CN107012247A - The real-time fluorescence PCR detection method of goat derived component in food and feed is detected using single-copy nuclear gene - Google Patents
The real-time fluorescence PCR detection method of goat derived component in food and feed is detected using single-copy nuclear gene Download PDFInfo
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Abstract
The invention discloses the real-time fluorescence PCR detection method of goat derived component in a kind of utilization single-copy nuclear gene detection food and feed, comprise the following steps：The first step, using the DNA of testing sample as template, carries out fluorescent quantitative PCR, obtains pcr amplification product；Second step, detects the fluorescence signal of amplified production；3rd step, judges whether contain goat derived component in sample with the Ct values of testing result；Wherein, the specific probe of specific primer pair and goat derived component containing amplification goat derived component in the reaction system expanded for PCR.The specific primer pair and probe of the real-time fluorescent PCR amplification of the goat derived component of the present invention are not only specific good, and sensitivity is high, rapidly and accurately to detect in food and feed whether provide a kind of quantitative detecting method containing goat derived component, with preferable application prospect.
Description
Technical field
The invention belongs to bioengineering field, specifically, be on one kind using single-copy nuclear gene detection food and
The real-time fluorescence PCR detection method of goat derived component in feed.
Background technology
Because there is very big difficulty always in the differentiation of chevon and meat of a sheep, therefore in the inspection of many food and feed
Do not go generally to distinguish goat and sheep among surveying, only detect whether containing sheep composition, this is buried virtually food-safe
Hidden danger, also causes some criminals to carry out adulterated make profit and had opportunity.At the beginning of 2013, multiple Europe such as Sweden, Britain
Continent country is involved in " horseflesh disturbance " scandal, causes concern of the various countries to meat and the adulterated problem of meat products.
In China, " adulterated " problem is always the focus of the focus that consumer complains and social concerns.Some illegal retailers
With enterprise because interests are driven, the low cost feedstocks such as pig, duck are mixed in the high price meat such as ox, sheep and meat products.These behaviors are not only
Consumer's interests are encroached on, it is also possible to because causing dispute containing non-Islamic composition in some religion food, if also had without quarantine
The propagation of epidemic disease may be caused, be unfavorable for maintaining social stability and people's health, if defective product outlet is gone abroad more
The overall image of China's food enterprise can be damaged.In order to hit the illegal activities of meat adulteration, consumers' rights and interests and life are safeguarded
Safety, ensures the sound development of food industries, and it is pole to formulate behavior of the strict laws and regulations to constrain the producer and operator
To be important, while being also required to work out accurate, sensitive, easy detection method.
Common meat and meat products true and false authentication technique mainly includes：(1) immunoassay based on protein molecular structure
Technology etc.；Immunoassay method based on protein macromolecule structure have high sensitivity, high flux, it is easy to operate the characteristics of, can
Realize the quick detection to a large amount of samples.For example, Macedo-Silva etc. by prepare anti-ox, chicken, pig, horse albumin anti-blood
The clear ELISA method that establishes is used to differentiate the cheap Meat ingredients that may be mixed in hamburger, and sensitivity is up to 0.6%.But, base
Easily there is cross reaction for the nearer species of affiliation in the immunoassay method specifically bound in Ag-Ab.Albumen
Matter tertiary structure may be destroyed in food preparation process and influence the important weak point that antibody identification is also this method.
(2) Protocols in Molecular Biology based on nucleic acid, such as PCR, real-time fluorescence PCR and molecular fingerprint technology；It is special based on DNA sequence dna
Detection target spot that the Protocols in Molecular Biology of property differentiates using the difference of hereditary information between animal species as meat kind and made extensively
With.Compared to protein, DNA is high due to heat endurance, although there is a certain degree of degraded in process, remains to carry
Taking out small fragment DNA is used for the analysis such as PCR.DNA has that specificity is high, sensitivity is high, by tissue class is not limited etc. all simultaneously
Many advantages, have higher resolution capability for the nearer species of affiliation.In recent years, meat based on DNA detection techniques and
The research of meat products authenticity discrimination method is more and more.All kinds of analysis methods based on PCR are focused primarily upon at present, its
Include DNA sequencing, multiplex PCR, real-time fluorescence quantitative PCR etc..Particularly Real-Time Fluorescent Quantitative PCR Technique it is animal derived into
The detection field divided, increasingly as main flow detection technique.
