CN104877995A - Primer pairs capable of amplifying multiple components of meat and meat products and application of primer pairs - Google Patents

Primer pairs capable of amplifying multiple components of meat and meat products and application of primer pairs Download PDF

Info

Publication number
CN104877995A
CN104877995A CN201510232307.9A CN201510232307A CN104877995A CN 104877995 A CN104877995 A CN 104877995A CN 201510232307 A CN201510232307 A CN 201510232307A CN 104877995 A CN104877995 A CN 104877995A
Authority
CN
China
Prior art keywords
primer
primer pair
sequence
species
primer pairs
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201510232307.9A
Other languages
Chinese (zh)
Inventor
杜红丽
赵毅
王菊芳
柳明玉
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China University of Technology SCUT
Original Assignee
South China University of Technology SCUT
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China University of Technology SCUT filed Critical South China University of Technology SCUT
Priority to CN201510232307.9A priority Critical patent/CN104877995A/en
Publication of CN104877995A publication Critical patent/CN104877995A/en
Pending legal-status Critical Current

Links

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses primer pairs capable of amplifying multiple components of meat and meat products and an application of the primer pairs. Four primer pairs are provided in all; sequences of the primer pairs are as shown in SEQ ID NO:1 to SEQ ID NO:8; sequences of meat foods of cattle, sheep, dog, pig, chicken, rabbit, duck and goose can be amplified by each primer pair under the same condition and the sequences are as shown in SEQ ID NO:9 to SEQ ID NO:48; a certain difference exists between species of the sequences which are amplified by the same primer pair so that different species can be distinguished. The primer pairs can be applied to development of kits based on fluorescent quantitative PCR (polymerase chain reaction) or other methods for meat and meat product component detection and species identification; the cost can be greatly reduced; and the operation is simplified.

