CN106148559A - The multiple PCR primer system of a kind of five kinds of animal derived materials of synchronous detecting and detection method - Google Patents
The multiple PCR primer system of a kind of five kinds of animal derived materials of synchronous detecting and detection method Download PDFInfo
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Abstract
The multiple PCR primer system of a kind of five kinds of animal derived materials of synchronous detecting and detection method, with the STb gene of measuring samples as template, utilize primer that to the multiple PCR primer system that V is formed by IV and primer, I, primer are carried out multiplexed PCR amplification experiment to III, primer to II, primer, react after terminating according to agarose gel electrophoresis result of determination；Wherein, one or more during described sample total DNA template is cattle, pig, sheep, chicken, duck species meat product.The present invention is utilized to detect the true and false of cattle,pig and sheep chicken and duck meat products and whether have adulterated, detection is quick, specificity is high, highly sensitive, can be greatly improved detection efficiency, saves the time, and save testing cost, it is particularly well-suited to the quick authentication of cattle,pig and sheep chicken and duck goods.Additionally, the multiple PCR primer system of the present invention coordinates related reagent to can be made into test kit, convenient use, provide possibility for industrialized production and application simultaneously.
Description
Technical field
The present invention relates to biology field, be specifically related to the multiple of a kind of five kinds of animal derived materials of synchronous detecting
PCR primer system and detection method.
Background technology
Animal is carried out species identification, On the Origin of Species and diversity evaluation and to animal derived from nucleic acid molecules level
Food is tested research, is the most effective means of current animal derived materials research.Molecular biology method mainly by
DNA analysis technology, determines species according to the DNA sequence in genome, and for remaining to very well through hot worked food
Area point animal origin.The hereditary information amount of DNA is more than protein, and DNA is the most stable, through the sample that autoclaving processes
Product, remain to detect the DNA fragmentation of about 300-400bp.Meanwhile, DNA is as the basis of molecular biology research, nucleic acid
Isolation and purification technology be developed the most further.Have in recent years the research much extracted about animal complete genome DNA and
How report, owing to different materials and the difference of extracting method, the genomic DNA purity of extraction and amount are the most different, select
Take suitable organization material, utilize appropriate method to extract high-quality genomic DNA, be a problem being worth inquiring into.
DNA is using its stability good after hot-working as the important evidence of qualification animal derived materials, high-quality, high-purity and knot
The acquisition of the genomic DNA that structure is complete is the premise carrying out this aspect research work, is to ensure that each quality inspection organization detection work obtains
With the essential condition being smoothed out.And round pcr based on this is extensively used to identify the animal derived materials in food,
Succeed in microorganism and animal feed detect application, used this technology to establish discriminating many animals
The method of derived component, demonstrates good application prospect, compensate for the routines such as electrophoretic techniques, immunological technique, spectral technique
Detection method sensitivity is low, detect cycle length, the meat products after deep processing process is differentiated many deficiencies such as reliability is low.Mesh
Before, the method based on detecting DNA mainly has: Standard PCR-gel electrophoresis, multiplex PCR-gel electrophoresis, random amplification
Polymorphic dna analyzes (PCR-RAPD), length polymorphism analysis (PCR-RFLP), DNA bar code (DNA barcoding), reality
Time fluorescent PCR etc..
For problem present in animal derived food, China the most also strengthens the understanding of meat safety, formulates
The method law that Law on the Prevention and Control of Infectious Diseases, " animal epidemic prevention method ", " food hygiene law ", " import-export commodity inspection method " etc. are relevant
Rule.The animal component detection method of national standard, industry standard and the provincial standard promulgated at present mainly uses routine
Qualitative PCR method and real-time fluorescence PCR method are to single animal derived qualitative detection.Wherein, based on Standard PCR-gel electrophoresis
Animal component qualitative analysis standard have 7 national standards, 4 industry standards and 2 provincial standards.Based on real-time fluorescence PCR
The animal component qualitative analysis standard of method has 3 national standards, 7 industry standards and 4 provincial standards.
But, the Standard PCR-gel electrophoresis the most promulgated and the standard method of real-time fluorescence PCR method, can only be to dynamic
Thing source property meat and meat products carry out the qualitative detection of single kind, and or the fabricated product of mixing indefinite for composition, need logical
Crossing repeated detection or Multiple detection test just can determine that, its cycle length, workload is big, cost is high.At following animal derived meat
And in the detection of meat products, several even ten several animal sources systems of the more and more not competent single treatment of this kind of detection technique
The special requirement of product, when the converted products of the biggest quantity enters merchandized handling, needs more effectively, the quick side of detection
Method.
