CN109355402A - A kind of Rapid identification yak, ox, pien niu meat method - Google Patents
A kind of Rapid identification yak, ox, pien niu meat method Download PDFInfo
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- CN109355402A CN109355402A CN201811601841.2A CN201811601841A CN109355402A CN 109355402 A CN109355402 A CN 109355402A CN 201811601841 A CN201811601841 A CN 201811601841A CN 109355402 A CN109355402 A CN 109355402A
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Abstract
The invention belongs to molecular biosciences identification method technical fields, in particular to a kind of Rapid identification yak, ox, pien niu meat method, including acquiring beef sample to be identified and extracting DNA sample, it designs specific primer and PCR amplification is carried out to sample to be identified, electrophoresis is carried out with Ago-Gel to the product after amplification, comparison electrophoresis result judges beef type.It is at low cost the present invention provides a kind of easy, quick, efficient, accurate beef Identification of Species method, there is application prospect.
Description
Technical field
The invention belongs to molecular biosciences identification method technical field, in particular to a kind of Rapid identification yak, ox, pien niu
The method of meat.
Background technique
Instantly the good and bad jumbled together in meat products market, and meat products is adulterated to propose very big difficulty to the supervision of meat products Market Quality
With require.Meat products is adulterated for example has and pretends to be mutton or beef with pork, and with duck, chicken, What is more is filled with mouse meat
When the higher beef and mutton of price, the problems such as this is not only related to consumer health and is related to religious belief, it is unknown come
Therefore the health of the consumer in source is faced with huge threat.
Beef is the meat that the majority of consumers prefer, especially big in the beef consumption figure such as Qinghai, Xinjiang, Tibet
Provinces and cities.However, also having Preference for different beef people due to eating habit, yak is because it is lived mostly in cold
Pastoral area, and it is most of herded based on, so its pro-gaze of green, organic environment by numerous consumers, catering market yak
Beef sales are extremely hot, this therefrom plays tricks with regard to someone, serve as yak meat with common beef (such as Carnis Bovis seu Bubali, pien niu meat), take advantage of
Deceive the majority of consumers.
Currently, common and effective meat identification method has organoleptic detection to be identified such as: color, fiber thickness,
This identification method need appraiser have experience abundant and simultaneously accuracy it is low；Another kind is exactly by specific DNA
Segment is expanded, and is recycled to purpose band, digestion, electrophoresis detection identification, and step is slightly cumbersome.For this purpose, our needles
A kind of simple and quick identification method is had studied to ox, pien niu, pien niu meat.
Summary of the invention
It is at low cost the present invention provides a kind of easy, quick, efficient, accurate beef Identification of Species method, there is application
Prospect.
The present invention provides a kind of Rapid identification yak, ox, pien niu meat method, comprising the following steps:
S1 extracts DNA sample；
S2 designs specific primer and carries out PCR amplification to sample to be identified；
Wherein, the specific primer sequence are as follows:
F:5 '-tgctctctggccttctccagtcagaa-3 ',
R:5 '-gctgcagtaattctcctgtgac-3 '；
S3, to the product after amplification with 1.5% Ago-Gel the electrophoresis 30-35min at 110V, compare electrophoresis result
Judge beef type；When clip size is 962bp, sample is yak；When clip size is 1047bp, sample is ox；
When clip size is two kinds of 962bp, 1047bp, sample is pien niu.
Preferably, the step of said extracted DNA sample is specific as follows:
S11 collects beef sample to be identified, sequentially adds cell pyrolysis liquid and the Proteinase K of 20mg/mL, 56 DEG C of digestion
Overnight, digestion solution is obtained；
S12, addition are equivalent to the isometric Tris saturated phenol of S11 digestion solution, mix, and 12000r/min is centrifuged 10min,
Retain supernatant, be then added and be equivalent to the isometric phenol of supernatant, chloroform and isoamyl alcohol three's mixed solution, mixes,
12000r/min is centrifuged 10min, retains supernatant,
Wherein, the volume ratio of phenol, chloroform and isoamyl alcohol three is 25:24:1；
Chloroform is added in S13, S12 supernatant, mixes, 12000r/min is centrifuged 10min, retains supernatant and is added suitable
In the cold ethyl alcohol of 2 times of volumes, precipitating is isolated and with 75% ethanol washing, 12000r/min is centrifuged 10min, precipitating is taken to dry,
Obtain solid DNA sample；
Solid DNA sample adds ddH in S14, S132O or TE dissolution, is adjusted to 500ng/ μ L.