Description

The primer pair of the multiple SDS in broiler chickens composition of energy amplification and application thereof
Technical field
The present invention relates to and utilize full-length genome analysis of biological information technical superiority, and develop the simplification primer pair and application thereof that can be used for multiple processed meat food stuff composition detection by the method for pcr amplification and sanger sequence verification primer pair.
Background technology
Meat-based food is one of the most polytrophic food, containing a large amount of full potassium protein, fat, carbohydrate, minerals and vitamins, and delicious flavour, specific absorption is high, so very popular.But meat-based food in process of production, source of pollution are complicated, and approach is many.Except common residue of veterinary drug, heavy metal, microorganism etc. pollute, it is also of common occurrence that meat and its products mixes pseudo-phenomenon.
Along with the further raising of all trades and professions transparency, the quality problems of processed meat food stuff also again and again expose by media, make processed meat food stuff become safely the problem of people's general concern.Various food-safety problem emerges in an endless stream, and the diet of the people has but been coverd with one deck shade.Therefore processed meat food stuff detects becomes food safety and ensures indispensable important component part.
Usually chromatographic technique, spectroscopic techniques, mass spectrum etc. are had at present for processed meat food stuff detection technique.And Measurement for Biotechnique is develop rapidly in recent years, and receive much concern in food inspection.Application more widely method has Enzyme-linked Immunosorbent Assay technology, round pcr, biosensor technology and biochip technology etc., often kind of technology has its relative merits, wherein main drawback testing cost is higher, and the cycle is longer, needs new method to detect processed meat food stuff.Along with the development of quantitative fluorescent PCR, gold mark and sequencing technologies, cost reduces gradually, the method of application quantitative fluorescent PCR, Jin Biao, sequencing technologies detects processed meat food stuff will become possibility, these technology have special, result is accurate, the advantage such as sensitive, single-minded, micro-and quick.Detect at present the fluorescent quantitative PCR technique of SDS in broiler chickens composition on the market, have employed multipair primer and to increase respectively different species, the higher and complicated operation of relative cost.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, the object of the present invention is to provide a kind of simplification primer pair of the multiple SDS in broiler chickens composition that can increase.The primer pair provided is totally 4 right; Often pair of primer can both increase the sequence of these common meat based foods of ox, sheep, dog, pig, chicken, rabbit, duck and goose under similarity condition.And there is certain otherness between the sequence species gone out with a pair primer amplification.Primer pair may be used on processed meat food stuff detection and species identification.
Another object of the present invention is to provide the application of described primer pair.
Object of the present invention is achieved through the following technical solutions:
First the present invention has done comparison to the genome of these seven species of ox, sheep, dog, pig, rat, mouse and chicken, filtered out homology be greater than 75% and be less than 100%, region that length is greater than 200bp.In order to design primer, from these regions, filtering out complete serial homology region be less than 17bp and the complete homology region number sequence region that is less than 2.Then from these homology region, design primer, require that pair of primers can amplify each species, and the sequence difference of each species is larger.With the genome sequence of the 4 pairs of primer PCRs amplification oxen of design, sheep, dog, pig, rat, mouse, chicken, rabbit, duck and goose ten species, agarose gel electrophoresis is determined successfully to amplify sequence, and then sanger order-checking, obtains sequence dna fragment.Analyze sequence and original kind genome sequence consistence that each species amplify, then analyze homology and otherness between each species sequence.Determine that the sequence amplified can be used for distinguishing the source of processed meat food stuff.
Simplification primer pair of the present invention can be applied and accelerate the exploitation of the food inspection such as quantitative fluorescent PCR or high-flux sequence test kit or method.Innovation of the present invention is also not inquire at present pair of primers and can increases above 10 species, and ensures that the sequence between species is variant.
Described primer pair is in the auxiliary application carried out in SDS in broiler chickens composition or Animal species identification.
Described primer pair is preparing the application in test kit or detection method; The purposes of described test kit or detection method is auxiliary SDS in broiler chickens Components identification or Animal species identification.
Described DNA fragmentation is in the auxiliary application carried out in SDS in broiler chickens Components identification or Animal species identification.
The present invention, relative to prior art, has following advantage and effect:
The present invention adopts biology information technology advantage to develop to increase the simplification primer pair of multiple processed meat food stuff composition, and namely the 1 pair of primer can amplify the object fragment of 10 species, and each species target DNA fragment is variant, can distinguish different species.The primer pair simplified can be applicable to the quantitative fluorescent PCR of SDS in broiler chickens composition detection and species identification or the kit developing of additive method, can greatly reduce costs and simplify the operation.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
1, target animal sample
The meat tissue of ox (cattle/bos), sheep (goat), dog (dog), pig (pig), chicken (chicken), rabbit (rabbit), rat (rat), mouse (mouse), duck (duck), goose (goose).
2, Animal genome source
The genome of this species latest edition that the genomic data of ten animal specimen is downloaded on Genebank, genomic data source is as table 1.
Table 1 genomic data is originated
Species Genome network address
Dog http://www.ncbi.nlm.nih.gov/genome/85
Pig http://www.ncbi.nlm.nih.gov/genome/84
Rat http://www.ncbi.nlm.nih.gov/genome/73
Mouse http://www.ncbi.nlm.nih.gov/genome/52
Sheep http://www.ncbi.nlm.nih.gov/genome/83
Ox http://www.ncbi.nlm.nih.gov/genome/82
Chicken http://www.ncbi.nlm.nih.gov/genome/111
3, animal tissues source: Agricultural University Of South China's animal genetic laboratory.
4, target sequence screening
The present invention adopts lastz to come comparison and analyzing gene group as comparison software.
Comparison strategy: using the genome of chicken (chicken) as with reference to genome, compare with the genome of ox, sheep, dog, pig, rat, mouse respectively.
Linux runs alignment programs, and alignment parameters is arranged as table 2, for the genome alignment of chicken and ox.
