CN105296476B - Buffalo specific primer, kit and its application in the identification of buffalo derived component - Google Patents
Buffalo specific primer, kit and its application in the identification of buffalo derived component Download PDFInfo
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- CN105296476B CN105296476B CN201510814871.1A CN201510814871A CN105296476B CN 105296476 B CN105296476 B CN 105296476B CN 201510814871 A CN201510814871 A CN 201510814871A CN 105296476 B CN105296476 B CN 105296476B
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Abstract
The present invention provides a kind of buffalo specific primer, kit and its applications in the identification of buffalo derived component, belong to animal introduces a collection component molecules detection technique field.In the present invention, buffalo specific primer is the primer pair SEQ ID No.2&3 of nucleotide sequence or the specific fragment of the sequence shown in energy specific amplified SEQ ID No.1.Kit of the present invention includes the buffalo specific primer, reaction reagent and the positive and blank control.Primer amplification sample DNA using the present invention, analyzes pcr amplification product, can determine that in sample whether contain buffalo derived component.The present invention is meat products, the detection of buffalo derived component provides effective, accurate, reliable means in hide and feed, have the characteristics that it is easy, quickly, comprehensively, high specificity, and cost is relatively low, and applicability is wide.
Description
Technical field
The present invention relates to Animal molecular biology fields, and in particular to buffalo specific primer, kit and its in buffalo
Application in derived component identification.
Background technology
With the improvement of people's living standards, the increase of meat consumption figure, it is adulterated also to there are various meat products in the market
The phenomenon that.In the foreign trade of meat, since China more falls behind the method for inspection of meat quality, false retrieval, missing inspection
Phenomenon happens occasionally.It adulterates, doping is adulterated to have seriously affected fair deal, food security, even relates to religious belief
Problem.Therefore the safety in meat products source becomes one of the focal issue that consumer is concerned about, and is carried out to the source of meat products
It is the key that solve the problems, such as this to identify and establish easily detection method.
Include in the prior art mainly protein assay, such as electrophoresis, immunization etc. for the identification of meat source food
Technology, this has certain feasibility for the discriminating of raw meat, but poor specificity, accuracy rate are low.What the country had delivered is directed to buffalo
The detection method for belonging to specificity is by a large amount of examining order and to compare analysis, species-specific gene site between being belonged to,
Thus it designs specific primer and differentiates for species.However it can only be used to water for the specific primer of target sequence design
The discriminating of ox, ox and yak, and do not make a search to whether other species are also suitable.Therefore, how efficiently and accurately to differentiate
Go out whether meat products belongs to buffalo meat, and establish a kind of simple and practicable detection method there is urgency.
Invention content
It is an object of the invention in view of the shortcomings of the prior art, providing one kind can be used for buffalo source property product specific detection
Primer；
Second object of the present invention is to provide the detection kit detected for buffalo source property product；
The present invention also aims to provide the identification method of buffalo derived component.
To achieve the above object, present invention consistency and stability, inter-species specificity, copy number etc. out of kind carries out
The screening of buffalo genome specific DNA sequence dna, by common ox, sheep (sheep, goat), pig, horse, donkey, mouse (rat, mouse, storehouse
Mouse), cavy, chicken, duck, goose, rabbit, dog, fox, the genomic DNA of mink and recoon dog and the detection in different buffalo kind DNA,
Screening is special in buffalo, be not present in other species or DNA fragmentation that homology is low is as detection molecules.By a large amount of
Analysis and experimental work, it is final to obtain for detecting the specific DNA sequence used, nucleotide sequence such as sequence table SEQ ID
Shown in No.1.
Based on this, present invention firstly provides a kind of buffalo specific primers, shown in energy specific amplification SEQ ID No.1
Nucleotide sequence or the sequence specific fragment.
Preferably, primer length is 18~27bp.The design needs of primer consider whether to be easy to happen mispairing, amplified fragments
The various aspects such as length, reaction temperature.
Preferably, the primer sequence is as follows：
BUBALUS-F：5′-GAAGAAGAGGGAGGGGTAGACAA-3′
BUBALUS-R：5′-CAGAACTAGTGAAGGCGGCA-3′；
Or the primer extends to 5 ', 3 ' ends or what modification obtained remains to sequence shown in specific amplification SEQ ID No.1
Primer.
Further, the present invention provides the detection kit containing above-mentioned specific primer.
Preferably, the kit further includes one or more in following reagents：Taq enzyme, dNTPs, MgCl2, PCR it is slow
Fliud flushing, positive control dna template, distilled water；Or further include one or more in following reagents：Taq DNA
MasterMix, positive control dna template, distilled water.