Preferably, the composition of the cell pyrolysis liquid are as follows: Tris-HCl, 36mL's of the 150mmol/L of 12mL
The 10%SDS solution of EDTA, 9mL of 0.5mmol/L mix, and water is added to be settled to 180mL.
Preferably, the beef sample to be checked, cell pyrolysis liquid, Proteinase K, in S13 chloroform amount ratio are as follows: 1g:
1400 μ L:40 μ L:1000 μ L.
Preferably, the PCR amplification system are as follows: Mix solution 5 μ L, each 0.5 μ L of two primer sequence solution, primer sequence
Solution concentration is 10 μm of ol/L, sample DNA templates 0.5 μ L, ddH2O supplies 10 μ L.
Preferably, the PCR amplification condition are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C
Extend 30s, totally 30 circulations；72 DEG C of extension 10min, 4 DEG C of amplified production preservations.
Compared with prior art, beneficial effects of the present invention:
It is simple and easy the present invention provides a kind of easy, quick, efficient, accurate beef Identification of Species method, it mentions significantly
The high speed and accuracy of detection, and it is at low cost, application prospect is all had in fields such as livestock culturing, food safeties.
Detailed description of the invention
Fig. 1 is the electrophoresis result figure of the embodiment of the present invention 1.
Specific embodiment
1 pair of the specific embodiment of the present invention is described in detail with reference to the accompanying drawing, it is to be understood that of the invention
Protection scope be not limited by the specific implementation.
Embodiment 1
A kind of Rapid identification yak, ox, pien niu meat method, comprising the following steps:
S1 extracts DNA sample；
S11 collects 0.5g beef sample to be identified, shreds as much as possible, sequentially add 700 μ L cell pyrolysis liquids and 20 μ L
20mg/mL Proteinase K, 56 DEG C digest overnight (more than 12h), obtain digestion solution；
The composition of cell pyrolysis liquid are as follows: the Tris-HCl (pH8.0) of the 150mmol/L of 12mL, the 0.5mmol/L of 36mL
The mass concentration 10%SDS solution mixing of EDTA (pH8.0), 9mL, add water to be settled to 180mL；
Wherein, EDTA is prepared are as follows: 186.12g EDTA.2Na.2H is added in 800mL water2O, magnetic stirring apparatus are vigorously stirred,
PH value of solution is adjusted to 8.0 with NaOH, and then plus water is settled to 1L, and high pressure sterilization is spare after packing；
10%SDS solution is prepared are as follows: 100g electrophoresis grade SDS is added in 900mL water, is heated to 68 DEG C of hydrotropies, and it is dense that several drops are added
Salt acid for adjusting pH is to 7.2, and then plus water is settled to 1L, and packing is spare, without sterilizing.
S12, addition are equivalent to the isometric Tris saturated phenol of S11 digestion solution, turn upside down and mix 10min, 12000r/
Min is centrifuged 10min, retains supernatant, is then added and is equivalent to the isometric phenol of supernatant, chloroform and isoamyl alcohol three mixing
Solution is mixed by inversion, and 12000r/min is centrifuged 10min, retains supernatant,
Wherein, the volume ratio of phenol, chloroform and isoamyl alcohol three is 25:24:1；
500 μ L chloroforms are added in S13, S12 supernatant, turns upside down and mixes 10min, 12000r/min is centrifuged 10min, protects
It stays supernatant and the cold ethyl alcohol (100% ethyl alcohol ice bath) for being equivalent to 2 times of volumes is added, isolate precipitating and washed with 75% ethyl alcohol
It washs, 12000r/min is centrifuged 10min, takes precipitating to dry, obtains solid DNA sample；
Solid DNA sample adds ddH in S14, S132O or TE buffer solution takes 1 μ L point sample electrophoresis, checks the extraction of DNA
Effect is adjusted to 500ng/ μ L；
S2 designs specific primer and carries out PCR amplification to sample to be identified；
Wherein, specific primer sequence are as follows:
F:5 '-tgctctctggccttctccagtcagaa-3 ', as shown in SEQ ID NO.1；
R:5 '-gctgcagtaattctcctgtgac-3 ', as shown in SEQ ID NO.2；
PCR amplification system are as follows: Mix solution 5 μ L, each 0.5 μ L of two primer sequence solution, primer sequence solution concentration are
10 μm of ol/L, sample DNA templates 0.5 μ L, ddH2O supplies 10 μ L；
PCR amplification condition are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 30
Secondary circulation；72 DEG C of extension 10min, 4 DEG C of amplified production preservations；
S3, to the product after amplification, with 1.5% Ago-Gel, the electrophoresis 30min at 110V, comparison electrophoresis result are sentenced
Disconnected beef type.The clip size of yak is 962bp；The clip size of ox is 1047bp；Pien niu has 962bp, 1047bp two
Band.Fig. 1 is the electrophoresis result figure of embodiment 1, and 1,2,3 swimming lanes are yak as we know from the figure；4,5,6 swimming lanes are ox；7,8,9
Swimming lane is pien niu.
