CN105274246A - Reagent kit for bovine-derived component identification and detection of multi-species provenance components in products of bovine-derived components - Google Patents
Reagent kit for bovine-derived component identification and detection of multi-species provenance components in products of bovine-derived components Download PDFInfo
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Abstract
The invention provides a reagent kit for bovine-derived component identification and detection of multi-species provenance components in products of bovine-derived components, and belongs to the technical field of animal provenance component molecular detection. The reagent kit comprises specific primers shown in the SEQ ID No.2 and the SEQ ID No.3, a reaction reagent and positive and blank control, and further comprises specific primers of pigs, horses, rabbits, foxes, dogs, minks, mice, chickens and ducks, and the nucleotide sequences of the specific primers are as shown from SEQ ID No.4 to SEQ ID No.21. By means of the primers of the reagent kit, sample DNA is amplified, a PCR amplification product is analyzed, whether a sample contains bovine-derived components or not can be judged and whether components of other animals such as pigs, horses, rabbits, foxes, dogs, minks, mice, chickens and ducks are doped in the sample or not can be judged. The reagent kit provides an efficient, precise and reliable means for detection of animal provenance components in meat, pelts and feed, and has the advantages of being easy and convenient to use, quick, comprehensive, high in specificity, low in cost and wide in adaptability.
Description
Technical field
The present invention relates to Animal molecular biology field, be specifically related to the detection kit of several species kind derived components in calf-derived Cyclospora qualification and goods thereof.
Background technology
In the consumption of China's animal food, beef is only second to pork, and it is of high nutritive value, and what Gu had " beef tonifying Qi, the same Radix Astragali of merit " says.Beef contains rich in protein, and amino acid ratio of components pork needs closer to human body, and lipid content is low, therefore delicious flavour, enjoy the laudatory title of " in meat favourite son ".Beef is rich in the nutritive ingredients such as sarkosine, VITAMIN, carnitine, L-Ala, the micronutrient levelss such as iron, calcium, potassium, magnesium enrich, and the lipid acid containing a kind of conjugated linolic acid, fatty deposits can be reduced, have effect of weight reducing, therefore beef items is subject to the favor of more and more human consumer.
Along with fast development and the growth in the living standard of economy, the requirement of people to food also changes thus, pursues quantitatively to meet from the past, turns to present to stress delicious and nourishing, health and safety.But it is many that meat product industry relates to field, industry chain length, strong with the relational degree of agricultural, contact closely with world market, its security control then becomes a great problem, therefore the security of meat product has become the social concern that human consumer shows great attention to.In recent years, worldwide meat product security incident occurs again and again, and in " the horseflesh disturbance " in Europe, the producer pretends to be top grade beef with horseflesh, the interests of grievous injury human consumer.In addition, the phenomenon that animal derived materials is adulterated in feed, hide also happens occasionally.Therefore, set up a set of science for ox source property goods, accurate, quick, cheap detection method is very necessary.
In daily life, people rely on sense organ and experience to carry out animal product discriminating usually, but along with the diversification of adulterated composition in the improvement of working method and each based article, the complicated of kind, this method far can not reach carries out to meat product adulteration the object that controls Yu supervise.Along with the development of modern molecular biology, the technology based on polymerase chain reaction (PCR) has become the core methed of animal derived materials qualification.Meanwhile, DNA also analyzes target spot because its high stability and the identity in different tissues are selected as than protein more reliably.At present, according to the existing a large amount of report (GhovvatiS of PCR method that Mitochondrial Genome Overview DNA sequence dna difference design Species-specific primer is set up, NassiriMR, MirhoseiniSZ, HeraviMA, JavadmaneshA.Fraudidentificationinindustrialmeatproducts bymultiplexPCRassay.FoodControl, 2009,20 (8): 696-699; GirishPS, AnjaneyuluASR, ViswasKN, ShivakumarBM, AnandM, PatelM, SharmaB.Meatspeciesidentificationbypolymerasechainreacti on-restrictionfragmentlengthpolymorphism (PCR-RFLP) ofmitochondrial12SrRNAgene.MeatScience, 2005,70 (1): 107-112; DooleyJJ, PaineKE, GarrettSD, BrownHM.DetectionofmeatspeciesusingTaqManreal-timePCRass ays.MeatScience, 2004,68 (3): 431-438.), and enter China authority detection technique standard (walk fast, Zhang Quanfang, Liu Yanyan. a kind of fluorescent marker gene composite amplification method [P] simultaneously identifying goat, sheep, pig and duck source property, Chinese