CN105274099B - The primer of 9 kinds of animal derived materials, probe compositions, kit and its detection method and application in Rapid identification food or feed - Google Patents

The primer of 9 kinds of animal derived materials, probe compositions, kit and its detection method and application in Rapid identification food or feed Download PDF

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CN105274099B
CN105274099B CN201510695558.0A CN201510695558A CN105274099B CN 105274099 B CN105274099 B CN 105274099B CN 201510695558 A CN201510695558 A CN 201510695558A CN 105274099 B CN105274099 B CN 105274099B
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probe
source property
primer
kinds
seq
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CN105274099A (en
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步迅
张全芳
刘艳艳
范阳阳
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Biotechnology Research Center of Shandong Academy of Agricultural Sciences
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Biotechnology Research Center of Shandong Academy of Agricultural Sciences
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Abstract

The present invention provides primer, probe compositions, kit and its detection method of 9 kinds of animal derived materials and application in a kind of Rapid identification food or feed, Rapid identification can go out the derived components of the food containing above-mentioned multiple species or feed simultaneously using primer, probe compositions and the real-time fluorescence PCR combined detection kit of the present invention.Belong to technical field of molecular biology.Universal primer is designed by target gene of 16SrDNA, the specific probe of 9 kinds of species is separately designed, design construction amplification interior label designs specific probe for interior label sequence, divide 3 groups of PCR reaction systems to carry out, passes through corresponding fluorescence probe signal and each species derived component of the direct interpretation of Ct values.This method is at low cost, time saving, efficient, and several species discriminating can be achieved at the same time.

Description

The primer of 9 kinds of animal derived materials, probe combinations in Rapid identification food or feed Object, kit and its detection method and application
Technical field
The present invention relates to the primer of 9 kinds of animal derived materials, probe compositions, kits in Rapid identification food or feed And its detection method and application, belong to technical field of molecular biology.
Background technology
European horseflesh disturbance in 2012 causes various countries for the adulterated great attention of the meat true and false, since 2013, The event of the inferior matter meat of domestic top grade meat adulteration also occurs again and again, media report in succession Shandong Binzhou, Inner Mongol, on Investigate and prosecute false mutton event in the ground such as sea, Xi'an, Shenzhen.Through investigation, the main problem of China's meat adulteration is：In mutton, beef etc. Incorporation pork, chicken, duck, horseflesh, mouse meat, fox meat, mink meat etc. are easy to cultivation and valence in high-grade expensive meat The less expensive meat of lattice, adulterates, cheats consumer.This not only serious infringement consumer's interests, but also more seriously Severe social influence is caused, huge injury is also resulted in entire meat industry.Meanwhile the adulterated feelings of serious international meat product Condition brings great security risk to China, if without reliable quickly true and false authentication technique, it will make China's meat into Mouth enterprise suffers huge economic loss.
Feed safety is closely related with animal productiong, environmental pollution and human health.There is the first rabid ox disease from Britain Afterwards, many countries all control the production and use of feed products from animal sources by law, regulation etc. in the world.China Also corresponding laws and regulations have been put into effect, such as《Feedstuff catalogue》(No. 1773 bulletins of the Ministry of Agriculture), it is desirable that produce animal derived feeding The raw material of material must derive from edible animal, and must derive from same animal.But animalsderived feedstuffs produce at present The raw material sources of enterprise are extremely complex, and there are prodigious security risks.It is mainly shown as the life of feed products from animal sources Non- edible animal raw material has been used in production.Such as produce pig, chicken, duck, fish, beef or mutton powder or bone meal used mink, fox and Recoon dog butchers leftover bits and pieces.Animalsderived feedstuffs raw material type is various at present, and the scale of manufacturing enterprise differs, the pipe of enterprise Reason level is also very different.Therefore, it is necessary to carry out the detection of animal derived materials to feed and single animalsderived feedstuffs, So as to the production and use of stronger specification and supervision feed products from animal sources.
Being presently used for the protein identification that mainly has of animal derived identification method, (isoelectric focusing electropho- resis, ELASA are enzyme-linked Immunoabsorption, high performance liquid chromatography, Liquid Chromatography/Mass Spectrometry etc.) and two class of DNA identification technologies, wherein with gene difference between species Based on DNA identification technologies be Recent study hot spot, reason is that biological gene has unique repeatability, What DNA sequence dna will not change in bion growth course, the DNA sequence dna information of same organism different growing stages is identical , even across processing, form changes, and DNA sequence dna information will not change.Therefore introduces a collection identification is carried out using DNA With very high accuracy, it is presently the most ideal method.And the nearly 5 years real-time fluorescent PCR technologies occurred, it is high special with it Property, high sensitivity, stopped pipe operation it is pollution-free the advantages that, be increasingly used in animal derived discriminating.It is currently based on multiple The animal derived materials detection of round pcr relies primarily on the amplification for telling different length after PCR using agarose gel electrophoresis Segment realizes that application No. is 201510061793.2 and 201510300666.3 patents to be all made of the molten of multiple RT-PCR Solution curve analytical technology reaches the quick discriminating of a variety of derived components in meat and meat products, but this method only passes through melting curve Temperature TM value ranges cannot distinguish between the close species of the equal animal homology of such as fox, recoon dog, and in addition this method is to design Primer requires very strong specificity, multipair primer to easily cause cross reaction and non-specific amplification.
Therefore, efficiently extensive several species animal derived materials associated detecting method is established, is answered improving accident Anxious processing capacity, promoting food, animal products and feed safety level monitoring and ability to supervise has important practice significance.
Invention content
In view of the deficienciess of the prior art, the present invention provide ox in a kind of Rapid identification food or feed, horse, sheep, pig, Chicken, duck, fox, the primer of 9 kinds of animal derived materials of mink and mouse, probe compositions and the examination of real-time fluorescence PCR joint-detection Agent box, using the present invention primer, probe compositions and real-time fluorescence PCR combined detection kit can simultaneously Rapid identification Go out the derived components of the food containing above-mentioned multiple species or feed.
The invention is realized by the following technical scheme：
Identify the primer of 9 kinds of animal derived materials, probe compositions in food or feed, including following primer and probe, Its nucleotide sequence is as follows：
(1) primer
Universal primer sequence：
Upstream sequence：UN-F：AAGACGAGAAGACCCATGGACTCTA, as shown in SEQ ID NO.1；
Downstream sequence：UN-R：GATTGCGCGGTTATCCCTAGGGTA, as shown in SEQ ID NO.2；
Horse source special primer：
Upstream sequence：LMF：TTTCTCCTCGCATAAGCCTATATC, as shown in SEQ ID NO.3；
Downstream sequence：LMR：GTTCCTTTTACTTCTTTTAATCTTTCCT, as shown in SEQ ID NO.4；
(2) 9 kinds of animal derived materials probes：
Sheep source property specific probe (MYP)：5 '-CCACCAAGGGATAACAACACTCTGGTGG-3 ', such as SEQ ID NO.5 It is shown；
Ox source property specific probe (NP)：5 '-CGGCGGTAACTAACCAACCCAAAGAGAATCCGCCG-3 ', such as SEQ ID Shown in NO.6；
Horse source property specific probe (MP)：5 '-CAGCGAAGATAGGGATAATCCAAAAACTAATCACGCTG-3 ', such as SEQ Shown in ID NO.7；
Fox source property probe (HP)：5 '-CAGACCGATGAAATCCAAACCCCTCGGTCTG-3 ', such as SEQ ID NO.8 It is shown；
Mink source probe (SDP)：5 '-CGGCGAGGTCTAACACAACATTATTACTGGTCGCCG-3 ', such as SEQ ID Shown in NO.9；
Pig source property probe (ZP)：5 '-CAGTGATGTTATGGTTATTTTGACTGGTTTGCATCACTG-3 ', such as SEQ ID Shown in NO.10；
Mouse probe (SP)：5'-CGCGCGTCCTCCGAATGATTTTAACCTAGACTCGCGCG-3 ', such as SEQ ID Shown in NO.11；
Chicken source property probe (JP)：5 '-CGGGTCTGGTTACTGTTGGTACTTTGAGAGACCCG-3 ', such as SEQ ID Shown in NO.12；
Duck source property probe (YP)：5 '-CGCGAACCTAAGACTAAACCCACCGGGTTCGCG-3 ', such as SEQ ID NO.13 It is shown；
Wherein, the 5 ' of all probes it is terminal modified have a fluorescent reporter group, the fluorescent reporter group of each probe be FAM, Any one in JOE, CY3, CY5 or ROX, each probe 3 ' it is terminal modified have quenching group be TAMRA, DABCYL, BHQ1 and Any one in BHQ2.
