CN101519682B - Method for detecting cat source components in food and fodder and kit - Google Patents
Method for detecting cat source components in food and fodder and kit Download PDFInfo
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- CN101519682B CN101519682B CN2009100482234A CN200910048223A CN101519682B CN 101519682 B CN101519682 B CN 101519682B CN 2009100482234 A CN2009100482234 A CN 2009100482234A CN 200910048223 A CN200910048223 A CN 200910048223A CN 101519682 B CN101519682 B CN 101519682B
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Abstract
The invention discloses a method for detecting cat source components in food and fodder and a kit. Specifically, the invention discloses a method for quickly detecting cat source components, wherein apolymerase chain reaction is carried out in a polymerase reaction system which contains specific primer pairs of the expanded cat source components and cat source component specific probes. The inven tion also provides a corresponding kit. The method can quickly and conveniently detect and identify the cat source components.
Description
Technical field
The present invention relates to molecular biology and nucleic acid detection technique field.More specifically, the method and the test kit that relate to a kind of rapid detection cat source components.
Background technology
In animal product raw material and converted products, mingling of animal component often occur and fake and the unintentional pollution phenomenon.The people mainly is that illegal merchant will reduce cost or increases quality for mingling; To seek huge economic interests; And in the course of processing of food and feed, with the raw material of the raw material substitution high price of low price or be incorporated in the raw material of high price, this is a kind of fraud.Mingle the fraud of fraud, forbidden by China's multi-section laws and regulations.
Thisly mingle fraud, shoddy behavior except influencing China's economy, most importantly another influence HUMAN HEALTH and fowl poultry safety.The generation of mad cow disease and spread to countries in the world economy such as European Union and brought heavy losses, the route of transmission of mad cow disease mainly is with Mammals source feed feeding of ruminant animals.
Various countries such as European Union, the U.S., Japan all make laws and regulations at present, forbid with Mammals source feed feeding of ruminant animals, with generation and the propagation that prevents mad cow disease.In order thoroughly to cut off the route of transmission of mad cow disease; Prevent that mad cow disease from taking place in China within the border; Ministry of Agriculture's issue in 2002 " about forbidding in ruminant feed, adding and using the notice of animal-feed "; Issued and implemented " animal derived feeds product safety and sanitation management method " in 2004, all forbidden in ruminant feed, using the Mammals derived component.Cat is as a kind of Mammals, a kind of people domesticated animal/pet, and quantity is very big.If not strict control, the tissue of dead cat may be used for the processing of animal-feed.If be mixed with cat source property animal component in the animal feedstuff, may bring the biological risk of Animal diseases eruption and prevalence.
Cat is the seldom edible animals of people, and itself carries zoonosiss such as rabies, has pathophorous risk.If edible seldom edible cat meat may cause spreading disease.
China does not also have correlation method that the cat source components in food and the feed is detected at present.That is to say that the cat source components in food and the feed detects and still is in space state.Set up cat source components detection method in food and the feed; For hitting fake and forged food, feed; Protect consumers' interests with healthy; Protection China's livestock industry safety and economic interests avoid aspect such as international trade dispute to have crucial meaning, are extremely urgent food-safety problem that will solve and inspection and quarantine problem.
In sum, in view of the detection technique that does not still detect and identify at present cat source components quickly and easily, therefore, this area presses for the new technology that detects and identify cat source components quickly and easily of exploitation.
Summary of the invention
The object of the invention just provides a kind of technology that whether has cat source components in food or the feed that detects quickly and easily and identify.
In first aspect of the present invention, a kind of polymerase chain reaction method is provided, it comprises step:
In the polymeric enzyme reaction system, carry out the polymerase chain reaction; The Auele Specific Primer that contains the cat source components that increases in the described reaction system to the cat source components specific probe; And the amplified production that said primer amplification goes out has the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQID NO:5, and described cat source components specific probe is incorporated into the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5.
In another kind of preference, described cat source components specific probe has the nucleotide sequence shown in the SEQ ID NO:3.
In another kind of preference, the Auele Specific Primer of described amplification cat source components is to being selected from down group:
Primer 1:5 '-agcacgaaag taactttaac acctcc-3 ', SEQ ID NO:1;
Primer 2: 5 '-tgctagtagt tctctggcgg atag-3 ', SEQ ID NO:2.
In another kind of preference, described cat source components specific probe is Taqman probe (an especially Taqman MGB probe).