The positive control dna template is the buffalo genomic DNA template of Bovidae Bubalus；
Further, the present invention provides above-mentioned buffalo specific primer or detection kit in the identification of buffalo derived component
Application.
The present invention also provides buffalo specific primer, kit and its detection sides PCR in the identification of buffalo derived component
Method, it is described that its step are as follows：
1) extraction sample gene group DNA；
2) PCR amplification is carried out with above-mentioned primer, and using buffalo DNA profiling as positive control, using distilled water as blank pair
According to；
3) pcr amplification product is detected and result judgement.
Preferably, the wherein reaction system of step 2) PCR amplification such as table 1
The reaction system of 1 PCR amplification of the present invention of table
PCR response procedures are as follows：
94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C extend 13s, 30 cycles；4 DEG C of coolings
2min。
Result judgement method is：When PCR reaction products are consistent with positive control amplified production, and blank control is produced without amplification
When object, judge to detect buffalo derived component in sample；When PCR reaction products without amplified production or with positive control amplified production
It is not inconsistent, and when blank control is without amplified production, judges not detecting buffalo derived component in sample；If positive does not detect
Go out expected segment, illustrates reagent failure or operation error；If blank control is detected with positive, illustrate reagent contamination or behaviour
It slips up.
Testing result can be presented with agarose gel electrophoresis in the present invention, it is also possible to sequencing or the detection of other effective ways；
The method of extraction sample DNA can use phenol chloroform extraction method, it is possible to use other extraction sides generally acknowledged, with identical effect
Method.
The present invention provides it is effective, accurately, reliable buffalo derived component method for identifying molecules, the reagent assembled
Whether contain buffalo derived component in box energy Rapid identification sample.The method of the present invention can be detected in meat, feed and hide
It is middle to be used as standard detecting method.Kit and detection method of the present invention have easy, quick, comprehensive, high specificity spy
Point, and cost is relatively low, applicability is wide.
Description of the drawings
Fig. 1 is SEQ ID NO.1 nucleotide sequences testing result with species specificity in buffalo.With Bovidae
The non-aqueous bovine animals of the buffalo of Bubalus, Bovidae (common ox, sheep, goat) and non-bovine mammal (mouse, rat, storehouse
Mouse, cavy, pig, horse, donkey, rabbit, fox, dog, mink, recoon dog) and nonmammalian (chicken, duck, goose) genomic DNA be mould
Plate expands specific fragment shown in SEQ ID No.1, amplified production, non-buffalo is only obtained in buffalo in all test samples
Without amplified production in species.The result shows that institute's amplified fragments have specificity in buffalo.Number is described as follows in swimming lane：M：
For DL2000Plus DNA Marker；1-3：Blank control；4-6：Buffalo；7-9：Mouse；10-12：Rat；13-15：Hamster；
16-18：Cavy；19-21：Common ox；22-24：Sheep；25-27：Goat；28-30：Pig；31-33：Horse；34-36：Donkey；37-
39：Chicken；40-42：Duck；43-45：Goose；46-48：Rabbit；49-51：Fox；52-54：Dog；55-57：Mink；58-60：Recoon dog.
Fig. 2：It is sensitivity technique knot of the SEQ ID NO.1 nucleotides sequences in buffalo difference DNA template concentration
Fruit.Respectively using 0.1ng/ μ L, 1ng/ μ L, 10ng/ μ L, 100ng/ μ L buffalo DNA as template, expand SEQ ID NO.1 nucleosides
The template concentrations of sour specific fragment, 1ng/ μ L have amplification, show that specific primer institute amplified fragments have preferable sensitivity.
Number is described as follows in swimming lane：M：For DL2000Plus DNA Marker；1-4：Blank control；5-8：The template of 0.1ng/ μ L
Concentration；9-12：The template concentrations of 1ng/ μ L；13-16：The template concentrations of 10ng/ μ L；17-20：The template concentrations of 100ng/ μ L.
Specific implementation mode
Following embodiment should not be construed as limiting the invention for further illustrating the present invention, without departing substantially from this
Under the premise of spirit and essence, the modification or polishing made to the present invention all belong to the scope of the present invention.
Buffalo derived component specific detection in 1 sample of embodiment
1. the preparation and preservation of sample
1.1 sampling
Buffalo meat sample 1g is acquired, is saved backup at -20 DEG C.
It is prepared by 1.2 DNA profilings
DNA profiling is prepared using common phenol chloroform thick formulation (Pehanorm Brooker J, not Ritchie E F, Manny A Disi
T. the golden winter wild geese of Molecular Cloning:A Laboratory guide [M] second editions, the Beijing Li Meng maple:Science Press, 1999.465-467) or it is public
Other extracting methods recognizing, with identical effect, these methods are all the common methods of report.
