CN101196463B - Identification method for animal derived materials - Google Patents
Identification method for animal derived materials Download PDFInfo
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- CN101196463B CN101196463B CN2007101448335A CN200710144833A CN101196463B CN 101196463 B CN101196463 B CN 101196463B CN 2007101448335 A CN2007101448335 A CN 2007101448335A CN 200710144833 A CN200710144833 A CN 200710144833A CN 101196463 B CN101196463 B CN 101196463B
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Abstract
An identification method of animal source character component is provided, which relates to an identification method of food component. The invention overcomes that the current identification method of animal source character component has the defects of low testing sensitivity and unable to test the heat-processed manufactured meat. The animal source character component is identified by the following method: extracting the source character sample DNA of the animal for testing, PCR testing and gel electrophoresis, and then identifying the component of animal source character. The method in the invention has high testing sensitivity, and the identification number limit can be below 1 percent. The identification method in the invention can test the heat-processed manufactured meat, and mix manufactured meat and bone meal product. The identification number limit also can reach below 1 percent. The identification method in the invention doesn't use liquid nitrogen in identification process, which not only lowers the cost, but also improves the safety of the operation.
Description
Technical field
The present invention relates to a kind of discrimination method of food composition.
Background technology
Because animal sources infectious diseases such as aftosa, rabid ox disease, bird flu, scrapie frequently take place, therefore the discriminating from the animal derived materials in this epidemic situation zone is just seemed very important; In order to ensure consumers in general's vital interests, stop to adulterate, pretend to be the meat products of (dope or pretend to be fully) to flow into the consumption link, and consider national and religious belief factor with other animal flesh, all need animal derived food is carried out the composition discriminating.
Because of therefore the homology of meat is difficult to direct resolution, the method differentiated of animal derived materials mainly contains micro measurement method, immunological detection method and near infrared spectrum detection method at present.
The micro measurement method is the animal derived food detection method of European Union's approval, mainly judge according to the architectural feature and the cell characteristic of various tissues in the food, by forming by the center of end is key, this end links to each other with other tissue usually, and comprises the growth cartilage as typical long bone.Cell in the cartilage is little cavity, being spherical in shape, does not have pipeline configuration in the cartilage, in addition, also has some other tissue specificity in the cartilage.Because the special shape and the tissue signatures such as distinctive lacuna, rill of fish-bone its easy bone with mammal, bird is separated, but the bone of mammal and bird then are not easily distinguishable.The micro measurement method is merely able to detect digested tankage, bone meal, meat meal tankage, blood meal, fish meal and hydrolysis, unhydrolysed feather meal at present, can't distinguish animal species wherein.
Immunology detection is a kind of convenient detection method based on soluble antigen and antibody response, and develops business-like kit.If the business-like ELISA kits of employing such as nineteen ninety-five Hofmann detect the back to food or feed sample and find that meat meal tankage has passed through sterilization treatment earlier and has been added into feed again, then can not detect.Although the distinct antibodies of heat-stable protein that adopts Ansileld detects the sample through stringent condition processing, can detect mammal in the animal feed and composition (" importing and exporting the detection method of ox and sheep components in the animal derived product " Chen Ying, Wu Yajun, Xu Baoliang, Wang Jing, money enhanced sensitivity, the Suning of pig, " food industry science and technology " 2004 25 8 phases of volume, P144～146), but ammonium sulfate precipitation technology is loaded down with trivial details in this method, and can not distinguish and forbid and allow albumen, sensitive specificity is low.
(Near infrared spectroscopic method NIRS) is widely used for the quality of the fodder control analysis, as the mensuration to moisture, albumen, fat, ash content, sugar, starch, fiber etc. to near-infrared method in feed industry.The principle of this method is that molecule in the composition analysis sample is in different absorption of electromagnetic wave differences.But the disadvantage right and wrong of this method are direct, therefore need have authoritative standard model in a large number with formation standard or discrimination model, i.e. the library of spectra of setting up at different animals composition, different component.In addition, the detection sensitivity of this method still is higher than 1% at present, and its testing result can not be separately as legal argument.
Summary of the invention
The method that above animal derived materials is differentiated all exists detection sensitivity to hang down and can't detect the defective of the meat products of hot-working processing.
The objective of the invention is to hang down and can't detect the defective of the meat products of hot-working processing for there is detection sensitivity in the method that solves present animal derived materials discriminating, and the discrimination method of a kind of animal derived materials that provides.