In another kind of preference, probe sequence is following:
5’-actacacgac?agctaaga-MGB-3’，SEQID?NO：3。
In another kind of preference, said method also comprises step: in the process of polymerase chain reaction or afterwards, detect the fluorescent signal that the cat source components specific probe sends.
In second aspect of the present invention, a kind of detection kit is provided, it contains following reagent:
(a) Auele Specific Primer of amplification cat source components is right, and said primer has the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5 to the amplified production that amplifies; With
(b) cat source components specific probe, and described cat source components specific probe is incorporated into the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5.
In another kind of preference, described cat source components specific probe has the nucleotide sequence shown in the SEQ ID NO:3.
In another kind of preference, the Auele Specific Primer of described amplification cat source components is to being selected from down group:
Primer 1:5 '-agcacgaaag taactttaac acctcc-3 ', SEQ ID NO:1
Primer 2: 5 '-tgctagtagt tctctggcgg atag-3 ', SEQ ID NO:2.
In another kind of preference, described cat source components specific probe is a Taqman MGB probe.
In the third aspect of the invention, a kind of method that whether contains cat source components in the detected sample that detects is provided, comprise step:
(a) extracting goes out nucleic acid component from detected sample;
(b) to extractive nucleic acid component; In the polymeric enzyme reaction system, carry out the polymerase chain reaction; The Auele Specific Primer that contains the cat source components that increases in the described reaction system to the cat source components specific probe; And the amplified production that said primer amplification goes out has the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5, and described cat source components specific probe is incorporated into the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5;
Wherein, if amplify amplified production, then represent to contain cat source components in the described detected sample corresponding to the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5.
In another kind of preference; If do not amplify amplified production corresponding to the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5; Then represent not contain cat source components in the described detected sample, or the content of cat source components is lower than detection sensitivity lower limit (as 0.0001%).
In another kind of preference, described polymerase chain reaction is a fluorescent real time PCR.
In another kind of preference, the Auele Specific Primer of amplification cat source components is to being selected from down group:
Primer 1:5 '-agcacgaaag taactttaac acctcc-3 ', SEQ ID NO:1
Primer 2: 5 '-tgctagtagt tctctggcgg atag-3 ', SEQ ID NO:2.
In another kind of preference, described detected sample is food or feed.
In fourth aspect of the present invention, but a kind of sequence right purposes of primer that nucleic acid molecule or the specific amplification shown in SEQ ID NO:5 goes out said nucleic acid molecule is provided, it is used to prepare the test kit that detects cat source components.
In another kind of preference, described primer is to being selected from down group:
Primer 1:5 '-agcacgaaag taactttaac acctcc-3 ', SEQID NO:1
Primer 2: 5 '-tgctagtagt tctctggcgg atag-3 ', SEQ ID NO:2.
In another kind of preference, described test kit also contains the probe of sequence shown in SEQ ID NO:3.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and specifically described each technical characterictic can mutual combination in (like embodiment) hereinafter, thus constitute new or optimized technical scheme.As space is limited, this tired no longer one by one stating.
Description of drawings
Fig. 1 has shown the nucleotide sequence (SEQ ID NO:4) of cat 12S rRNA gene and upstream and downstream thereof and the sequence of Auele Specific Primer and probe.
Fig. 2 has shown in instance of the present invention, detects collection of illustrative plates with cat source property primer probe specificity degree real-time fluorescence PCR.
Fig. 3 has shown in instance of the present invention, detects collection of illustrative plates with cat source property primer probe sensitivity real-time fluorescence PCR.Wherein,
A: specific detection, amplification appears in cat DNA; B: sensitivity detects, and amplification curve is respectively from left to right: 10% cat DNA, 1% cat DNA, 0.1% cat DNA, 0.01% cat DNA, 0.001% cat DNA, 0.0001% cat DNA.
Fig. 4 has shown the linear relationship between Ct value and the template amount.
Fig. 5 has shown in instance of the present invention, and meat product is carried out the collection of illustrative plates that the cat source components real-time fluorescence PCR detects.
Fig. 6 has shown in instance of the present invention, to carrying out the collection of illustrative plates that the cat source components real-time fluorescence PCR detects in the plant feed.
Embodiment
Inventor's process thinks that to the extensive and deep research of the range gene sequence of cat source components the nucleotides sequence column region of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5 is particularly suitable for detecting and identifying cat source components.Not only can design the primer of the high amplification cat source components of specificity to this zone, also can design the high cat source components probe of specificity.Like this, in a PCR reaction,, just can identify cat source components quickly and easily through detecting the fluorescent signal of TaqMan MGB probe.