Round pcr is combined with target dna sequence by one section of special oligonucleotides and synthesized in vitro millions of
DNA copy.DNA fragmentation is expanded by Species-specific primer, is separated by agarose electrophoresis and observes specificity
Band is the most basic method that round pcr differentiates Species composition.Mitochondrial DNA number of copies in cell is big, sensitivity is high, enter
Change speed fast, with higher inter-species diversity and relatively low intraspecific variablity, be widely used in design of primers and target sequence
Amplification.Chondriogen as conventional has cytochrome b gene, 12S and 16S rRNAs subunit, D-loop areas.But,
Because the copy number of mtdna sequence is high and non-constant, the qualitative detection of animal derived materials is can be only applied to, it is difficult to using
In quantitative detection.“Species identification and quantification in meat and meat
products using droplet digital PCR(ddPCR)Analytical Methods”,C.Floren,
I.Wiedemann, B.Brenig, E.Sch ü tz, J.Beck, Food Chemistry 173 (2015) 1054-1058, this article
Conclusion specify that and can cause result error using the primer and probe in mitochondria source quantitative when.
The primer and probe of detection animal component is typically all to be designed for chondriogen both at home and abroad at present, mitochondria
Copy number of the gene in cell is in 5000-6000 copies or more.But found in practical study work：Utilize multicopy
When chondriogen is designed a set of primer and probe and detected, it is 13 or so that its Ct value, which low can reach, so low Ct values, when
Animal derived materials are at 0.00000001% in sample, and Ct values are still 37 or so.In the such low detection of target detection thing
, there are such Ct values in concentration, and it is difficult to judge that actual sample is really to contain target component to make testing staff, or sample by
Very slight pollution.So using chondriogen design primer and probe detected, easily occur false positive results or
It is difficult to judge the testing result under low target content.If providing positive detection report according to so low Ct values, it can draw
Very big trade dispute.
Therefore, it is necessary to set up that a set of specific good, sensitivity is high, the food of false positive results etc. can be avoided the occurrence of and
The real-time fluorescence quantitative PCR detection method of goat derived component in feed.
The content of the invention
The present invention is had found by studying：The animal derived materials in the pure meat sample product of some animal are detected, single copy is utilized
Karyogene design primer and probe detected, its Ct value for 22-24 when, when animal derived materials exist in sample
When 0.001%, Ct values are 37 or so.Species discriminating is carried out using the karyogene of single copy to detect, as a result find for this present invention
The situation generation that can be avoided the occurrence of false positive results or be difficult judged result is detected using single copy gene, and utilizes single copy
Karyogene detected, moreover it is possible to quantitatively detected, marked so the karyogene for finding single copy detect and set up accordingly
Quasi- method seems very necessary.
Primary and foremost purpose of the present invention is the real-time PCR detection side for providing goat derived component in a kind of food and feed
Method, with overcome the shortcomings of present in prior art be difficult to it is quantitative detection, easily there are false positive results etc..The second of the present invention
Individual purpose is the application for providing a kind of detection kit and detection kit.Third object of the present invention is offer one
Plant the primer pair and probe of the real-time fluorescence PCR detection method of goat derived component in food and feed.
To achieve the above object, the present invention uses following technical scheme：
As the first aspect of the invention, one kind using single-copy nuclear gene detect food and goat source property in feed into
The real-time fluorescence PCR detection method divided, this method comprises the following steps：
The first step, using the DNA of testing sample as template, carries out fluorescent quantitative PCR, obtains pcr amplification product；
Second step, detects the fluorescence signal of amplified production；
3rd step, judges whether to contain goat derived component in sample and quantitatively detects sample with the Ct values of testing result
In goat derived component content；
Wherein, specific primer pair and goat containing amplification goat derived component in the reaction system expanded for PCR
The specific probe of derived component, the sequence such as SEQ ID NO.1 of the specific primer pair of the amplification goat derived component and
Shown in SEQ ID NO.2, the sequence of the specific probe of the goat derived component is as shown in SEQ ID NO.3.
