CN104894263A - Multiple-species-component real-time fluorescence PCR (polymerase chain reaction) combined detection method - Google Patents
Multiple-species-component real-time fluorescence PCR (polymerase chain reaction) combined detection method Download PDFInfo
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- CN104894263A CN104894263A CN201510300666.3A CN201510300666A CN104894263A CN 104894263 A CN104894263 A CN 104894263A CN 201510300666 A CN201510300666 A CN 201510300666A CN 104894263 A CN104894263 A CN 104894263A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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Abstract
The invention discloses a real-time fluorescence PCR (polymerase chain reaction) combined detection method for simultaneously determining multiple animal-derived components in a meat product. The method can design species-specific primers according to the mitochondrion gene polymorphism difference among the animal species and simultaneously identify more than 10 species components in one PCR reaction system. The CT value is inversely proportional to the logarithm of the initial DNA (deoxyribonucleic acid) concentration, and thus, the difference of 7 above on the CT value indicates that the template DNA content difference is more than a hundred times, thereby eliminating the false positive result caused by contamination according to such judgment standard. The method can complete all animal-derived component identification on any unknown animal-derived component meat product or multiple-animal-derived-component mixed meat product within the identification rage by a single test, and is suitable for detection and identification of raw and cooked meat products and other meat products processed by various processing methods. The method does not need to perform electrophoresis, thereby shortening the analysis time and reducing the product pollution.
Description
Invention field
The present invention relates to the real-time fluorescence PCR detection method of Meat ingredients in meat product, particularly relate to the real-time fluorescence PCR associated detecting method of many animals derived components in a kind of Simultaneously test meat product, belong to food safety detection applied technical field.
Background technology
Meat product is mixed
forge vacationbe common phenomenon, some illegal retailer replaces high price meat to sell with low price meat, compromises the interests of human consumer, causes illegitimate competition; Along with the globalization of the popular of the disease such as mad cow disease and bird flu and food trade, human consumer requires to know food more urgently and to be really cut into point, therefore must set up meat product cultivar identification method fast and accurately.
At present, meat product cultivar identification method mainly contains identification of proteins (isoelectric focusing electrophoresis, enzyme linked immunosorbent assay, chromatogram etc.) and molecules identifies two classes, molecules authentication method wherein by between species based on gene difference is the focus of research, major cause is: 1. DNA is stronger than the thermotolerance of protein, the DNA of small segment still can be extracted in the food that pyroprocessing is crossed, and sex change can occur most protein, qualification needs can not be met; 2. molecules authentication method does not rely on the type of tissue and cell; 3. different gene fragment efficiency of evolution is different, can experimentally object select different goal gene to study; 4. DNA has higher interspecies variation than protein, is conducive to cultivar identification.
At present for main molecules detection method and the relative merits thereof of meat product qualification:
1.PCR-FINS: the method identifies species based on the sequence difference of specific gene fragment, be made up of 4 steps: 1. set up the method from DNA isolation biological sample widely, comprise the food (can, half processing, compacting, salt marsh, smoke) processed; 2. apply universal primer and amplify specific DNA fragmentation; 3. the DNA fragmentation of amplification is checked order; 4. this nucleotide sequence and submitted database are carried out sequence alignment, thus Direct Identification goes out species or finds species immediate with its sibship in database.Advantage: the Species origin that directly can judge non-principal component in food.Shortcoming: owing to employing universal primer, usually has Multiple components to be increased simultaneously, can cause the mixed and disorderly of sequencing result, therefore not be suitable for the qualification of mixture in mixture.
2.PCR-RFLP: be also called the sequential analysis of cutting amplification polymorphism, its basic step comprises: pcr amplification specific gene fragment, digestion with restriction enzyme, the electrophoresis of digestion products, enzyme are cut
figurethe comparison of spectrum and species identification.Advantage: simple, quick, cheap, is more suitable for conventional sense.Shortcoming: the impact being subject to Interspecific polymorphism, if restriction enzyme site there occurs sudden change, will change enzyme and cut
figurespectrum, causes occurring
mistakequalification result.
3. species specificity PCR: species specificity PCR is the difference design Auele Specific Primer according to gene order between species, make this primer can only amplify the fragment of length-specific in specific species, in other any species, all do not have the appearance of respective segments, the presence or absence according to amplified fragments realizes species identification.Advantage: directly can judge source of species by electrophoresis result, does not need follow-up order-checking or enzyme to cut process, more simply, fast, can be used for the conventional sense of large sample.Shortcoming: PCR primer easily Aerosol Pollution occurs.
