CN107699613A - The multiple PCR method and kit of a kind of 5 kinds of animal derived materials of synchronous detection - Google Patents
The multiple PCR method and kit of a kind of 5 kinds of animal derived materials of synchronous detection Download PDFInfo
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Abstract
The invention discloses a kind of multiple PCR method of 5 kinds of animal derived materials of synchronous detection, this method comprises the following steps：The DNA of animal tissue to be checked is extracted, and multiplexed PCR amplification is carried out to it；Gel electrophoresis analysis are carried out to the product of multiplex PCR, according to PCR primer stripe size and whether there is judge whether contain 5 kinds of animal derived materials in sample.The PCR method of five kinds of animal derived materials of synchronous identification of the present invention has the advantages that simple to operate, high specificity, high sensitivity, cost-effective and detection time, more conventional PCR can be realized to being detected while five kinds of animal derived materials, effectively to differentiate the true and false offer technical support of meat products.
Description
Technical field
It is dynamic more particularly to a kind of synchronous 5 kinds of detection the present invention relates to meat products molecular biology identification technique field
The multiple PCR method and kit of thing derived component.
Background technology
Due to using a large amount of flavor enhancements and toner in adulterated product, only from the color and luster and smell of product, pass through tradition
Sense organ judges to be difficult to judge whether adding other animal derived materials in food.In order to strictly carry out food security prison
Pipe, it is severe to hit the illegal activities such as adulterated sell-fake-products of meat products in food, in developing food products animal derived materials source differentiate and
Trace-back technique is particularly important.The detection of animal derived materials is a kind of adulterated means of important identification, is advantageous to safeguard
Consumer's interests, people's life safety is ensured, avoids the appearance of fake and forged food, skill is provided to hit such illegal activities
Art supports, and is also beneficial to the sound development of meat industry.Meanwhile differentiate that animal derived materials are some because food is asked for solving
National conflict caused by topic and dispute are all particularly significant.Therefore, animal derived materials in fast and accurately food are established to detect
Method has become key subjects of the pendulum in face of researcher.
The content of the invention
The purpose of the present invention first consists in the multiple PCR method for providing a kind of synchronous 5 kinds of animal derived materials of detection and examination
Agent box, this method comprise the following steps：
(1) DNA of animal tissue to be checked is extracted, and multiplexed PCR amplification is carried out to it, PCR reaction systems are：5×PCR
Buffer 10 μ L, dNTP Mix 0.2mmol/L, Taq archaeal dna polymerases 2.5U, MgCl22mmol/L, primer proportioning is ox：
Sheep：Chicken：Pig：Duck=2:1:1:1:0.5, with dd H2O supplies system to 50 μ L.Ox in end reaction system, sheep, chicken, pig and
The positive and negative primer concentration of duck is respectively 1mmol/L, 0.5mmol/L, 0.5mmol/L, 0.5mmol/L and 0.25mmol/L.It is more
Weight PCR response procedures are as follows：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 40s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 are followed
Ring；72 DEG C of extension 5min；
(2) gel electrophoresis analysis are carried out to the product of multiplex PCR, according to PCR primer stripe size and whether there is judgement sample
In whether contain 5 kinds of animal derived materials, wherein obtaining purpose band size after ox, sheep, chicken, pig and duck amplification is respectively：
493bp、302bp、400bp、277bp、239bp；
Wherein described primer is：
Ox：Sense primer：SEQ NO.1 5′-tagcaattgccatagtccac c-3′
Anti-sense primer：SEQ NO.2 5′-taggatgaggagaaagtataggaca-3′；
Sheep：Sense primer：SEQ NO.3 5′-cgccatagttcacctactct tc-3′
Anti-sense primer：SEQ NO.4 5′-ttactaggactaggattgag aggat-3′；
Chicken：Sense primer：SEQ NO.5 5′-cttccttcacatcggacgag-3′
Anti-sense primer：SEQ NO.6 5′-gggtgagtatgagagttaag cc-3′；
Pig：Sense primer：SEQ NO.7 5′-aatttttggggatgcttaga ct-3′
Anti-sense primer：SEQ NO.8 5′-tattttgggaggttattgtg ttgta-3′；
Duck：Sense primer：SEQ NO.9 5′-cgaggcttctactacggc-3′
Anti-sense primer：SEQ NO.10 5′-gggtgattcctgcgatta-3′
Above-mentioned primer is the gene order that ox, sheep, chicken, pig and duck mtDNA are searched in GeneBank, is used
The special strategy of forward primer, reverse primer, will after ensuring to belong between interior sequence by Blast functions and having higher homology
Sequence is imported into DNAMAN softwares, and the otherness of genome, on the basis of this otherness, uses Primer between analysis category
What Premier 6.0 was designed.