According to the present invention, the condition of the PCR amplifications is as follows：
Reaction system is 25 μ L：The μ L of real-time fluorescence PCR reagent (2 ×) 12.5, each 10 μM of primer, 5 μM of probe, DNA profiling 5
μL；
PCR reaction conditions：95℃10min；95℃15s；60℃1min；Totally 45 circulations.
As the second aspect of the invention, a kind of detection kit of goat derived component, the detection kit contains
Following reagent：
(a) specific primer pair of goat derived component is expanded；
(b) specific probe of goat derived component；
Wherein, sequence such as SEQ ID NO.1 and the SEQ ID of the specific primer pair of the amplification goat derived component
Shown in NO.2, the sequence of the specific probe of the goat derived component is as shown in SEQ ID NO.3.
Further, the detection kit also includes：
(c) marker, the content for quantitatively detecting the goat derived component in sample.
As the third aspect of the invention, a kind of application of described detection kit, the detection kit is used for
Goat derived component in qualitative or quantitative detection food or feed.
As the fourth aspect of the invention, a kind of specific primer pair and probe of goat derived component are described special
The sequence of property primer pair is as shown in SEQ ID NO.1 and SEQ ID NO.2, the sequence such as SEQ ID of the specific probe
Shown in NO.3.
The beneficial effects of the invention are as follows：
1st, there is provided it is a kind of from karyogene can specificity identification goat derived component primer pair and probe, it is special
It is different in nature good, specific amplification can be realized for goat derived component, and then can not be special for other compositions beyond goat
Specific amplification；Moreover, the primer pair and probe have good repeatability, result reliable and stable.
2nd, it can quickly, in large quantity be detected using described primer pair and probe and whether there is goat source in food or feed
Property the composition and quantitative content of detection food or the goat derived component in feed, and required sample size is few, simple to operate,
Sensitivity is high.
3rd, the real-time fluorescence PCR detection method of goat derived component of the invention, its is applied widely, is particularly suitable for use in each
The detection of goat derived component in based food or feed, therefore can be to be applicable, the quality to ensureing product, protection consumption
Person's right to know and right to choose, safeguard that normal economic order etc. provides technical support, are market surveillance department and the inspection of food
Quarantine departments are tested there is provided technical support.
Brief description of the drawings
Fig. 1 is first group of primer pair of embodiment 1 and the real-time fluorescence PCR sensitivity test amplification curve diagram of probe.Its
In, amplification curve is from left to right 40ng/ μ L, 20ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L mountains respectively
Sheep DNA sample.
Fig. 2 is second group of primer pair of embodiment 1 and the real-time fluorescence PCR sensitivity test amplification curve diagram of probe.Its
In, amplification curve is from left to right 40ng/ μ L, 20ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L mountains respectively
Sheep DNA sample.
Fig. 3 for embodiment 1 first group of primer pair and probe real-time fluorescence PCR primer specific test amplification curve
Figure.Wherein, amplification curve is goat standard items.
Fig. 4 is the quantitation curves of goat derived component.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following examples are merely to illustrate this
Invention is not for restriction the scope of the present invention.The experimental method of unreceipted actual conditions in the following example, generally according to normal
Rule condition, such as《Molecular cloning：Laboratory manual》(New York：Cold Spring Harbor Laboratory Press,
1989) condition that condition or manufacturer described in are provided is carried out.
The experiment material of the present invention is as follows：
(1) goat (Capra hircus), sheep (Ovis aries), family ox (Bos taurus), donkey (Equus
Asinus), horse (Equus caballus), chicken (Gallus gallus), duck (Anas platyrhynchos), goose (Anser
Anser), turkey (Meleagris gallopavo), pig (Sus scrofa), quail (Coturnix coturnix), camel
The DNA standard items of (Camelus dromedarius), domestic cat (Felis catus) are purchased from U.S. Zyagen
Laboratories companies.
(2) animal component such as chevon, beef, bream meat, butterfish meat, yellow croaker meat, dog salmon meat is commercially available prod.
(3) big rat meat, small rat meat are bought in the western pul-Bi Kai experimental animals Co., Ltd in Shanghai.