Preferably, 5 ' terminal modified fluorescent reporter groups of all probes and 3 ' terminal modified quenching group difference It is：
The fluorescent reporter group of sheep source property specific probe (MYP), ox source property specific probe (NP) and pig source property probe (ZP) Marked with JOE；The fluorescence report of horse source property specific probe (MP), mouse probe (SP) and fox source property specific probe (HP) Group is marked with FAM；The fluorescence report base of mink source probe (SDP), chicken source property probe (JP) and duck source property probe (YP) Group is marked with CY3；The quenching group at 3 ' ends of all probes is modified with DABCYL.
Preferably：Further include internal standard gene and corresponding exogenous interior label primer (IMF) and exogenous internal standard probe (IMP), internal standard gene nucleotide sequence is as shown in SEQ ID NO.14；
Exogenous interior label primer (IMF)：
Upstream sequence：5 '-AGACCACCAAATGCCCCTATC-3 ', as shown in SEQ ID NO.15；
Downstream sequence：5 '-GTTCCTTTTACTTCTTTTAATCTTTCCT-3 ', as shown in SEQ ID NO.16；
Exogenous internal standard probe (IMP)：5 '-GCGAGGTCCCCTAGAAGAAGAACTCCCTCGC-3 ', such as SEQ ID Shown in NO.17；
Wherein, the 5 ' of exogenous internal standard probe (IMP) terminal modified have the fluorescent reporter group, the fluorescent reporter group to be FAM, JOE, CY3, CY5 or ROX, exogenous internal standard probe (IMP) 3 ' it is terminal modified have quenching group, be TAMRA, DABCYL, BHQ1 or BHQ2.
Preferably, the interior form for being marked with recombinant plasmid exists, and the recombinant plasmid is to be connected to internal standard segment It is formed on PMD18-T carriers.
The present invention provides primed probe mixture, Taq thermal starting enzyme 1U, 10 × PCR buffer solution, positive criteria product, the moon The kit of property standard items, the kit are easy to use, quick, efficient.
A kind of real-time fluorescence PCR combined detection kit for identifying 9 kinds of animal derived materials in food or feed, including institute Primer, probe compositions are stated, the recombinant plasmid and the corresponding exogenous interior label primer (IMF/R) and exogenous internal standard are visited Reaction raw materials and reagent necessary to needle (IMP) and PCR reactions.
Preferably, the PCR combined detection kits are divided into three PCR reaction systems, are A systems, B systems and C respectively System, each system share the universal primer in the kit, recombinant plasmid and the corresponding exogenous interior label primer pair With reaction raw materials and reagent necessary to exogenous internal standard probe (IMP) and PCR reactions, A systems include above-mentioned sheep source property Specific probe (MYP), fox source property probe (HP) and chicken source property probe (JP), B systems include include that above-mentioned ox source property is special Probe (NP), horse source property specific probe (MP) and duck source property probe (YP) and horse source special primer, C systems include Above-mentioned mink source probe (SDP), pig source property probe (ZP) and mouse probe (SP), wherein three kinds in each system The fluorophor that specific probe is marked is different.
Reaction raw materials and reagent necessary to the PCR reactions include：10 × PCR buffer solutions, dNTPs, Taq enzyme and ultrapure Water；
Each component constitutes the reaction system of a 20 μ l in the kit：Taq thermal starting enzyme 1U, 10 × PCR buffer solutions (include 600mmol/L trishydroxymethylaminomethanes-hydrochloric acid solution Tris-Cl (pH8.9), 500mmol/L potassium chloride (KCl), 25mmol/L magnesium chlorides (MgCl2), 10mmol/L dithiothreitol (DTT)s (DTT), 2.5mmol/L dNTPs), each primer pair final concentration For 0.2~0.5 μm of ol/L, each concentration and probe concentration is 0.05~0.5 μm of ol/L, recombinant plasmid dna concentration containging interior traget 0.1~ 1pg。
The present invention also provides using above-mentioned primer, probe compositions or kit simultaneously differentiate ox, horse, sheep, pig, Chicken, duck, fox, the application in 9 kinds of animal derived materials of mink and mouse and multiple real time fluorescence PCR detection method, the party Method is quick and convenient, accuracy is high, and flux is big.
A kind of real-time fluorescence PCR associated detecting method for identifying 9 kinds of animal derived materials in food or feed, including it is following Step：
(1) genomic DNA of measuring samples is extracted, it is spare；
(2) using the primer, probe compositions or the PCR combined detection kits to extraction in step (1) Genomic DNA carries out PCR amplification, collects fluorescence signal, is judged, the PCR amplification program is：95 DEG C of 3min pre-degenerations, 95 DEG C of 5s, 60 DEG C of 20s~60s, collect fluorescence signal herein, amount to 45 cycles, amplification elementary reaction can be in any of 5 channels It carries out on the fluorescence quantitative PCR instrument of model, can be adjusted accordingly according to the different instrument reaction time；
(3) result interpretation：The fluorescence signal during PCR is collected, is reflected by corresponding fluorescence signal amplification curve, Ct values Whether contain 9 kinds of animal derived materials in sample not to be checked.
In the above method, the sample to be checked can be cold fresh meat, frozen meat, meat packing product, animal fat, feed original Expect meat meal tankage etc..
It is design universal primer and specific probe based on fluorescence probe label multiple real time fluorescence PCR detection techniques, leads to Different fluorescence probe signals are crossed to differentiate animal derived materials, add exogenous internal standard (IAC) quality control system pair in the reaction system Reaction system is monitored, and be can avoid the generation of false negative result, can be improved detection accuracy.In order to reach while detect food And ox in feed, horse, goat, sheep, pig, chicken, duck, fox, 9 kinds of animal derived materials of mink and mouse purpose, select special Property good high sensitivity primer and probe be the key that the present invention, in order to overcome multipair primer pair multi-PRC reaction system to bring Primer between the drawbacks of interfering with each other, the present invention has selected the shared pair of primers of 9 kinds of animals.According to food and feed depth The characteristics of processing causes DNA height to be degraded, using animal mitochondria DNA as target gene, reason is that mitochondrial DNA is being lost Relatively independent, the features such as relatively core gene copy number is quite high is caught, therefore is differentiated with mitochondrial DNA molecular labeling animal derived Ingredient have high sensitivity, accuracy are high, degradation small (process Mitochondria DNA keeps relatively complete), stablize it is easy to operate Etc. advantages.It reports that the 16SrDNA genes selected on mitochondria have highly conserved type according to pertinent literature, is widely used in more objects Introduces a collection identifies, using Primer5.0 softwares to the 16SrDNA gene orders of above-mentioned species in conserved regions design universal primer, Corresponding specific probe is designed in variable region, the probe of design must satisfy conservatism in special kind of inter-species.Additionally, due to Horse and donkey homology are very close in the universal primer amplification range of selection, only Individual base difference, so needing individually designed The primer and specific probe of exclusive horse and donkey.