Present invention also offers a kind of kit of 5 kinds of animal derived materials of synchronous detection, the kit contains in following
Hold：
PCR A liquid：PCR Buffer, dNTP Mix, Taq archaeal dna polymerases, MgCl2, five pairs of primer and distilled water；
PCR B liquid：Contain positive DNA profiling, including ox, sheep, chicken, pig, the genomic DNA of duck；
Operation instructions.
Compared with prior art, the PCR method of synchronous five kinds of animal derived materials of identification of the invention and kit have
Following beneficial effect：
The technology for the identification animal derived materials being previously reported by is more using universal primer or using identical sense primer, downstream
Primer is different, and species specificity is not high.The method of the present invention is for the animal derived materials such as ox, sheep, chicken, pig and duck, difference
Design 5 pairs of specific primers of synthesis, inter-species specificity is high, mutual no cross reaction, can accurately differentiate five kinds it is animal derived
Composition.Individually every kind of animal component need not be detected, greatly save detection time and cost, improve detection
Efficiency.
The method and kit of the present invention has simple to operate, high specificity, high sensitivity, cost-effective and detection time
The advantages that, more conventional PCR can be realized to being detected while five kinds of animal derived materials, effectively to differentiate that the true and false of meat products carries
For technical support.
The present invention tightly aims at animal derived in food using the illegal adulterated problem of animal derived materials in food to be oriented to
The cutting edge technology of composition detection, foundation can differentiate simultaneously beef in food, mutton, chicken, pork and duck etc. it is animal derived into
Animal derived materials in delicatessen food are further tested and analyzed, are by the sixfold PCR detection techniques divided with the technology
Ensure that China's Safety of Food Quality provides key technology support.
Brief description of the drawings
The present invention is described in more detail with reference to the accompanying drawings and examples
Fig. 1 is the specificity verification result of ox primer.
Fig. 2 is the specificity verification result of sheep primer.
Fig. 3 is the specificity verification result of chicken primer.
Fig. 4 is the specificity verification result of pig primer.
Fig. 5 is the specificity verification result of duck primer.
Indicate in wherein Fig. 1-5：
M：2kb DNA marker；1st, Taihu pigs；2nd, ternary pigs；3rd, duroc；4th, good miscellaneous pig；5th, the black pig in Yimeng；6、
Nanyang cattle；7th, Luxi Yellow cattle；8 and ox；9th, Anduo County ox；10th, the white goat in Chongming；11st, boer goat；12nd, Sanhuang chicken；13rd, it is black
Bone chicken；14th, careless duck；15th, donkey；16th, rabbit；17th, dog.
Fig. 6 is the amplification of the heavy PCR reaction systems of substance PCR and five.
Note：M：2kb DNA marker；1st, DNA of the ox primer amplification containing ox, sheep, chicken, duck, pig mixes template；2nd, sheep is drawn
DNA of the thing amplification containing ox, sheep, chicken, duck, pig mixes template；3rd, DNA of the chicken primer amplification containing ox, sheep, chicken, duck, pig mixes mould
Plate；4th, DNA of the pig primer amplification containing ox, sheep, chicken, duck, pig mixes template；5th, the amplification of ox primer is containing ox, sheep, chicken, duck, pig
DNA mixes template；6th, ox, sheep, chicken, pig duck, the DNA profiling of primer amplification ox；7th, ox, sheep, chicken, pig duck, primer expand sheep
DNA profiling；8th, ox, sheep, chicken, pig duck, the DNA profiling of primer amplification chicken；9th, ox, sheep, chicken, pig duck, the DNA of primer amplification pig
Template；10th, ox, sheep, chicken, pig, the DNA profiling of duck primer amplification duck.