Summary of the invention
It is an object of the invention to provide multiple PCR primer system and the detection of a kind of five kinds of animal derived materials of synchronous detecting
Method, when utilizing the method detection cattle, pig, sheep, chicken, the true and false of Duck Products and qualification whether to have adulterated, highly sensitive, quickly
And specificity is high, be greatly improved detection efficiency, save the time, and save testing cost, be particularly well-suited to cattle, pig, sheep, chicken,
The quick authentication of duck product.
In order to achieve the above object, technical scheme is as follows:
A kind of multiple PCR primer system of five kinds of animal derived materials of synchronous detecting, including following five pairs of primers pair:
(1) for cattle species-specific gene design primer to I:
Forward primer: 5 '-GATTGGACTAGCATTAGCTGCAACC-3 '；
Reverse primer: 5 '-CTTGAAGGCGTTTGAGGGGTAGTGAT-3 '.
(2) for pig species-specific gene design primer to II:
Forward primer: 5 '-GCCTAAATCTCCCTCAATGGTA-3 '；
Reverse primer: 5 '-ATGAAAGAGGCAATAGATTTTCG-3 '；
(3) for sheep species-specific gene design primer to III:
Forward primer: 5 '-CAATGAAAATAACCGTTCCTAAT-3 '；
Reverse primer: 5 '-GTAAGGTTGTACTATGAGGATCGTG-3 '；
(4) for chicken species-specific gene design primer to IV:
Forward primer: 5 '-AGCAATTCCTACATTGGACACA-3 '；
Reverse primer: 5 '-GATGATAGTATACCTGCGATTGCA-3 '；
(5) for duck species-specific gene design primer to V:
Forward primer: 5 '-CACAATCTAGCCTGGACAC-3 '；
Reverse primer: 5 '-TCGTCTTTAGTCTGGAGTT-3 '；
The multi-PCR detection method of five kinds of animal derived materials of a kind of synchronous detecting of the present invention, it includes as follows
Step:
1) with measuring samples STb gene as template, 5 weight PCR primer systems described in utilization carry out multiplexed PCR amplification experiment,
React after terminating according to agarose gel electrophoresis result of determination；Wherein, described sample total DNA template is cattle, pig, sheep, chicken, duck thing
One or more in kind of meat product；
2) result judges: measuring samples amplifies 733bp band, it is determined that positive for calf-derived Cyclospora；Measuring samples expands
Increase and 212bp band, it is determined that be positive for pig derived component；Measuring samples amplifies 468bp band, it is determined that become for sheep source property
Divide the positive；Measuring samples amplifies 133bp band, it is determined that positive for chicken derived component；Measuring samples amplifies 292bp band
, it is determined that positive for duck derived component.
Further, the PCR reaction system of described multiplexed PCR amplification experiment includes:
The specific PCR response procedures of described multiplexed PCR amplification experiment is: denaturation 5-at carrying out 90-100 DEG C successively
20min, 90-100 DEG C of degeneration 20-60s, 50-60 DEG C of annealing 30-60s, 70-74 DEG C extends 60-150s, carries out 30-60 time following
Ring, extends 5-20min, terminates after finally keeping ＞ 1min at 4-20 DEG C at 70-74 DEG C.
Preferably, the PCR reaction system of described multiplexed PCR amplification experiment includes:
Preferably, the specific PCR response procedures of described multiplexed PCR amplification experiment is: carry out denaturation at 95 DEG C successively
10min, 94 DEG C of degeneration 30s, 52 DEG C of annealing 45s, 72 DEG C extend 90s, carry out 35 circulations, extend l0min, finally exist at 72 DEG C
Terminate after keeping 1min at 4 DEG C.
It is furthermore preferred that the PCR reaction system of described multiplexed PCR amplification experiment includes:
For in all detection methods of the nucleic acid amplification of animal derived materials, the specificity of its testing result and susceptiveness
Main source by target nucleic acid sequence is affected, and therefore, the target sequence selecting target gene is key point.The present invention's is multiple
PCR system can carry out efficient amplification to five kinds of animal derived materials in same PCR system with same response procedures, and these 5 kinds are moved
Thing specific primer to be selected to it crucial.
The present invention by analyzing the heredity not conservative region gene in cattle, pig, sheep, chicken and 5 kinds of animal mitochondria DNAs of duck,
After carrying out sequencing and comparison, the specific PCR having separately designed each species according to the specific base site in each sequence draws
Thing to i.e. primer to I, primer to II, primer to III, primer to IV, primer to V, designed specific primer is to only to phase
The species STb gene template answered has amplified signal, does not has purpose amplified signal, meanwhile, the sheet of five kinds of amplified productions to other species
Section is not of uniform size, and can be separated by electrophoresis, is especially suitable for the multiplex PCR detection of cattle, pig, sheep, chicken, duck.