patent: CN103397101A, 2013-11-20; Zhang Yingjie, Liu Yueqin, Chen Rui are subtle, Liu Yanqin, Guo Yunxia, LI XUEMEI, Yang Jiadong, Xi Jianzhong. the PCR detection method of disposable discriminating mutton, chicken, duck, pork and test kit [P], Chinese patent: CN104099405A, 2014-10-15.).But mitochondria DNA copy number is many, in different tissues, content is different, poor stability and be difficult to carry out quantitative analysis.And zooblast nuclear DNA sequence has the species specificity of height, non-specific amplification can be avoided, be more suitable as PCR qualitative and quantitative analysis.At present, be target sequence in the world with Nuclear DNA sequences, for differentiating that the PCR detection method in meat source can only distinguish the common meat product (Huang Ming such as ox, sheep, pig, chicken, duck mostly, Cheng Xin, Yang Jing, Huang Jichao, Zhou Xinghu. the method [P] of the prosperous middle chicken and duck pig blood composition of a kind of rapid detection blood, Chinese patent: CN103525908A, 2014-01-22; Huang Ming, Huang Weiling, Yang Jing, Xu Xinglian, period-luminosity is grand. and a kind ofly add pork or chicken composition Taqman fluorescence probe quantitative PCR method for quick [P] in the food of amplification interior label, Chinese patent: CN102864243B, 2013-9-25).And the complicacy that meat product market is adulterated (such as, " mixing mutton " event on the many ground of China, lawless person, by animal meat such as fox, mink, mouse, is added to the market of farm produce selling to the ground such as Jiangsu, Shanghai in mutton; Top grade beef etc. is pretended to be with horseflesh in Europe " horseflesh disturbance ") show, the detection method of quicker, easy, accurate, comprehensive meat adulteration becomes a urgent demand, and for can differentiate the PCR detection method of the animal derived materials such as horse, fox, mink, mouse simultaneously, there is not been reported at present.In addition, the phenomenon that animal derived materials is adulterated in feed, hide also happens occasionally.Therefore, set up for differentiating that the supervision of the method for quick of several species to food safety has important practice significance.
In addition, with regard to detection method, the fluorescent PCR that adopts detects (Cao Chenfu more at present, Zong Hui, Zhang Caihong etc. donkey or donkey derived component real-time fluorescence PCR detection method and detection primer and probe [P], Chinese patent: CN102311999A, 2012-1-11; Huang Ming, Huang Weiling, Yang Jing, Xu Xinglian, period-luminosity is grand. a kind ofly add pork or chicken composition Taqman fluorescence probe quantitative PCR method for quick [P] in the food of amplification interior label, Chinese patent: CN102864243B, 2013-9-25), enzyme cuts detection (Zhang An, You Jinhua, Xie Fusheng, Shi Xiumei. a kind of method [P] identifying animal hide, Chinese patent: CN1294277C, 2007-1-10; Yao Yonggang, Chen Shiyi, Liu Yiping. the method [P] of a kind of quick discriminating 5 kinds of livestock meats and dried meat product kind, Chinese patent: CN101712996,2010-5-26) or by amplified fragments size (Qiao Jianjun, Sun Yanhua, Zhang Zhiyu, Guan Congxiao. multiplex PCR detects primer sets and the test kit [P] of meat sources in food, Chinese patent: CN101962675B, 2013-2-6) etc. judge.But the requirement of fluorescence detection method to equipment is high and cost is larger; Enzyme cuts detection side's rule wastes time and energy; And by amplified fragments size, multiplex PCR differentiates that animal provenance seriously limits the quantity that can detect species, and often mutually disturb between primer or form dimer, affecting the confidence level of detected result.
Summary of the invention
The object of the invention is to for prior art not enough, a kind of primer that can be used for ox source property goods specific detection is provided;
Second object of the present invention is the detection kit being provided for the property goods detection of ox source;
The present invention also aims to provide ox source property goods Adulteration identification method.
For achieving the above object; the present invention from planting between consistence and stability, kind the aspect such as specificity, copy number carry out the screening of cow genome group specific DNA sequence; through sheep, the goat of the non-ox subfamily of Bovidae; detection in the genomic dna of the pig of non-Bovidae, horse, donkey, mouse (rat, mouse, hamster), cavy, chicken, duck, goose, rabbit, dog, fox, mink and recoon dog etc. and different cattle breeds DNA, screen special in ox, do not exist in other species or DNA fragmentation that homology is low as detection molecules.Through large component analysis and cut-and-try work, final acquisition can for the ox species specific DNA sequence detected, and its nucleotide sequence is as shown in sequence table SEQ IDNo.1.
Based on this, first the present invention provides a kind of bovine primer, the specific fragment of the nucleotide sequence shown in its specific amplification SEQIDNo.1 or this sequence.
Preferably, primer length is 18 ~ 27bp.The design of primer needs to consider whether the many-sides such as mispairing, expanding fragment length, temperature of reaction easily occur.