4. fluorescence real-time quantitative PCR: this technology uses fluorescent energy Transfer Technology on Standard PCR basis, adds fluorescence labeling probe or fluorescence dye, detect PCR primer by means of fluorescent signal.According to fluorescently-labeled difference, two classes can be divided into: a class is the detection to specific sequence, quantitative with fluorescently-labeled specific hybridization probes, as TaqMan.Another kind of is detection to non-specific sequences, application DNA binding dye, and as SYBR green, it is combined fluorescent signal afterwards and strengthens with the sulculus of double-stranded DNA, cohesive process and sequence irrelevant.
The advantage of fluorescence real-time quantitative PCR is: 1. can realize detection by quantitative.2. its fluorescence data can pass through real-time PCR instrument or fluorimetric detector direct-detection, does not need to carry out electrophoresis, shortens analysis time, reduces product pollution.3. be applicable to the amplification of small segment, this is particularly important when the food that the food that check processing is crossed especially DNA degradation is serious.
Above method can be applied to the qualification of single species, but the PCR testing cost of single species is high, and other kind in meat, may be there is, substance PCR can not meet existing requirement, and then there is multiple PCR technique, in same reaction system, namely add multipair primer to increase the method for many target DNA fragments simultaneously.Have been reported the technology utilizing multiple PCR technique to detect 18 kinds of common animals kinds, but to vary in size according to its PCR primer, with capillary electrophoresis, its fragment is separated to the kind of rear judgement species, subsequent disposal is loaded down with trivial details, and multiplex PCR also comes with some shortcomings, the amplification efficiency caused as there is multipair primer in system is lower, and the amplification efficiency between different templates is inconsistent, and these all limit the commercial applications of technique.Therefore, the detection method setting up the unknown raw meat kind of quick, sensitive, the special detection of a kind of energy is have a difficult problem to be solved always, and associated detecting method will be expected to solve this difficult problem.
So-called joint-detection, namely use identical reaction system and reaction conditions, one-time detection can obtain all possible result.This method is different from multiple PCR technique, and each reaction carries out in different reaction tubess, avoids the mutual interference between primer.The difficult point of joint-detection is that the primer designed wants species specificity strong, can not have cross reaction, and want to be optimized to same PCR reaction conditions.The application Taqman probe that has of joint-detection qualification meat product kind that utilizes of current report measures beef, pork, mutton, chicken, turkey meat, ostrich meat composition in mixture, also has report SYBR Green method successfully to differentiate to mix the red deer of meat kind, European deer, roe deer, chamois and Pyrénées Mont Perdu goat.But also there are some problems in above detection: probe method to probe design require higher, and because of detection sensitivity too high, higher to the requirement of experimental implementation, some small pollutions also may be detected.The design requirements of dye method to primer is higher, easily has non-specific amplification, and only can not get rid of false positive with solubility curve sentence read result.
For above problem, the present invention proposes the real-time fluorescence PCR associated detecting method of many animals derived components in a kind of Simultaneously test meat product: by designing Auele Specific Primer respectively to multiple species, can increase to the specific fragment of multiple species in a PCR reaction system simultaneously, be specially adapted to detecting of mixing element in current meat product.Detecting fluorescent signal by adding fluorescence dye, need not design specific probe, cost-saving, the CT value finally by amplification determines yin and yang attribute with contrasting of internal reference, gets rid of because being infected with the false positive results caused.The feature of the method: 1, by designing Auele Specific Primer respectively to multiple species, can increase to the specific fragment of multiple species in a PCR reaction system simultaneously, and noiseless to fluorescent signal, be specially adapted to the qualification of mixing element in meat and its products.2, reaction tubes is integrated, and only needing to add template during use can carry out PCR detection, easy and simple to handle.3, judging yin and yang attribute by contrasting with internal reference, getting rid of because being infected with the false positive results caused.4, can as required flexible combination qualification meat product kind and detect the number of sample.
Summary of the invention
Technical problem to be solved by this invention overcomes the deficiencies in the prior art, the real-time fluorescence PCR associated detecting method of many animals derived components in a kind of Simultaneously test meat product is provided, design Auele Specific Primer respectively for multiple species, and the specific fragment of multiple species is increased in a PCR reaction system simultaneously.The use of binding fluorescent dyes, can direct interpretation yin and yang attribute.The method cost is low, save time, efficiently, can differentiate Multiple components simultaneously.
For solving above technical problem, the present invention adopts following technical scheme: 1. the real-time fluorescence PCR associated detecting method of several species composition in a Simultaneously test product, according on Mitochondrial DNA between species the difference of gene pleiomorphism design the primer of each species specificity, and by reaction condition optimization to same system, namely can identify multiple Species composition in a PCR system simultaneously, detecting fluorescent signal by adding fluorescence dye, finally determining yin and yang attribute by the CT value of amplification with contrasting of internal reference; The method detects more than 10 kinds animal derived materials in SDS in broiler chickens simultaneously.