In multi-PRC reaction system, the too high meeting of annealing temperature causes the combination that primer and template can not be stable, impact to be expanded
Potentiation fruit.Annealing temperature is too low, causes primer and template generation non-specific binding.The present invention is directed to these 5 kinds of animal designs
The melting temperature of specific primer pair is close, and the gradient of annealing temperature is arranged on upper and lower 5 DEG C of primer theory melting temperature value
In the range of, and differ between amplified production length at more than 80bp, specificity is high.
The present invention is provided with suitable primer concentration, and each primer concentration is in 0.1-0.4 parts by volume, at multi-PRC reaction body
In system, when primer concentration is less, the amount of amplified production increases along with the increase of primer concentration, when primer concentration arrives one
During saturation value, the amount of amplified production no longer increases along with the increase of primer concentration, but is held essentially constant.When primer is too much
Time, primer and template generation non-specific binding can be caused, and between primer, form dimer, affect expanding effect.Primer
The saturation value of concentration and the amount of template are about (amount of template is the biggest, and the saturation value of primer is the highest), also with the knot of primer Yu template
Forming dimeric ability between conjunction ability, primer relevant, primer is the strongest with the binding ability of template, it is the fewest to form dimer,
Saturation value is the lowest, otherwise the highest.
Multi-PCR detection method of the present invention carries out the species of cattle, pig, sheep, chicken, duck for single species STb gene template
Detect, and described multi-PCR detection method carries out cattle and/or pig and/or sheep and/or chicken for several species mixing STb gene template
And/or the species detection of duck.Described multi-PCR detection method be particularly suited for detect cattle, pig, sheep, chicken, the true and false of duck product and
Adulteration identification.
The present invention also provides for a kind of cattle,pig and sheep chicken and duck animal derived materials containing multiple PCR primer system of the present invention
The multiple PCR detection kit of detection.
Multiple PCR detection kit of the present invention can be by PCR primer system of the present invention and related reagent group
Dress up test kit, to facilitate use.Wherein, except always during described related reagent can be heretofore described PCR reaction system
Other reagent beyond DNA profiling；The reagent in addition to sample total DNA template can also being suitable for for other, as reacted for PCR
Some conventional reagent, or the compositions etc. of conventional reagent obtained through limited number of time experiment by those skilled in the art.In addition originally
Invent and described multiple PCR detection kit also includes the basic apparatus implemented needed for the present invention.
Multiple PCR reagent kit of the present invention carries out the species inspection of cattle, pig, sheep, chicken, duck for single species STb gene template
Survey, and described multiple PCR reagent kit for several species mixing STb gene template carry out cattle and/or pig and/or sheep and/or chicken and/or
The species detection of duck.Described test kit is particularly suited for detecting cattle, pig, sheep, chicken, the true and false of duck product and Adulteration identification.
The invention has the beneficial effects as follows:
1. the primer in the multiple PCR primer system of the present invention is to the most only the target fragment of respective species being carried out specificity
Amplification, will not expand other region and will not react with other species, therefore, uses the multiple PCR primer body of the present invention
Cattle, pig, sheep, chicken, 5 animal species of duck can disposably detect in system, thus effectively shortens the detection time.
2. utilize multi-PCR detection method of the present invention, 5 kinds of animal derived materials can be differentiated through a PCR, be not required to
Every kind of animal derived materials is carried out respectively confirmation detection, substantially reduces experimental period, accelerate detection speed.The present invention
The specific PCR reaction system optimized and response procedures, use unified PCR detection method, simplify testing process, establishes fast
The detection mode that speed is efficient and specificity is high, is particularly suited for the quick authentication of cattle, pig, sheep, chicken, duck product.
3. the multiple PCR primer system of the present invention and PCR detection method have highly sensitive, high specificity, easy and simple to handle
Etc. advantage, relatively regular-PCR is the quickest, easy, can realize quick, accurate, the special inspection simultaneously to 5 kinds of animal derived materials
Survey and analyze, thus effectively identify the true and false of cattle,pig and sheep chicken and duck meat products and adulterated situation, for cattle, pig, sheep, chicken, duck meat system
The quality control of product provides the detection means of modern molecular biology.
Multiple PCR primer system the most of the present invention coordinates related reagent to make multiple PCR reagent kit, conveniently uses, with
Time be industrialized production and application provides possibility, its application prospect is fabulous.