Table 2 lastz alignment parameters
Genome alignment obtains 6 comparison result files altogether, is respectively the comparison result of chicken and ox, sheep, dog, pig, rat, mouse.Because 6 species are all the comparisons carried out as reference sequences with the genome of chicken, so each species and the genomic homology region of chicken can be obtained.According to the overlap of these 6 species and chicken homology region, and then filter out region and the genome sequence of these seven species homologies of ox, sheep, dog, pig, rat, mouse and chicken.Partial results is as shown in table 3.Table 3 is the details of one group of homology region, lists the chromosome position of seven species homology region and homologous sequence length and homologous base sequence.
Table 3 species homologous sequence information
Species Karyomit(e) Homologous position Sequence long (bp) Positive minus strand Chromosome length Sequence
Chicken chrZ 60295890 420 + 82363669 CTTTGTGAA**
Dog chr3 20000602 420 + 91889043 CTTTGTGAA**
Pig chr2 63827565 420 - 162569375 CTTTGTGAA**
Ox chr7 22713126 420 - 113029157 CTTTGTGAA**
Rat chr2 245950825 420 - 258207540 CTTTGTGAA**
Mouse chr13 83741702 420 + 120421639 CTTTGTGAA**
Sheep chr5 22352555 420 - 107901688 CTTTGTGAA**
Altogether filter out homology that seven species have and be greater than 75% and homology region length is greater than the sequence 8035 groups of 200bp.Design primer needs consecutive identical sequence more than 18bp, therefore, has filtered out in these 8035 groups of sequences the sequence at least comprising two consecutive identical zone lengths and be less than 23 further, obtains 127 groups of homologous sequences that can be used for designing primer.
In order to ensure that sequence can distinguish the difference of species, choose the larger sequence of interspecies differences degree to design PCR primer.
5, design of primers
After target fragment has been screened, primer-design software Priemer 5.0 is utilized to carry out design of primers.Choose the primer can containing target fragment completely, primer length between 18 ~ 24bp, annealing temperature 55 DEG C.Design four pairs of primers altogether: No. 48, No. 70, No. 114, No. 1063, primer sequence is as table 4.Trust Beijing Hua Da genome company synthesis after all design of primers complete.
Table 4 primer sequence information
6, target fragment pcr amplification
By the gene order of four pairs of primer amplification rabbits of design, ox, sheep, dog, pig, rat, mouse, chicken, duck and goose.
Extracting genome DNA operates with reference to the specification sheets of the Animal genome DNA extraction kit (centrifugal column method) that Pu Boxin bio tech ltd manufactures.Primer is synthesized by Shanghai biotechnology company limited.Synthetic primer carries out of short duration centrifugal, and adding aseptic deionized water, to be fully dissolved into concentration be 10 μMs, and room temperature leaves standstill 30 minutes, 4 DEG C of preservations.
Carry out PCR reaction using the genomic dna of each animal specimen as template, PCR reaction system (20 μ L) is as table 5.
Table 5 PCR reaction system
Title Concentration Volume
Genomic dna 10ng/μL 5μL
Upstream primer 10μM 0.8μL
Downstream primer 10μM 0.8μL
PCR kit(2×) 10μL
ddH 2O 3.4μL
Of short duration centrifugal after mixing, be placed in PCR instrument, arranging cycling program is:
Above-mentioned PCR reaction product carries out 1.0% agarose gel electrophoresis qualification.
Take 0.5g agarose and be added to heating for dissolving in 50mL TAE reaction solution, when temperature is down to 60 DEG C, add ethidium bromide analogue 3 μ L and mix, pour into be inserted with 2mm comb electrophoresis chamber in, wait rear use to be solidified.Every hole adds 5 μ L PCR primer, adopts 121V constant voltage, after electrophoresis 15min under ultraviolet transilluminator observations, target stripe is whole single bright.
The successful PCR primer of all amplifications, with 4 DEG C of preservations, directly carries out DNA sequencing, and whole examining order is completed by order-checking portion of Hua Da genome company, Beijing.
7, sequencing sequence analysis
7.1 sequencing sequence splicings
Because the sequence initiating terminal sequencing quality of Sanger order-checking is lower, accuracy is relatively poor, adopts forward primer and reverse primer to check order respectively, and according to order-checking peak figure, sequence is supplemented mutually, is spliced into complete sequencing sequence.Detailed sequence information is shown in shown in sequence table SEQ ID NO:9 ~ SEQ ID NO:48.
7.2PCR Sequence Identification
Sequence alignment in the sequence and protogene group that obtain checking order, by determining whether by this primer PCR sequence obtained that increases be target sequence, the results are shown in Table 6,7,8,9 (duck and goose do not have complete original genomic sequence) with the Identity (homology) of original series.
Table 6, table 7, table 8 and table 9 are the comparison result of four pairs of primer PCR product sequencing results and original genomic sequence respectively, and in table, corresponding genome sequence is returned in the whole well comparison of sequence.Show that the primer designed can amplify the genome sequence of these ten classes species really accurately in target area.
Table 6 No. 48 primer PCR sequences and primary sequence homology (%)
Species Chicken Dog Pig Ox Rat Mouse Sheep Rabbit Duck Goose
Homology (%) 100 99.50 100 99.80 100 100 99.80 99.80 *** ***
Table 7 No. 70 primer PCR sequences and primary sequence homology (%)
Species Chicken Dog Pig Ox Rat Mouse Sheep Rabbit Duck Goose
Homology (%) 100 99.50 100 99.20 100 100 99.50 99.20 *** ***
Table 8 No. 114 primer PCR sequences and primary sequence homology (%)
Species Chicken Dog Pig Ox Rat Mouse Sheep Rabbit Duck Goose
Homology (%) 100 98.30 100 99.80 100 100 100 99.60 *** ***
Table 9 No. 1063 primer PCR sequences and primary sequence homology (%)
Species Chicken Dog Pig Ox Rat Mouse Sheep Rabbit Duck Goose
Homology (%) 99.80 99.20 100 98.90 100 100 99.20 99.70 *** ***
The interspecies differences of 7.3PCR sequence compares
Below, the otherness between the sequence of ten species of com-parison and analysis four pairs of primer amplifications, result is as shown in table 10, table 11, table 12, table 13.
In table, base homology between numeric representation corresponding two species in upper triangle, represents with per-cent.In lower triangle, the diversity factor of numeric representation distinguishing base, also represents with per-cent.
Four forms are the otherness of the target sequence increased between ten species, when homology not higher than 95% time, extension increasing sequence institute species can be differentiated from homology.When homology is higher, by specifically comparing homology and the diversity factor of base sequence, extension increasing sequence institute species can be differentiated.
Table 10 No. 48 primer PCR sequence Sanger order-checking homologys and diversity factor (%)
Table 11 No. 70 primer PCR sequence Sanger order-checking homologys and diversity factor (%)
Table 12 No. 114 primer PCR sequence Sanger order-checking homologys and diversity factor (%)
Table 13 No. 1063 primer PCR sequence Sanger order-checking homologys and diversity factor (%)
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (10)