As used herein, term " cat source components specific probe " refer to such primer (to), the amplified production that it amplifies has the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5.A kind of preferred primer is to having the sequence shown in SEQ ID NO:1 and 2.
As used herein, term " cat source components specific probe " refers to be incorporated into the amplified production of cat source components, but debond is in the probe of the amplified production of other species (like pig, ox, sheep etc.).More preferably, described cat source components specific probe specificity is incorporated into the nucleotide sequence of cat plastosome 12S rRNA gene cat source components, shown in SEQ ID NO:5.A kind of preferred probes has the sequence shown in the SEQ IDNO:3.
Real-time fluorescence PCR is airtight amplification detection method, after the amplification system application of sample finishes, can increase and detect, and does not analyze after need not increasing again; Not only reduce the possibility of environmental pollution, also owing to there is primer pair to hybridize jointly, higher than regular-PCR specificity with probe.
The TaqMan polymerase chain reaction technique utilizes Taq enzyme 5 ' → 3 ' 5 prime excision enzyme activity, in the process of extending, realizes the cut-out to hybridization probe, eliminates the cancellation effect of probe 3 ' end fluorophor to 5 ' end mark report fluorophor signal.The quantity of the power of fluorescent signal and PCR product is proportional, and detection system can be inferred the quantity of PCR product through detecting fluorescence.
Preferred amplification region is the nucleotide sequence (corresponding to the 374-507 bit sequence among the SEQ ID NO:4) of cat plastosome 12S rRNA gene cat source components, shown in SEQ ID NO:5 in the inventive method; This zone is that cat source components is distinctive, can be used for detecting cat source components.
According to instruction of the present invention, those skilled in the art can design and synthetic primer and cat source components specific probe, and is used for fluorescent real time PCR qualitative and quantitative detection technology.
In the present invention; Except detecting the target sequence be directed against and prior art be different; Such as all identical with prior art from the condition of steps such as the total extracting DNA of sample, pcr amplification and fluoroscopic examination, those skilled in the art can carry out above-mentioned various step with the condition of routine fully.
In a preferred embodiment of the invention, electrophoresis result shows that the Auele Specific Primer of amplification cat source components is to increasing to the nucleic acid of cat source components.The real-time fluorescence detected result shows, only containing the sample performance positive of cat source components, produces a desired effect.This shows that the cat source components specific probe that goes out through choose reasonable is very effective and special.
When using the quantitative PCR appearance to carry out the real-time fluorescence detection,, can detect at 530nm or 640nm equiwavelength according to the difference of fluorescent marker (FAM, TET).
Major advantage of the present invention is:
(1) can detect and identify cat source components easy, apace.
(2) based on the primer of the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5 to the nucleotide sequence of specific amplification cat source components effectively, the cat source components specific probe then can be distinguished cat source components more specifically.
(3) the detected result good linearity can be used for quantitative analysis.
(4) can detect amplified production in the real-time fluorescence PCR amplification procedure, not need the electrophoresis detection process, both shorten detection time, again because of not adopting the ethidium bromide carcinogenic substance, to environment and personal security.In addition, owing to add specific probe, therefore can directly prove conclusively detected result.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; People such as Sambrook for example; Molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
One, experiment material and method
1.1 material
Animal species: 29 kinds of animals such as cat meat, cat blood, dog meats, beef, pork, Goral mutton, meat of a sheep, Cervus nippon meat, Carnis Cameli, horseflesh, donkey meat, rabbit meat, hare meat, Wild boar as food, racoon dog meat, chicken, duck, goose, turkey, pigeon, quail, meat of snake, bullfrog, frog meat, grass shrimp, crab, grass carp, oyster, mussel, snail are available from market, Shanghai.
Plant species: 10 kinds of frequently seen plants materials such as soybean, corn, barley, wheat, rice, yam, tomato, pea, cotton, rape.
1.2 reagent
(1) primer and TaqMan MGB fluorescent probe sequence:
12Scat-1：5’-AGCACGAAAGTAACTTTAACACCTCC-3’(SEQ?ID?NO：1)
12Scat-2：5’-TGCTAGTAGTTCTCTGGCGGATAG-3’(SEQ?ID?NO：2)
12Scat-P：5’-ACTACACGACAGCTAAGA-MGB-3’(SEQ?ID?NO：3)
(2) liquid nitrogen;
(3)
Magnetic DNA Purification system for Food; Paramagnetic particle method DNA extraction test kit, the CAT.#FF3750 of Promega company;
(4)
Genomic DNA Purification Kit; The DNA extraction test kit, the CAT.#A1250 of Promega company;
(5) Virahol;
(6) 70% ethanol;
(7) TE damping fluid, pH8.0
(8)ABI?TaqMan?Universal?PCR?Master?Mix(Part?Number?4304437)
(9) ultrapure water (ddH
2O).