Accompanying drawing explanation
Fig. 1 is the Different adding amount gained agarose of duck species primer in multiplexed PCR amplification system in the embodiment of the present invention 1
Gel electrophoresis figure；Wherein, 1-3: cattle 1.0 μ l, pig 1.0 μ l, sheep 1.0 μ l, chicken 1.0 μ l, duck 1.0 μ l；4-6: cattle 1.0 μ l, pig 1.0
μ l, sheep 1.0 μ l, chicken 1.0 μ l, duck 0.8 μ l；7-9: cattle 1.0 μ l, pig 1.0 μ l, sheep 1.0 μ l, chicken 1.0 μ l, duck 0.6 μ l；10-12:
Cattle 1.0 μ l, pig 1.0 μ l, sheep 1.0 μ l, chicken 1.0 μ l, duck 0.4 μ l；13-15: cattle 1.0 μ l, pig 1.0 μ l, sheep 1.0 μ l, chicken 1.0 μ
L, duck 0.2 μ l, M represent standard nucleic acid molecules amount label (1000 be DNA standard molecular weight, be from up to down followed successively by 1000bp,
700bp, 500bp, 400bp, 300bp, 200bp and 100bp).
Fig. 2 is the Different adding amount gained agarose of chicken species primer in multiplexed PCR amplification system in the embodiment of the present invention 2
Gel electrophoresis figure；Wherein, 1-3: cattle 1.0 μ l, pig 1.0 μ l, sheep 1.0 μ l, duck 0.6 μ l, chicken 1.0 μ l；4-6: cattle 1.0 μ l, pig 1.0
μ l, sheep 1.0 μ l, duck 0.6 μ l, chicken 0.8 μ l；7-9: cattle 1.0 μ l, pig 1.0 μ l, sheep 1.0 μ l, duck 0.6 μ l, chicken 0.6 μ l；10-12:
Cattle 1.0 μ l, pig 1.0 μ l, sheep 1.0 μ l, duck 0.6 μ l, chicken 0.4 μ l；13-15: cattle 1.0 μ l, pig 1.0 μ l, sheep 1.0 μ l, duck 0.6 μ
L, chicken 0.2 μ l, M represent standard nucleic acid molecules amount label (1000 be DNA standard molecular weight, be from up to down followed successively by 1000bp,
700bp, 500bp, 400bp, 300bp, 200bp and 100bp).
Fig. 3 is the Different adding amount gained agarose of pig species primer in multiplexed PCR amplification system in the embodiment of the present invention 3
Gel electrophoresis figure；Wherein, 1-3: cattle 1.0 μ l, sheep 1.0 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 1.0 μ l；4-6: cattle 1.0 μ l, sheep 1.0
μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.8 μ l；7-9: cattle 1.0 μ l, sheep 1.0 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.6 μ l；10-12:
Cattle 1.0 μ l, sheep 1.0 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.4 μ l；13-15: cattle 1.0 μ l, sheep 1.0 μ l, duck 0.6 μ l, chicken 0.6 μ
L, pig 0.2 μ l, M represent standard nucleic acid molecules amount label (1000 be DNA standard molecular weight, be from up to down followed successively by 1000bp,
700bp, 500bp, 400bp, 300bp, 200bp and 100bp).
Fig. 4 is the Different adding amount gained agarose of cattle species primer in multiplexed PCR amplification system in the embodiment of the present invention 4
Gel electrophoresis figure；Wherein, 1-3: sheep 1.0 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.6 μ l, cattle 1.0 μ l；4-6: sheep 1.0 μ l, duck 0.6
μ l, chicken 0.6 μ l, pig 0.6 μ l, cattle 0.8 μ l；7-9: sheep 1.0 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.6 μ l, cattle 0.6 μ l；10-12:
Sheep 1.0 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.6 μ l, cattle 0.4 μ l；13-15: sheep 1.0 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.6 μ
L, cattle 0.2 μ l, M represent standard nucleic acid molecules amount label (1000 be DNA standard molecular weight, be from up to down followed successively by 1000bp,
700bp, 500bp, 400bp, 300bp, 200bp and 100bp).
Fig. 5 is that in the embodiment of the present invention 5, in multiplexed PCR amplification system, sheep species primer Different adding amount gained agarose coagulates
Gel electrophoresis figure；Wherein, 1-3: sheep 1.0 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.6 μ l, cattle 1.0 μ l；4-6: sheep 1.2 μ l, duck 0.6 μ
L, chicken 0.6 μ l, pig 0.6 μ l, cattle 1.0 μ l；7-9: sheep 1.4 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.6 μ l, cattle 1.0 μ l；10-12:
Sheep 1.6 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.6 μ l, cattle 1.0 μ l；13-15: sheep 1.8 μ l, duck 0.6 μ l, chicken 0.6 μ l, pig 0.6 μ
L, cattle 1.0 μ l, M represent standard nucleic acid molecules amount label (1000 be DNA standard molecular weight, be from up to down followed successively by 1000bp,
700bp, 500bp, 400bp, 300bp, 200bp and 100bp).
