CN106480203A - For detecting internal standard gene and its application of chicken derived components - Google Patents
For detecting internal standard gene and its application of chicken derived components Download PDFInfo
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- CN106480203A CN106480203A CN201610976239.1A CN201610976239A CN106480203A CN 106480203 A CN106480203 A CN 106480203A CN 201610976239 A CN201610976239 A CN 201610976239A CN 106480203 A CN106480203 A CN 106480203A
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- 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
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The present invention is provided to the internal standard gene of detection chicken derived components and its application, it is related to living species identification and PCR detection technique field.The present invention filters out the general internal standard gene Actb in chicken source first, as shown in SEQ ID NO.1, it is located on chromosome its nucleotide sequence, and single copy is easily quantitative, in chicken species, copy number is constant does not have allelic variation, can be used as the target gene of identification Ji Yuan.Establish qualitative PCR, quantitative PCR and the digital PCR method detecting this internal standard gene with this gene for target sequence respectively, the chicken derived components in food and feedstuff can enzyme rapidly and sensitively be detected, sensitivity is high, high specificity, simple to operate, with low cost, reaction result is easy to observe, and is highly suitable for live real-time detection, animal derived food supervision with resist the adulterated aspect of meat productss and have a extensive future.
Description
Technical field
The present invention relates to living species identification and PCR detection technique field, it is used for detecting Ji Yuan more particularly to one kind
The internal standard gene of composition and its application.
Background technology
With the development of social economy and product technology processing, a lot of food are illegally added various chemical raw materials, interpolation
Agent, hormone etc., or even much businessman is to seek great number interests to carry out food adulteration behavior or directly adulterate, this serious harm
Interests of consumer and healthy are therefore most important to the precise Identification of food variety.
No matter at home or external, Carnis Gallus domesticus are all the main meat of current consumption, and its importance is self-evident.In Europe
Continent, Carnis Gallus domesticus occupy staple market, and supermarket supplies 60% Carnis Gallus domesticus processed goods, and turkey is even more by popular consumer.In China, chicken
Meat is traditional poultry meat, enters Chinese market with miscellaneous external fast food, Carnis Gallus domesticus have been processed to various products.So
And more and more businessman is to obtain great number interests, feeding chicken with hormone and antibody makes its accelerated growth, in chicken in feeding process
Add Ji Cheng branch in feedstuff and cause " crazy fowl disease ", more have illegal businessman with the cheap adulterated beef and mutton of Carnis Gallus domesticus, to seek violence
Deng.And Carnis Gallus domesticus pollution may cause global food-safety problem, internal standard gene and molecular engineering using chicken detect Carnis Gallus domesticus
Adulterated is a kind of effective detection meanss.
At present, internal standard gene is widely used for differentiating food adulteration, but how to filter out suitable internal standard base
Because being particularly important.Mostly meat productss detection of adulterations technology both domestic and external is specifically to draw for the gene design on mitochondrion at present
Carrying out real-time fluorescence quantitative PCR amplification, because mitochondrial gene is multi-copy gene, its detection sensitivity is high for thing, but simultaneously
Distinguishing due to there is puzzlement when the unconscious cross-contamination produced by process such as selling, transport with conscious illegal interpolation.
In addition, the concentration of high copy number mitochondrial DNA cannot be corresponding with the concentration of genomic DNA, therefore essence cannot be carried out to sample
Really quantitative analyses, simultaneously because mitochondrial gene homology high it is difficult to qualitative detection is realized by regular-PCR.Therefore, should
The screening that method can only realize meat adulteration by quantitative fluorescent PCR differentiates.To differentiate low concentration be not intended to cross-contamination and
Have a mind to illegal interpolation and clearly adulterated ratio realizes rapid screening, then want the low copy gene on selective staining body as in meat
Standard gene.
Content of the invention
It is an object of the invention to provide for the chicken source general internal standard gene Actb detecting chicken derived components.
Another object of the present invention is to provide the detection method set up with this internal standard gene Actb for target gene.
A kind of gene for detecting chicken derived components in food and feedstuff, it is internal standard gene Actb, has SEQ ID
Sequence shown in NO.1.
The invention provides application in detection chicken derived components for the above-mentioned internal standard gene Actb.