1. can increase the primer pair of multiple SDS in broiler chickens composition, it is characterized in that comprising at least one pair of in primer pair 48F/R, primer pair 70F/R, primer pair 114F/R and primer pair 1063F/R:
Primer 48F:GAGGACTCCAAAGAAAGTGG;
Primer 48R:TCTTTGTCTTTGCCAATCCC;
Primer 70F:TGTGCCCCAACATGATCG;
Primer 70R:CAGCAGCAGCGTCTTCAT;
Primer 114F:GTAAATTGCCCAGTGACAGT;
Primer 114R:CACAGATTATAGCTTAGGGC;
Primer 1063F:GCCATGAGTGCCTTCAC;
Primer 1063R:CTCCATTGTAATGGATGTAG;
Described multiple SDS in broiler chickens, refers to the SDS in broiler chickens of ox, sheep, dog, pig, rat, mouse, chicken, rabbit, duck or goose.
2. the DNA sequence dna utilizing the primer pair amplifies described in claim 1 to obtain, is characterized in that:
The DNA sequence dna utilizing the primer pair 48F/R described in claim 1 to increase to obtain is as shown in SEQ IDNO:9 ~ SEQ ID NO:18.
3. the DNA sequence dna utilizing the primer pair amplifies described in claim 1 to obtain, is characterized in that:
The DNA sequence dna utilizing the primer pair 70F/R described in claim 1 to increase to obtain is as shown in SEQ IDNO:19 ~ SEQ ID NO:28.
4. the DNA sequence dna utilizing the primer pair amplifies described in claim 1 to obtain, is characterized in that:
The DNA sequence dna utilizing the primer pair 114F/R described in claim 1 to increase to obtain is as shown in SEQ IDNO:29 ~ SEQ ID NO:38.
5. the DNA sequence dna utilizing the primer pair amplifies described in claim 1 to obtain, is characterized in that:
The DNA sequence dna utilizing the primer pair 70F/R described in claim 1 to increase to obtain is as shown in SEQ IDNO:39 ~ SEQ ID NO:48.
6. primer pair according to claim 1 is in the auxiliary application carried out in SDS in broiler chickens Components identification.
7. primer pair according to claim 1 is in the auxiliary application carried out in Animal species identification.
8. primer pair according to claim 1 is preparing the application in test kit, it is characterized in that: the purposes of described test kit is auxiliary SDS in broiler chickens Components identification or Animal species identification.
9. the application of primer pair according to claim 1 in detection method, is characterized in that: the purposes of described detection method is auxiliary SDS in broiler chickens Components identification or Animal species identification.
10. the DNA sequence dna described in any one of claim 2 ~ 5 is in the auxiliary application carried out in SDS in broiler chickens Components identification or Animal species identification.
CN201510232307.9A 2015-05-08 2015-05-08 Primer pairs capable of amplifying multiple components of meat and meat products and application of primer pairs Pending CN104877995A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510232307.9A CN104877995A (en) 2015-05-08 2015-05-08 Primer pairs capable of amplifying multiple components of meat and meat products and application of primer pairs