Preferably, this primer sequence is as follows:
BOS_BUB-F：5′-CAGACAAAGGTCAGGAAGTAATC-3′
BOS_BUB–R：5′-AGATGAGGGAAGAGCAGGTCTG-3′；
This primer extend to 5 ', 3 ' end or modify obtain still can the primer of sequence shown in specific amplification SEQIDNo.1.
Further, the invention provides the detection kit containing above-mentioned Auele Specific Primer.
Preferably, described test kit also comprises one or more in following primer further:
(1) primer pair I that amplification generates pig specific amplification fragment is carried out to pig genomic dna:
SUS-F：5′-GCAATGCTCCAAGGACTTAGTGA-3′
SUS-R：5′-TGTGCTCAAATAGGAAGGTTGTCA-3′；
(2) primer pair II that amplification generates Equus specific amplification fragment is carried out to Equus (horse, donkey) genomic dna:
EQUUS-F：5′-TGGCTCTGAAGATGATGAGGCTG-3′
EQUUS-R：5′-GAGGCAATGATTTTGTTCTGCGT-3′；
(3) primer pair III that amplification generates rabbit specific amplification fragment is carried out to rabbit genomic dna:
OCU-F：5′-TAGCAGTAGGGATGACAGGGTTT-3′
OCU-R：5′-GCCTTAGAGTAGGGTCTTTTTGG-3′；
(4) primer pair IV that amplification generates fox specific amplification fragment is carried out to fox genomic dna:
VULPES-F：5′-ACAGGGGAGAAAACGCTGTTC-3′
VULPES-R：5′'-GGCCTCCCCGAGATGAATC-3′；
(5) primer pair V that amplification generates dog specific amplification fragment is carried out to dog genomic dna:
CANIS-F：5′-GCGGCAAAGTTAACGGCAGT-3′
CANIS-R：5′-GGATGGGAAGCAAACTCCGA-3′；
(6) primer pair VI that amplification generates mink specific amplification fragment is carried out to mink genomic dna:
MUSTLA-F：5′-TTCGGCGCTGCCTTAATTGTC-3′
MUSTLA-R：5′'-AAGACCTGGGACCCGAGAGTT-3′；
(7) primer pair VII that amplification generates mouse specific amplification fragment is carried out to mouse (mouse, rat, hamster) genomic dna:
MUS_RAT_CRI-F：5′-CATCGTTGACAAGAAGGTGCTC-3′
MUS_RAT_CRI-R：5′-GAAAAAGCTGTACTTGGTGGGG-3′；
(8) primer pair VIII that amplification generates specific chicken amplified fragments is carried out to chicken genomic dna:
GALLUS-F：5′'-GCAAGGAGATGTAACCCAGTAAAG-3′
GALLUS-R：5′-AGAATCAATCAGAAAAATGAAGCC-3′；
(9) primer pair Ⅸ that amplification generates duck specific amplification fragment is carried out to Duck genome DNA:
ANAS-F：5′'-GTCAACGATTGCCCCGAAAC-3′
ANAS-R：5′'-GGGGACTTTGACGGCATCTT-3′。
Preferably, described test kit also comprises one or more in following reagent: Taq enzyme, dNTPs, MgCl
2, PCR damping fluid, positive control dna template, distilled water; Or one or more also comprising in following reagent: TaqDNAMasterMix, positive control dna template, distilled water.
Described positive control dna template is for comprising ox positive control dna template, and it is Bos or the Bubalus genomic DNA template of Bovidae Niu Yake;
Preferably, described positive control dna template also comprises one or more in pig, horse, donkey, mouse, rat, hamster, chicken, duck, rabbit, dog, fox or mink DNA profiling.
Further, the invention provides above-mentioned bovine primer or the application of detection kit in calf-derived Cyclospora qualification or ox source property goods detection of adulterations.
The present invention also provides the PCR detection method of several species kind derived components in the qualification of a kind of calf-derived Cyclospora and goods thereof, and its step is as described below:
1) sample gene group DNA is extracted;
2) pcr amplification:
A) carry out pcr amplification with above-mentioned bovine primer, and with cow genome group DNA profiling for positive control, take distilled water as blank;
B) with above-mentioned test kit, carry out pcr amplification, and with one or more in cow genome group DNA profiling, pig, Equus, rabbit, fox, dog, mink, mouse, chicken or duck DNA profiling for positive control, take distilled water as blank;
3) electrophoresis detection and result judgement are carried out to pcr amplification product.