The invention provides the application in above-mentioned internal standard gene Actb chicken Components identification in food or feedstuff.
The invention provides a kind of specific primer detecting above-mentioned internal standard gene Actb for PCR method, its nucleotide
Sequence is as shown in SEQ ID NO.2,3.
Application in detection chicken derived components for the above-mentioned specific primer that the present invention provides.
The invention provides the test kit containing above-mentioned specific primer.
Test kit containing above-mentioned 2 primers and 1 probe belongs to protection scope of the present invention, the core of described 1 probe
Nucleotide sequence is as shown in SEQ ID NO.4 or as shown in SEQ ID NO.7.
Further, present invention also offers the test kit containing 2 primers and 2 probes, described 2 primers and nucleoside
, as shown in SEQ ID NO.2,3, the nucleotide sequence of described 2 probes is as shown in SEQ ID NO.4,7 for acid sequence.
Application in detection chicken derived components for the mentioned reagent box that the present invention provides.
Digital pcr method is generally used for the absolute quantification analysis to nucleic acid, present invention discover that digital pcr can also be used to examine
The copy number of cls gene.Therefore this discovery falls within protection scope of the present invention.
In one embodiment of the invention, using digital pcr method method identify chicken source internal standard gene Actb, result with
Southern blot method detects that the result of internal standard gene Actb copy number is consistent.The nucleotide sequence of primer used such as SEQ
ID NO.2, shown in 3, the nucleotide sequence of the probe using cooperatively with primer is as shown in SEQ ID NO.7.
The present invention filters out chicken source internal standard gene first on chromosome, and copy number is single copy.The present invention uses many
Individual kind chicken checking internal standard gene is it was demonstrated that there is not allelic variation in this internal standard stable gene.The choosing of internal standard gene
Select and typically require copy number low and stable, general animal internal standard gene often selects on mitochondrion, and such reference gene is copied
Shellfish number is many, is not easy to quantitation.As internal standard gene, its copy number ground is easy to quantitative to gene on selective staining body of the present invention,
Compared to the gene on mitochondrion, mutation rate is lower.The present invention takes digital pcr and southern hybridization two ways, enters
The identification of row copy number.In conjunction with Ensembl gene information database data it is determined that the copy number of Actb gene is single copy,
Determine accuracy on checking copy number for the digital pcr simultaneously, other species can be extended to.
Based on present invention determine that internal standard Gene A ctb for detecting chicken derived components in food and feedstuff, the present invention sets
Count the primer of qualitative PCR, quantitative PCR and digital pcr and/or the probe for detecting this gene, established detection Ji Yuan and become
Point corresponding PCR method, these methods are time-consuming few, and specificity is good, and sensitivity is high, simple to operate it is not necessary to professional's operation,
Result is easy to observe, and is very suitable for basic unit's food supervision and inspection and uses.
Brief description
Fig. 1 is Genetic homology of carbapenem-resistant.
Fig. 2 is common family chicken, the Southern results of hybridization of turkey, the common family chicken of 1, BamH1 enzyme action；2, Nhe1 enzyme action are general
Long and deep friendship between two families chicken；3, BamH1 enzyme action turkeys 4, Nhe1 enzyme action turkey.
Fig. 3 is that Actb and Gcg expands hotspot graph.
Fig. 4 is the Actb/Gcg ratio figure under common 5 Concentraton gradient of family chicken, and concentration is respectively 100,10,1,0.1,
0.01ng/ul.
Fig. 5 is that the qualitative PCR of Actb gene verifies specificity, swimming lane 1-21:1, common family chicken；2, pheasant；3, turkey；4,
Gallus Domesticuss；5, pig；6, cattle；7, sheep；8, horse；9, donkey；10, duck；11, goose；12, Canis familiaris L.；13, rabbit；14, Mus；15, fish；16, yak；
17, Babalus bubalis L.；18, camel；19, ermine；20, deer；21, negative；M:DNA Marker DL 2000.
Fig. 6 is that the quantitative PCR of Actb gene verifies specificity, and wherein 1 is pheasant, and 2 is Gallus Domesticuss, and 3 is common family chicken, and 4 are
Turkey.