The Auele Specific Primer of more than 10 species that described real-time fluorescence PCR method comprises and the sequence of 1 internal reference primer.
Described real-time fluorescence PCR detection method once completes in a PCR reaction system, on same reaction conditions the detection of many animals derived components.
The fluorescence dye SYBR Green used of described real-time fluorescence PCR method, but be not limited to SYBR Green, also can be the fluorescence dye such as Eva Green, LC Green, and by the specificity analysing amplified to the interpretation of solubility curve.
The interpretation method of the result of described real-time fluorescence PCR method: determine yin and yang attribute with contrasting of internal reference by the CT value of amplification; Direct interpretation without CT value is negative; There is comparing with internal reference primer amplification CT value of amplification CT value, be more than or equal to the interpretation of 7 for negative, be less than the interpretation of 7 for positive.
Arbitrary species that described real-time fluorescence PCR method is applied in more than 10 species detect.
Described real-time fluorescence PCR method is applied to the detection of any mixture kind in more than 10 species.
Advantage of the present invention and beneficial effect are:
1, by designing Auele Specific Primer respectively to multiple species, can increase to the specific fragment of multiple species in a PCR reaction system simultaneously, and noiseless to fluorescent signal, be specially adapted to the qualification of mixing element in meat and its products.
2, reaction tubes is integrated, and only needing to add template during use can carry out PCR detection, easy and simple to handle.
3, judging yin and yang attribute by contrasting with internal reference, getting rid of because being infected with the false positive results caused.
4, can as required flexible combination qualification meat product kind and detect the number of sample.
Embodiment
Be clearly and completely described to the technical scheme in the embodiment of the present invention below, obviously, described embodiment is only a part of embodiment of the present invention, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making other embodiments all obtained under creative work prerequisite, belong to the scope of protection of the invention.
The real-time fluorescence PCR associated detecting method of many animals derived components in a kind of Simultaneously test meat product, its authentication method is: design the primer of each species specificity according to the difference of animal interspecies variation on chondriogen, and by reaction condition optimization to same system, namely can identify the Species composition of more than 10 in a PCR system simultaneously, detecting fluorescent signal by adding fluorescence dye SYBR, finally determining yin and yang attribute by the CT value of amplification with contrasting of internal reference.This RT-PCR method can carry out Testing and appraisal to the single meat product of many animals derived component in SDS in broiler chickens (pig, ox, sheep, dog, rabbit, cat, chicken, duck, goose, mouse etc.) or the mixing meat product of many animals simultaneously.
Further, the Species-specific primer of described real-time fluorescence PCR method and the sequence of 1 internal reference primer.
Further, the PCR reaction system of described real-time fluorescence PCR method and condition.
PCR system:
PCR condition:
The fluorescence dye SYBR used of described real-time fluorescence PCR method, and by the specificity analysing amplified to the interpretation of solubility curve.The interpretation method of the result of described real-time fluorescence PCR method: determine yin and yang attribute with contrasting of internal reference by the CT value of amplification.Direct interpretation without CT value is negative; There is comparing with internal reference primer amplification CT value of amplification CT value, be more than or equal to the interpretation of 7 for negative, be less than the interpretation of 7 for positive.The logarithm of CT value and initiate dna concentration is inversely proportional to, therefore CT value difference 7 with
upper tableshowing that template DNA content differs more than hundred times, can getting rid of because being infected with the false positive results caused with this judging criterion.The arbitrary species be applied in multiple species of described real-time fluorescence PCR method detect.The detection being applied to any mixture kind in multiple species of described real-time fluorescence PCR method.
Embodiment 1
Single component detects:
1. material:
| Numbering | Sample | Composition |
| 1 | Meat processed goods | Pig |
| 2 | Meat processed goods | Ox |
| 3 | Meat processed goods | Sheep |
| 4 | Meat processed goods | Chicken |
| 5 | Meat processed goods | Duck |
| 6 | Meat processed goods | Goose |
| 7 | Tissue | Dog |
| 8 | Tissue | Rabbit |
[0042]
| 9 | Tissue | Cat |
| 10 | Tissue | Mouse |
2. extracting genome DNA: extract the genomic dna that test kit extracts each material above with OMEGA tissue DNA.
3.PCR amplified reaction:
Prepare PCR reaction solution respectively by following component, finally add sample DNA.