1.3 plant and instrument
(1) ABI 7300 real-time fluorescence PCR appearance
(2) balance
(3) liquid-transfering gun
(4) whizzer
(5) constant temperature couveuse
(6) freezing runner milling
(7) DNA lyophilize whizzer
(8) nucleic acid-protein concentration analyzer
(9) Eppendorf centrifuge tube
(10) PCR reaction tubes
1.4 the preparation of sample
1.4.1 the grinding of sample
With freeze grinding machine (SPEX 6850) above-mentioned meat sample is ground to form powdery.
1.4.2 sample preparation
Use the freeze grinding machine with mutton and the wheat shape of claying into power, and 120 ℃ dry 6h.Using mutton powder and wheat-flour to be made into cat digested tankage content respectively is the biased sample of 0.01% (W/W).
1.5DNA extraction
Extract above-mentioned sample DNA according to
Magnetic DNA Purification system for Food (CAT.#FF3750 of Promega company) DNA extraction test kit.Extract cat blood DNA with
Genomic DNAPurification Kit (CAT.#A1250 of Promega company) DNA extraction test kit.
1.6DNA concentration determination and preparation
Concentration with German Eppendorf Biophotometer type nucleic acid-protein analysis-e/or determining extracting sample DNA.
(concentration: 100ng/ μ L) (concentration: 100ng/ μ L) be diluted to the mixed solution of 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L, 0.001ng/ μ L, 0.0001ng/ μ L, 0.00001ng/ μ L, its relative dna content is 10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001% and 0.00001% with extractive cat blood dna solution with the wheat dna solution.
1.7PCR test
1.7.1PCR reaction system
Carry out the reaction system of real-time fluorescence PCR detection and see table 1.
The reaction system that table 1 real-time fluorescence PCR detects
1.7.3PCR reaction cycle parameter
Real-time fluorescence PCR reaction cycle parameter: 50 ℃, 2min; 95 ℃, 10min; 95 ℃ of 15s, 60 ℃ of 1min circulate 40 times.
1.8 sensitivity test
LOD is meant the minimum sample concentration that can detect when detection probability surpasses 95%.Can carry out the duplicate detection of capacity through setting up a series of samples of different concentrations, perhaps through detecting the reference substance of a series of various objectives gene template content repeatedly, to confirm LOD.
The expression form of LOD can be the copy number of dna molecular, the relative percentage of dna molecular or the quality of DNA.This experiment is with the quality of the relative percentage of dna molecular and the DNA expression form as sensitivity test.
2 results and analysis
2.1 the design of the primer probe of cat native gene
(GenBank is numbered: the nucleotide sequence (936bp-1895bp) of 12S rRNA gene U20753) and upstream and downstream thereof (shown in SEQ ID NO:4) according to the cat Mitochondrial Genome Overview; On primer design to the consistence of the annealing temperature of cat 12S rRNA gene test primer and probe; The factors such as similarity of GC content have been carried out sufficient consideration; Adopted DNAMAN 8.0 software designs the cat source components primer and the probe that detect, the nucleotide sequence and the primer probe sequence of cat 12S rRNA gene and upstream and downstream thereof are seen Fig. 1.Blast function through GenBank is sought the homology of this primer probe, and this gene has very high specificity, and the homology of other genes and this gene is very little.
Primer and probe
12Scat-1：5’-AGCACGAAAGTAACTTTAACACCTCC-3’(SEQ?ID?NO：1)
12Scat-2：5’-TGCTAGTAGTTCTCTGGCGGATAG-3’(SEQ?ID?NO：2)
12Scat-P：5’-ACTACACGACAGCTAAGA-MGB-3’(SEQ?ID?NO：3)
The PCR product that this primer increases and obtains (SEQ ID NO:1 and 2), its sequence is shown in SEQ ID NO:5, corresponding to the 374-507 position among the SEQ ID NO:4.
2.2 cat source components real-time fluorescence PCR species specificity detected result
Supply examination animal and plant sample DNA to detect cat DNA with other with cat source property detection primer, the result is as shown in Figure 2, amplification (Fig. 2) wherein only in cat DNA, occurs.