Fig. 7 is the fluorescent quantitative PCR curve of Actb gene, and wherein 1 is 100ng/ul, and 2 is 10ng/ul, and 3 is 1ng/
Ul, 4 is 0.1ng/ul, and 5 is 0.01ng/ul, and 6 is feminine gender.Each concentration do three parallel.
Fig. 8 is the quantitative fluorescent PCR standard curve of Actb gene.
Specific embodiment
Following examples further illustrate present disclosure, but should not be construed as limitation of the present invention.Without departing substantially from
In the case of present invention spirit and essence, the modification that the inventive method, step or condition are made or replacement, belong to the present invention
Scope.
If not specializing, the conventional meanses that in embodiment, technological means used are well known to those skilled in the art.
Pig (Sus scrofa), cattle (Bos taurus), sheep (Ovis aries), common family chicken (Gallus gallus),
Pheasant (Phasianuscolchicus), turkey (Meleagris gallopavo), Gallus Domesticuss (Gallus domesticus
Brisson), duck (Anas platyrhynchos), goose (Goose calicivirus), Canis familiaris L. (Canis lupus
Familiaris), rabbit (Oryctolagus cuniculus), yak (Bos mutus), Channa argus (Pseudosciaena
Polyactis) buy for supermarket.Horse (Equus caballus), donkey (Equus asinus) is bought for Beijing market of farm produce.Always
Mus (Mus musculus) are provided with Molecular Biology Lab by China Agricultural University's food safety.Babalus bubalis L. (Bubalus
Bubalis), ermine (Martes zibellina), camel (Camelus ferus), deer (Cervus) is entered and left the border by Tianjin and checks
Doctor Li Zongmeng of office provides.
The screening of the general internal standard gene Actb in embodiment 1 chicken source
By searching for the gene information with regard to chicken in GenBank, target gene group is downloaded from NCBI, and saves as
" .FASTA " form.Full-length genome information for chicken is analyzed, and is entered using BLAST and DNAMAN Version 4.0 software
Row homology analysis, filter out Actb gene, and this gene is located on chromosome, is one of actin cytoskeleton.By 20 kinds
Meat (it is common family chicken (Gallus gallus) respectively, pheasant (Phasianuscolchicus), turkey (Meleagris
Gallopavo), Gallus Domesticuss (Gallus domesticus brisson), pig (Sus scrofa), cattle (Bos taurus), sheep
(Ovis aries), duck (Anas platyrhynchos), goose (Goose calicivirus), Canis familiaris L. (Canis lupus
), familiaris rabbit (Oryctolagus cuniculus), yak (Bos mutus), Channa argus (Pseudosciaena
Polyactis), horse (Equus caballus), donkey (Equus asinus), mouse (Mus musculus), Babalus bubalis L.
(Bubalus bubalis), ermine (Martes zibellina), camel (Camelus ferus), deer (Cervus)) Actb
Channel genes DNAMAN Version 4.0, carries out multisequencing specificity analyses, and sequence alignment result saves as " .seq " form.
The fragment selecting specificity high carries out BLAST analysis again, sequence homology and specificity in searching data storehouse.Finally by sequence
Row are integrated, and determine that final specific targets gene β-Actin (Actb) gene can be used as internal standard gene.This Actb
The nucleotide sequence of fragment is as shown in SEQ ID NO.1.
The checking of the general internal standard gene in embodiment 2 chicken source
1st, it is directed to internal gene Actb of embodiment 1 screening, (U.S. gives birth to by Primer Express 3.0 design software
Life technology company) it is directed to Actb gene design PCR and quantification PCR primer and probe.
Table 1 qualitative PCR, quantitative PCR and digital pcr the primer and probe sequence
Note：FAM, TAMRA, VIC and BHQ1 are 4 kinds of fluorophors
2nd, copy number measures
Take digital pcr and southern hybridization two ways, carry out the identification of copy number.In conjunction with Ensembl gene
Information data database data, it is determined that the copy number of Actb gene is single copy, determines digital pcr on checking copy number simultaneously
Accuracy, other species can be extended to.