Animal derived materials is differentiated by the following method: one, animal derived sample to be identified is put into mortar and grind; Two, the animal derived sample powder of 50mg is put into centrifuge tube, and add 200 μ L TE solution and 400 μ L cell pyrolysis liquids, the vortex mixing adds 300 μ L water-saturated phenols, 275 μ L chloroforms and 25 μ L isoamylol again, and concussion shakes up; Four, centrifugal 10min under the condition of 14000r/min; Five, get centrifuged supernatant and add isopyknic chloroform and isoamylol mixed liquor, chloroform is mixed by 24: 1 volume ratio by chloroform and isoamylol with the isoamylol mixed liquor and forms; Six, centrifugal 10min under the condition of 14000r/min; Seven, get the step 6 centrifuged supernatant, add the isopropyl alcohol of 0.8 times of supernatant volume, then centrifugal 10min under the condition of 14000r/min; Eight, discard centrifuged supernatant, with mass concentration be the washing of 70% ethanol once, centrifugal 5min under the condition of 10000r/min discards ethanol more then, and be positioned over and dry up remaining ethanol in the centrifuge tube in the super-clean bench, in centrifuge tube, add 100 μ L TE solution afterwards and make the sediment dissolving; Nine, PCR detects and gel electrophoresis; Can differentiate the composition of animal derived sample; Mortar in the step 1 is frozen before use in-20 ℃ environment; The pH value of TE solution is 8.0 in step 2 and the step 8.
The discrimination method detection sensitivity height of animal derived materials of the present invention differentiates that inspection number limit can be below 1%.The discrimination method of animal derived materials of the present invention can detect meat products, mixing meat products and the bone meal goods of handling through hot-working, differentiates that inspection number limit reaches below 1% equally.
The discrimination method of animal derived materials of the present invention does not use liquid nitrogen in discrimination process, not only reduced cost, has improved the security of operation yet.
Description of drawings
Present embodiment is not used liquid nitrogen, and can shorten detection time, and whole testing process can be finished in 5 hours, has to detect fast advantage.
Embodiment
Embodiment two: the difference of present embodiment and embodiment one is: the step 4 repetitive operation once.Other step and parameter are identical with embodiment one.
The present embodiment identification result is better, and the amplified production band is more clear.
Embodiment three: the difference of present embodiment and embodiment one is: the PCR detection architecture is respectively pig, ox, sheep, horse, chicken and vertebrate discriminating group in the step 9, every group of PCR detection architecture is 50 μ L, is made up of 0.5 μ LTakara Taq archaeal dna polymerase, 5 μ L10 * PCR Buffer damping fluid, 2 μ L animal derived sample DNA to be identified, 4 μ L dNTP, 1 μ L upstream primer, 1 μ L downstream primer and 36.5 μ L double distilled waters.Other step and parameter are identical with embodiment one.
Present embodiment can be distinguished animality and vegetable food product composition, and whether can distinguish this composition be vertebrate, and can confirm whether contain pig, ox, sheep, horse or chicken composition in the animal derived sample to be identified.Present embodiment is differentiated by the double check of vertebrate and concrete animal, improves detection efficiency and accuracy, saves and detects cost.
Embodiment four: the difference of present embodiment and embodiment one is: the PCR response procedures is 94 ℃ of preheating 4min in the step 9,94 ℃ of sex change 30s, and 58 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate 35 times, and last 72 ℃ are extended 10min.Other step and parameter are identical with embodiment one.
Embodiment five: the difference of present embodiment and embodiment three is: the concentration of dNTP is 2.5nmol in the step 9.Other step and parameter are identical with embodiment three.
Embodiment six: the difference of present embodiment and embodiment one is: the cell pyrolysis liquid in the step 2 is a guanidinium isothiocyanate.Other step and parameter are identical with embodiment one.