Detailed description of the invention
Below in conjunction with specific embodiments and the drawings, technical scheme is described in further detail.
Embodiment 1
Under the different primers addition of duck species, with cattle, pig, sheep, chicken, the STb gene sample mixed in equal amounts of 5 kinds of animals of duck
After, being diluted to final DNA concentration is 0.5ng/ μ l, as sample template, with cattle, pig, sheep, chicken, duck specific primer many
Weight PCR system and reaction condition, expand the genome of cattle,pig and sheep chicken and duck respectively, and each reaction is repeated 3 times.Specifically comprise the following steps that
1) process of mixed in equal amounts STb gene sample: by cattle, pig, sheep, chicken, 5 kinds of animals of duck STb gene sample according to equivalent
After mixing, being diluted to final DNA concentration is 0.5ng/ μ l, as sample template, and primer is diluted to 10 μm ol/L；
2) preparation of reaction system: add following each reactive component in PCR light-wall pipe, reaction system 50 μ l/ manages.Double steamings
Water 28-29.6 μ l, the Taq enzyme 2 μ l of dNTPs 4 μ l, the 2.5U/ μ l of l0 × PCR reaction buffer 5 μ l, 2.5mM, STb gene template
1 μ l, 10 μMs of primers to I, primer to II, primer to III, primer to IV, primer to the forward primer of V and reverse primer, cattle 1.0
μ l, pig 1.0 μ l, sheep 1.0 μ l, chicken 1.0 μ l, duck 1.0 μ l, 0.8 μ l, 0.6 μ l, 0.4 μ l, 0.2 μ l, wherein Taq enzyme is conventional Taq
Enzyme (sees the Taq enzyme description of Tiangen company).
3) formulation of PCR response procedures: carry out denaturation 10min at 95 DEG C, 94 DEG C of degeneration 30s, 60 DEG C of annealing successively
45s, 72 DEG C extend 90s, carry out 35 circulations, extend l0min, terminate, obtain PCR after finally keeping 1min at 4 DEG C at 72 DEG C
Product.
4) 2wt% agarose gel electrophoresis: by 3) in gained PCR product carry out 2wt.% agarose gel electrophoresis,
Adding ethidium bromide (EB) in gel to dye, electrophoresis 30 minutes under 120V the most again, when bromophenol blue band migration is to gel
Electrophoresis, then gel imaging is stopped during edge.Gained gel imaging result is as shown in Figure 1.
As shown in Figure 1, in certain multiplex PCR system and amplification condition, the primer addition of the different duck species of change,
When the consumption of duck is 0.4-0.8 μ l (0.08-0.16 parts by volume), all can amplify its homologue from STb gene hybrid template
Kind the band of specificity size, demonstrate in the PCR system of the different primers addition of different duck species and amplification condition each
The effectiveness of Species-specific primer and specificity.
Embodiment 2
Under the different primers addition of chicken species, with cattle, pig, sheep, chicken, the STb gene sample mixed in equal amounts of 5 kinds of animals of duck
After, being diluted to final DNA concentration is 50ng/ μ l, as sample template, with cattle, pig, sheep, chicken, specific primer multiple of duck
PCR system and reaction condition, expand the genome of cattle,pig and sheep chicken and duck respectively, and each reaction is repeated 3 times.Specifically comprise the following steps that
(1) process of mixed in equal amounts STb gene sample: by cattle, pig, sheep, chicken, 5 kinds of animals of duck STb gene sample according to equivalent
After mixing, being diluted to final DNA concentration is 50ng/ μ l, as sample template, and primer is diluted to 10 μm ol/L；
(2) preparation of reaction system: add following each reactive component in PCR light-wall pipe, reaction system 50 μ l/ manages.Double
Steam water 28.8-30.4 μ l, the Taq enzyme 2 μ l of dNTPs4 μ l, the 2.5U/ μ l of l0 × PCR reaction buffer 5 μ l, 2.5mM, STb gene
Template 1 μ l, 10 μMs of primers to I, primer to II, primer to III, primer to IV, primer to the forward primer of V and reverse primer,
Cattle 1.0 μ l, pig 1.0 μ l, sheep 1.0 μ l, duck 0.6 μ l, chicken 1.0 μ l, 0.8 μ l, 0.6 μ l, 0.4 μ l, 0.2 μ l, wherein Taq enzyme is normal
Rule Taq enzyme (seeing the Taq enzyme description of Tiangen company).