Preferably internal standard gene should copy number be constant there is not allelic variation in corresponding species.According to
Actb gene sets up chicken source species specificity PCR detection system using primer Actb-1F/1R, for analyzing internal standard gene
Middle specificity and allelic variability, to ensure the accuracy of internal standard genescreen.Using primer carry out qualitative and
Find after quantitative analyses, Actb gene does not have allelic variation in chicken species.Template DNA is carried out with 10 times of gradients dilute
Release, carry out quantitative fluorescent PCR to sensitive analysis using primer Actb-1F/1R and probe Actb-1P, when DNA profiling concentration
During as little as 10pg, fluorescence signal still can be detected, obtaining good linear equation according to standard curve is：Y=-3.196x+
32.481, R2=0.994.Understand that Actb gene is in common family chicken, turkey, Gallus Domesticuss and pheasant by the identification of above copy number
Single copy, thus it could be speculated that this reference gene Actb and quantitative PCR system equally can be also suitably used for turkey, Gallus Domesticuss and pheasant
Detection by quantitative, its detection is limited to 8 copy numbers.
In order to identify copy number in chicken for the Actb, the present invention devises pair of primers Actb-2F/2R and expands one section
436bp DNA fragmentation, this fragment is used for Southern blot as probe.It is respectively cut chicken, turkey with Nhe1 and BamH1
DNA, then hybridizes with 436bp DNA fragmentation.One rule all only occurs in the DNA fragmentation of Nhe1 and BamH1 enzyme action as shown in Figure 2
Band, this shows that Actb gene is single copy in chicken and turkey.
Additionally, carrying out digital pcr using primer Actb-1F/1R and probe Actb-2P to do copy number analysis.System configurations
After the completion of, reaction system is transferred in the system well that Droplet Generator Cartridge microdroplet generates card, and
The digital pcr reaction oil of 70 μ L is added in reaction oil well；After the completion of sample-adding, microdroplet generation card is transferred to
In Droplet Generator drop generators, start instrument；Microdroplet generates after terminating, by the micro system after the completion of generating
It is transferred to sealer in 96 holes；96 orifice plates are placed in PCR instrument, PCR amplification program is 50 DEG C of hot activation 5min；95 DEG C of denaturations
5min；50 circulations：95 DEG C of degeneration 15s, 60 DEG C of annealing and extension 60s.After amplification terminates, 96 orifice plates are taken out, is placed in
Droplet Reader microdroplet reads in instrument and carries out microdroplet fluorescence reading；Microdroplet signals collecting adopts FAM/VIC dual pathways fluorescence
By analyzing hotspot graph, collection, after fluorescent collecting, determines that fluorescence threshold limit (2995) determines negative microdroplet and positive microdroplet.
According to Poisson distribution, calculate the copy number of each passage using formula.Concrete formula is as follows：
Formula 1：N (reference gene)=- ln [(N-X)/N] × N
Formula 2：N (specific gene)=- ln [(N-Y)/N] × N
Wherein, N (reference gene) and N (specific gene) represents reference gene (the i.e. single copy Gcg base calculating respectively
Cause) and specific gene (being this experiment Actb internal standard gene) copy number；N is total hole count, and X represents the corresponding sun of reference gene
Property hole count, Y represent specific gene corresponding the positive hole count.
Chicken template concentrations are arrived 100ng/ul surely, dilutes 5 Concentraton gradient successively, then calculate Actb/ with digital pcr
The value of Gcg, from the figure 3, it may be seen that the value of Actb/Gcg is 1:1.The ratio of Actb/Gcg and the limits of error are automatically generated by hotspot graph.
As shown in Figure 4, when template concentrations are very high, the stability of reaction result is still fine, and result fluctuation range is little, ratio and error
Limit is near 1.Reduce with concentration although result fluctuation shows larger difference, the limits of error substantially broadens, but Actb/Gcg
Value still 1 about.Show that the result of Actb/Gcg value is not affected by template concentrations.Southern results of hybridization and numeral
PCR result is consistent, illustrates that digital pcr is a kind of efficient, quick, method of precise Identification copy number of target genes, can apply
Digital pcr method identifies the copy number of target gene.