Fig. 7 is the sample that different animals derived component is mixed with using PCR amplifications.
Note：M：2kb DNA marker；1st, Luxi Yellow cattle：Chongming Aries：Sanhuang chicken=1:1:1；2nd, Sanhuang chicken：Chongming
Bai Shanyang=1:1；3rd, Luxi Yellow cattle：Chongming Aries=1:1；4-6 is respectively Sanhuang chicken, the white goat of Luxi Yellow cattle and Chongming
Positive sample.
Fig. 8 is the sample that different animals derived component is mixed with using PCR amplifications.
Note：M：2kb DNA marker；1st, Luxi Yellow cattle：The white goat in Chongming：Sanhuang chicken：Taihu pigs：Careless duck=1:1:1:
1:0.5；2nd, Luxi Yellow cattle：The white goat in Chongming：Sanhuang chicken：Taihu pigs：Careless duck=0:1:0:0:0；3rd, Luxi Yellow cattle：Chongming is white
Goat：Sanhuang chicken：Careless duck=1:1:1:1.
Embodiment
The present invention is further illustrated for example given below, it is accordingly required in particular to is pointed out that all similar replacements
Or change, it is regarded as being included in the present invention.
The source of material used, reagent and instrument is as follows in the following example：
Collect totally 17 kinds of the livestock and poultry sample of different cultivars.Wherein, Luxi Yellow cattle and ox, Nanyang cattle, Anduo County yak, Taihu Lake
Pig, ternary pigs, duroc, good miscellaneous pig, the meat of the black pig in Yimeng are provided by Chinese Academy of Agricultural Sciences's matter mark.The white goat in Chongming
Shanghai plant is come from the meat of Sanhuang chicken.Boer goat, white feather chicken, black-bone chicken, hen, careless duck, the meat of rabbit and dog meats
Purchased from the supermarket of District of Shanghai.
Paramagnetic particle method genome DNA extraction kit：Sangon Biotech (Shanghai) Co., Ltd.
20mg/mL protein kinase Ks Sangon Biotech (Shanghai) Co., Ltd.
10mg/mL ribonuclease A solution：Sangon Biotech (Shanghai) Co., Ltd.
DNA Marker D：Sangon Biotech (Shanghai) Co., Ltd.
4S Red Plus nucleic acid stainings agent (the 10000X aqueous solution)：Sangon Biotech (Shanghai) Co., Ltd.
Agarose H：Sangon Biotech (Shanghai) Co., Ltd.
PCR instrument：BIO-RAD companies of the U.S.
Electronic thermostatic water-bath：Shanghai Yarong Biochemical Instrument Plant
Electrophoresis apparatus：BIO-RAD companies of the U.S.
Gel imager：BIO-RAD companies of the U.S.
Embodiment 1：Animal tissue DNA extraction
(1) salt extraction method takes sample 50mg liquid nitrogen grindings to be transferred in 1.5mL centrifuge tubes into powder, add 400 μ L
Lysate I (10mM Tris-HCl, pH 8.0,2mM EDTA, pH 8.0,0.4M NaCl) and the SDS of 80 μ L 10%.Acutely shake
After dynamic, 2 μ L 20mg/mL protein kinase Ks are added, 1h is incubated under the conditions of 65 DEG C.After vortex, 20 μ L 10mg/mL are added
Rnase enzymes, 10min is incubated at 65 DEG C.Add 300 μ L 5M NaCl, vortex 30s.10min is centrifuged under 10000rpm, on
Clear liquid is transferred to new centrifuge tube, adds equivalent isopropanol, -20 DEG C of refrigerator 30min are placed in after vortex, under 15000rpm from
After heart 5min, supernatant is removed.The ethanol waters of 700 μ L 70% are added into centrifuge tube, mixing that suction is beaten or point shakes,
After centrifuging 5min under 15000rpm, supernatant is removed.The step is repeated, room temperature is uncapped dry 15-20min or 55 DEG C of constant temperature
The dry 5-7min that uncapped in case is remained to no liquid in pipe.100 μ L ddH are added into centrifuge tube2O, acutely concussion obtain
Animal genome DNA.