The present invention the advantage is that compared with existing detection technique：
A. the present invention uses multiple real time fluorescence round pcr to reflecting in meat, processed meat food and feedstuff meat meal tankage 9 kinds of animal derived materials such as other ox, horse, sheep, pig, chicken, duck, fox, mink and mouse.It is set using mitochondria 16SrDNA as target gene Universal primer is counted, the specificity of 9 kinds of species and strong molecular beacon probe, design construction amplification interior label, for interior are separately designed Sequence design specific probe is marked, point 3 groups of PCR reaction systems carry out, each by corresponding fluorescence probe signal and the direct interpretation of Ct values Species derived component.This method is at low cost, time saving, efficient, and several species discriminating can be achieved at the same time.
B. only contain a pair of of universal primer in kit of the invention, reduce multipair primer pair quantitative fluorescent PCR system Generate the risk of interference；The addition of internal standard quality control system can effectively indicate that false negative occurs, and improve the accuracy of detection；The reagent 4 heavy multicolor fluorescence quantitative PCR detection of box, 9 kinds of animal derived qualitative detections can be completed by 3 groups of PCR systems, be particularly suitable for mixing Pattern detection.
C. the whole stopped pipe operation of the present invention, it is not easy to pollute, there is accurate stable, easy to operate, high sensitivity, specificity By force, the advantages such as flux is big.
Description of the drawings
Specific amplification curve figures of Fig. 1 multiplex PCR system A to ovine genome DNA.
Specific amplification curve figures of Fig. 2 multiplex PCR system A to goat genomic DNA.
Specific amplification curve figures of Fig. 3 multiplex PCR system A to fox genomic DNA.
Specific amplification curve figures of Fig. 4 multiplex PCR system A to chicken genomic DNA.
Fig. 5 multiplex PCR system A are to the genomic DNA of other species without specific amplification curve, only internal standard Quality Control curve.
Specific amplification curve figures of Fig. 6 multiplex PCR system B to cow genome group DNA.
Specific amplification curve figures of Fig. 7 multiplex PCR system B to Duck genome DNA.
Specific amplification curve figures of Fig. 8 multiplex PCR system B to horse genomic DNA.
Fig. 9 multiplex PCR system B are to the genomic DNA of other species without specific amplification curve, only internal standard Quality Control curve.
Specific amplification curve figures of Figure 10 multiplex PCR system C to pig genomic DNA.
Specific amplification curve figures of Figure 11 multiplex PCR system C to mink genomic DNA.
Specific amplification curve figures of Figure 12 multiplex PCR system C to musculus cdna group DNA.
Figure 13 multiplex PCR system C are to the genomic DNA of other species without specific amplification curve, only internal standard Quality Control curve.
Figure 14 Ct values when the Genome DNA content of horse is 0.01ng, 0.005ng<35 as DNA content 0.0025ng Some Ct values ＞ 35, so the sensitivity DNA content of this method is 0.005ng.
Figure 15 Ct values when the Genome DNA content of ox is 0.01ng, 0.005ng<35 have when containing 0.0025ng Ct values ＞ 35, so the sensitivity DNA content of this method is 0.005ng.
Figure 16 Ct values when the Genome DNA content of duck is 0.01ng, 0.005ng<35 have when containing 0.0025ng Ct values ＞ 35, so the sensitivity DNA content of this method is 0.005ng.
Figure 17 Ct values when the Genome DNA content of pig is 0.01ng, 0.005ng<35 have when containing 0.0025ng Ct values ＞ 35, so the sensitivity DNA content of this method is 0.005ng.
Figure 18 Ct values when the Genome DNA content of mink is 0.01ng, 0.005ng<35 have when containing 0.0025ng Ct values ＞ 35, so the sensitivity DNA content of this method be 0.005ng.
Ct values when the Genome DNA content that Figure 19 surely belong to is 0.01ng, 0.005ng<35 have when containing 0.0025ng Ct values ＞ 35, so the sensitivity DNA content of this method is 0.005ng.
Figure 20 Ct values when the Genome DNA content of fox is 0.01ng, 0.005ng<35 have when containing 0.0025ng Ct values ＞ 35, so the sensitivity DNA content of this method be 0.005ng.
Figure 21 Ct values when the Genome DNA content of sheep is 0.01ng, 0.005ng<35 have when containing 0.0025ng Ct values ＞ 35, so the sensitivity DNA content of this method is 0.005ng.
Figure 22 Ct values when the Genome DNA content of chicken is 0.01ng, 0.005ng<35 have when containing 0.0025ng Ct values ＞ 35, so the sensitivity DNA content of this method is 0.005ng.
Figure 23 are to detect sheep, chicken and fox derived component simultaneously in sample.
Figure 24 are to detect ox, horse and duck derived component simultaneously in sample.
Figure 25 are to detect pig, mink and mouse derived component simultaneously in sample.
Figure 26 are to detect chicken, duck and sheep derived material simultaneously in sample.
Specific implementation mode
The following drawings illustrate the preferred embodiments to the present invention to be described in detail.These embodiments are only used for It is bright rather than limit the scope of the invention.To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with Present invention is further described in detail for attached drawing.
Experiment material：Ox, horse, sheep, pig, chicken, duck standard items have the herding of academy of agricultural sciences of Shandong Province to be provided, fox, mink and Mouse come from feeding quality inspection institute of Shandong Province, samples sources to be checked in food and medicine Inspection Research institute of Shandong Province and purchased from Jinan it is each Big supermarket, the market of farm produce, nearly 300 parts of samples of sampling, including fresh meat, chilled meat (monoblock meat, meat mincing, compression roulade, sliced meat etc. Various types), processed meat food, feedstuff meat meal tankage.
Instrument：Above real-time fluorescence quantitative PCR instrument such as ABI7500 in 4 channels etc., ultraviolet specrophotometer, electronic balance, most High rotating speed 15000rpm desk centrifuges, water-bath, the conventional instruments such as high-pressure sterilizing pot.
Embodiment 1
1. primed probe designs：According to announced in Genebank databases ox, sheep, pig, chicken, fox, mink, horse, The mitochondrial genomes sequence information of duck and mouse reports the 16SrDNA genes selected on mitochondria according to pertinent literature, uses Primer5.0 softwares, in conserved regions design universal primer, use ABI Primer to the 16SrDNA gene orders of above-mentioned species Express softwares design corresponding specific molecular beacon probe in variable region, and the probe of design must satisfy special kind of inter-species Interior conservatism.In the present invention, the ox, horse, goat, sheep, pig, chicken, duck, fox, mink and mouse specific probe and internal standard 5 ' ends of probe are modified with reporter group, and the reporter group can be FAM, JOE, CY3, CY5 etc., and 3 ' ends, which are modified with, quenches Go out group, and the quenching group can be TAMRA, BHQ1, BHQ2, DABCYL etc..