Table 2 Bio-Rad digital pcr flat reaction system
3rd, the analysis of the specificity identification of Actb gene and its allelic variation
Using primer pair Actb-1F/1R (being shown in Table 1) designing to 4 breeder species (common family chicken, turkey, Gallus Domesticuss, pheasant)
With 16 kinds of non-chicken species (pig, cattle, sheep, duck, goose, Canis familiaris L., rabbit, yak, horse, donkey, Mus, Babalus bubalis L., ermine, camel, deer, fish) carry out qualitative
PCR expands and with 2% agarose gel, amplified production is carried out with electrophoretic analysiss, pcr amplification reaction program：95 DEG C of denaturations
5min；95 DEG C of 30s, 60 DEG C of 45s, 72 DEG C of 1min, 35 circulations；72 DEG C of extension 10min.Carry out quantitative PCR analysis, reaction simultaneously
Program：95 DEG C of denaturations 10min；95 DEG C of 15s, 60 DEG C of 1min, 40 circulations；60 DEG C of collection signals.Result shows, all 4 kinds
Chicken species all can amplify the fragment of 113bp, meets the corresponding clip size of designed primer.And 16 kinds of non-chicken species of other are equal
No specific amplification band (Fig. 5).Carry out quantitative PCR with primer Actb-1F/1R and probe Actb-1P this 20 kinds of species are entered
Row detection, 4 breeder species can detect signal and 16 kinds of non-chicken species are not detected by signal (Fig. 6).This show this gene with
And corresponding primer can be used to specifically detect chicken and chicken source property product.
Table 3 qualitative PCR reaction system
Table 4 quantitative PCR reaction system
4th, quantitative PCR sensitivity checking
In order to evaluate the susceptiveness of quantitative fluorescent PCR, by genomic DNA concentration 7 gradients of 10 times of dilutions of family chicken,
100ng, 10ng, 1ng, 0.1ng, 10pg, 1pg, 0.1pg, carry out quantitative fluorescent PCR, amplification curve such as Fig. 7, take 5 point-renderings
Standard curve, the standard curve obtaining is as shown in figure 8, as DNA profiling concentration the most as little as 10pg, fluorescence signal can be detected.
Therefore, the test limit of this fluorescent quantitation system is 10pg DNA, that is, 8 copy numbers.Give with regard to Cq value and base simultaneously
Because of linear equation between the logarithm of group concentration.Linear equation is：Y=-3.196x+32.481, R2=0.994.Y represents Cq value,
X represents the logarithm value of genome concentration, R2Represent linearly dependent coefficient.According to R2Value understands copy number and the Ct of chicken genomic DNA
There is between value good linear relationship.Understand Actb gene in common family chicken, turkey, Gallus Domesticuss and pheasant by the identification of above copy number
In be single copy, thus it could be speculated that this gene and quantitative PCR system can be also suitably used for turkey, Gallus Domesticuss and pheasant quantitation inspection
Survey, its detection is limited to 8 copy numbers.
5th, Carnis Gallus domesticus processed goods detection
Determined by the checking present invention, the quantitative PCR detection technique of internal standard gene and foundation detects in actual sample
During accuracy, this research to commercially available 14 kinds of Carnis Gallus domesticus processed goods and 6 kinds mixing meat productss detect, result shows 4
There is the phenomenon collected less than fluorescence signal in kind of food and 2 kinds of feedstuffs, this with KOKA Chicken satay taste instant noodles, close taste chicken
Meat flavour cup face, Mr. Zhang's refined little sister charcoal roasted chicken juice dessert face, in Mexico's chicken with several spices dessert surface compositions display do not contain chicken derived component
And be consistent without chicken derived component practical situation in chicken feed, show that the fluorescence quantifying PCR method that this research is set up can be used
Detection in actual sample.
Table 5 Carnis Gallus domesticus processed goods and mixing meat testing result
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
For member, on the premise of without departing from the technology of the present invention principle, some improvements and modifications can also be made, these improvements and modifications
Also should be regarded as protection scope of the present invention.