Embodiment 2
A kind of Rapid identification yak, ox, pien niu meat method, comprising the following steps:
S1 extracts DNA sample；
0.5g beef sample to be identified is collected, according to bibliography: Bao Yixin, Sun Bo, Zhang Longlong, to animal tissue DNA
The improvement of extracting method and PCR detect Zhejiang Normal University journal: the method for natural science edition 2009 obtains solid DNA sample；
Solid DNA sample is added into ddH2O or TE buffer solution takes 1 μ L point sample electrophoresis, checks the extraction effect of DNA, be adjusted to
500ng/μL；
S2 designs specific primer and carries out PCR amplification to sample to be identified；
Wherein, specific primer sequence are as follows:
F:5 '-tgctctctggccttctccagtcagaa-3 ', as shown in SEQ ID NO.1；
R:5 '-gctgcagtaattctcctgtgac-3 ', as shown in SEQ ID NO.2；
PCR amplification system are as follows: Mix solution 5 μ L, each 0.5 μ L of two primer sequence solution, primer sequence solution concentration are
10 μm of ol/L, sample DNA templates 0.5 μ L, ddH2O supplies 10 μ L；
PCR amplification condition are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 30
Secondary circulation；72 DEG C of extension 10min, 4 DEG C of amplified production preservations；
S3, to the product after amplification, with 1.5% Ago-Gel, the electrophoresis 35min at 110V, comparison electrophoresis result are sentenced
Disconnected beef type；As a result segment band size is 962bp, and sample is yak.
It should be noted that the step method used in claims of the present invention is same as the previously described embodiments, in order to anti-
Only repeating, the present invention describes preferred embodiment, once a person skilled in the art knows basic creative concept,
Then additional changes and modifications can be made to these embodiments.So it includes being preferably implemented that the following claims are intended to be interpreted as
Example and all change and modification for falling into the scope of the invention.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Sequence table
<120>a kind of Rapid identification yak, ox, pien niu meat method
<141> 2018-12-19
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213>artificial sequence
<400> 1
tgctctctgg ccttctccag tcagaa 26
<210> 2
<211> 22
<212> DNA
<213>artificial sequence
<400> 2
gctgcagtaa ttctcctgtg ac 22
Claims (6)
1. a kind of Rapid identification yak, ox, pien niu meat method, which comprises the following steps:
S1 extracts DNA sample；
S2 designs specific primer and carries out PCR amplification to sample to be identified；
Wherein, the specific primer sequence are as follows:
F:5 '-tgctctctggccttctccagtcagaa-3 ',
R:5 '-gctgcagtaattctcctgtgac-3 '；
S3, to the electrophoresis 30-35min at 110V, the comparison electrophoresis result judgement with 1.5% Ago-Gel of the product after amplification
Beef type；When clip size is 962bp, sample is yak；When clip size is 1047bp, sample is ox；Work as piece
When Duan great little is two kinds of 962bp, 1047bp, sample is pien niu.