(3) formulation of PCR response procedures: carry out denaturation 10min at 95 DEG C, 94 DEG C of degeneration 30s, 56 DEG C of annealing successively
50s, 72 DEG C extend 90s, carry out 45 circulations, extend l0min, terminate, obtain PCR after finally keeping 1min at 4 DEG C at 72 DEG C
Product.
(4) 2wt% agarose gel electrophoresis: by 3) in gained PCR product carry out 2wt.% agarose gel electrophoresis,
Adding ethidium bromide (EB) in gel to dye, electrophoresis 30 minutes under 120V the most again, when bromophenol blue band migration is to gel
Electrophoresis, then gel imaging is stopped during edge.Gained gel imaging result is as shown in Figure 2.
As shown in Figure 2, in certain multiplex PCR system and amplification condition, the primer addition of the different chicken species of change,
When the consumption of chicken is 0.2-1.0 μ l, (0.04-0.20 parts by volume) all can amplify its corresponding species from STb gene hybrid template
The band of specificity size, demonstrate each thing in the PCR system of the different primers addition of different chicken species and amplification condition
The effectiveness of species-specific primer and specificity.
Embodiment 3
Under the different primers addition of pig species, with cattle, pig, sheep, chicken, the STb gene sample mixed in equal amounts of 5 kinds of animals of duck
After, being diluted to final DNA concentration is 100ng/ μ l, as sample template, with cattle, pig, sheep, chicken, duck specific primer many
Weight PCR system and reaction condition, expand the genome of cattle,pig and sheep chicken and duck respectively, and each reaction is repeated 3 times.Specifically comprise the following steps that
(1) process of mixed in equal amounts STb gene sample: by cattle, pig, sheep, chicken, 5 kinds of animals of duck STb gene sample according to equivalent
After mixing, being diluted to final DNA concentration is 100ng/ μ l, as sample template, and primer is diluted to 10 μm ol/L；
(2) preparation of reaction system: add following each reactive component in PCR light-wall pipe, reaction system 50 μ l/ manages.Double
Steam water 29.6-31.2 μ l, the Taq enzyme 2 μ l of dNTPs4 μ l, the 2.5U/ μ l of l0 × PCR reaction buffer 5 μ l, 2.5mM, STb gene
Template 1 μ l, 10 μMs of primers to I, primer to II, primer to III, primer to IV, primer to the forward primer of V and reverse primer cattle
1.0 μ l, chicken 0.6 μ l, sheep 1.0 μ l, duck 0.6 μ l, pig 1.0 μ l, 0.8 μ l, 0.6 μ l, 0.4 μ l, 0.2 μ l, wherein Taq enzyme is conventional
Taq enzyme (sees the Taq enzyme description of Tiangen company).
(3) formulation of PCR response procedures: carry out denaturation 10min at 95 DEG C, 94 DEG C of degeneration 50s, 64 DEG C of annealing successively
50s, 72 DEG C extend 90s, carry out 45 circulations, extend 10min, terminate, obtain PCR after finally keeping 1min at 4 DEG C at 72 DEG C
Product.
(4) 2wt% agarose gel electrophoresis: by 3) in gained PCR product carry out 2wt.% agarose gel electrophoresis,
Adding ethidium bromide (EB) in gel to dye, electrophoresis 30 minutes under 120V the most again, when bromophenol blue band migration is to gel
Electrophoresis, then gel imaging is stopped during edge.Gained gel imaging result is as shown in Figure 3.
From the figure 3, it may be seen that in certain multiplex PCR system and amplification condition, the primer addition of the different pig species of change,
When the consumption of pig is 0.4-0.8 μ l, (0.08-0.16 parts by volume) all can amplify its corresponding species from STb gene hybrid template
The band of specificity size, demonstrate each thing in the PCR system of the different primers addition of different pig species and amplification condition
The effectiveness of species-specific primer and specificity.
Embodiment 4
Under the different primers addition of cattle species, with cattle, pig, sheep, chicken, the STb gene sample mixed in equal amounts of 5 kinds of animals of duck
After, being diluted to final DNA concentration is 150ng/ μ l, as sample template, with cattle, pig, sheep, chicken, duck specific primer many
Weight PCR system and reaction condition, expand the genome of cattle,pig and sheep chicken and duck respectively, and each reaction is repeated 3 times.Specifically comprise the following steps that
(1) process of mixed in equal amounts STb gene sample: by cattle, pig, sheep, chicken, 5 kinds of animals of duck STb gene sample according to equivalent
After mixing, being diluted to final DNA concentration is 150ng/ μ l, as sample template, and primer is diluted to 10 μm ol/L；
(2) preparation of reaction system: add following each reactive component in PCR light-wall pipe, reaction system 50 μ l/ manages.Double
Steam water 30.4-32 μ l, the Taq enzyme 2 μ l of dNTPs4 μ l, the 2.5U/ μ l of l0 × PCR reaction buffer 5 μ l, 2.5mM, STb gene mould
Plate 1 μ l, 10 μMs of primers to I, primer to II, primer to III, primer to IV, primer to the forward primer of V and reverse primer, pig
0.6 μ l, chicken 0.6 μ l, sheep 1.0 μ l, duck 0.6 μ l, cattle 1.0 μ l, 0.8 μ l, 0.6 μ l, 0.4 μ l, 0.2 μ l, wherein Taq enzyme is conventional
Taq enzyme (sees the Taq enzyme description of Tiangen company).