PCR reaction is carried out by following condition:
4. interpretation of result:
Whether normally to check amplification curve and solubility curve, the need of manual setting baseline and threshold value.The CT value of amplification curve is read after good curve to be adjusted.
5. result judges:
Result judges as follows: 1-pig 2-ox 3-sheep 4-chicken 5-duck 6-goose 7-dog 8-rabbit 9-cat 10-mouse
Embodiment 2
Mixing element detects:
1. material:
| Numbering | Sample | Composition |
| 1 | Meat meal tankage 1 | Pig, ox source mixing element |
| 2 | Meat meal tankage 2 | Ox, sheep source mixing element |
| 3 | Meat meal tankage 3 | Pig, sheep source mixing element |
| 4 | Ham sausage | Pig, chicken source mixing element |
2. extracting genome DNA: extract the genomic dna that test kit extracts each material above with OMEGA tissue DNA.
3.PCR amplified reaction:
Prepare PCR reaction solution respectively by following component, finally add sample DNA.
PCR reaction is carried out by following condition:
4. interpretation of result: whether normally to check amplification curve and solubility curve, the need of manual setting baseline and threshold value.The CT value of amplification curve is read after good curve to be adjusted.
5. result judges:
Result judges as follows: 1-pig, ox mixing element 2-ox, sheep mixing element 3-pig, sheep mixing element 4-pig, chicken mixing element.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should example be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.
Claims (7)
1. the real-time fluorescence PCR associated detecting method of several species composition in a Simultaneously test product, it is characterized in that: according on Mitochondrial DNA between species the difference of gene pleiomorphism design the primer of each species specificity, and by reaction condition optimization to same system, namely can identify multiple Species composition in a PCR system simultaneously, detecting fluorescent signal by adding fluorescence dye, finally determining yin and yang attribute by the CT value of amplification with contrasting of internal reference; The method detects more than 10 kinds animal derived materials in SDS in broiler chickens simultaneously.
2. the real-time fluorescence PCR associated detecting method of many animals derived components in a kind of Simultaneously test meat product according to claim 1, is characterized in that: the Auele Specific Primer of more than 10 species that described real-time fluorescence PCR method comprises and the sequence of 1 internal reference primer.
3. the real-time fluorescence PCR associated detecting method of many animals derived components in a kind of Simultaneously test meat product according to claim 1 and 2, is characterized in that: described real-time fluorescence PCR detection method once completes in a PCR reaction system, on same reaction conditions the detection of many animals derived components.
4. the real-time fluorescence PCR associated detecting method of many animals derived components in a kind of Simultaneously test meat product according to claim 3, it is characterized in that: the fluorescence dye SYBR Green used of described real-time fluorescence PCR method, but be not limited to SYBR Green, also can be the fluorescence dye such as Eva Green, LC Green, and by the specificity analysing amplified to the interpretation of solubility curve.
5. the real-time fluorescence PCR associated detecting method of many animals derived components in a kind of Simultaneously test meat product according to claim 1 or 4, is characterized in that: the interpretation method of the result of described real-time fluorescence PCR method: determine yin and yang attribute by the CT value of amplification with contrasting of internal reference; Direct interpretation without CT value is negative; There is comparing with internal reference primer amplification CT value of amplification CT value, be more than or equal to the interpretation of 7 for negative, be less than the interpretation of 7 for positive.
6. the real-time fluorescence PCR associated detecting method of many animals derived components in a kind of Simultaneously test meat product according to claim 5, is characterized in that: arbitrary species that described real-time fluorescence PCR method is applied in more than 10 species detect.
7. the real-time fluorescence PCR associated detecting method of many animals derived components in a kind of Simultaneously test meat product according to claim 6, is characterized in that: described real-time fluorescence PCR method is applied to the detection of any mixture kind in more than 10 species.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907856A (en) * | 2016-04-30 | 2016-08-31 | 江苏省家禽科学研究所 | COI (oxidase subunit I) gene based quail-meat-derived ingredient PCR (polymerase chain reaction) amplification method and special primer |
| CN106244698A (en) * | 2016-08-22 | 2016-12-21 | 四川华汉三创生物科技有限公司 | A kind of animal derived materials detection kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105907856A (en) * | 2016-04-30 | 2016-08-31 | 江苏省家禽科学研究所 | COI (oxidase subunit I) gene based quail-meat-derived ingredient PCR (polymerase chain reaction) amplification method and special primer |
| CN106244698A (en) * | 2016-08-22 | 2016-12-21 | 四川华汉三创生物科技有限公司 | A kind of animal derived materials detection kit |
| CN106244698B (en) * | 2016-08-22 | 2017-10-17 | 四川华汉三创生物科技有限公司 | A kind of animal derived materials detection kit |
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