Preferably, wherein step 2) reaction system of pcr amplification is as follows:
The reaction system of table 1 pcr amplification of the present invention
Described PCR response procedures is as follows:
95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 62 DEG C of annealing 30s, 72 DEG C extend 15s, 30 circulations; 4 DEG C of cooling 2min;
Result decision method is: when PCR reaction product conforms to positive control amplified production, and blank without amplified production time, judge to detect calf-derived Cyclospora in sample; When PCR reaction product is not inconsistent without amplified production or with positive control amplified production, and blank without amplified production time, judge not detect calf-derived Cyclospora in sample; If positive does not detect expection fragment, illustrate that reagent lost efficacy or misoperation; If blank and positive all detect, reagent contamination or misoperation are described.
Be to increase measuring samples DNA with above-mentioned pig, Equus, rabbit, fox, dog, mink, mouse, chicken, duck primer pair I ~ Ⅸ for adulterated inspection method, detect and whether there is corresponding amplified production, and judge whether to there is corresponding composition, judgment mode is the same.
In the present invention, detected result can present with agarose gel electrophoresis, also can detect with order-checking or other effective ways; The method extracting sample DNA can use phenol chloroform extraction method, also can use other extracting method that are generally acknowledged, that have identical effect.
The invention provides effectively, accurately, the method for reliably calf-derived Cyclospora qualification or ox source property goods detection of adulterations, whether containing calf-derived Cyclospora in the test kit energy Rapid identification sample assembled, and whether be mixed with the animal components such as pig, Equus, rabbit, fox, dog, mink, mouse, chicken, duck.Method of the present invention can use as standard detecting method in meat, feed and hide detect.Test kit of the present invention and detection method have feature that is easy, quick, comprehensive, high specificity, and cost is lower, and suitability is wide.
Accompanying drawing explanation
Fig. 1: be the detected result that the nucleotide sequence of SEQIDNo.1 has species specificity in ox.With the DNA of ox (Bos, Bubalus), sheep (sheep, goat), pig, horse, donkey, mouse (rat, mouse, hamster), cavy, chicken, duck, goose, rabbit, dog, fox, mink and recoon dog for template, increase described specific fragment, only in ox (Bos, Bubalus), amplified production is obtained, all without amplified production in non-ox sample in all test samples.Result shows that institute's amplified fragments has specificity in ox.In swimming lane, numbering is described as follows: M: be BM2000DNAMarker; 1-3: blank; 4-6: common ox; 7-9: buffalo; 10-12: mouse; 13-15: rat; 16-18: hamster; 19-21: cavy; 22-24: sheep; 25-27: goat; 28-30: pig; 31-33: horse; 34-36: donkey; 37-39: chicken; 40-42: duck; 43-45: goose; 46-48: rabbit; 49-51: fox; 52-54: dog; 55-57: mink; 58-60: recoon dog.
Fig. 2: the sensitivity technique result of polynucleotide sequence in Niu Butong DNA profiling concentration being SEQIDNo.1.Respectively with the ox DNA of 0.1ng/ μ L, 1ng/ μ L, 10ng/ μ L, 100ng/ μ L for template, the specific nucleotide acid fragment of amplification, namely the template concentrations of 1ng/ μ L has amplification, shows that Auele Specific Primer institute amplified fragments has good susceptibility.In swimming lane, numbering is described as follows: M: be BM2000DNAMarker; 1-4: blank; The template concentrations of 5-8:0.1ng/ μ L; The template concentrations of 9-12:1ng/ μ L; The template concentrations of 13-16:10ng/ μ L; The template concentrations of 17-20:100ng/ μ L.
Fig. 3: the conservative property detected result of polynucleotide sequence in multiple bodies of common ox different varieties and buffalo being SEQIDNo.1.With the DNA of holstein cow, Qinchuan Cattle, Simmental, Dabie Mountain ox, Luxi Yellow cattle, Bohai Black Cattle, Burma ox, Foochow ox, peak ox, yak and buffalo for template, increase described specific fragment, different varieties and the buffalo of all test samples and common ox all obtain specific amplification, and consistence is good, shows that institute's amplified fragments has conservative property in ox different varieties.In swimming lane, numbering is described as follows: M: be DL2000plusDNAMarker; E: blank; N: the negative control (without ox DNA) of several species mixing; 1-3: holstein cow; 4-6: Qinchuan Cattle; 7-9: Simmental; 10-12: Dabie Mountain ox; 13-15: Luxi Yellow cattle; 16-18: Bohai Black Cattle; 19-21: Burma ox; 22-24: Foochow ox; 25-27: peak ox; 28-30: yak; 31-44: buffalo.