(3) formulation of PCR response procedures: carry out denaturation 10min at 95 DEG C, 94 DEG C of degeneration 30s, 58 DEG C of annealing successively
35s, 72 DEG C extend 90s, carry out 50 circulations, extend 10min, terminate, obtain PCR after finally keeping 1min at 4 DEG C at 72 DEG C
Product.
(4) 2wt% agarose gel electrophoresis: by 3) in gained PCR product carry out 2wt.% agarose gel electrophoresis,
Adding ethidium bromide (EB) in gel to dye, electrophoresis 30 minutes under 120V the most again, when bromophenol blue band migration is to gel
Electrophoresis, then gel imaging is stopped during edge.Gained gel imaging result is as shown in Figure 4.
As shown in Figure 4, in certain multiplex PCR system and amplification condition, the primer addition of the different cattle species of change,
When the consumption of cattle is 0.4-1.0 μ l, (0.08-0.20 parts by volume) all can amplify its corresponding species from STb gene hybrid template
The band of specificity size, demonstrate each thing in the PCR system of the different primers addition of different cattle species and amplification condition
The effectiveness of species-specific primer and specificity.
Embodiment 5
Under the different primers addition of sheep species, with cattle, pig, sheep, chicken, the STb gene sample mixed in equal amounts of 5 kinds of animals of duck
After, being diluted to final DNA concentration is 200ng/ μ l, as sample template, with cattle, pig, sheep, chicken, duck specific primer many
Weight PCR system and reaction condition, expand the genome of cattle,pig and sheep chicken and duck respectively, and each reaction is repeated 3 times.Specifically comprise the following steps that
(1) process of mixed in equal amounts STb gene sample: by cattle, pig, sheep, chicken, 5 kinds of animals of duck STb gene sample according to equivalent
After mixing, being diluted to final DNA concentration is 200ng/ μ l, as sample template, and primer is diluted to 10 μm ol/L；
(2) preparation of reaction system: add following each reactive component in PCR light-wall pipe, reaction system 50 μ l/ manages.Double
Steam water 29.6-31.2 μ l, the Taq enzyme 2 μ l of dNTPs4 μ l, the 2.5U/ μ l of l0 × PCR reaction buffer 5 μ l, 2.5mM, STb gene
Template 1 μ l, 10 μMs of primers to I, primer to II, primer to III, primer to IV, primer to the forward primer of V and reverse primer,
Pig 0.6 μ l, chicken 0.6 μ l, cattle 0.6 μ l, duck 0.6 μ l, sheep 1.0 μ l, 1.2 μ l, 1.4 μ l, 1.6 μ l, 1.8 μ l, wherein Taq enzyme is normal
Rule Taq enzyme (seeing the Taq enzyme description of Tiangen company).
(3) formulation of PCR response procedures: carry out denaturation 10min at 95 DEG C, 94 DEG C of degeneration 30s, 56 DEG C of annealing successively
50s, 72 DEG C extend 90s, carry out 35 circulations, extend 10min, terminate, obtain PCR after finally keeping 1min at 4 DEG C at 72 DEG C
Product.
(4) 2wt% agarose gel electrophoresis: by 3) in gained PCR product carry out 2wt.% agarose gel electrophoresis,
Adding ethidium bromide (EB) in gel to dye, electrophoresis 30 minutes under 120V the most again, when bromophenol blue band migration is to gel
Electrophoresis, then gel imaging is stopped during edge.Gained gel imaging result is as shown in Figure 5.
As shown in Figure 5, in certain multiplex PCR system and amplification condition, the primer addition of the different sheep species of change,
When the consumption of sheep is 1.0-1.8 μ l, (0.20-0.36 parts by volume) all can amplify its corresponding species from STb gene hybrid template
The band of specificity size, demonstrate each thing in the PCR system of the different primers addition of different sheep species and amplification condition
The effectiveness of species-specific primer and specificity.