2. Rapid identification yak as described in claim 1, ox, pien niu meat method, which is characterized in that extract DNA sample
The step of it is specific as follows:
S11 collects beef sample to be identified, sequentially adds cell pyrolysis liquid and the Proteinase K of 20mg/mL, and 56 DEG C of digestion are stayed overnight,
Obtain digestion solution；
S12, addition are equivalent to the isometric Tris saturated phenol of S11 digestion solution, mix, and 12000r/min is centrifuged 10min, retain
Then supernatant is added and is equivalent to the isometric phenol of supernatant, chloroform and isoamyl alcohol three's mixed solution, mix, 12000r/
Min is centrifuged 10min, retains supernatant,
Wherein, the volume ratio of phenol, chloroform and isoamyl alcohol three is 25:24:1；
Chloroform is added in S13, S12 supernatant, mixes, 12000r/min is centrifuged 10min, retains supernatant and addition is equivalent to 2
The cold ethyl alcohol of times volume, isolates precipitating and with 75% ethanol washing, and 12000r/min is centrifuged 10min, takes precipitating to dry, must consolidate
Body DNA sample；
Solid DNA sample adds ddH in S14, S132O or TE dissolution, is adjusted to 500ng/ μ L.
3. Rapid identification yak as claimed in claim 2, ox, pien niu meat method, which is characterized in that the cell cracking
The composition of liquid are as follows: the 10%SDS solution of EDTA, 9mL of the 0.5mmol/L of Tris-HCl, 36mL of the 150mmol/L of 12mL are mixed
It closes, water is added to be settled to 180mL.
4. Rapid identification yak as claimed in claim 2, ox, pien niu meat method, which is characterized in that the beef to be checked
Sample, cell pyrolysis liquid, Proteinase K, in S13 chloroform amount ratio are as follows: 1g:1400 μ L:40 μ L:1000 μ L.
5. Rapid identification yak as described in claim 1, ox, pien niu meat method, which is characterized in that the PCR amplification
System are as follows: 5 μ L of Mix solution, each 0.5 μ L of two primer sequence solution, primer sequence solution concentration is 10 μm of ol/L, sample
DNA profiling 0.5 μ L, ddH2O supplies 10 μ L.
6. Rapid identification yak as described in claim 1, ox, pien niu meat method, which is characterized in that the PCR amplification
Condition are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 63 DEG C of annealing 30s, 72 DEG C of extension 30s, totally 30 times circulation；72 DEG C of extensions
10min, 4 DEG C of amplified production preservations.
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Cited By (10)
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| CN110106258A (en) * | 2019-05-14 | 2019-08-09 | 西南民族大学 | A kind of yak meat identification kit |
| US10986817B2 (en) | 2014-09-05 | 2021-04-27 | Intervet Inc. | Method and system for tracking health in animal populations |
| US10986816B2 (en) | 2014-03-26 | 2021-04-27 | Scr Engineers Ltd. | Livestock location system |
| US11071279B2 (en) | 2014-09-05 | 2021-07-27 | Intervet Inc. | Method and system for tracking health in animal populations |
| USD990062S1 (en) | 2020-06-18 | 2023-06-20 | S.C.R. (Engineers) Limited | Animal ear tag |
| USD990063S1 (en) | 2020-06-18 | 2023-06-20 | S.C.R. (Engineers) Limited | Animal ear tag |
| US11832584B2 (en) | 2018-04-22 | 2023-12-05 | Vence, Corp. | Livestock management system and method |
| US11832587B2 (en) | 2020-06-18 | 2023-12-05 | S.C.R. (Engineers) Limited | Animal tag |
| US11864529B2 (en) | 2018-10-10 | 2024-01-09 | S.C.R. (Engineers) Limited | Livestock dry off method and device |
| US11960957B2 (en) | 2020-11-25 | 2024-04-16 | Identigen Limited | System and method for tracing members of an animal population |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US10986816B2 (en) | 2014-03-26 | 2021-04-27 | Scr Engineers Ltd. | Livestock location system |
| US11963515B2 (en) | 2014-03-26 | 2024-04-23 | S.C.R. (Engineers) Limited | Livestock location system |
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| USD990063S1 (en) | 2020-06-18 | 2023-06-20 | S.C.R. (Engineers) Limited | Animal ear tag |
| US11832587B2 (en) | 2020-06-18 | 2023-12-05 | S.C.R. (Engineers) Limited | Animal tag |
| US11960957B2 (en) | 2020-11-25 | 2024-04-16 | Identigen Limited | System and method for tracing members of an animal population |
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