Fig. 4: be Equus (horse, donkey), mouse (rat, mouse, hamster), pig, chicken, duck, rabbit, dog, fox and mink the distinguished sequence that increases of each Auele Specific Primer there is the detected result of species specificity in these species.Adopt above-mentioned primer pair I ~ Ⅸ, with the DNA of pig, common ox, buffalo, sheep (sheep, goat), horse, donkey, mouse (rat, mouse, hamster), cavy, chicken, duck, goose, rabbit, dog, fox, mink and recoon dog for template, increase the specific fragment of above-mentioned each species, all primers only obtain amplified production in these species, all without amplified production in non-species sample, result shows that each species primer and institute's amplified fragments have species specificity.As figure: A: Equus (horse and donkey) primer amplified result; B: mouse (rat, mouse, hamster) primer amplified result; C: pig primer amplified result; D: specific chicken primer amplification result; E: duck primer amplified result; F: rabbit primer amplified result; G: dog primer amplified result; H: fox primer amplified result; I: mink primer amplified result; .
Fig. 5: be that assembled test kit is to the detected result of beef product adulterated product in market.Detect obtain in market 29 groups of samples with test kit of the present invention, finding that there is 5 set products is adulterated product.In swimming lane, numbering is described as follows: M: be BM2000DNAMarker; E: blank; N: ox positive control; J: chicken positive control; H: fox positive control; Z: pig positive control; 1: detect chicken derived component in beef items adulterated; 2: detect fox derived component in beef items adulterated; 3-5: detect pig derived component in beef items adulterated.
Embodiment
Following examples are used for further illustrating the present invention, but should not be construed as limitation of the present invention, and under the prerequisite not deviating from the present invention's spirit and essence, the amendment make the present invention or polishing all belong to scope of the present invention.
Calf-derived Cyclospora specific detection in embodiment 1 sample
1. the preparation of sample and preservation
1.1 sampling
Gather beef sample 1g, save backup at-20 DEG C.
1.2DNA Template preparation
DNA profiling preparation adopts the conventional thick formulation of phenol chloroform (Pehanorm Brooker J, not Ritchie EF, Manny A Disi T. Molecular Cloning: A Laboratory guide [M]. the 2nd edition. Jin Dongyan, Li Mengfeng. Beijing: Science Press, 1999.465-467) or generally acknowledge, other extracting method with identical effect, these methods are all the common methods of report.
2. design of primers
The primers DNA sequences of the present embodiment is as shown in table 2 and sequence table SEQ IDNO:2 and 3.
The ox pcr amplification primer of table 2 the present invention design
Expection amplified fragments size is 223bp, and its nucleotide sequence is as shown in sequence table SEQ IDNO:1.
Experiment shows, this primer specificity is strong, in common ox and each kind of buffalo, all can obtain specific amplification, and all without object fragment amplification in other non-ox species; And the sensitivity of primer is higher, can increase when template DNA concentration is 1ng/ μ L.Extend a base and two bases with above-mentioned primer respectively to 3 ', 5 ' or modify the primer pair formed and test, experiment shows still can carry out specific amplification, from economy and net effect best with the primer table 2.
3.PCR detects
3.1 sample PCR react
3.1.1PCR reaction system is with table 1.
3.1.2 mix, add 25 μ L paraffin oils (having the PCR instrument of hot lid equipment not add), on whizzer, the centrifugal 10s of 500g ~ 3000g, then takes out PCR pipe, puts into PCR instrument.
3.1.3 carry out PCR reaction.Program is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 62 DEG C of annealing 30s, 72 DEG C extend 15s, 30 circulations; 4 DEG C of cooling 2min.；
3.1.4 reaction terminates rear taking-up PCR pipe, carries out electrophoresis detection to PCR reaction product.
3.2 contrast PCR reaction
3.2.1, while sample PCR reacts, negative control, positive control, blank are set.In each contrast PCR reaction system, removing template all the other components outer and PCR reaction conditions identical with 3.1, and feminine gender, positive control dna template concentrations also should reach sample DNA profiling concentration requirement.
3.2.2 using sheep (sheep, goat), pig, horse, donkey, mouse (rat, mouse, hamster), cavy, chicken, duck, goose, rabbit, dog, fox, mink and recoon dog genomic dna as the negative control template of PCR reaction system.
3.2.3 using ox (Bos, Bubalus) genomic dna as the positive control template of PCR reaction system.
3.2.4 using distilled water as the blank template of PCR reaction system.
The detection of 4.PCR amplified production and result judge
Weigh agarose by the mass concentration of 30g/L, add in 1 × TAE damping fluid, heating for dissolving, is mixed with agarose solution.Add 5 μ LEB solution in every 100mL agarose solution, mixing, after slightly suitable cooling, be poured on electrophoresis plate, plug pecten, after set at room temperature becomes gel, put into 1 × TAE damping fluid, take out pecten gently vertically upward.Get 12 μ LPCR products and 3 μ L sample loading buffers (take 250.0mg tetrabromophenol sulfonphthalein, add 10mL water, at room temperature dissolve 12h; Take the blue FF of 250.0mg diformazan cyanophenyl, add 10mL water dissolution; Take 50.0g sucrose, add 30mL water dissolution.The above three kinds of solution of mixing, add water and are settled to 100mL, preserve at 4 DEG C) add gel loading wells after mixing, add DNA molecular amount standard in a loading wells wherein simultaneously, switch on power and to detect after electrophoresis 15 ~ 30min under 2V/cm ~ 5V/cm condition.