(4) plant component such as rice, corn, soya bean, millet, mung bean, sweet potato, potato, Semen Phaseoli Vulgaris, apple, celery is
Commercially available prod.
(5) it is used for chicken and ham roll sausage (import), pork ham sausage (import), the beef ham sausage of actual verification
(import), goat Pork sausage, goat row, beef pork luncheon meat, pork luncheon meat, beef with brown sauce can, dried beef, dried pork slice, ox
Burger, fried curry beef sauce, mutton taste dog food, chicken flavor dog food (import), beef flavour dog food (import), tuna fish cat can (enter
Mouthful) it is commercially available prod.
(6) Australian pet level chicken bone meal is provided by Shanghai customs port.
The design of the primer pair of embodiment 1 and probe
The design of (1) first group of primer pair and probe
According to goat (the Capra hircus breed Yunnan black goat) chromosome 9 delivered in NCBI
DNA sequence dna (accession number：NC_022301), choose suitable sequence fragment and carry out primed probe design.For goat derived component
Purpose fragment length be 87bp.The sequence of fluorescence PCR primer pair and probe is as follows：
Goat-87bp-F：5’-GGAAGGAAAGAGAATGGGGATATGG-3’(SEQ ID NO：1)
Goat-87bp-R：5’-TCTCCACACACAGCCAAAACC-3’(SEQ ID NO：2)
Goat-87bp-P：FAM-ATCCATCTCTCCCTCCACTCCCTGCCTAA-TAMRA(SEQ ID NO：3)
Wherein, FAM represents fluorescent reporter group, and TAMRA represents quenching group.The present invention uses fluorescence probe method, and it is examined
It is come recognition template using fluorescence labeling specific probe to survey principle.Compared with SYBR dye methods in the prior art, the present invention is glimmering
Signal specific probe it is specific stronger, ambient interferences are lower.
The design of (2) second groups of primer pairs and probe
According to goat (the Capra hircus breed Yunnan black goat) chromosome 9 delivered in NCBI
DNA sequence dna (accession number：NC_022301), suitable fragment design primer pair and probe among choosing, primer pair and probe are such as
Under：
Goat-105bp-F：5’-ATGAGTGTCTGTGTCTCTGTGTAAG-3’(SEQ ID NO：4)
Goat-105bp-R：5’-ACAAATTTCTTCACATGCACAATAAGC-3’(SEQ ID NO：5)
Goat-105bp-P:FAM-ACCCACAGCCCAGTCTCTACAAACACACA-TAMRA(SEQ ID NO：6)
Goat-105bp-P:FAM-ACCCACAGCCCAGTCTCTACAAACACACA-TAMRA(SEQ ID NO：6)
Wherein, FAM represents fluorescent reporter group, and TAMRA represents quenching group.The present invention uses fluorescence probe method, and it is examined
It is come recognition template using fluorescence labeling specific probe to survey principle.Compared with SYBR dye methods in the prior art, the present invention is glimmering
Signal specific probe it is specific stronger, ambient interferences are lower.
The reaction system of the real-time fluorescence PCR of (3) first groups of primer pairs and probes and second group of primer pair and probe and anti-
Answer program
Real-time fluorescence PCR reaction system is shown in Table 1：
Wherein, real-time fluorescence PCR response procedures are：95℃10min；95℃15s,60℃1min；Totally 45 circulations.
The goat derived component real-time fluorescence PCR reaction system of table 1
The preparation of the test sample of embodiment 2
Animal meat sample is clayed into power shape after tearing up, drying using refrigeration grinding machine (SPEX 6850).Plant sample
Product and sample for actual verification are directly clayed into power shape using refrigeration grinding machine (SPEX 6850).
Embodiment 3DNA extracting
Use Animal genome extracts kit (Tiangeng biochemical technology Co., Ltd；Catalog number (Cat.No.)：DP323 animal sample) is extracted
Product DNA, Plant Genome extracts kit (Tiangeng biochemical technology Co., Ltd；Catalog number (Cat.No.)：DP305 plant sample DNA) is extracted,
Animal derived plant feed genome extracts kit (Tiangeng biochemical technology Co., Ltd；Catalog number (Cat.No.)：DP323 aggregate sample) is extracted
Product and actual verification sample DNA, extracting method refer to kit operational manual.DNA solution is placed in -20 DEG C and saved backup.