Sequence table
<110>China Agricultural University
<120>For detecting internal standard gene and its application of chicken derived components
<130> KHP161117577.3
<160> 10
<170> PatentIn version 3.5
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cacagcccta cacacagcac cctccccctc ccaccccaac tcacagccag cacttaatct 180
cattactcac atggctgagt ggctgccacc ctgcccagag cgaggccata aggctccacc 240
actaagacaa agcagtttcc atcttctgta gtagtttagc tcagcctcct ccacccactc 300
agcattaaga ggtgtgctaa gagcagccta gggctccagc ctaccttgat tttcattgtg 360
ctaggtgcca gggctgtgat ctccttctgc atcctgtcag caatgccagg gtacattgtg 420
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atgatggagt tgaaggtagt ttcatggata ccacaggact ccatacctgc aagcaaaaag 540
cagtgtcaag tcagtaaatg gttcagggac aaggaagaca gaggacctac tgctatggga 600
aaacaagaga atcaaacacc ccatagtggg aaacaacaca gacaaaggca tggctgacag 660
gctcagcctc ctgttctact gacctaggta cctcaatcat cctcaaactg aggaagaaaa 720
aagactgcag tggaatggga cagacctgct aagggccagc aatattgtgg ccagtctcta 780
agtcgctcta ggcagaaggg gcatggaatc tcctcaaagg agtcagactt acccaagaaa 840
gatggctgga agagggcctc ggggcacctg aacctctcat tgccaatggt gatgacctga 900
ccatcaggga gttcatagct cttctctagg gaagagctag aggcagctgt ggccatctcc 960
tgctcgaaat ccagtgcgac gtagcacagc ttctccttga tgtcacgcac aatttctctc 1020
tcggctgtgg tggtgaagct gtagcctctc tctgtcagga tcttcatgag gtagtccgtc 1080
aggtcacggc cagccagatc cagacggagg atggcatggg ggagggcgta gccttcatag 1140
atgggcacag tgtgggtaac accatcacca gagtccatca caataccagt ggtacgacca 1200
gaggcataca gggacagcac agcctggatg gctacataca tggctggggt gttgaaggtc 1260
tcaaacatga tctagaaaga taaaaaaagc actgagtcaa gcagaataaa ccaaggttct 1320
cactgcacac ctacatcggt ccatgcatgt ggtttacatt tgtccctaga taagaggctt 1380
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Claims (10)
1. a kind of internal standard gene for detecting chicken derived components, it is named as Actb, has the sequence shown in SEQ ID NO.1.
2. application in detection chicken derived components for the gene described in claim 1.
3. the application in the chicken Components identification in food or feedstuff of the gene described in claim 1.
4. for the specific primer of gene described in PCR method test right requirement 1, its nucleotide sequence such as SEQ ID NO.2,
Shown in 3.
5. the test kit containing specific primer described in claim 4.
6. test kit as claimed in claim 5 is it is characterised in that also contain a probe, its nucleotide sequence such as SEQ ID
Shown in NO.4.
7. the test kit as described in claim 5 or 6 is it is characterised in that also contain a probe, its nucleotide sequence such as SEQ
Shown in ID NO.7.
8. application in detection gene copy number for the digital pcr method.
9. application as claimed in claim 8 is it is characterised in that identify chicken source internal standard gene Actb, institute using digital pcr method
The nucleotide sequence of primer as shown in SEQ ID NO.2,3, the nucleotide sequence such as SEQ of the probe using cooperatively with primer
Shown in ID NO.7.
10. the specific primer described in claim 4 or the arbitrary described test kit of claim 5-7 are in detection chicken derived components
Application.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610976239.1A CN106480203A (en) | 2016-11-07 | 2016-11-07 | For detecting internal standard gene and its application of chicken derived components |
Applications Claiming Priority (1)
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| CN110373455A (en) * | 2018-04-12 | 2019-10-25 | 奎克生技光电股份有限公司 | The nucleic acid samples measurement method of digital quantitative PCR |
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| CN109337991A (en) * | 2018-11-20 | 2019-02-15 | 昆明理工大学 | The application and its kit of a kind of gene Gcg in detection family's chicken derived component |
| CN109457034A (en) * | 2018-11-20 | 2019-03-12 | 昆明理工大学 | The application and its kit of a kind of gene Gcg in detection turkey derived component |
| CN109337991B (en) * | 2018-11-20 | 2021-09-14 | 昆明理工大学 | Application of gene Gcg in detection of chicken-derived components and kit thereof |
| CN113046469A (en) * | 2021-05-07 | 2021-06-29 | 昆明理工大学 | Application of gene TSA in detection of termitomyces albuminosus derived components |
| CN113046469B (en) * | 2021-05-07 | 2024-05-24 | 昆明理工大学 | Application of gene TSA in detection of collybia albuminosa source component |
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