After electrophoresis terminates, take out sepharose, be placed on gel imaging instrument or imaging on ultraviolet transilluminator.Judge the size of amplified band according to DNA molecular amount standard, electrophoresis result is formed electronic file and file or take pictures by photographic system.
5 interpretations of result and statement
5.1 control test interpretations of result
As shown in Figure 1, in the PCR reaction of positive control, bovine sequence is increased, and amplified fragments size is consistent with expection clip size, and in negative control and blank except primer dimer without any amplified fragments, show that PCR reaction system is working properly.
5.2 sample detection interpretation of result and statements
If 5.2.1 the specific sequence shown in ox SEQIDNo.1 is increased, and amplified fragments size is consistent with expection clip size, shows the specific sequence detecting Niu Suoshu in sample, is expressed as " detecting calf-derived Cyclospora in sample ".
If 5.2.2 the specific sequence shown in ox SEQIDNo.1 is increased, or amplified fragments size is inconsistent with expection clip size, shows the specific sequence not detecting Niu Suoshu in sample, is expressed as " not detecting calf-derived Cyclospora in sample ".
Embodiment 2 sensitivity test
Respectively with the ox DNA of 0.1ng/ μ L, 1ng/ μ L, 10ng/ μ L, 100ng/ μ L for template, according to the condition of embodiment 1, amplification SEQIDNO.1 nucleotide fragment.As shown in Figure 2, namely the template concentrations of 1ng/ μ L has amplification to result, shows that Auele Specific Primer institute amplified fragments has good susceptibility.
Embodiment 3 detects the primer sets of mixing non-calf-derived Cyclospora in the property goods of ox source
The present invention is the detection for animal derived materials, by the situation of the corresponding primer PCR amplification of each species, determines whether or contains the DNA of certain species, and then determine whether that the animal derived materials of certain species mixes.
The extraction of DNA in 1 sample
Described in the extraction of each species DNA and the same embodiment 1 of store method.
The design of 2 primers and screening
In order to find the species specific detection sequence of each thing, the present invention from planting between consistence and stability, kind the aspect such as specificity, copy number carry out the screening of distinguished sequence, by the nucleic acid sequence alignment with other species and these species different varieties, nucleotide sequence special between comparatively conservative in screening kind, species is as detection molecules.Choose the good sequence of wherein result and design respective species primer sequence respectively, and the good primer of experiment screening specificity.The PCR qualification primer system of one group of pig, ox, Equus, rabbit, fox, dog, mink, mouse, chicken, duck, is made up of following primer pair:
(1) primer pair that amplification generates pig specific amplification fragment is carried out to pig genomic dna:
SUS-F：5′-GCAATGCTCCAAGGACTTAGTGA-3′
SUS-R：5′-TGTGCTCAAATAGGAAGGTTGTCA-3′；
(2) primer pair that amplification generates sheep specific amplification fragment is carried out to ox (common ox, buffalo) genomic dna:
BOS_BUB-F：5′-CAGACAAAGGTCAGGAAGTAATC-3′
BOS_BUB-R：5′-AGATGAGGGAAGAGCAGGTCTG-3′；
(3) primer pair that amplification generates Equus specific amplification fragment is carried out to Equus (horse, donkey) genomic dna:
EQUUS-F：5'-TGGCTCTGAAGATGATGAGGCTG-3'
EQUUS-R：5'-GAGGCAATGATTTTGTTCTGCGT-3'；
(4) primer pair that amplification generates rabbit specific amplification fragment is carried out to rabbit genomic dna:
OCU-F：5’-TAGCAGTAGGGATGACAGGGTTT-3’
OCU-R：5’-GCCTTAGAGTAGGGTCTTTTTGG-3’；
(5) primer pair that amplification generates fox specific amplification fragment is carried out to fox genomic dna:
VULPES-F：5'-ACAGGGGAGAAAACGCTGTTC-3'
VULPES-R：5'-GGCCTCCCCGAGATGAATC-3'；
(6) primer pair that amplification generates dog specific amplification fragment is carried out to dog genomic dna:
CANIS-F：5’-GCGGCAAAGTTAACGGCAGT-3’
CANIS-R：5’-GGATGGGAAGCAAACTCCGA-3’；
(7) primer pair that amplification generates ermine specific amplification fragment is carried out to mink genomic dna:
MUSTLA-F：5'-TTCGGCGCTGCCTTAATTGTC-3'
MUSTLA-R：5'-AAGACCTGGGACCCGAGAGTT-3'；
(8) primer pair that amplification generates mouse specific amplification fragment is carried out to mouse (mouse, rat, hamster) genomic dna:
MUS_RAT_CRI-F：5’-CATCGTTGACAAGAAGGTGCTC-3’
MUS_RAT_CRI-R：5’-GAAAAAGCTGTACTTGGTGGGG-3’；
(9) primer pair that amplification generates specific chicken amplified fragments is carried out to chicken genomic dna:
GALLUS-F：5'-GCAAGGAGATGTAACCCAGTAAAG-3'
GALLUS-R：5'-AGAATCAATCAGAAAAATGAAGCC-3'；
(10) primer pair that amplification generates duck specific amplification fragment is carried out to Duck genome DNA:
ANAS-F：5'-GTCAACGATTGCCCCGAAAC-3'
ANAS-R：5'-GGGGACTTTGACGGCATCTT-3'。
3PCR detects
3.1 conveniently detect, and reaction system, when each primer of guarantee effectively increases and specificity is good, is unified, with above-mentioned table 1 by the present invention.