(2) phenol-chloroform extraction method takes sample 50mg liquid nitrogen grindings to be transferred in 1.5mL centrifuge tubes, add into powder
500 μ L lysates II (50mM Tris-HCl pH 8.0,10mM EDTA pH 8.0,0.1M NaCl), 30 μ L 10%SDS and
2 μ L 20mg/mL protein kinase Ks, 1h is incubated under the conditions of 50 DEG C.After vortex, the mg/mL Rnase enzymes of 20 μ L 10 are added, 50
10min is incubated at DEG C.Isometric phenol/chloroform/isoamyl alcohol (24/24/1, v/v/v) is added into reaction system.It is vortexed
3min, 5min is centrifuged under 10000rpm.Supernatant is transferred to new centrifuge tube, adds isometric chloroform/isoamyl alcohol (24/
1, v/v), acutely after concussion, 10000rpm centrifugations 5min.Take supernatant to be transferred to new test tube, add the second that equivalent ice bath is crossed
Alcohol, after 10000rpm centrifuges 5min, remove supernatant.DNA is rinsed using 70% ethanol solution, supernatant is removed after centrifugation,
Remove supernatant.100 μ L ddH of residue2O dissolves, that is, obtains animal DNA.
(3) using magnetic bead genome DNA extraction RNA isolation kit extraction DNA, comprise the following steps that：Take sample 50mg liquid
Nitrogen grind into powder is added in 1.5mL centrifuge tubes, and 400 μ L Buffer MACL, 200 μ L are then added into centrifuge tube
Buffer MCL and 20 μ L protein kinase Ks, concussion shake up, 65 DEG C of water-bath 20-30min.Take out centrifuge tube, 12000 rpm centrifugations
5-10min, take the μ L of supernatant 500 to move to new 1.5mL centrifuge tubes, the DNA as no RNA need to be obtained, 20 can be added in supernatant
μ L ribonuclease As (10mg/mL) solution, mix, room temperature places 5min.Added into centrifuge tube 500 μ L Buffer MA and
15 μ L MagicMag Beads, mixing of shaking is blown and beaten or put repeatedly, is stored at room temperature 1 min.Centrifuge tube is placed in 30s on magnetic frame,
Treat that MagicMag Beads are drawn to after tube wall completely, supernatant is abandoned in suction, and centrifuge tube is taken out from magnetic frame.Into centrifuge tube
The ethanol waters of 700 μ L 70% are added, mixing that suction is beaten or point shakes, centrifuge tube is placed in 30s on magnetic frame, treats MagicMag
Beads is drawn to after tube wall completely, and supernatant is abandoned in suction, and centrifuge tube is taken out from magnetic frame.Repeat step 5 once, uncap by room temperature
The dry 5-7min that uncaps in 15-20min or 55 DEG C of insulating box is dried to remain to no liquid in pipe.100 are added into centrifuge tube
μ L TE Buffer (pH 8.0), 65 DEG C of water-bath 5-10min, or mix.Take out centrifuge tube and be placed in 30s on magnetic frame, treat
MagicMag Beads are drawn to after tube wall completely, careful Aspirate supernatant obtains genomic DNA to new centrifuge tube.
Example 2：Substance and multi-PRC reaction
(1) design primer and synthesized by Sangon Biotech (Shanghai) Co., Ltd., primer sequence is as follows：
Ox：Sense primer：SEQ No.1 5′-tagcaattgccatagtccacc-3′
Anti-sense primer：SEQ No.2 5′-taggatgaggagaaagtataggaca-3′；
Sheep：Sense primer：SEQ No.3 5′-cgccatagttcacctactcttc-3′
Anti-sense primer：SEQ No.4 5′-ttactaggactaggattgagaggat-3′；
Chicken：Sense primer：SEQ No.5 5′-cttccttcacatcggacgag-3′
Anti-sense primer：SEQ No.6 5′-gggtgagtatgagagttaagcc-3′；
Pig：Sense primer：SEQ No.7 5′-aatttttggggatgcttagact-3′
Anti-sense primer：SEQ No.8 5′-tattttgggaggttattgtgttgta-3′；
Duck：Sense primer：SEQ No.9 5′-cgaggcttctactacggc-3′
Anti-sense primer：SEQ No.10 5′-gggtgattcctgcgatta-3′
(2) PCR reaction systems
Substance PCR reacts：μ L, dNTP Mix 0.2mmol/L, Taq the archaeal dna polymerase 2.5U of 5 × PCR Buffer 10,
MgCl22mmol/L, primer 1mmol/L, the μ g of DNA profiling 1, with dd H2O supplies system to 50 μ L.PCR reaction conditions are：94
DEG C pre-degeneration 5min；94 DEG C of denaturation 40s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulate；72 DEG C of extension 5min.