2. design of primers
The primer sequence of the present embodiment such as table 2 and sequence table SEQ ID NO：Shown in 2 and 3.
The PCR amplification primer that 2 present invention of table designs
It is expected that amplified fragments size is 231bp, nucleotide sequence such as sequence table SEQ ID NO：Shown in 1.
Experiment shows that the primer specificity is strong, can effectively be expanded in each kind of buffalo, and in other 18 non-buffalos
It is all expanded without target fragment in species；And the sensitivity of primer is higher, in a concentration of 1ng/uL of template DNA, that is, amplifiable.With
The primer pair that above-mentioned primer is formed to 3 ', 5 ' one base of extension and two bases or modification respectively is tested, and experiment shows
Specific amplification is can still provide for, it is best with the primer in table 2 from the point of view of economical and resultant effect.
3.PCR is detected
3.1 sample PCR reactions
3.1.1 reaction reagent (being shown in Table 1), mixing are sequentially added in PCR reaction tubes, then 25 μ L paraffin oils is added (to have hot lid
The PCR instrument of equipment can be not added with).
3.1.2 by PCR pipe, 500g~3000g centrifuges 10s on centrifuge, then takes out PCR pipe, is put into PCR instrument.
3.1.3 carrying out PCR reactions.Program is：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C are prolonged
13s is stretched, carries out 30 cycles altogether；4 DEG C of cooling 2min.
3.1.4 PCR pipe is taken out after reaction, and electrophoresis detection is carried out to PCR reaction products.
3.2 control PCR reactions
3.2.1 while sample PCR reacts, setting blank control, negative control and positive control.Each control PCR is anti-
It answers in system, remaining outer component of removing template and PCR reaction conditions are identical as 3.1, and negative, positive control dna template concentrations
The requirement of sample DNA template concentration should be reached.
3.2.2 with the non-aqueous bovine animals of Bovidae (common ox, sheep, goat) and non-bovine mammal (mouse, rat,
Hamster, cavy, pig, horse, donkey, rabbit, fox, dog, mink, recoon dog) and nonmammalian (chicken, duck, goose) genomic DNA conduct
The negative control template of PCR reaction systems.
3.2.3 using the buffalo genomic DNA of Bovidae Bubalus as the positive control template of PCR reaction systems.
3.2.4 using distilled water as the blank control template of PCR reaction systems.
The detection of 4.PCR amplified productions and result judgement
Agarose is weighed by the mass concentration of 20g/L, is added in 1 × TAE buffer solutions, is dissolved by heating, be configured to agarose
Solution.5 μ L EB solution are added in per 100mL agarose solutions, mixing is poured on electrophoresis plate, plugs after slightly suitable cooling
Pecten after being frozen into gel at room temperature, is placed in 1 × TAE buffer solutions, gently takes out pecten vertically upward.Take 5 μ L PCR products
With 1 μ L sample loading buffers (250.0mg bromophenol blues is weighed, 10mL water is added, dissolves 12h at room temperature；Weigh 250.0mg diformazans
Cyanophenyl indigo plant FF, adds 10mL water dissolutions；50.0g sucrose is weighed, 30mL water dissolutions are added.Three of the above solution is mixed, water is added to be settled to
100mL is preserved at 4 DEG C) mixing after be added gel loading wells, while in a wherein loading wells be added DNA molecular amount mark
Standard powers on and is detected after 15~30min of electrophoresis under the conditions of 2V/cm~5V/cm.
After electrophoresis, Ago-Gel is taken out, is placed in gel imager or is imaged on ultraviolet transilluminator.According to DNA
Molecular weight standard judges the size of amplified band, and electrophoresis result is formed electronic document archive or is taken pictures with photographic system.
5 interpretations of result and statement
5.1 control test interpretations of result
As shown in Figure 1, in the PCR reactions of positive control, specific sequence is expanded shown in buffalo SEQ ID No.1
Increase, and amplified fragments size is consistent with expected clip size, and does not have in addition to primer dimer in negative control and blank control
Any amplified fragments show that PCR reaction systems are working properly.
5.2 sample detection interpretations of result and statement
If 5.2.1 specific sequence shown in buffalo SEQ ID No.1 is expanded, and amplified fragments size and expected piece
Section it is in the same size, show to detect the specific sequence described in buffalo in sample, be expressed as " detected in sample buffalo source property at
Point ".