3.2 programs of carrying out PCR reaction are:
95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 62 DEG C of annealing 30s, 72 DEG C extend 15s, 30 circulations; 4 DEG C of cooling 2min.
3.3, while sample PCR reacts, arrange negative control, positive control, blank.
The species related in 3.4 tests have: common ox, buffalo, sheep (sheep, goat), pig, horse, donkey, mouse (rat, mouse, hamster), cavy, chicken, duck, goose, rabbit, dog, fox, mink and recoon dog etc.
3.5 using distilled water as the blank template of PCR reaction system.
3.6 reactions terminate rear taking-up PCR pipe, carry out electrophoresis detection: this electrophoresis process and condition are with the electrophoresis in embodiment 1 to PCR reaction product.
Gained gel imaging result as shown in Figure 4.The method carries out pcr amplification reaction with each species DNA profiling respectively with each Species-specific primer, well demonstrates validity and the specificity of each Species-specific primer.As seen from Figure 4, each Species-specific primer all well can amplify the band of the specificity size of its corresponding species, and does not all amplify object band in non-species.
Embodiment 4 one kinds detects the test kit of animal derived materials
The composition of this test kit comprises:
Primer pair (each species primer pair SEQIDNo.2 & 3 and I ~ Ⅸ each portion);
2×TaqDNAMasterMix；
Positive control dna template (common ox, buffalo, pig, horse, donkey, rabbit, fox, dog, mink, mouse, rat, hamster, chicken, each portion of Duck genome DNA profiling);
Distilled water.
This test kit-20 DEG C stores does not affect result of use in 12 months.
The application method of test kit:
1., according to following PCR reaction system application of sample, manage in the EP of 200 μ L:
2 × TaqDNAMasterMix (purchased from Ai Delai bio tech ltd, Beijing) is by Taq enzyme, dNTP mixture, the MgCl needed for PCR reaction
2and reaction buffer is pre-configured to the mixture of 2 times of concentration.
2. the pcr amplification condition loading of foundation embodiment 1 or embodiment 3.
3. reaction gets 5 μ LPCR amplified productions, the agarose gel electrophoresis (technical parameter: 2V/cm ~ 5V/cm, electrophoresis 15min ~ 30min) of 3.0% after terminating, with ethidium bromide staining, and gel imaging system detected result.
Each reaction need arrange blank and positive control, and reaction system and amplification condition are with tested sample.
Embodiment 5 one kinds detects the application of animal derived materials test kit
1. sample DNA extraction, PCR and electrophoresis method are identical with embodiment 1.
2. this detection method is applied to the detection of beef product in market, from market, buys the various beef products such as beef sausage, dried beef, beef granules, beef noodle, and record the information such as its brand, extract STb gene, carry out PCR reaction, detect true component wherein.
3. with test kit of the present invention, the 29 groups of samples (comprising dried beef, beef roll, beef sausage etc.) obtained in market are detected, it is beef that all products all indicate composition on label, result shows, wherein 24 is qualified product, but have the PCR detected result of 5 groups and food labelling different, as shown in Figure 5, be the detected result of these 5 products.Detected result shows, has 3 to be that beef pretended to be by pork in these 5 adulterated products, and 1 is ox chicken mixing meat sample, 1 ox fox mixing meat sample.
Method of the present invention is applied to food inspection and will has broad prospects.Except can detecting meat product, also can be widely used in the fields such as feed, hide goods and traditional Chinese medicine ingredients detection.
Sequence table illustrates:
SEQIDNO.1 is the nucleotide sequence of target nucleotide acid fragment, and sequence length is 223bp;
SEQIDNO.2 & 3 is the primer pairs of above-mentioned nucleotide fragments of increasing;
SEQIDNO.4 ~ 21 are primer pair I ~ Ⅹ respectively.
