' end carries out mark respectively so that detect.
3. detection method as claimed in claim 1 is used and completes the chromatography in test strip with the expansion damping fluid of nucleic acid denaturation agent, and its composition is: 10mM Tris, 1% BSA, 1% Tween 20 and concentration are respectively the NaOH of 0.05mol/L to 0.5mol/L.
4. the test strip detecting for nucleic acid amplification product claimed in claim 1, wherein coloured particle is colloid gold particle or latex particle, described tunica fibrosa is nitrocellulose filter or nylon membrane.
5. PCR process as claimed in claim 2, its primer pair can be selected two kinds of diverse ways marks in biotin molecule (Biotin), fluorescein isothiocyanate (FITC) or digoxin (Digoxin).
6. PCR process as claimed in claim 2, its reaction conditions is: 95 ℃ of denaturation 5 min, 94 ℃ of sex change 30 sec; 55 ℃ of annealing 30 sec; 72 ℃ are extended 30sec, 30 circulations of increasing, and last 72 ℃ are extended 5 min.
7. PCR-immunity colloidal gold test paper strip claimed in claim 1 is used for food and feeds from the zoogenous amplification of ox and the purposes detecting by immune colloidal gold technique.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310670600.4A CN103773847B (en) | 2013-12-10 | 2013-12-10 | Lateral flow test strip detection kit for detection of bovine-derived components in foods and feeds and application of kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310670600.4A CN103773847B (en) | 2013-12-10 | 2013-12-10 | Lateral flow test strip detection kit for detection of bovine-derived components in foods and feeds and application of kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103773847A true CN103773847A (en) | 2014-05-07 |
| CN103773847B CN103773847B (en) | 2017-02-08 |
Family
ID=50566604
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310670600.4A Expired - Fee Related CN103773847B (en) | 2013-12-10 | 2013-12-10 | Lateral flow test strip detection kit for detection of bovine-derived components in foods and feeds and application of kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103773847B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017872A (en) * | 2014-05-29 | 2014-09-03 | 天津出入境检验检疫局动植物与食品检测中心 | Lateral flow test paper strip detection kit for detection of fox source components in food and feed and application thereof |
| CN106967843A (en) * | 2017-04-06 | 2017-07-21 | 苏州点晶生物科技有限公司 | Canine parvovirus nucleic acid rapid detection kit |
| CN109576379A (en) * | 2019-01-16 | 2019-04-05 | 北京同仁堂科技发展股份有限公司 | Identify specific primer, kit and the discrimination method of donkey derived component in donkey-hide gelatin |
| CN109825612A (en) * | 2019-04-11 | 2019-05-31 | 中国农业大学 | Bovine material quick detection kit and its application in a kind of food |
| CN110055340A (en) * | 2019-04-11 | 2019-07-26 | 中国农业大学 | Bovine material rapid detection method and kit in a kind of food |
| CN113088531A (en) * | 2021-03-25 | 2021-07-09 | 四川省食品药品检验检测院(四川省药品质量研究所、四川省医疗器械检测中心) | Bovine-derived component quantitative analysis standard plasmid, preparation and detection method and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101659996A (en) * | 2009-07-27 | 2010-03-03 | 花群义 | Fluorescence PCR detection reagent capable of discriminating source components of ruminant animal, preparation method and application thereof |
| CN102994637A (en) * | 2012-11-26 | 2013-03-27 | 中国肉类食品综合研究中心 | Method for detecting bovine-derived materials in meat and meat products |
-
2013
- 2013-12-10 CN CN201310670600.4A patent/CN103773847B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101659996A (en) * | 2009-07-27 | 2010-03-03 | 花群义 | Fluorescence PCR detection reagent capable of discriminating source components of ruminant animal, preparation method and application thereof |
| CN102994637A (en) * | 2012-11-26 | 2013-03-27 | 中国肉类食品综合研究中心 | Method for detecting bovine-derived materials in meat and meat products |
Non-Patent Citations (1)
| Title |
|---|
| 金凌艳等: "应用两种PCR方法检测饲料中牛、羊源性成分", 《中国农业科技导报》, vol. 10, 31 December 2008 (2008-12-31), pages 76 - 80 * |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017872A (en) * | 2014-05-29 | 2014-09-03 | 天津出入境检验检疫局动植物与食品检测中心 | Lateral flow test paper strip detection kit for detection of fox source components in food and feed and application thereof |
| CN104017872B (en) * | 2014-05-29 | 2017-02-08 | 天津出入境检验检疫局动植物与食品检测中心 | Lateral flow test paper strip detection kit for detection of fox source components in food and feed and application thereof |
| CN106967843A (en) * | 2017-04-06 | 2017-07-21 | 苏州点晶生物科技有限公司 | Canine parvovirus nucleic acid rapid detection kit |
| CN106967843B (en) * | 2017-04-06 | 2020-07-14 | 苏州点晶生物科技有限公司 | Rapid detection kit for canine parvovirus nucleic acid |
| CN109576379A (en) * | 2019-01-16 | 2019-04-05 | 北京同仁堂科技发展股份有限公司 | Identify specific primer, kit and the discrimination method of donkey derived component in donkey-hide gelatin |
| CN109576379B (en) * | 2019-01-16 | 2022-05-17 | 北京同仁堂科技发展股份有限公司 | Specific primer, kit and identification method for identifying donkey-derived component in donkey-hide gelatin |
| CN109825612A (en) * | 2019-04-11 | 2019-05-31 | 中国农业大学 | Bovine material quick detection kit and its application in a kind of food |
| CN110055340A (en) * | 2019-04-11 | 2019-07-26 | 中国农业大学 | Bovine material rapid detection method and kit in a kind of food |