The primer pair of the goat derived component of embodiment 4 and the sensitivity technique of probe
With goat DNA content 40ng/ μ L, 20ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L, 0.01ng/ μ L,
0.001ng/ μ L and 0.0001ng/ μ L are that sample DNA is template, carry out real-time PCR detection, and experiment is repeated 6 times.
As a result：(1) first group of primer pair and probe：In 6 times are detected, 40ng/ μ L, 20ng/ μ L, 10ng/ μ L, 1ng/ μ
Occur amplification curve in L, 0.1ng/ μ L, 0.01ng/ μ L goat sample DNA, and 0.001ng/ μ L and 0.0001ng/ μ L mountains
Sheep sample DNA does not occur amplification curve, such as Fig. 1.
(2) second groups of primer pairs and probes：In 6 times are detected, 40ng/ μ L, 20ng/ μ L, 10ng/ μ L, 1ng/ μ L,
Occur amplification curve in 0.1ng/ μ L, 0.01ng/ μ L goat sample DNA, but amplification curve and DNA concentration not into than
Example, such as Fig. 2.
Conclusion：1st, first group of primer pair and probe have obvious S types amplification curve when template usage amount is 0.01ng/ μ L,
Sensitivity reaches 0.01ng/ μ L, and the primer pair and probe of first group of goat derived component have preferable accuracy；2nd,
There is notable difference in two groups of primer pairs and probe and first group of primer pair and probe.Second group of primer pair and probe are examined in sensitivity
DeGrain in survey.
Due to the DeGrain in sensitivity technique of primer pair and probe of second group of goat derived component, therefore after
Primer pair and probe of the continuous experiment only for first group of goat derived component.
The primer pair of the goat derived component of embodiment 5 and the specific detection of probe
The present embodiment supplies embodiment 3 32 kinds of examination to move using the primer pair and probe of the goat derived component of embodiment 1
Vegetable material DNA sample is detected, detects the fluorescence signal of amplified production.The summary of testing result is shown in Table 2 and Fig. 3.
The specific detection result of the goat derived component of table 2
| Sequence number | Sample ID | Result of the test | Sequence number | Sample ID | Result of the test |
| 1 | Goat DNA | + | 17 | Beef | — |
| 2 | Sheep DNA | — | 18 | Bream meat | — |
| 3 | Donkey DNA | — | 19 | Butterfish meat | — |
| 4 | Family ox DNA | — | 20 | Yellow croaker meat | — |
| 5 | Horse dna | — | 21 | Dog salmon meat | — |
| 6 | Chicken DNA | — | 22 | Big rat meat | — |
| 7 | Duck DNA | — | 23 | Small rat meat | — |
| 8 | Goose DNA | — | 24 | Rice | — |
| 9 | Quail DNA | — | 25 | Corn | — |
| 10 | Turkey DNA | — | 26 | Soya bean | — |
| 11 | Pig DNA | — | 27 | Millet | — |
| 12 | Camel DNA | — | 28 | Mung bean | — |
| 13 | Domestic cat DNA | — | 29 | Sweet potato | — |
| 14 | Dog dna | — | 30 | Potato | — |
| 15 | Deer DNA | — | 31 | Semen Phaseoli Vulgaris | — |
| 16 | Celery | — | 32 | Apple | — |
From Fig. 3 and table 2 as can be seen that primer pair and probe are positive only to goat DNA meat sample amplification, there are obvious S types to expand
Increase curve, and other multiple species DNA samples are expanded with feminine gender, without obvious S types amplification curve.
Conclusion：The detection method of the present invention has good species specificity.
The primer pair of the goat derived component of embodiment 6 and the actual verification of probe
Using the detection method of embodiment 5, the 17 parts of processed meat food stuffs collected in the market, foreign trade approach and animal
The samples such as feed are detected.It the results are shown in Table 3.