All general primer and probe sequence are as follows：
2. the present invention also provides the multiple of a kind of discriminating ox, horse, goat, sheep, pig, chicken, duck, fox, mink and mouse Real-time fluorescence PCR assay kit, the kit include：A. a pair of of universal primer, a pair of of horse source special primer, a pair of of internal standard are special Different Primer composition；B. ox, horse, sheep, pig, chicken, duck, fox, mink and mouse specific probe and internal standard probe composition；C. internal standard Recombinant plasmid；D.10 × TaqMan Master mix (include Taq thermal startings enzyme, MgCL2, the components such as dNTP)；E. include ox, Horse, goat, sheep, pig, chicken, duck, fox, mink and mouse positive reference substance；F. include other species DNA conducts other than 9 kinds Negative controls；G. distilled water.
In the kit, the dosage of each ingredient is referred to dosage rule disclosed in the prior art to select, and works as examination When agent box PCR reaction systems are 20 μ l, each ingredient can be selected according to following dosage in system：10×TaqMan Master 2 μ l of mix, universal primer are 0.05~0.5 μm of ol/L to final concentration of 0.2~0.5 μm of ol/L, concentration and probe concentration, contain internal standard matter Grain DNA concentration 0.1~1pg/ μ l.
The optimum reaction condition is 95 DEG C of 3min；95 DEG C of 5s, 60 DEG C of 35s (collecting fluorescence signal), 45cycles.
Embodiment 2
It is former using kit of the present invention and multiple real time fluorescence PCR detection method detection meat, processed meat food and feed Expect that the method and step of the animal derived joint-detection of several species in meat meal tankage is as follows：
1.DNA is extracted：
According to sample type difference, pre-processed one of in the following manner：
A) when sample is monoblock meat, after sample preparation methods mixing, appropriate meat piece is taken, sample physiological saline is clear After washing, it is fully ground into powder in liquid nitrogen, weighs 0.03g and is fitted into the centrifuge tube of 2mL, the DNA that 400 μ L are added extracts cell During which lysate, 10 μ L Proteinase K mixings, 56 DEG C of water-bath 2h slightly shake up frequently.10 μ L RNA enzyme, 37 DEG C of enzymolysis 1h are added.
B) sample is that meat mincing, sliced meat or roulade etc. freeze meat mixture or meat packing product and feedstuff meat meal tankage When, after sample preparation methods mixing, appropriate amount of sample is weighed, after sample is cleaned with physiological saline, is fully ground in liquid nitrogen It clays into power, weighs 5g samples in 50mL centrifuge tubes, 10mLDNA extraction cell pyrolysis liquids and 200 μ L Proteinase Ks are added, mix Even, 56 DEG C of water-bath digestion are overnight.It takes the lysate of 3 1mL to be added separately in the EP pipes of 3 1.5mL, 10 μ L RNA is added Enzyme, 37 DEG C of enzymolysis 1h.
C) isometric chloroform is added into lysate：Isoamyl alcohol (24: 1), slow reverse mixing 10min, 4 DEG C, 12000g 10min is centrifuged, supernatant is taken to be transferred in new 1.5mL centrifuge tubes.Isometric chloroform is added, mixes well, 4 DEG C, 12000g Centrifuge 10min.Supernatant is gone in new EP pipes, the absolute ethyl alcohols and 0.1 times of volume of 4 DEG C of 2.5 times of volumes precoolings are added Sodium acetate buffer gently overturns mixing, it is seen that white DNA flocculent deposits are precipitated, 4 DEG C, 12000g, centrifuge 5min, precipitation DNA abandons supernatant.The 70% ethyl alcohol washing of 1mL4 DEG C of precooling is added, 12000g centrifuges 3min, discards ethanol washes, repeats It washed once.Placing at room temperature makes residual ethanol volatilize completely, 50 μ LTE dissolving DNAs is added, -20 DEG C save backup.
2 nucleic acid concentrations and purity testing
Nucleic acid concentration and purity detecting, OD are carried out to the DNA of extraction with ultraviolet specrophotometer260Nm and OD280It is surveyed at nm Its fixed light absorption value, OD260/2801.7~1.9, experiment is controlled with DNA concentration between 0.1~50ng/ μ L Ratio control.
3 real-time fluorescence PCRs react
The present invention divides 3 groups of PCR reaction systems of A, B, C to carry out multiple real time fluorescence PCR reactions, is shown in Table 1.Every group of PCR reaction Include 3 kinds of animal derived specific probes in system, and external source quality control system is added, excludes the false negative as caused by inhibition ingredient Possibility, realize 9 kinds of animal derived materials qualitative detection.
1 PCR reaction systems of table
A group systems：
Reagent name Final concentration
Taq enzyme 1U
10×PCRBuffer 1×
UNF 0.2~0.5 μm of ol/L
UNR 0.2~0.5 μm of ol/L
MY-P-(JOE) 0.05~0.2 μm of ol/L
H-P(FAM) 0.20~0.4 μm of ol/L
J-Probe(CY3) 0.25~0.5 μm of ol/L
Internal standard sense primer IMF 0.05~0.2 μm of ol/L
Internal standard downstream primer IMR 0.05~0.2 μm of ol/L
Internal standard probe IMP (CY5) 0.05~0.2 μm of ol/L
10-3Internal standard plasmid (original concentration is 1ng/ μ L) 1μL
DNA profiling 2μL
ddH2O To20μL
B group systems：
Reagent name Final concentration
Taq enzyme 1U
10×PCRBuffer 1×
UNF 0.2~0.5 μm of ol/L
UNR 0.2~0.5 μm of ol/L
LMF1 0.2~0.5 μm of ol/L
LMR1 0.2~0.5 μm of ol/L
N-P(JOE) 0.05~0.2 μm of ol/L
MP(FAM) 0.05~0.1 μm of ol/L
Y-Probe(CY3) 0.25~0.5 μm of ol/L
Internal standard sense primer IMF 0.05~0.2 μm of ol/L
Internal standard downstream primer IMR 0.05~0.2 μm of ol/L
Internal standard probe IMP (CY5) 0.05~0.2 μm of ol/L
10-3Internal standard plasmid (original concentration is 1ng/ μ L) 1μL
DNA profiling 2μL
ddH2O To20μL
C group systems：
Reagent name Final concentration
Taq enzyme 1U
10×PCRBuffer 1×
UNF 0.2~0.5 μm of ol/L
UNR 0.2~0.5 μm of ol/L
Rat-P2(FAM) 0.05~0.2 μm of ol/L
SD-Probe(CY3) 0.2~0.5 μm of ol/L
Z-Probe(JOE) 0.05~0.2 μm of ol/L
Internal standard sense primer IMF 0.05~0.2 μm of ol/L
Internal standard downstream primer IMR 0.05~0.2 μm of ol/L
Internal standard probe IMP (CY5) 0.05~0.2 μm of ol/L
10-3Internal standard plasmid (original concentration is 1ng/ μ L) 1μL
DNA profiling 2μL
ddH2O To20μL
Each PCR reactions setting 3 times is parallel.While sample PCR reacts, setting negative control, blank control and sun Property control.