If 5.2.2 specific sequence shown in buffalo SEQ ID No.1 is not expanded or amplified fragments size and expection
Clip size is inconsistent, shows not detecting the specific sequence described in buffalo in sample, is expressed as " not detecting water outlet in sample
Calf-derived Cyclospora ".
2 sensitivity test of embodiment
Respectively using 0.1ng/ μ L, 1ng/ μ L, 10ng/ μ L, 100ng/ μ L buffalo DNA as template, according to the item of embodiment 1
Part expands SEQ ID NO.1 nucleotide segments.The results are shown in Figure 2, and the template concentrations of 1ng/ μ L have amplification, show
Specific primer institute amplified fragments have preferable sensitivity.
The assembling of 3 kit of embodiment
The composition of the kit includes：
Sense primer (SEQ ID No.2)；
Downstream primer (SEQ ID No.3)；
2×Taq DNA MasterMix；
Positive control dna template (buffalo DNA profiling)；
Distilled water.
- 20 DEG C of storages of this kit do not influence using effect in 12 months.
The application process of kit：
1. being loaded according to PCR reaction systems below, managed in the EP of 200 μ L：
2 × Taq DNA MasterMix (being purchased from Beijing bio tech ltd Ai Delai) are will be needed for PCR reactions
Taq enzyme, dNTP mixtures, MgCl2And reaction buffer is pre-configured to the mixture of 2 times of concentration.
2. the PCR amplification condition loading according to embodiment 1
3. taking 5 μ L pcr amplification products, 2.0% agarose gel electrophoresis (technical parameter after reaction：2V/cm~
5V/cm, electrophoresis 15min~30min), with ethidium bromide staining, gel imaging system testing result.
Blank control and positive control need to be arranged in each reaction, and reaction system and amplification condition are the same as by sample.
Sequence table explanation：
SEQ ID NO.1 are the nucleotide sequence of target nucleotide acid fragment, sequence length 231bp；
SEQ ID NO.2 are the forward primer sequences for expanding above-mentioned nucleotide fragments；
SEQ ID NO.3 are the reverse primer sequences for expanding above-mentioned nucleotide fragments.
Claims (7)
1. buffalo specific primer, nucleotide sequence shown in specific amplification SEQ ID No.1, the primer sequence is such as
Under：
BUBALUS-F：5′-GAAGAAGAGGGAGGGGTAGACAA-3′
BUBALUS-R：5′-CAGAACTAGTGAAGGCGGCA-3′.
2. the detection kit containing specific primer described in claim 1.
3. detection kit according to claim 2, which is characterized in that it further includes one kind or more in following reagents
Kind：Taq enzyme, dNTPs, MgCl2, PCR buffer solutions, positive control dna template, distilled water；Or further include in following reagents
It is one or more：Taq DNA MasterMix, positive control dna template, distilled water.
4. detection kit according to claim 3, which is characterized in that the positive control dna template is Bovidae buffalo
The buffalo genomic DNA template of category, distilled water are blank control.
5. buffalo specific primer according to claim 1 or claim 2~4 any one of them detection kit exist
Application in the identification of buffalo derived component.
6. the PCR detection method of buffalo derived component, it is described that its step are as follows：
1) extraction sample gene group DNA；
2) PCR amplification is carried out with primer described in claim 1, and using buffalo DNA profiling as positive control, is sky with distilled water
White control；
3) pcr amplification product is detected and result judgement.
7. the reaction system of PCR detection method according to claim 6, wherein step 2) PCR amplification is as follows：
PCR response procedures are as follows：
94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C extend 13s, 30 cycles；4 DEG C of cooling 2min.
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Citations (3)
| 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 |
| CN101724701A (en) * | 2009-12-23 | 2010-06-09 | 天津市农业科学院中心实验室 | Method for rapidly detecting cow and sheep derived components |
| CN102559919A (en) * | 2012-02-29 | 2012-07-11 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Real-time PCR (Polymerase Chain Reaction) detection method of buffalo components in food and feed |
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2015
- 2015-11-20 CN CN201510814871.1A patent/CN105296476B/en active Active
Patent Citations (3)
| 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 |
| CN101724701A (en) * | 2009-12-23 | 2010-06-09 | 天津市农业科学院中心实验室 | Method for rapidly detecting cow and sheep derived components |
| CN102559919A (en) * | 2012-02-29 | 2012-07-11 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | Real-time PCR (Polymerase Chain Reaction) detection method of buffalo components in food and feed |
Non-Patent Citations (1)
| Title |
|---|
| Bos indicus breed Nelore isolate QUIL7308 chr15_contig6354,whole genome shotgun sequence,AGFL01133914;Canavez,F.C. et al.;《Genbank》;20120216;全文 * |
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