The testing result of the goat derived component of 3 17 parts of testing samples of table
From table 3 it can be seen that detecting goat derived component in 2 parts of processed meat food stuffs (goat Pork sausage, goat row)；Remaining
Food and feed in do not detect goat derived component.
Conclusion：The meat source for extracting sample is consistent with testing result, illustrates that this method can accurately detect food
Whether contain goat derived component with feed.Also explanation can be detected in large quantity using the detection method of embodiment 4 simultaneously
It whether there is goat derived component in food or feed.
The foundation of the quantitation curves of embodiment 7
(1) the quantitative detection of goat derived component primer pair and probe
The goat DNA that embodiment 3 is extracted into acquisition be diluted to 100ng/ μ L, 10ng/ μ L, 1ng/ μ L, 0.1ng/ μ L,
0.01ng/ μ L, totally five concentration gradients, are detected using the primer pair and probe of embodiment 1, each 6 repetitions of concentration, are obtained
Obtain Ct values.Negative control is water.As shown in table 4, wherein Ct values are the average value of 6 repetition experiments.
The real-time fluorescence PCR of table 4 quantitatively detects the Ct values of experiment
| Template consumption (ng/ul) | Ct values |
| 100 | 22.736 |
| 10 | 26.471 |
| 1 | 30.206 |
| 0.1 | 34.099 |
| 0.01 | 37.492 |
| Negative control | - |
Conclusion：It can be seen that being reduced with the concentration of goat derived component, amplification Ct values increase in gradient, and are in certain
Linear relationship (R2=0.999).
(2) processing and calculating of data
As a result as shown in figure 4, using the logarithm value of DNA concentration as abscissa, with the flat of the Ct values of 6 repetition experiments of sample
Average is ordinate, takes and a little carries out curve fitting, and obtains quantitative criterion straight line, and linear equation is y=3.714x+19.05, and R2=
0.999.For the sample that need to be quantified, the respective Ct values of testing sample are measured every time, are tried to achieve accordingly using calibration curve formula
X values.The content of the goat source constituent of testing sample can be drawn.
(3) checking of quantitation curves
Extract DNA as described in Example 3, dilute quantitative 6 independent samples respectively, concentration be respectively 50ng/ μ L and
0.5ng/ μ L's, respective Ct values are detected, and the content of goat source constituent therein is calculated by quantitation curves, it is used to
Verify the repeatability of this method.
As a result show, the average value of 50ng sample measured values is 52.3ng, and the average value of 0.5ng sample measured values is
0.44ng。
Conclusion：Illustrate that there is the ability of accurate quantification using the method for the present embodiment in certain dynamic range.So as to demonstrate,prove
The bright actually measured value of method of the invention is consistent with theoretical value, reproducible.
The present invention establishes the real-time fluorescence PCR that goat derived component in food and feed is detected using single-copy nuclear gene
Method, the karyogene for caprine species designs primer pair and probe, it is only necessary to reference to real-time fluorescence amplification curve diagram, no more than two
Individual hour, which just can accurately detect, whether there is goat source constituent in sample, its sensitivity is 0.01ng/ μ L.
In summary, the specific primer pair and probe for the goat source constituent real-time fluorescent PCR amplification that the present invention is designed
It is not only specific good, and sensitivity is high, and whether one kind is provided very well containing goat derived component rapidly and accurately to distinguish
Detection method, field of food safety detection shutting, have a good application prospect.Moreover, the specific primer
Pair and probe can use method as known in the art, detection kit is further made, the detection kit is except described
Outside specific primer pair and probe, marker can also be included, this is aobvious and easy for those skilled in the art
See.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, various changes and modifications of the present invention are possible without departing from the spirit and scope of the present invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent is defined.