Claims (9)
1. bovine primer, the nucleotide sequence shown in its specific amplification SEQIDNo.1.
2. Auele Specific Primer according to claim 1, is characterized in that, this primer sequence is as follows:
BOS_BUB-F：5′-CAGACAAAGGTCAGGAAGTAATC-3′
BOS_BUB-R：5′-AGATGAGGGAAGAGCAGGTCTG-3′；
This primer extend to 5 ', 3 ' end or modify obtain still can the primer of sequence shown in specific amplification SEQIDNo.1.
3. the detection kit containing Auele Specific Primer described in claim 1 or 2.
4. detection kit according to claim 3, is characterized in that, it also comprises one or more in following primer:
(1) primer pair that amplification generates pig specific amplification fragment is carried out to pig genomic dna:
SUS-F：5′-GCAATGCTCCAAGGACTTAGTGA-3′
SUS-R：5′-TGTGCTCAAATAGGAAGGTTGTCA-3′；
(2) primer pair that amplification generates Equus specific amplification fragment is carried out to Equus genomic dna:
EQUUS-F：5'-TGGCTCTGAAGATGATGAGGCTG-3'
EQUUS-R：5'-GAGGCAATGATTTTGTTCTGCGT-3'；
(3) primer pair that amplification generates rabbit specific amplification fragment is carried out to rabbit genomic dna:
OCU-F：5′-TAGCAGTAGGGATGACAGGGTTT-3′
OCU-R：5′-GCCTTAGAGTAGGGTCTTTTTGG-3′；
(4) primer pair that amplification generates fox specific amplification fragment is carried out to fox genomic dna:
VULPES-F：5'-ACAGGGGAGAAAACGCTGTTC-3'
VULPES-R：5'-GGCCTCCCCGAGATGAATC-3'；
(5) primer pair that amplification generates dog specific amplification fragment is carried out to dog genomic dna:
CANIS-F：5′-GCGGCAAAGTTAACGGCAGT-3′
CANIS-R：5′-GGATGGGAAGCAAACTCCGA-3′；
(6) primer pair that amplification generates mink specific amplification fragment is carried out to mink genomic dna:
MUSTLA-F：5'-TTCGGCGCTGCCTTAATTGTC-3'
MUSTLA-R：5'-AAGACCTGGGACCCGAGAGTT-3'；
(7) primer pair that amplification generates mouse specific amplification fragment is carried out to musculus cdna group DNA:
MUS_RAT_CRI-F：5′-CATCGTTGACAAGAAGGTGCTC-3′
MUS_RAT_CRI-R：5′-GAAAAAGCTGTACTTGGTGGGG-3′；
(8) primer pair that amplification generates specific chicken amplified fragments is carried out to chicken genomic dna:
GALLUS-F：5'-GCAAGGAGATGTAACCCAGTAAAG-3'
GALLUS-R：5'-AGAATCAATCAGAAAAATGAAGCC-3'；
(9) primer pair that amplification generates duck specific amplification fragment is carried out to Duck genome DNA:
ANAS-F：5'-GTCAACGATTGCCCCGAAAC-3'
ANAS-R：5'-GGGGACTTTGACGGCATCTT-3'。
5. the detection kit according to claim 3 or 4, is characterized in that, it also comprises one or more in following reagent: Taq enzyme, dNTPs, MgCl
2, PCR damping fluid, positive control dna template, distilled water; Or one or more also comprising in following reagent: TaqDNAMasterMix, positive control dna template, distilled water.
6. detection kit according to claim 5, is characterized in that, described positive control dna template is Bos or the Bubalus genomic DNA template of Bovidae Niu Yake, and blank is distilled water.
7. test kit according to claim 6, is characterized in that, described positive control dna template also comprise in pig, horse, donkey, mouse, chicken, duck, rabbit, dog, fox or ermine DNA profiling one or more.
8. bovine primer according to claim 1 and 2 or the application of the detection kit described in any one of claim 3 ~ 7 in calf-derived Cyclospora qualification or ox source property goods detection of adulterations.
9. the PCR detection method of several species kind derived components in calf-derived Cyclospora qualification and goods thereof, its step is as described below:
1) sample gene group DNA is extracted;
2) pcr amplification:
A) carry out pcr amplification with the primer described in claim 1 or 2, and with cow genome group DNA profiling for positive control, take distilled water as blank;
B) with the test kit described in any one of claim 4-7, carry out pcr amplification, and with one or more in cow genome group DNA profiling, pig, Equus, rabbit, fox, dog, mink, mouse, chicken or duck DNA profiling for positive control, take distilled water as blank;
3) pcr amplification product is detected and result judge.
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