Last it should be noted that, above example is only in order to illustrate technical scheme and unrestricted, although ginseng
According to preferred embodiment, the present invention is described in detail, it will be understood by those within the art that, can be to invention
Technical scheme is modified or equivalent, and without deviating from the spirit and scope of technical solution of the present invention, it all should be contained
In scope of the presently claimed invention.
Claims (10)
1. a multiple PCR primer system for five kinds of animal derived materials of synchronous detecting, it includes following five pairs of primers pair:
1) for cattle species-specific gene design primer to I:
Forward primer: 5 '-GATTGGACTAGCATTAGCTGCAACC-3 '；
Reverse primer: 5 '-CTTGAAGGCGTTTGAGGGGTAGTGAT-3 '；
2) for pig species-specific gene design primer to II:
Forward primer: 5 '-GCCTAAATCTCCCTCAATGGTA-3 '；
Reverse primer: 5 '-ATGAAAGAGGCAATAGATTTTCG-3 '；
3) for sheep species-specific gene design primer to III:
Forward primer: 5 '-CAATGAAAATAACCGTTCCTAAT-3 '；
Reverse primer: 5 '-GTAAGGTTGTACTATGAGGATCGTG-3 '；
4) for chicken species-specific gene design primer to IV:
Forward primer: 5 '-AGCAATTCCTACATTGGACACA-3 '；
Reverse primer: 5 '-GATGATAGTATACCTGCGATTGCA-3 '；
5) for duck species-specific gene design primer to V:
Forward primer: 5 '-CACAATCTAGCCTGGACAC-3 '；
Reverse primer: 5 '-TCGTCTTTAGTCTGGAGTT-3 '.
2. a multi-PCR detection method for five kinds of animal derived materials of synchronous detecting, it comprises the steps:
1) with the STb gene of measuring samples as template, multiple PCR primer system as claimed in claim 1 is utilized to carry out multiplex PCR
Amplification experiment, after reaction terminates, according to agarose gel electrophoresis result of determination；Wherein, the STb gene template of described measuring samples is
One or more in cattle, pig, sheep, chicken, duck species meat product；
2) result judges: measuring samples amplifies 733bp band, it is determined that positive for calf-derived Cyclospora；Measuring samples amplifies
212bp band, it is determined that positive for pig derived component；Measuring samples amplifies 468bp band, it is determined that for sheep derived material sun
Property；Measuring samples amplifies 133bp band, it is determined that positive for chicken derived component；Measuring samples amplifies 292bp band,
It is judged to that duck derived component is positive.
The multi-PCR detection method of five kinds of animal derived materials of synchronous detecting the most according to claim 2, its feature exists
In, the PCR reaction system of described multiplexed PCR amplification experiment includes:
4. according to the multi-PCR detection method of the five kinds of animal derived materials of synchronous detecting described in Claims 2 or 3, its feature
Being, the PCR reaction system of described multiplexed PCR amplification experiment includes:
5. according to the multi-PCR detection method of the five kinds of animal derived materials of synchronous detecting described in Claims 2 or 3, its feature
Being, the PCR reaction system of described multiplexed PCR amplification experiment includes:
The multi-PCR detection method of five kinds of animal derived materials of synchronous detecting the most according to claim 2, its feature exists
In, the specific PCR response procedures of described multiplexed PCR amplification experiment is: denaturation 5-20min at carrying out 90-100 DEG C successively, 90-
100 DEG C of degeneration 20-60s, 50-64 DEG C of annealing 30-60s, 70-74 DEG C extends 60-150s, carries out 30-60 time and circulates, 70-74 DEG C
Lower extension 5-20min, terminates after finally keeping ＞ 1min at 4-20 DEG C.
7. according to the multi-PCR detection method of the five kinds of animal derived materials of synchronous detecting described in claim 2 or 5, its feature
Being, the specific PCR response procedures of described multiplexed PCR amplification experiment is: carry out denaturation 10min at 95 DEG C, 94 DEG C of changes successively
Property 30s, 60 DEG C annealing 45s, 72 DEG C extend 90s, carry out 40 times circulation, at 72 DEG C extend l0min, finally at 4 DEG C keep
Terminate after 1min.
8. the application in detecting for species of the multi-PCR detection method as described in any one of claim 2-7, described thing
Planting detection object is one or more in cattle, pig, sheep, chicken and duck.
9. the multi-PCR detection method as described in any one of claim 2-7 in cattle, pig, sheep, chicken and the true and false of duck product and is mixed
Application in false qualification.
10. the cattle,pig and sheep chicken and duck animal derived materials detection containing multiple PCR primer system as claimed in claim 1 is many
Weight PCR detection kit.
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