Negative controls:Using the mammalian genome DNA except 9 kinds of animals as negative control；
Blank control product：To replace sample setting extraction as blank control using water in DNA extraction process；
Positive reference substance：Using sheep, fox, chicken, ox, horse, duck, pig, mink and musculus cdna group DNA as positive control, It is middle that sheep, fox and chicken genomic DNA isoconcentration are mixed into the positive reference substance as A systems in equal volume, by ox, horse and duck gene Group DNA isoconcentrations mix the positive reference substance as B systems in equal volume, and pig, mink and musculus cdna group DNA isoconcentrations is isometric Mix the positive reference substance as C systems.
4 real-time fluorescence PCR reaction cycle parameters
Real-time fluorescence PCR reaction condition is slightly changed according to instrument model difference, generally：42℃5min；95 DEG C of pre- changes Property 3min；95 DEG C of denaturation 10s, 60 DEG C of annealing extend 35s (collecting fluorescence signal), 45 cycles.
5 interpretations of result and statement
The availability deciding of 5.1PCR
Blank control：A, the corresponding reacting hole of 3 groups of PCR systems of B, C is without FAM, JOE, CY3 fluorescence signals, corresponding Ct values >40.0, there is the fluorescence signal of CY5 Quality Control probes, and value≤35.0 Ct illustrate PCR system validity.
Negative control：A, the corresponding reacting hole of 3 groups of PCR systems of B, C is without FAM, JOE, CY3 fluorescence signals, corresponding Ct values >40.0, there is the fluorescence signal of CY5 Quality Control probes, and value≤35.0 Ct illustrate PCR system validity.
Positive control：A, there are FAM, JOE, the amplification of CY3 fluorescence signal S types in the corresponding reacting hole of 3 groups of PCR systems of B, C Curve, and value≤35.0 Ct illustrate PCR system validity.
5.2DNA extracts availability deciding
In the case of the blank, negative control and the positive control experiment result that carry out at the same time are normal, tested sample is answered There is corresponding fluorescence signal to detect, and apparent amplification curve occurs in corresponding fluorescence channel, meets value≤35.0 Ct.
5.3 sample detection interpretations of result and statement
In the case where meeting 5.1 and 5.2, using 3 groups of PCR reaction systems of A, B, C to test sample carry out sheep, fox, 9 kinds of chicken, ox, horse, duck, pig, mink and mouse animal derived materials identifications：
If any FAM, JOE, the detection of CY3 fluorescent amplification curves, and value≤35.0 Ct then judge that sample detects corresponding animal Derived component；
Such as Ct values>40.0, then it is judged to that corresponding animal derived materials are not detected；
Such as 35.0<Value≤40.0 Ct then need to repeat to test, and the Ct values after expanding again are still<40, and negative control, sky White control and positive control result are normal, then judge that the sample detects corresponding animal derived materials；Again expand after Ct values >= 40, and negative control, blank control and positive control result are normal, then judge the sample be not detected it is corresponding it is animal derived at Point.
3 kit specificity verification of embodiment
With established multiplex PCR system to ox (N), goat (SY), sheep (MY), fox (H), mink (Sd), duck (Ya), chicken (J), pig (Z), horse (M) and rat meat (S), donkey (L), rabbit (R), recoon dog (HZ), dog (G), fish (Yu) totally 15 kinds of species Genomic DNA is detected, and with Nanodrop UV spectrophotometer measuring concentration, is uniformly diluted to 10ng levels, is carried out special Anisotropic confirmatory experiment, amplification show：
(1) multiplex PCR system A (including sheep, fox and chicken mixed probe), only accordingly to sheep, fox and chicken DNA are mould Just there is corresponding amplification curve when plate, other species are in addition to the amplification curve of Quality Control probe, without corresponding amplification, show that PCR expands Increasing system has good specific amplification, sees Fig. 1 to Fig. 5；
(2) multiplex PCR system B (including ox, horse and duck mixed probe), only accordingly to ox, when horse and duck DNA are template Just there is corresponding amplification curve, other species, without corresponding amplification, show PCR amplification body in addition to the amplification curve of Quality Control probe System has good specific amplification, sees Fig. 6 to Fig. 9；
(3) multiplex PCR system C (include mink, pig and mouse mixed probe), only accordingly to mink, the DNA of pig and mouse is Just there is corresponding amplification curve when template, other species, without corresponding amplification, show PCR in addition to the amplification curve of Quality Control probe Amplification system has good specific amplification, sees Figure 10 to Figure 13.
4 kit sensitivity of embodiment is verified
By beef (N), mutton (Y), fox (H), mink (Sd), duck (Ya), chicken (J), pork (Z), horseflesh (M) and mouse The genomic DNA of meat (R) totally 9 kinds of meat quantitatively arrives 0.002ng/ μ L, 0.001ng/ μ L, 0.0005ng/ μ L, 0.0002ng/ μ respectively It is 5 μ L that L, which each reacts dosage, i.e., the Genome DNA contents of 9 kinds meat be respectively 0.01ng, 0.005ng, 0.0025ng, 0.001ng, the sample of each concentration gradient of 9 kinds of meat are respectively provided with 5 Duplicate Samples and carry out PCR amplification, further evaluate detection side The sensitivity of method.Shown in Figure 14-Figure 22, Ct values when 9 kinds of species meat Genome DNA content 0.01ng, 0.005ng<35, when containing There is the Ct values ＞ 35 having when 0.0025ng, so the sensitivity DNA content of this method is 0.005ng.
5 adulterated detection limit of embodiment is verified
Detection of adulterations is simulated according to the adulterated situation of common meat product, 1% is mixed respectively in beef,
2%, 5%, 10%, 20%, 50% horseflesh, duck, pork, mink meat；
Mix 1% in mutton respectively, 2%, 5%, 10%, 20%, 50% fox meat, rat meat, duck, chicken are shown in Table 2.Above-mentioned sample is mixed well, extract genomic DNA, carry out multiple real time fluorescence PCR detections, each sample set 4 it is parallel Sample.
2 cattle and sheep meat adulteration ratio table of table
Interpretation of result, when mixing 1% in beef respectively, 2%, 5%, 10%, 20%, 50% horseflesh, duck, pork, Mink meat；Mix 1% in mutton respectively, when 2%, 5%, 10%, 20%, 50% fox meat, rat meat, duck, chicken, equal energy It is detected, so meat adulteration detection is limited to 1%.1% adulterated meat additive amount, for illegal trade company, without any addition Meaning, therefore, 1% detection limit are suitable for the authenticity identification of the meat true and false, are shown in Table 3- tables 6.