Multi-PRC reaction：μ L, dNTP Mix 0.2mmol/L, Taq the archaeal dna polymerase 2.5U of 5 × PCR Buffer 10,
MgCl22mmol/L, primer proportioning is ox：Sheep：Chicken：Pig：Duck=2:1:1:1:0.5, the μ g of DNA profiling 1 of each species, use dd
H2O supplies system to 50 μ L.Ox in end reaction system, sheep, chicken, the positive and negative primer concentration of pig and duck are respectively 1mmol/L,
0.5mmol/L, 0.5mmol/L, 0.5mmol/L and 0.25mmol/L.PCR reaction conditions are：94 DEG C of pre-degeneration 5min；94℃
40s is denatured, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulate；72 DEG C of extension 5min.
(3) primer specificity is verified
Expand the 17 kinds of animal DNA templates extracted in example 1 respectively using five kinds of primers of design.Using substance PCR
Expanded, product carries out gel electrophoresis analysis after amplification.According to PCR primer stripe size and whether there is judge sample in whether
Contain animal derived materials.As shown in figure 1,17 kinds of animal tissues are expanded respectively using the primer of ox, wherein 6,7 and 8 bands point
Not Wei Nanyang cattle, Luxi Yellow cattle and ox, Anduo County ox do not occur specific band, shows primer pair Anduo County ox without specificity
Primer.The result shows that designed ox primer has higher specificity.Equally, drawing using sheep, chicken, pig and duck respectively
Thing expands 17 kinds of animal DNA templates, and electrophoresis result is as shown in Fig. 2 to 5.Four kinds of primers can be to the animal tissue of identical kind
Specific band is amplified, band is not amplified to other kinds.
In addition, expanding the sample for being mixed with five kinds of animal DNA templates respectively using substance primer and use is mixed with five kinds and drawn
Thing expands ox, sheep, chicken, pig, duck equal samples respectively.As shown in fig. 6, all only produce specific purpose band, show
The detection method has higher species specificity.
(4) actual sample detects
The meat samples such as Luxi Yellow cattle, the white goat in Chongming and Sanhuang chicken are selected to carry out different quality ratio mixing：1. ox：Sheep：Chicken=
1:1:1；2. chicken：Sheep=1:1；3. ox：Sheep=1:1.Mixed sample, carrying for DNA is carried out according to the method for example 1 (3)
Take.Then, the amplification of target gene is carried out using multi-PRC reaction system in example 2 (2).Product carries out gel after amplification
Electrophoretic analysis.According to PCR primer stripe size and whether there is judge whether contain animal derived materials in sample.As shown in fig. 7,
Simultaneously containing ox, sheep, chicken source constituent sample it is amplifiable go out three kinds of compositions specific band；The sample mixed two-by-two is only
It can detect that two kinds of compositions.The result shows that this detection architecture accurately while can identify many animals derived component.
A kind of 3 kit of embodiment and application method
Have in the kit：PCR A liquid：PCR Buffer, dNTP Mix, Taq archaeal dna polymerases, MgCl2, five pairs draw
Thing and distilled water；PCR B liquid：Contain positive DNA profiling, including ox, sheep, chicken, pig, the genomic DNA of duck.PCR kit makes
Use specification.