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510232307.9A CN104877995A (en) 2015-05-08 2015-05-08 Primer pairs capable of amplifying multiple components of meat and meat products and application of primer pairs

Publications (1)

Publication Number Publication Date
CN104877995A true CN104877995A (en) 2015-09-02

Family

ID=53945672

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510232307.9A Pending CN104877995A (en) 2015-05-08 2015-05-08 Primer pairs capable of amplifying multiple components of meat and meat products and application of primer pairs

Country Status (1)

Country Link
CN (1) CN104877995A (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101712996A (en) * 2009-12-14 2010-05-26 中国科学院昆明动物研究所 Method for quickly identifying categories of meat and dried meat products of five domestic animals
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
CN103589801A (en) * 2013-11-20 2014-02-19 湖南师范大学 Specific primer sequence capable of being applied in method for identifying different fish, and DNA (Deoxyribose Nucleic Acid) molecular marker method for identifying different fish
CN103805703A (en) * 2014-02-13 2014-05-21 向华 Ingredients of animal origins in meat products identified by using mass spectrometry

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101712996A (en) * 2009-12-14 2010-05-26 中国科学院昆明动物研究所 Method for quickly identifying categories of meat and dried meat products of five domestic animals
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
CN103589801A (en) * 2013-11-20 2014-02-19 湖南师范大学 Specific primer sequence capable of being applied in method for identifying different fish, and DNA (Deoxyribose Nucleic Acid) molecular marker method for identifying different fish
CN103805703A (en) * 2014-02-13 2014-05-21 向华 Ingredients of animal origins in meat products identified by using mass spectrometry

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
M.T. BOTTERO, ET AL.: "Design of Universal Primers for the Detection of Animal Tissues in Feedstuff", 《VETERINARY RESEARCH COMMUNICATIONS》 *

Similar Documents

Publication Publication Date Title
CN108624659B (en) Real-time quantitative PCR method for detecting components of meat products
Alikord et al. Species identification and animal authentication in meat products: a review
Cheng et al. Multiplex real-time PCR for the identification and quantification of DNA from duck, pig and chicken in Chinese blood curds
Xu et al. Multiplex TaqMan locked nucleic acid real-time PCR for the differential identification of various meat and meat products
CA3079253A1 (en) Normalizing tumor mutation burden
CN105648046B (en) Method for identifying sheep, goat, mink, nutria and duck meat at one time
WO2019165858A1 (en) Multiplex pcr amplification method for species and human individual recognition and identification of unknown biological sample suspected to be from human
Liu et al. A multiplex PCR assay mediated by universal primers for the detection of adulterated meat in mutton
CN104946790A (en) PCR method for tracking source identification of origin components of eight kinds of animals
CN104328190A (en) PCR (polymerase chain reaction) product melting point analysis method for quantitatively detecting species composition of mixed-type sample
EP2841602B1 (en) A method of determining rna integrity
Dang et al. Development of a robust DNA quality and quantity assessment qPCR assay for targeted next-generation sequencing library preparation
CN102776289A (en) Reagent kit for distinguishing four components including pork, beef, mutton and chicken in food at same time and application thereof
CN106987647A (en) A kind of RPA primers, kit and detection method for detecting pig derived component
CN107345250B (en) PCR amplification identification method and special primer for quail meat source components based on 16SrRNA
CN110592268A (en) RAA constant temperature fluorescence detection method and reagent for lake luo virus (TiLV)
CN105177121B (en) Novel pigeon sex identification method
CN106995852B (en) Real-time fluorescence PCR detection method for detecting sheep-derived components in food and feed by using single-copy nuclear gene
CN107012247B (en) Real-time fluorescence PCR detection method for detecting goat-derived ingredients in food and feed by using single-copy nuclear gene
Gao et al. Multiplex-PCR method application to identify duck blood and its adulterated varieties
CN106811534B (en) Detection primer, method and kit for detecting multiple meat-derived components at one time
CN104877995A (en) Primer pairs capable of amplifying multiple components of meat and meat products and application of primer pairs
CN112501320B (en) Snake origin component rapid detection kit and application thereof
CN108251534B (en) Multiple PCR detection kit for rapidly detecting meat-derived food
WO2021250530A1 (en) Rapid method for genotyping sting variants in human individuals

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
EXSB Decision made by sipo to initiate substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20150902