3 beef of table distinguishes adulterated horseflesh and duck detection limit
Adulterated ratio Ox Horse Duck Result judgement
The horseflesh of incorporation 0% in beef 18.25 N N Detect calf-derived Cyclospora
The horseflesh of incorporation 1% in beef 25.62 33.56 N Detect ox source property and horse derived component
The horseflesh of incorporation 20% in beef 22.69 24.14 N Detect ox source property and horse derived component
The horseflesh of incorporation 50% in beef 20.17 19.46 N Detect ox source property and horse derived component
The duck of incorporation 0% in beef 19.45 N N Detect calf-derived Cyclospora
The duck of incorporation 1% in beef 18.75 N 30.65 Detect ox source property and duck derived component
The duck of incorporation 20% in beef 21.45 N 27.66 Detect ox source property and duck derived component
The duck of incorporation 50% in beef 20.41 N 21.52 Detect ox source property and duck derived component
4 beef of table distinguishes adulterated pork and mink meat detection limit
Adulterated ratio Ox Pig Mink Result judgement
The pork of incorporation 0% in beef 18.33 N N Detect calf-derived Cyclospora
The pork of incorporation 1% in beef 18.75 31.12 N Detect ox source property and pig derived component
The pork of incorporation 20% in beef 20.17 26.43 N Detect ox source property and pig derived component
The pork of incorporation 50% in beef 22.46 20.79 N Detect ox source property and pig derived component
The mink meat of incorporation 0% in beef 17.54 N N Detect calf-derived Cyclospora
The mink meat of incorporation 1% in beef 17.26 32.59 N Detect ox source property and mink source point
The mink meat of incorporation 20% in beef 20.37 25.46 N Detect ox source property and mink source point
The mink meat of incorporation 50% in beef 19.56 18.97 N Detect ox source property and mink source point
5 mutton of table distinguishes adulterated fox meat and chicken detection limit
Adulterated ratio Sheep Fox Chicken Result judgement
The fox meat of incorporation 0% in mutton 16.98 N N Detect sheep derived material
The fox meat of incorporation 1% in mutton 17.82 25.14 N Detect sheep source property and fox derived component
The fox meat of incorporation 20% in mutton 18.69 19.27 N Detect sheep source property and fox derived component
The fox meat of incorporation 50% in mutton 17.96 18.13 N Detect sheep source property and fox derived component
The chicken of incorporation 0% in mutton 17.09 N N Detect sheep derived material
The chicken of incorporation 1% in mutton 17.28 N 27.63 Detect sheep source property and chicken derived component
The chicken of incorporation 20% in mutton 17.25 N 20.69 Detect sheep source property and chicken derived component
The chicken of incorporation 50% in mutton 18.68 N 19.14 Detect sheep source property and chicken derived component
6 mutton of table distinguishes adulterated duck and rat meat detection limit
Adulterated ratio Sheep Duck Mouse Result judgement
The duck of incorporation 0% in mutton 16.33 N N Detect sheep derived material
The duck of incorporation 1% in mutton 17.26 31.26 N Detect sheep source property and duck derived component
The duck of incorporation 20% in mutton 19.66 23.56 N Detect sheep source property and duck derived component
The duck of incorporation 50% in mutton 18.64 20.46 N Detect sheep source property and duck derived component
The rat meat of incorporation 0% in mutton 17.99 N N Detect sheep derived material
The rat meat of incorporation 1% in mutton 17.45 N 30.21 Detect sheep source property and mouse ingredient
The rat meat of incorporation 20% in mutton 19.22 N 25.91 Detect sheep source property and mouse ingredient
The rat meat of incorporation 50% in mutton 18.75 N 20.37 Detect sheep source property and mouse ingredient
6 actual sample of embodiment detects
Unknown sample from market sampling observation sampling nearly 300 parts of samples, including fresh meat, chilled meat (monoblock meat, meat mincing, Compress the various types such as roulade, sliced meat), processed meat food, feedstuff meat meal tankage are used as blind sample after removing packaging, using this 300 parts of samples of method pair are detected, and testing result passes through sequence verification.Wherein detect mutton and pork derived component simultaneously 1 part of sample, while detecting 1 part of pork and chicken components Sample, while detecting sheep and 3 parts of duck components Sample (is shown in Table 7 and Figure 23- Figure 26).
7. unknown sample testing result of table
Sample is compiled Sheep Fox Chicken Ox Duck Horse Mouse Mink Ct Pig Result judgement
1 N N N 22.72 N N N N N Detect ox source property
2 N N N 16.18 N N N N N Detect ox source property
3 N N N 15.24 N N N N N Detect ox source property
4 N N N 29.51 N N N N N Detect ox source property
10 N N 19.15 N N N N N N Detect chicken source property
11 N N N 15.43 N N N N N Detect ox source property
12 N N N 15.39 N N N N N Detect ox source property
27 N N N 15.57 N N N N N Detect ox source property
29 N N N N 17.27 N N N N Detect duck source property
30 N N N 15.87 N N N N N Detect ox source property
31 N N N 15.34 N N N N N Detect ox source property
32 18.53 N N N N N N N N Detect sheep source property
33 17.54 N N N N N N N N Detect sheep source property
39 21.09 N N N N N N N N Detect sheep source property
40 N N N 20.17 N N N N N Detect ox source property
41 N N N 14.06 N N N N N Detect ox source property
46 N N N 13.83 N N N N N Detect ox source property
47 N N N N N N N N 15.66 Detect pig source property
48 N N N N 15.54 N N N N Detect ox source property
49 N N N N 13.54 N N N N Detect ox source property
50 N N N N 34.46 N N N N Detect ox source property
51 N N N 32.38 N N N N N Detect ox source property
52 N N N 23.74 N N N N N Detect ox source property
53 26.43 N N N N N N N N Detect sheep source property
524 31.99 N N N N N N N N Detect sheep source property
525 N N N 25.13 N N N N N Detect ox source property
526 N N N 34.99 N N N N N Detect ox source property
538 20.16 N N N N N N N N Detect sheep source property
539 25.96 N N N N N N N N Detect sheep source property
545 21.21 N N N N N N N N Detect sheep source property
546 N N N N N N N N 20.40 Detect pig source property
547 21.85 N N N N N N N N Detect sheep source property
548 N N N N N N N N 18.73 Detect pig source property
553 N N N 23.01 N N N N N Detect ox source property
554 N N N 17.76 N N N N N Detect ox source property
575 22.11 N N N N N N N N Detect sheep source property
579 N N N 17.29 N N N N N Detect ox source property
580 N N N N N N N N 21.36 Detect pig source property
581 22.60 N N N N N N N N Detect sheep source property
582 16.20 N N N N N N N N Detect ox source property
583 N N 21.33 N N N N N 21.81 Detect chicken source property
584 23.17 N N N N N N N N Detect sheep source property
585 22.71 N N N 22.97 N N N N Detect sheep source property