Embodiment seven: the difference of present embodiment and embodiment three is: differentiate pig animality sample downstream primer Pork-lower:5 '-GCTGATAGTAGATTTGTGATGACCGTA-3 ' in the step 9, differentiate ox animality sample downstream primer beef-lower:5 '-CTAGAAAAGTGTAAGACCCGTAATATAAG-3 ', differentiate sheep animality sample downstream primer sheep-lower:5 '-CTATGAATGCTGTGGCTATTGTCGCA-3 ', differentiate horse animality sample downstream primer horse-lower:5 '-CTCAGATTCACTCGACGAGGGTAGTA-3 ', differentiate chicken animality sample downstream primer chicken-lower:5 '-AAGATACAGATGAAGAAGAATGAGGCG-3 ', differentiate pig, ox, sheep, the shared upstream primer SIM:5 ' of horse and chicken animality sample-GACCTCCCAGCTCCATCAAACATCTCATCTTGATGAAA-3 ', differentiate vertebrate property sample upstream primer General-upper:5 '-AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA-3 ', differentiate vertebrate property sample downstream primer General-lower: 5 '-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3 '.Other step and parameter are identical with embodiment three.
Present embodiment is with differentiating that vertebrate property sample upstream primer General-upper and downstream primer General-lower detect pig, ox, sheep, horse and chicken meat sample, amplified production gel electrophoresis result as shown in Figure 1, pig, ox, sheep, horse and chicken or bone meal sample all obviously have the amplified production band of 370bp among Fig. 1." M " swimming lane standard specimen is Marker among Fig. 1, " 1 " swimming lane standard specimen is pork (or bone meal) sample, " 2 " swimming lane standard specimen is beef (or bone meal) sample, " 3 " swimming lane standard specimen is mutton (or bone meal) sample, " 4 " swimming lane standard specimen is horseflesh (or bone meal) sample, and " 5 " swimming lane standard specimen is chicken (or bone meal) sample.
With present embodiment pig, ox, sheep, horse and chicken discriminating group primer the meat products of principal component is detected, meat products is respectively pure pork, pure beef, pure mutton, pure horseflesh and pure chicken products.Testing result as shown in Figure 2, " M " swimming lane standard specimen is Marker among Fig. 2, " 1 " swimming lane standard specimen is pure pork amplified production band, " 2 " swimming lane standard specimen is pure beef amplified production band, " 3 " swimming lane standard specimen is pure mutton amplified production band, " 4 " swimming lane standard specimen is pure horseflesh amplified production band, and " 5 " swimming lane standard specimen is pure chicken amplified production band.Fig. 2 is an animal derived sample reference standard sample to be identified (at pig, ox, sheep, Ma Heji).
The meat products of principal component is detected the pure beef that meat products is respectively the pure pork handled through hot-working, handle through hot-working, the pure mutton of handling through hot-working, the pure horseflesh of handling through hot-working and the pure chicken products of handling through hot-working with present embodiment pig, ox, sheep, horse and chicken discriminating group primer.Testing result as shown in Figure 3, " M " swimming lane standard specimen is Marker among Fig. 3, " 1 " swimming lane standard specimen is the pure pork amplified production band of handling through hot-working, " 2 " swimming lane standard specimen is the pure beef amplified production band of handling through hot-working, " 3 " swimming lane standard specimen is the pure mutton amplified production band of handling through hot-working, " 4 " swimming lane standard specimen is the pure horseflesh amplified production band of handling through hot-working, and " 5 " swimming lane standard specimen is the pure chicken amplified production band of handling through hot-working.
With present embodiment pig, ox, sheep, horse and chicken discriminating group primer the meat products of principal component is detected, comprise pig, ox, sheep, horse and chicken in this meat products, and the content of pig, ox, sheep, horse and chicken all is lower than 1%.This meat products testing result as shown in Figure 4, " M " swimming lane standard specimen is Marker among Fig. 4, " 1 " swimming lane standard specimen is pure pork amplified production band, " 2 " swimming lane standard specimen is pure beef amplified production band, " 3 " swimming lane standard specimen is pure mutton amplified production band, " 4 " swimming lane standard specimen is pure horseflesh amplified production band, " 5 " swimming lane standard specimen is pure chicken amplified production band, " 6 " swimming lane standard specimen is a pig animal derived materials diagnostic primers amplified production band, " 7 " swimming lane standard specimen is an ox animal derived materials diagnostic primers amplified production band, " 8 " swimming lane standard specimen is a sheep animal derived materials diagnostic primers amplified production band, " 9 " swimming lane standard specimen is a horse animal derived materials diagnostic primers amplified production band, and " 10 " swimming lane standard specimen is a chicken animal derived materials diagnostic primers amplified production band.
The discrimination method that experimental results show that the present embodiment animal derived materials can detect meat products, mixing meat products and the bone meal goods of handling through hot-working, differentiates that inspection number limit reaches below 1%.