Luxi Yellow cattle, the white goat in Chongming, Sanhuang chicken, Taihu pigs and the sample of careless duck are selected, carries out different quality ratio
Mixing：1. ox：Sheep：Chicken：Pig：Duck=1:1:1:1:0.5；2. ox：Sheep：Chicken：Pig：Duck=0:1:0:0:0；3. ox：Chicken：Sheep：Duck
=1:1:1:1.Sample simulates actual sample after being sufficiently mixed, carry out following steps：
(1) take sample 50mg to be added to liquid nitrogen grinding into powder in 1.5mL centrifuge tubes, 400 are then added into centrifuge tube
μ L Buffer MACL, 200 μ L Buffer MCL and 20 μ L protein kinase Ks, concussion shake up, 65 DEG C of water-bath 20-30 min.Take
Go out centrifuge tube, 12000rpm centrifugation 5-10min, take the μ L of supernatant 500 to move to new 1.5mL centrifuge tubes, as no RNA need to be obtained
DNA, 20 μ L RNase A (10mg/mL) solution can be added in supernatant, mix, room temperature place 5min.Add into centrifuge tube
Enter 500 μ L Buffer MA and 15 μ L MagicMag Beads, blow and beat or put mixing of shaking repeatedly, be stored at room temperature 1min.Will be from
Heart pipe is placed in 30s on magnetic frame, treats that MagicMag Beads are drawn to after tube wall completely, and supernatant is abandoned in suction, is taken from magnetic frame
Go out centrifuge tube.The ethanol waters of 700 μ L 70% are added into centrifuge tube, mixing that suction is beaten or point shakes, centrifuge tube are placed in magnetic
30s on power frame, treat that MagicMag Beads are drawn to after tube wall completely, supernatant is abandoned in suction, and centrifuge tube is taken out from magnetic frame.
Once, room temperature is uncapped uncaps dry 5-7min in dry 15-20min or 55 DEG C of insulating box to no liquid in pipe to repeat step 5
Residual.100 μ L TE Buffer (pH 8.0), 65 DEG C of water-bath 5-10min are added into centrifuge tube, or mix.Take out centrifugation
Pipe is placed in 30s on magnetic frame, treats that MagicMag Beads are drawn to after tube wall completely, careful Aspirate supernatant to new centrifugation
Pipe, that is, obtain genomic DNA.
(2) expanded using following PCR reaction systems：μ L, dNTP the Mix 0.2mmol/L of 5 × PCR Buffer 10,
Taq archaeal dna polymerases 2.5U, MgCl22mmol/L, primer proportioning is ox：Sheep：Chicken：Pig：Duck=2:1:1:1:0.5, use dd
H2O supplies system to 50 μ L.Ox in end reaction system, sheep, chicken, the positive and negative primer concentration of pig and duck are respectively 1mmol/L,
0.5mmol/L, 0.5mmol/L, 0.5mmol/L and 0.25mmol/L.PCR reaction conditions are：94 DEG C of pre-degeneration 5min；94℃
40s is denatured, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulate；72 DEG C of extension 5min.
(3) gel electrophoresis analysis are carried out to the product of multiplex PCR, according to PCR primer stripe size and whether there is judgement sample
In whether contain animal derived materials.As shown in figure 8, using the multiplex PCR detection technique, sample 1 can amplify simultaneously ox,
Sheep, chicken, the specific purpose band of five kinds of animal derived materials of pig and duck, sample 2 only amplify the purpose band of sheep, sample 3
Amplify the purpose band of ox, sheep, chicken and duck.In summary, the kit method has higher Stability and veracity,
It can be applied to identify five kinds of animal derived materials in food simultaneously.