Sequence table
<110>Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Entry-Exit Inspection and Quarantine Bureau
<120>The real time fluorescent PCR method of goat derived component in food and feed is detected using single-copy nuclear gene
<130> 171009
<160> 6
<170> PatentIn version 3.3
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Claims (6)
1. the real-time fluorescence PCR detection method of goat derived component in a kind of utilization single-copy nuclear gene detection food and feed,
It is characterised in that it includes following steps：
The first step, using the DNA of testing sample as template, carries out fluorescent quantitative PCR, obtains pcr amplification product；
Second step, detects the fluorescence signal of amplified production；
Whether the 3rd step, judged in sample containing goat derived component and quantitatively in detection sample with the Ct values of testing result
The content of goat derived component；
Wherein, specific primer pair and goat source property containing amplification goat derived component in the reaction system expanded for PCR
The specific probe of composition, the sequence such as SEQ ID NO.1 and SEQ of the specific primer pair of the amplification goat derived component
Shown in ID NO.2, the sequence of the specific probe of the goat derived component is as shown in SEQ ID NO.3.
2. real-time fluorescence PCR detection method as claimed in claim 1, it is characterised in that the condition of the PCR amplifications is as follows：
Reaction system is 25 μ L：The μ L of real-time fluorescence PCR reagent (2 ×) 12.5, each 10 μM of primer, 5 μM of probe, the μ L of DNA profiling 5；
PCR reaction conditions：95℃10min；95 DEG C of 15s, 60 DEG C of 1min；Totally 45 circulations.
3. a kind of detection kit of goat derived component, it is characterised in that contain following reagent：
(a) specific primer pair of goat derived component is expanded；
(b) specific probe of goat derived component；
Wherein, the sequence such as SEQ ID NO.1 and SEQ ID NO.2 institutes of the specific primer pair of the amplification goat derived component
Show, the sequence of the specific probe of the goat derived component is as shown in SEQ ID NO.3.
4. detection kit according to claim 3, it is characterised in that the detection kit also includes：
(c) marker.
5. the application of a kind of detection kit as described in claim 3 or 4, it is characterised in that the detection kit is used for
Goat derived component in qualitative or quantitative detection food or feed.
6. a kind of specific primer pair and probe of goat derived component as claimed in claim 1, it is characterised in that the spy
The sequence of specific primer pair is as shown in SEQ ID NO.1 and SEQ ID NO.2, the sequence such as SEQ ID of the specific probe
Shown in NO.3.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825602A (en) * | 2019-03-26 | 2019-05-31 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | The real-time fluorescence PCR detection method of horse derived component in food and feed |
| CN109825608A (en) * | 2019-04-11 | 2019-05-31 | 中国农业大学 | The kit of goat derived components and its application in a kind of Rapid identification food |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673900A (en) * | 2015-02-05 | 2015-06-03 | 中国肉类食品综合研究中心 | Method for identifying animal-derived ingredients in meat or meat product |
| CN105296647A (en) * | 2015-11-20 | 2016-02-03 | 华中农业大学 | Detection kit for sheep origin component identification and detection of multi-species origin components in products |
-
2017
- 2017-05-12 CN CN201710335321.0A patent/CN107012247B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104673900A (en) * | 2015-02-05 | 2015-06-03 | 中国肉类食品综合研究中心 | Method for identifying animal-derived ingredients in meat or meat product |
| CN105296647A (en) * | 2015-11-20 | 2016-02-03 | 华中农业大学 | Detection kit for sheep origin component identification and detection of multi-species origin components in products |
Non-Patent Citations (3)
| Title |
|---|
| MIGUEL A.RODRIGUEZ ET AL.: "PCR identification of beef，sheep，goat，and pork in raw and heat-treated meat mixtures", 《JOURNAL OF FOOD PROTECTION》 * |
| RENE KOPPEL ET AL.: "Multiplex real-time PCR for the detection and quantification of DNA from beef, pork, horse and sheep", 《EUR FOOD RES TECHNOL》 * |
| 曾少灵等: "多重实时荧光PCR检测牛、山羊和绵羊源性成分", 《生物工程学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109825602A (en) * | 2019-03-26 | 2019-05-31 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | The real-time fluorescence PCR detection method of horse derived component in food and feed |
| CN109825602B (en) * | 2019-03-26 | 2021-09-21 | 上海海关动植物与食品检验检疫技术中心 | Real-time fluorescent PCR detection method for equine derived components in food and feed |
| CN109825608A (en) * | 2019-04-11 | 2019-05-31 | 中国农业大学 | The kit of goat derived components and its application in a kind of Rapid identification food |
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| CN107012247B (en) | 2020-01-10 |
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