587 21.17 N N N N N N N N Detect sheep source property
588 N N N 16.99 N N N N N Detect ox source property
590 21.59 N N N N N N N N Detect sheep source property
591 23.58 N N N N N N N N Detect sheep source property
594 N N N 23.37 N N N N N Detect ox source property
595 23.38 N N N N N N N N Detect sheep source property
668 20.32 N N N N N N N N Detect sheep source property
670 N N N 18.82 N N N N N Detect ox source property
673 20.64 N N N N N N N N Detect sheep source property
674 21.37 N N N N N N N N Detect sheep source property
693 N N N 25.77 N N N N N Detect ox source property
716 24.18 N N N N N N N N Detect sheep source property
718 26.03 N N N 18.58 N N N N Detect sheep source property
719 N N N 16.36 N N N N N Detect ox source property
720 18.44 N N N N N N N N Detect sheep source property
721 N N N 15.94 N N N N N Detect ox source property
723 20.97 N N N N N N N N Detect sheep source property
724 20.35 N N N N N N N N Detect sheep source property
731 21.03 N N N N N N N N Detect sheep source property
738 23.49 N N N N N N N N Detect sheep source property
741 19.54 N N N N N N N N Detect sheep source property
742 21.95 N N N N N N N N Detect sheep source property
744 23.84 N N N N N N N N Detect sheep source property
746 N N N N N N N N 18.86 Detect pig source property
751 20.77 N N N N N N N N Detect sheep source property
752 19.77 N N N N N N N N Detect sheep source property
761 21.46 N N N N N N N N Detect sheep source property
767 20.00 N N N N N N N N Detect sheep source property
768 20.37 N N N N N N N N Detect sheep source property
769 N N N N 18.99 N N N N Detect duck source property
770 22.30 N N N N N N N N Detect sheep source property
776 19.42 N N N N N N N N Detect sheep source property
789 22.35 N N N N N N N N Detect sheep source property
790 20.63 N N N N N N N N Detect sheep source property
795 21.13 N N N N N N N N Detect sheep source property
809 N N N 25.57 N N N N N Detect ox source property
813 N N N N N N N N 16.34 Detect pig source property
814 19.24 N N N N N N N N Detect sheep source property
840 18.31 N N N N N N N N Detect sheep source property
842 24.60 N N N N N N N N Detect sheep source property
843 N N N N 21.50 N N N N Detect duck source property
851 N N N 17.28 N N N N N Detect ox source property
853 N N N 15.56 N N N N N Detect ox source property
855 N N N 17.78 N N N N N Detect ox source property
SEQUENCE LISTING
<110>Academy of agricultural sciences of Shandong Province biotech research center
<120>The primer of 9 kinds of animal derived materials in Rapid identification food or feed, probe compositions, kit and Its detection method and application
<130> 2015
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
aagacgagaa gacccatgga ctcta 25
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence
<400> 2
gattgcgcgg ttatccctag ggta 24
<210> 3
<211> 24
<212> DNA
<213>Artificial sequence
<400> 3
tttctcctcg cataagccta tatc 24
<210> 4
<211> 28
<212> DNA
<213>Artificial sequence
<400> 4
gttcctttta cttcttttaa tctttcct 28
<210> 5
<211> 28
<212> DNA
<213>Artificial sequence
<400> 5
ccaccaaggg ataacaacac tctggtgg 28
<210> 6
<211> 35
<212> DNA
<213>Artificial sequence
<400> 6
cggcggtaac taaccaaccc aaagagaatc cgccg 35
<210> 7
<211> 38
<212> DNA
<213>Artificial sequence
<400> 7
cagcgaagat agggataatc caaaaactaa tcacgctg 38
<210> 8
<211> 31
<212> DNA
<213>Artificial sequence
<400> 8
cagaccgatg aaatccaaac ccctcggtct g 31
<210> 9
<211> 36
<212> DNA
<213>Artificial sequence
<400> 9
cggcgaggtc taacacaaca ttattactgg tcgccg 36
<210> 10
<211> 39
<212> DNA
<213>Artificial sequence
<400> 10
cagtgatgtt atggttattt tgactggttt gcatcactg 39
<210> 11
<211> 38
<212> DNA
<213>Artificial sequence
<400> 11
cgcgcgtcct ccgaatgatt ttaacctaga ctcgcgcg 38
<210> 12
<211> 35
<212> DNA
<213>Artificial sequence
<400> 12
cgggtctggt tactgttggt actttgagag acccg 35
<210> 13
<211> 33
<212> DNA
<213>Artificial sequence
<400> 13
cgcgaaccta agactaaacc caccgggttc gcg 33
<210> 14
<211> 570
<212> DNA
<213>Artificial sequence
<400> 14
tgcactcctc ctgcatatag accaccaaat gcccctatct tatcaacact tccggaaact 60
actgttgtta gacgaagagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga 120
aggtctcaat cgccgcgtcg cagaagatct caatctcggg aatctcaatg ttagtattcc 180
ttggacacat aaggtgggaa actttacggg gctttattct tctacggtac cttgctttaa 240
tcctaaatgg caaactcctt cttttcctga cattcatttg caggaggaca ttgttgatag 300
atgtaagcaa tttgtggggc cccttacagt aaatgaaaac aggagactaa aattaattat 360
gcctgctagg ttttatccca atgttactaa atatttgccc ttagataaag ggatcaaacc 420
gtattatcca gagtatgtag ttaatcatta cttccagacg agacattatt tacacactct 480
ttggaaggcg gggatcttat ataaaagaga gtccacacgt agcgcctcat tttgcgggtc 540
accatattct tgggaacaaa gagctacaaa 570
<210> 15
<211> 21
<212> DNA
<213>Artificial sequence
<400> 15
agaccaccaa atgcccctat c 21
<210> 16
<211> 28
<212> DNA
<213>Artificial sequence
<400> 16
gttcctttta cttcttttaa tctttcct 28
<210> 17
<211> 31
<212> DNA
<213>Artificial sequence
<400> 17
gcgaggtccc ctagaagaag aactccctcg c 31

Claims (10)

1. identifying the primer of 9 kinds of animal derived materials, probe compositions in food or feed, which is characterized in that draw including following Object and probe, nucleotide sequence are as follows：
(1) primer
Universal primer sequence：
Upstream sequence：UN-F：AAGACGAGAAGACCCATGGACTCTA, as shown in SEQ ID NO.1；
Downstream sequence：UN-R：GATTGCGCGGTTATCCCTAGGGTA, as shown in SEQ ID NO.2；
Horse source special primer：
Upstream sequence：LMF：TTTCTCCTCGCATAAGCCTATATC, as shown in SEQ ID NO.3；
Downstream sequence：LMR：GTTCCTTTTACTTCTTTTAATCTTTCCT, as shown in SEQ ID NO.4；
(2) 9 kinds of animal derived materials probes：
Sheep source property specific probe MYP：5 '-CCACCAAGGGATAACAACACTCTGGTGG-3 ', as shown in SEQ ID NO.5；
Ox source property specific probe NP：5 '-CGGCGGTAACTAACCAACCCAAAGAGAATCCGCCG-3 ', such as SEQ ID NO.6 It is shown；
Horse source property specific probe MP：5 '-CAGCGAAGATAGGGATAATCCAAAAACTAATCACGCTG-3 ', such as SEQ Shown in IDNO.7；
Fox source property probe HP：5 '-CAGACCGATGAAATCCAAACCCCTCGGTCTG-3 ', as shown in SEQ ID NO.8；
Mink source probe SDP：5 '-CGGCGAGGTCTAACACAACATTATTACTGGTCGCCG-3 ', such as SEQ ID NO.9 It is shown；
Pig source property probe ZP：5 '-CAGTGATGTTATGGTTATTTTGACTGGTTTGCATCACTG-3 ', such as SEQ ID NO.10 It is shown；
Mouse probe SP：5'-CGCGCGTCCTCCGAATGATTTTAACCTAGACTCGCGCG-3 ', such as SEQ ID NO.11 It is shown；
Chicken source property probe JP：5 '-CGGGTCTGGTTACTGTTGGTACTTTGAGAGACCCG-3 ', such as SEQ ID NO.12 institutes Show；
Duck source property probe YP：5 '-CGCGAACCTAAGACTAAACCCACCGGGTTCGCG-3 ', as shown in SEQ ID NO.13；
Wherein, the 5 ' of all probes it is terminal modified have a fluorescent reporter group, the fluorescent reporter group of each probe be FAM, Any one in JOE, CY3, CY5 and ROX, each probe 3 ' it is terminal modified have quenching group, be TAMRA, DABCYL, Any one in BHQ1 and BHQ2.