Sequence table
＜110〉Northeast Agricultural University
＜120〉a kind of discrimination method of animal derived materials
<160>8
<210>1
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉differentiate pig animality sample downstream primer Pork-lower.
<400>1
gctgatagta?gatttgtgat?gaccgta 27
<210>2
<211>29
<212>DNA
＜213〉artificial sequence
<220>
＜223〉differentiate ox animality sample downstream primer beef-lower.
<400>2
ctagaaaagt?gtaagacccg?taatataag 29
<210>3
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉differentiate sheep animality sample downstream primer sheep-lower.
<400>3
ctatgaatgc?tgtggctatt?gtcgca 26
<210>4
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉differentiate horse animality sample downstream primer horse-lower.
<400>4
ctcagattca?ctcgacgagg?gtagta 26
<210>5
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉differentiate chicken animality sample downstream primer chicken-lower.
<400>5
aagatacaga?tgaagaagaa?tgaggcg 27
<210>6
<211>38
<212>DNA
＜213〉artificial sequence
<220>
＜223〉differentiate pig, ox, sheep, horse and the shared upstream primer SIM of chicken animality sample.
<400>6
gacctcccag?ctccatcaaa?catctcatct?tgatgaaa 38
<210>7
<211>35
<212>DNA
＜213〉artificial sequence
<220>
＜223〉differentiate vertebrate property sample upstream primer General-upper.
<400>7
aaaaagcttc?catccaacat ctcagcatga?tgaaa 35
<210>8
<211>34
<212>DNA
＜213〉artificial sequence
<220>
＜223〉differentiate vertebrate property sample downstream primer General-lower.
<400>8
aaactgcagc?ccctcagaat?gatatttgtc?ctca 34
Claims (6)
1. the discrimination method of an animal derived materials is characterized in that animal derived materials differentiates by the following method: one, animal derived sample to be identified is put into mortar and grind; Two, the animal derived sample powder of 50mg is put into centrifuge tube, and add 200 μ L TE solution and 400 μ L cell pyrolysis liquids, the vortex mixing adds 300 μ L water-saturated phenols, 275 μ L chloroforms and 25 μ L isoamylol again, and concussion shakes up; Four, centrifugal 10min under the condition of 14000r/min; Five, get centrifuged supernatant and add isopyknic chloroform and isoamylol mixed liquor, chloroform is mixed by 24: 1 volume ratio by chloroform and isoamylol with the isoamylol mixed liquor and forms; Six, centrifugal 10min under the condition of 14000r/min; Seven, get the step 6 centrifuged supernatant, add the isopropyl alcohol of 0.8 times of supernatant volume, then centrifugal 10min under the condition of 14000r/min; Eight, discard centrifuged supernatant, with mass concentration be the washing of 70% ethanol once, centrifugal 5min under the condition of 10000r/min discards ethanol more then, and be positioned over and dry up remaining ethanol in the centrifuge tube in the super-clean bench, in centrifuge tube, add 100 μ L TE solution afterwards and make the sediment dissolving; Nine, PCR detects and gel electrophoresis; Can differentiate the composition of animal derived sample; Mortar in the step 1 is frozen before use in-20 ℃ environment; The pH value of TE solution is 8.0 in step 2 and the step 8.
2. the discrimination method of a kind of animal derived materials according to claim 1 is characterized in that the step 4 repetitive operation once.
3. the discrimination method of a kind of animal derived materials according to claim 1, it is characterized in that the PCR detection architecture is respectively pig, ox, sheep, horse, chicken and vertebrate discriminating group in the step 9, every group of PCR detection architecture is 50 μ L, is made up of 0.5 μ LTakara Taq archaeal dna polymerase, 5 μ L10 * PCR Buffer damping fluid, 2 μ L animal derived sample DNA to be identified, 4 μ L dNTP, 1 μ L upstream primer, 1 μ L downstream primer and 36.5 μ L double distilled waters.
4. the discrimination method of a kind of animal derived materials according to claim 1 is characterized in that the PCR response procedures is 94 ℃ of preheating 4min in the step 9,94 ℃ of sex change 30s, and 58 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate 35 times, and last 72 ℃ are extended 10min.