Sequence table
<110>Academy of Agricultural Sciences, Shanghai City
<120>The multiple PCR method and kit of a kind of 5 kinds of animal derived materials of synchronous detection
<141> 2017-11-17
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
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<213>Artificial sequence (Artificial Sequence)
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tagcaattgc catagtccac c 21
<210> 2
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
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taggatgagg agaaagtata ggaca 25
<210> 3
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
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cgccatagtt cacctactct tc 22
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ttactaggac taggattgag aggat 25
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cttccttcac atcggacgag 20
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<212> DNA
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gggtgagtat gagagttaag cc 22
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aatttttggg gatgcttaga ct 22
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<213>Artificial sequence (Artificial Sequence)
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tattttggga ggttattgtg ttgta 25
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cgaggcttct actacggc 18
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<212> DNA
<213>Artificial sequence (Artificial Sequence)
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gggtgattcc tgcgatta 18
Claims (2)
1. the multiple PCR method of 5 kinds of animal derived materials of a kind of synchronous detection, it is characterised in that this method comprises the following steps
(1) DNA of animal tissue to be checked is extracted, and multiplexed PCR amplification is carried out to it, PCR reaction systems are：5×PCR
Buffer 10 μ L, dNTP Mix 0.2mmol/L, Taq archaeal dna polymerases 2.5U, MgCl22mmol/L, primer proportioning is ox：
Sheep：Chicken：Pig：Duck=2:1:1:1:0.5, with dd H2O supplies system to 50 μ L.Ox, sheep, chicken, pig and duck in end reaction system
Positive and negative primer concentration be respectively 1mmol/L, 0.5mmol/L, 0.5mmol/L, 0.5mmol/L and 0.25mmol/L.It is multiple
PCR response procedures are as follows：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 40s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 circulate；
72 DEG C of extension 5min；
(2) gel electrophoresis analysis are carried out to the product of multiplex PCR, judged according to PCR primer stripe size and whetheing there is be in sample
It is no containing 5 kinds of animal derived materials, wherein obtaining purpose band size after ox, sheep, chicken, pig and duck amplification is respectively：493bp、
302bp、400bp、277bp、239bp；
Wherein described primer is：
Ox：Sense primer：SEQ NO.1 5′-tagcaattgccatagtccac c-3′
Anti-sense primer：SEQ NO.2 5′-taggatgaggagaaagtataggaca-3′；
Sheep：Sense primer：SEQ NO.3 5′-cgccatagttcacctactct tc-3′
Anti-sense primer：SEQ NO.4 5′-ttactaggactaggattgag aggat-3′；
Chicken：Sense primer：SEQ NO.5 5′-cttccttcacatcggacgag-3′
Anti-sense primer：SEQ NO.6 5′-gggtgagtatgagagttaag cc-3′；
Pig：Sense primer：SEQ NO.7 5′-aatttttggggatgcttaga ct-3′
Anti-sense primer：SEQ NO.8 5′-tattttgggaggttattgtg ttgta-3′；
Duck：Sense primer：SEQ NO.9 5′-cgaggcttctactacggc-3′
Anti-sense primer：SEQ NO.10 5′-gggtgattcctgcgatta-3′.
2. the kit of 5 kinds of animal derived materials of a kind of synchronous detection, it is characterised in that the kit contains following content：
PCR A liquid：PCR Buffer, dNTP Mix, Taq archaeal dna polymerases, MgCl2, five pairs of primer and distilled water；
PCR B liquid：Contain positive DNA profiling, including ox, sheep, chicken, pig, the genomic DNA of duck；
Operation instructions；
Five pairs of wherein described primers are：
Ox：Sense primer：SEQ NO.1 5′-tagcaattgccatagtccac c-3′
Anti-sense primer：SEQ NO.2 5′-taggatgaggagaaagtataggaca-3′；
Sheep：Sense primer：SEQ NO.3 5′-cgccatagttcacctactct tc-3′
Anti-sense primer：SEQ NO.4 5′-ttactaggactaggattgag aggat-3′；
Chicken：Sense primer：SEQ NO.5 5′-cttccttcacatcggacgag-3′
Anti-sense primer：SEQ NO.6 5′-gggtgagtatgagagttaag cc-3′；
Pig：Sense primer：SEQ NO.7 5′-aatttttggggatgcttaga ct-3′
Anti-sense primer：SEQ NO.8 5′-tattttgggaggttattgtg ttgta-3′；
Duck：Sense primer：SEQ NO.9 5′-cgaggcttctactacggc-3′
Anti-sense primer：SEQ NO.10 5′-gggtgattcctgcgatta-3′.
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| CN108977552A (en) * | 2018-08-14 | 2018-12-11 | 北华大学 | Meat identifies positive reference substance and its preparation method and application |
| CN113215281A (en) * | 2021-06-11 | 2021-08-06 | 宁波大学 | PCR detection primer, kit and method for simultaneously detecting five animal-derived components in meat and meat products |
| CN116024364A (en) * | 2022-08-25 | 2023-04-28 | 上海市农业科学院 | Primer and method for identifying Fusarium goolgardi toxigenic genotype of PCR-RFLP |
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