2. the primer of 9 kinds of animal derived materials, probe compositions in identification food according to claim 1 or feed, It is characterized in that, 5 ' the terminal modified fluorescent reporter groups and 3 ' terminal modified quenching groups of all probes are respectively：
The fluorescent reporter group of sheep source property specific probe MYP, ox source property specific probe NP and pig source property probe ZP are marked with JOE Note；The fluorescent reporter group of horse source property specific probe MP, mouse probe SP and fox source property specific probe HP are marked with FAM Note；The fluorescent reporter group of mink source probe SDP, chicken source property probe JP and duck source property probe YP are marked with CY3；All spies The quenching group at 3 ' ends of needle is modified with DABCYL.
3. the primer of 9 kinds of animal derived materials, probe compositions in identification food according to claim 1 or 2 or feed, It is characterized in that, further include internal standard gene and corresponding exogenous interior label primer IMF and IMR and exogenous internal standard probe IMP, Internal standard gene nucleotide sequence is as shown in SEQ ID NO.14；
Exogenous interior label primer：
Upstream sequence IMF：5 '-AGACCACCAAATGCCCCTATC-3 ', as shown in SEQ ID NO.15；
Downstream sequence IMR：5 '-GTTCCTTTTACTTCTTTTAATCTTTCCT-3 ', as shown in SEQ ID NO.16；
Exogenous internal standard probe IMP：5 '-GCGAGGTCCCCTAGAAGAAGAACTCCCTCGC-3 ', such as SEQ ID NO.17 institutes Show；
Wherein, the 5 ' of exogenous internal standard probe IMP it is terminal modified have a fluorescent reporter group, the fluorescent reporter group be FAM, JOE, CY3, CY5 or ROX, exogenous internal standard probe IMP 3 ' it is terminal modified have a quenching group, the quenching group be TAMRA, DABCYL, BHQ1 or BHQ2.
4. the primer of 9 kinds of animal derived materials, probe compositions in identification food according to claim 3 or feed, It is characterized in that, the internal standard gene exists in the form of recombinant plasmid, and the recombinant plasmid is to be connected to interior label gene fragment It is formed on PMD18-T carriers.
5. the real-time fluorescence PCR combined detection kit of 9 kinds of animal derived materials, feature exist in a kind of identification food or feed In, including primer, probe compositions described in claims 1 or 2, the recombination matter in primer, probe compositions described in claim 4 Exogenous interior label primer IMF in primer, probe compositions described in grain and claim 3 and exogenous internal standard probe IMP, and Reagent necessary to PCR reactions.
6. the real-time fluorescence PCR joint-detection of 9 kinds of animal derived materials in identification food according to claim 5 or feed Kit, which is characterized in that the PCR combined detection kits are divided into three PCR reaction systems, are A systems, B systems respectively With C systems, each system shares the universal primer in the kit, recombinant plasmid and the corresponding exogenous interior label primer Pair and exogenous internal standard probe IMP and PCR reactions necessary to reagent, A systems include above-mentioned sheep source property specific probe MYP, fox source property probe HP and chicken source property probe JP, B system include that above-mentioned ox source property specific probe NP, horse source property are specifically visited Needle MP and duck source property probe YP and horse source special primer, C systems include above-mentioned mink source probe SDP, pig source property Probe ZP and mouse probe SP, wherein the fluorophor that three Species specific probes in each system are marked is different.
7. the real-time fluorescence PCR joint of 9 kinds of animal derived materials in identification food according to claim 5 or 6 or feed Detection kit, which is characterized in that reagent includes necessary to the PCR reactions：PCR buffer solutions, MgCl2, dNTPs, Taq enzyme And ultra-pure water；Each component constitutes the reaction system of a 20 μ l in the kit：10 × TaqMan Master mix, 2 μ l, Each final concentration of 0.2~0.5 μm of ol/L of primer pair, each concentration and probe concentration are 0.05~0.5 μm of ol/L, recombinant plasmid dna concentration 0.1 ~1pg/ μ l.
8. the primer of 9 kinds of animal derived materials, probe compositions are being reflected in identification food as claimed in claim 1 or 2 or feed Determine the application in 9 kinds of animal derived materials in food or feed.
9. the real-time fluorescence PCR joint of 9 kinds of animal derived materials in the food or feed described in any one of claim 5~7 Application of the detection kit in identification food or feed in 9 kinds of animal derived materials.
10. the real-time fluorescence PCR associated detecting method of 9 kinds of animal derived materials, feature exist in a kind of identification food or feed In including the following steps：
(1) genomic DNA of measuring samples is extracted, it is spare；
(2) it utilizes described in any one of any one of claim the 1-4 primer, probe compositions or claim 5-7 PCR combined detection kits carry out PCR amplification to the genomic DNA extracted in step (1), collect fluorescence signal, are judged, The PCR amplification program is：95 DEG C of 3min pre-degenerations, 95 DEG C of 5s, 60 DEG C of 35s collect fluorescence signal herein, and total 45 are followed Ring；
(3) result interpretation：The fluorescence signal during PCR is collected, is waited for by corresponding fluorescence signal amplification curve, the discriminating of Ct values Whether contain 9 kinds of animal derived materials in sample sheet.
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CN109355402A (en) * 2018-12-26 2019-02-19 中国科学院西北高原生物研究所 A kind of Rapid identification yak, ox, pien niu meat method
CN112646898B (en) * 2020-12-31 2021-10-19 吉林工商学院 Disc type micro-fluidic chip for identifying 7 animal-derived components
CN113088531B (en) * 2021-03-25 2023-10-17 四川省药品检验研究院(四川省医疗器械检测中心) Bovine-derived ingredient quantitative analysis standard plasmid, preparation and detection methods and application
CN114182027B (en) * 2022-01-26 2022-08-09 宁波市产品食品质量检验研究院(宁波市纤维检验所) Fusion curve technology-based animal-derived component universal screening kit and detection method
CN116103379A (en) * 2023-01-18 2023-05-12 广东医科大学附属医院 Detection method and application of human cells in animal body

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103397101A (en) * 2013-08-22 2013-11-20 山东省农业科学院生物技术研究中心 Fluorescently-labeled gene multiplex amplification method for identifying sources of goat, sheep, pig and duck meat simultaneously
CN104928391A (en) * 2015-06-26 2015-09-23 山东省农业科学院生物技术研究中心 Primer probe combination for identifying four components of canine animal origin, kit and multiple real-time fluorescence PCR (polymerase chain reaction) detection method

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI319813B (en) * 2007-04-14 2010-01-21 The detection method for differentiating between chickens and eggs in products

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103397101A (en) * 2013-08-22 2013-11-20 山东省农业科学院生物技术研究中心 Fluorescently-labeled gene multiplex amplification method for identifying sources of goat, sheep, pig and duck meat simultaneously
CN104928391A (en) * 2015-06-26 2015-09-23 山东省农业科学院生物技术研究中心 Primer probe combination for identifying four components of canine animal origin, kit and multiple real-time fluorescence PCR (polymerase chain reaction) detection method

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Development and validation of fast Real-Time PCR assays for species identification in raw and cooked meat mixtures;Cesare Camma等;《Food Control》;20120228;第23卷(第2期);第400-404页 *
一种基于实时荧光聚合酶链式反应的肉及肉制品中猪源性成分含量测定;周彤等;《肉类研究》;20131230;第27卷(第12期);第11-15页 *
利用多重PCR技术检测羊肉中掺杂狐狸肉的方法研究;张全芳等;《山东农业科学》;20141230;第46卷(第12期);第4-6页 *
羊肉产品中若干动物源性成分的七重PCR检测技术应用研究;冯海永等;《中国兽牧兽医》;20100920;第37卷(第9期);第85-90页 *

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