2.3 cat source components real-time fluorescence PCR sensitivity detected result
To 100ng/ μ L (100%), 10ng/ μ L (10%), 1ng/ μ L (1%); 0.1ng/ μ L (0.1%), 0.01ng/ μ L (0.01%), 0.001ng/ μ L (0.001%); 0.0001ng/ μ L (0.0001%), 0.00001ng/ μ L (0.00001%) cat DNA detects.
The result is as shown in Figure 3.Among Fig. 3, A: specific detection, amplification appears in cat DNA; B: sensitivity detects, and amplification curve is respectively from left to right: 10ng/ μ L (10%) cat DNA, 1ng/ μ L (1%) cat DNA; 0.1ng/ μ L (0.1%) cat DNA; 0.01n g/ μ L (0.01%) cat DNA, 0.001ng/ μ L (0.001%) cat DNA, 0.0001ng/ μ L (0.0001%) cat DNA.
Amplification (Fig. 3) all appears in greater than the cat DNA of 0.0001ng/ μ L (0.0001%).This shows that cat source property detection primer has species specificity, and detection sensitivity is 0.0001ng/ μ L (0.0001%) cat DNA.Logarithm (x) with the dna profiling amount is done typical curve with Ct value (y), and obtains equation of linear regression (Fig. 4); Linear relationship between Ct value and the template amount is good, R
2Be 0.9976.The result shows that this method can carry out quantitative analysis to cat DNA concentration (relative content).
2.4 the detection of cat source components in the meat product (animal product)
Adopt cat meat composition positive material to add, carry out the detection research of cat source components in the animal material product.Add in the mutton powder matrix that the animal that does not contain the cat composition is a raw material with cat digested tankage sample, be prepared into 0.01% interpolation sample.Use present method and detect, with the detection of cat source components in the simulated animal product.
Experimental result shows that amplification curve does not appear in PCR negative control result, and reaction result is normal, and reaction system is pollution-free.Detect cat source components in the animal material product, the result shows that this method can detect cat source components material (Fig. 5) in 0.01% the animal material product.
2.5 the detection of cat source components in the plant feed (plant prod)
Adopt cat meat composition positive material to add, carry out the detection research of cat source components in the plant material product.Add in the wheat-flour matrix that the plant that does not contain the cat composition is a raw material with cat digested tankage sample, be prepared into 0.01% interpolation sample.Use present method and detect, with the detection of cat source components in the simulating plant product.
Experimental result shows that amplification curve does not appear in PCR negative control result, and reaction result is normal, and reaction system is pollution-free.Detect cat source components in the plant material product, the result shows that this method can detect cat source components material (Fig. 6) in 0.01% the plant material product.
3 conclusions
The present invention exploitation be the detection that the real-time fluorescent PCR testing primer probe of object is applicable to raw material (cat meat) and the middle cat source components of converted products (food, feed etc.) to detect cat 12S rRNA gene, can be applied to the routine check quarantine.
Detection to food and feed
In addition, with sequenator the amplified production of detected sample has been carried out nucleotide sequencing, the result shows the nucleotides sequence column region of the cat plastosome 12S rRNA gene of cat source components just shown in SEQ ID NO:5 that amplifies.
The fluorescence real-time RT-PCR detected result of table 2 different batches sample
| Batch | The sample title | The source place | Detected |
|
| 1 | Cat | Shanghai | Positive | |
| 2 | | Shanghai | Positive | |
| 3 | Unknown cube meat | Game restaurant, | Positive | |
| 4 | Cube meat | Another game store of | Positive | |
| 5 | Cube meat | The restaurant, | Negative | |
| 6 | Cube meat | The restaurant, | Negative | |
| 7 | Spiced cube meat | The restaurant, | Negative | |
| 8 | Meat meal tankage | Australia | Negative | |
| 9 | Fish meal | Peru | Negative | |
| 10 | Fish meal | The | Negative | |
| 11 | Fodder additives | The | Negative | |
| 12 | | Shanghai | Negative | |
| 13 | Feed | Jiangsu | Negative | |
| 14 | Feed | Guangzhou | Negative | |
| 15 | Feed | Guangzhou | Negative |
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
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Claims (7)
1. polymerase chain reaction method is characterized in that it comprises step:
In the polymeric enzyme reaction system, carry out the polymerase chain reaction; The Auele Specific Primer that contains the cat source components that increases in the described reaction system to the cat source components specific probe; And the amplified production that said primer amplification goes out is the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5
The nucleotide sequence of wherein said cat source components specific probe is shown in SEQ ID NO:3, and the Auele Specific Primer of described amplification cat source components is to being:
Primer 1:5 '-agcacgaaag taactttaac acctcc-3 ', SEQ ID NO:1 with
Primer 2: 5 '-tgctagtagt tctctggcgg atag-3 ', SEQ ID NO:2.