5. the discrimination method of a kind of animal derived materials according to claim 3, it is characterized in that differentiating in the step 9 pig animality sample downstream primer Pork-lower:5 '-GCTGATAGTAGATTTGTGATGACCGTA-3 ', differentiate ox animality sample downstream primer beef-lower:5 '-CTAGAAAAGTGTAAGACCCGTAATATAAG-3 ', differentiate sheep animality sample downstream primer sheep-lower:5 '-CTATGAATGCTGTGGCTATTGTCGCA-3 ', differentiate horse animality sample downstream primer horse-lower:5 '-CTCAGATTCACTCGACGAGGGTAGTA-3 ', differentiate chicken animality sample downstream primer chicken-lower:5 '-AAGATACAGATGAAGAAGAATGAGGCG-3 ', differentiate pig, ox, sheep, the shared upstream primer SIM:5 ' of horse and chicken animality sample-GACCTCCCAGCTCCATCAAACATCTCATCTTGATGAAA-3 ', differentiate vertebrate property sample upstream primer General-upper:5 '-AAAAAGCTTCCATCCAACATCTCAGCATGATGAAA-3 ', differentiate vertebrate property sample downstream primer General-lower:5 '-AAACTGCAGCCCCTCAGAATGATATTTGTCCTCA-3 '.
6. the discrimination method of a kind of animal derived materials according to claim 1 is characterized in that the cell pyrolysis liquid in the step 2 is a guanidinium isothiocyanate.
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| CN2007101448335A CN101196463B (en) | 2007-12-14 | 2007-12-14 | Identification method for animal derived materials |
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| CN2007101448335A CN101196463B (en) | 2007-12-14 | 2007-12-14 | Identification method for animal derived materials |
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| CN101196463B true CN101196463B (en) | 2010-10-27 |
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Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MY149386A (en) * | 2008-06-26 | 2013-08-30 | Univ Putra Malaysia | A method for identifying a pork content in a food |
| MY148573A (en) * | 2009-05-15 | 2013-04-30 | Univ Sains Malaysia | A method to differentiate the gelatin capsule of the porcine source from the bovine source |
| CN102605090A (en) * | 2012-03-31 | 2012-07-25 | 南京市产品质量监督检验院 | Multiplex PCR method for distinguishing four meat components in food at the same time |
| CN104280468B (en) * | 2013-07-12 | 2016-04-27 | 山东东阿阿胶股份有限公司 | The detection method of horse kind and mule kind derived component in a kind of glue class Chinese medicine and goods thereof |
| CN104293899B (en) * | 2013-07-19 | 2018-08-10 | 神州基因科技（天津）有限公司 | A kind of method of horseflesh ingredient in detection food |
| CN103409519B (en) * | 2013-07-30 | 2016-01-20 | 新疆维吾尔自治区种羊与羊毛羊绒质量安全监督检验中心 | A kind of gene probe for differentiating sheep and goat derived component |
| CN104789647B (en) * | 2014-01-22 | 2019-05-14 | 深圳华大基因研究院 | Method, primer pair and kit for rapidly detecting animal-derived nucleic acid |
| CN103773846B (en) * | 2014-03-05 | 2016-05-18 | 天津出入境检验检疫局动植物与食品检测中心 | In a kind of food and feeds, goose derived components detects lateral flow ELISA test strip kit and application thereof |
| CN105087764A (en) * | 2014-05-16 | 2015-11-25 | 乌鲁木齐欧易生物医学科技有限公司 | Detecting method for safely and quickly identifying porcine-derived materials in food and kit for detecting method |
| CN104328194B (en) * | 2014-11-13 | 2016-08-17 | 西北民族大学 | Animal derived materials detection method in a kind of plant-derived food based on LAMP technology |
| CN104946788B (en) * | 2015-07-25 | 2018-02-09 | 舟山市食品药品检验检测研究院 | A kind of PCR primer and kit for differentiating 8 kinds of animal derived materials |
| CN107460248B (en) * | 2017-09-27 | 2021-01-12 | 黑龙江珍宝岛药业股份有限公司 | Primer combination for detecting animal-derived components, application and detection kit |
| CN108676896A (en) * | 2018-05-30 | 2018-10-19 | 黑龙江珍宝岛药业股份有限公司 | Detection reagent, detection kit and the application of calf blood protein-removed extraction animal derived components |
| CN111830218B (en) * | 2020-07-27 | 2021-03-09 | 江苏省家禽科学研究所 | Animal origin identification method for livestock and poultry meat |
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