| CN110055340B (en) * | 2019-04-11 | 2021-01-01 | 中国农业大学 | Method and kit for rapidly detecting bovine-derived components in food |
| CN109825612B (en) * | 2019-04-11 | 2021-01-01 | 中国农业大学 | Kit for rapidly detecting bovine-derived components in food and application thereof |
| CN113088531A (en) * | 2021-03-25 | 2021-07-09 | 四川省食品药品检验检测院(四川省药品质量研究所、四川省医疗器械检测中心) | Bovine-derived component quantitative analysis standard plasmid, preparation and detection method and application |
| CN113088531B (en) * | 2021-03-25 | 2023-10-17 | 四川省药品检验研究院(四川省医疗器械检测中心) | Bovine-derived ingredient quantitative analysis standard plasmid, preparation and detection methods and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103773847B (en) | 2017-02-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103255217A (en) | Side direction current test strip detection kit for detecting canis familiaris component in feed and application thereof | |
| CN103773847A (en) | Lateral flow test strip detection kit for detection of bovine-derived components in foods and feeds and application of kit | |
| Rahmati et al. | Identification of meat origin in food products–A review | |
| CN103773850B (en) | In a kind of food and feeds, chicken derived components detects lateral flow ELISA test strip test kit and application thereof | |
| CN103773846B (en) | In a kind of food and feeds, goose derived components detects lateral flow ELISA test strip kit and application thereof | |
| WO2001079534A1 (en) | A method of detecting neoplastic or non-neoplastic cells | |
| CN109136408A (en) | Detection reagent, kit and its application of African swine fever virus | |
| CN108486258A (en) | A kind of kit and its method of visual quickly detection food-borne pathogens | |
| CN104278079A (en) | Test strip and method for detecting nucleic acid through nucleic acid chromatographic technique | |
| CN103757098B (en) | Donkey derived components detection lateral flow ELISA test strip test kit and application thereof in a kind of food and feedstuff | |
| CN103805691A (en) | Preparation method and application of nucleic acid lateral flow test strip for detecting Shigella | |
| CN103757097A (en) | Lateral flow test paper strip detection kit for detection of duck source component in food and feed, and application thereof | |
| CN103740803A (en) | Food borne pathogenic organism nucleic acid lateral flow test strip detection kit and application thereof | |
| CN102094088B (en) | Kit for determining sex of sheep early embryo | |
| CN103773845A (en) | Side flow test strip detection kit for detecting Cervusaxis in rood and feed and application thereof | |
| CN104017872A (en) | Lateral flow test paper strip detection kit for detection of fox source components in food and feed and application thereof | |
| CN103773839A (en) | Lateral flow test strip detection kit for detection of felissilvestriscatus component in feed and application of lateral flow test strip detection kit | |
| CN110055339B (en) | Quick detection kit for duck source components in food and application thereof | |
| CN104017871B (en) | In a kind of food and feeds, rabbit derived components detects lateral flow ELISA test strip test kit and application thereof | |
| CN106399537A (en) | Test strip kit for detecting duck-derived materials in food and feed and application of test strip kit | |
| CN109852705B (en) | Method and kit for rapidly detecting horse-derived components in food | |
| CN103773849A (en) | Preparation and applications of nucleic acid lateral flow test strip kit for detecting EHEC O157 | |
| CN103725772A (en) | Preparation method and application of nucleic acid lateral flow test strip for detecting cronobacter sakazakii | |
| AU2021100416A4 (en) | Lateral flow test strip detection kit for detecting martes-derived ingredients in food and feed and application thereof | |
| CN109811069B (en) | Kit for rapidly detecting donkey-derived components in food and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Zheng Wenjie Inventor after: Liu Wei Inventor after: Zhang Xia Inventor after: Zhao Weidong Inventor after: Xiao Yabing Inventor after: Xi Wenhui Inventor after: Yin Changcheng Inventor before: Zheng Wenjie Inventor before: Yin Changcheng Inventor before: Liu Siqi Inventor before: Li Honghong Inventor before: Zhang Yalian Inventor before: He Yan Inventor before: Chen Qiyong Inventor before: Cheng Yu Inventor before: Zhang Hongwei Inventor before: Zhang Can Inventor before: Xi Wenhui Inventor before: Du Jing Inventor before: Han Yuning |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: ZHENG WENJIE HE YAN CHEN QIYONG CHENG YU ZHANG HONGWEI ZHANG CAN XI WENHUI DU JING HAN YUNING YIN CHANGCHENG LIU SIQI LI HONGHONG ZHANG YALIAN TO: ZHENG WENJIE LIU WEI ZHANG XIA ZHAO WEIDONG XIAO YABING XI WENHUI YIN CHANGCHENG |
|
| CB03 | Change of inventor or designer information |
Inventor after: Zheng Wenjie Inventor after: Yin Changcheng Inventor after: Liu Wei Inventor after: Zhao Liangjuan Inventor after: Xiao Yabing Inventor after: Li Zongmeng Inventor after: Hou Liping Inventor after: Zhang Hongwei Inventor after: Gao Qili Inventor after: Zhang Yalian Inventor before: Zheng Wenjie Inventor before: Liu Wei Inventor before: Zhang Xia Inventor before: Zhao Weidong Inventor before: Xiao Yabing Inventor before: Xi Wenhui Inventor before: Yin Changcheng |
|
| COR | Change of bibliographic data | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170208 Termination date: 20171210 |