2. the method for claim 1 is characterized in that, described cat source components specific probe is a Taqman MGB probe.
3. method as claimed in claim 2 is characterized in that, said method also comprises step: in the process of polymerase chain reaction or afterwards, detect the fluorescent signal that the cat source components specific probe sends.
4. a detection kit is characterized in that, it contains following reagent:
(a) Auele Specific Primer of amplification cat source components is right, and said primer is the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5 to the amplified production that amplifies; With
(b) cat source components specific probe, and the nucleotide sequence of described cat source components specific probe is shown in SEQ ID NO:3; The Auele Specific Primer of described amplification cat source components is to being:
Primer 1:5 '-agcacgaaag taactttaac acctcc-3 ', SEQ ID NO:1 with
Primer 2: 5 '-tgctagtagt tctctggcgg atag-3 ', SEQ ID NO:2.
5. test kit as claimed in claim 4 is characterized in that, said cat source components specific probe is a Taqman MGB probe.
6. one kind is detected the method that whether contains cat source components in the detected sample, it is characterized in that, comprises step:
(a) extracting goes out nucleic acid component from detected sample;
(b) to extractive nucleic acid component; In the polymeric enzyme reaction system, carry out the polymerase chain reaction; The Auele Specific Primer that contains the cat source components that increases in the described reaction system to the cat source components specific probe; And the amplified production that said primer amplification goes out is the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5, and the nucleotide sequence of described cat source components specific probe is shown in SEQ ID NO:3;
Wherein, if amplify amplified production, then represent to contain cat source components in the described detected sample corresponding to the nucleotide sequence of the cat plastosome 12S rRNA gene shown in SEQ ID NO:5;
The Auele Specific Primer of wherein said amplification cat source components is to being:
Primer 1:5 '-agcacgaaag taactttaac acctcc-3 ', SEQ ID NO:1 with
Primer 2: 5 '-tgctagtagt tctctggcgg atag-3 ', SEQ ID NO:2.
7. sequence nucleic acid molecule or the primer shown in SEQ ID NO:5 is characterized in that the purposes of SEQ ID NO:1 and SEQ ID NO:2, is used to prepare the test kit that detects cat source components.
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| CN2009100482234A CN101519682B (en) | 2009-03-26 | 2009-03-26 | Method for detecting cat source components in food and fodder and kit |
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| CN2009100482234A CN101519682B (en) | 2009-03-26 | 2009-03-26 | Method for detecting cat source components in food and fodder and kit |
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| CN101519682A CN101519682A (en) | 2009-09-02 |
| CN101519682B true CN101519682B (en) | 2012-01-11 |
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| CN105039540B (en) * | 2015-07-14 | 2018-07-03 | 山东省兽药质量检验所 | A kind of LAMP detection method for differentiating cat meat in beef and mutton using mitochondrial DNA |
| CN108676853A (en) * | 2017-12-01 | 2018-10-19 | 苏州百源基因技术有限公司 | A kind of real-time fluorescence quantitative PCR kit for mouse detection |
| CN113957172B (en) * | 2021-11-16 | 2022-11-15 | 广州蔚捷生物医药科技有限公司 | Kit for simultaneously detecting various cat pathogens and detection method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1779441A (en) * | 2004-11-19 | 2006-05-31 | 潘良文 | Real-time fluorescent inspection and reagent kit for Norwalk virus in mussel |
| CN101381767A (en) * | 2008-10-24 | 2009-03-11 | 东北农业大学 | Universal real time fluorescent PCR detection method of trichinella |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1779441A (en) * | 2004-11-19 | 2006-05-31 | 潘良文 | Real-time fluorescent inspection and reagent kit for Norwalk virus in mussel |
| CN101381767A (en) * | 2008-10-24 | 2009-03-11 | 东北农业大学 | Universal real time fluorescent PCR detection method of trichinella |
Non-Patent Citations (2)
| Title |
|---|
| I. Martin 等.Technical Note: Detection of cat, dog, and rat or mouse tissues in food and animal feed using species-specific polymerase chain reaction.《Journal of Animal Science》.2007,第85卷2734-2739. * |
| 张慧霞等.应用PCR技术检测禽类源性成分.《中国畜牧杂志》.2008,第44卷(第11期),第46-49页. * |
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