CN107043818A - A kind of method that meat products pig derived component is quantitatively detected based on real-time fluorescence PCR - Google Patents
A kind of method that meat products pig derived component is quantitatively detected based on real-time fluorescence PCR Download PDFInfo
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Abstract
The present invention relates to biological products detection technique field, particularly a kind of method that meat products pig derived component is quantitatively detected based on real-time fluorescence PCR.The selection of PCR specific primers, the foundation of quantitative measurement standard curve model, the extracting genome DNA of product to be measured including pig DNA, product PCR to be measured amplifications, PCR amplification sensitivity and amplification efficiency detection, the animal derived materials PCR detection method predominantly qualitative detection set up, and adulterated amount is also the problem of consumer pays close attention in the actual content and adulterated food of meat in meat products.Therefore, we use real-time fluorescence PCR relative quantification method by making standard curve, the method for quantitatively detecting pig derived component content is set up.Using mixing meat sample DNA known to pig derived component content as template, withβ‑actinGene is internal reference, detectionCyt bGene, sets up the standard curve of quantitative analysis, and standard curve is verified with the mixing meat sample DNA that pig derived component content is different, and detected value and actual content matching degree are high, and the rate of recovery is up to 99.85 102.60%.
Description
Technical field
The present invention relates to biological products detection technique field, particularly one kind quantitatively detects meat system based on real-time fluorescence PCR
The method of product pig derived component.
Background technology
With the continuous improvement of people's living standards, food security is received more and more attention with ethics, however, meat system
Product spurious tags and adulterated and low price pork, duck pretend to be the fraudulents such as beef, mutton replacement to happen occasionally, clenbuterol hydrochloride event,
Melamine milk event, dioxin event etc. are even more the worry for exacerbating society and consumer to animal production safety.It is based on
Factor in terms of economy, religion and health, it is imperative that animal derived materials are quickly and accurately detected.In recent years,
China has formulated the method and standard of animal derived materials in some detection food, cosmetics and feeds（GB/T 21101-
2007, SN/T 2051-2008), but its detection method still suffer from sensitivity not enough, can not quantify and the side such as be difficult to promote the use of
The defect in face.Therefore, this experiment detects the PCR specific primers of pig derived component by designing, and foundation utilizes real-time fluorescence PCR
The method of technology qualitative and quantitative analysis pig derived component.
The content of the invention
It is an object of the invention to provide a kind of sensitivity is high, specificity is high, the base that pork content is quantitatively detected can be achieved
The method for quantitatively detecting meat products pig derived component in real-time fluorescence PCR.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of method that meat products pig derived component is quantitatively detected based on real-time fluorescence PCR, is comprised the following steps：
A, pig DNA PCR specific primers selection
PCR primer selection livestock and poultry mitochondrial cytochrome b-cytochrome b, the Cyt b genes DNA of specific amplification pig DNA
Sequence, using its variable region as template design primer sequence, Cyt b sense primers be 5 '-
CATGCGTATCACCACCATTATAT-3 ', anti-sense primer is 5 '-TGCCAAGCGGGTTGCT-3 ', the bp of primer size 100；With
β-actin are internal reference, universal amplification primer, and sense primer is 5 '-GATCGTGCGGGACATCAA-3 ', anti-sense primer is 5 '-
AGGAAGGAGGGCTGGAAGA G-3 ', the bp of primer size 180；
B, quantitative measurement standard curve model are set up
Ox, sheep, chicken and duck sample press 1:1:1:After 1 mixing, pork sample, pork sample adding proportion are added in biased sample
For 0,1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, in reagent SYBR Premix Ex Taq II
Add PCR upstream and downstream primer, ROX reference dye ROX Reference Dye, DNA sample and distilled water ddH2O, adjusts DNA
Sample concentration is 50ng/μ L, and deformation process is 95 DEG C of pre-degeneration processing 30s, 95 DEG C of denaturation treatment 5s, 64 DEG C of annealings
34s, deformation process is circulated 40 times, using β-actin as internal reference, and the pork sample of different adding proportions is control, using relatively fixed
Amount method calculates DDCT methods and 2-DDCtValue, draws y=3075.5x+160.83 --- y: 2-DDCtValue, x:Pig derived component hundred
Divide content；
C, product to be measured extracting genome DNA
Take 20mg tested animal tissue samples to be put into 2ml centrifuge tubes, protease K digesting time 4h is added, from TIANGEN groups
Genome DNA extracting reagent kit is knitted, 200 μ l buffer solution GB are added, mixing 15 seconds of turning upside down, 70 DEG C of placement 13min, rotating speed 12
000rpm centrifugal treating 2min, the solution of centrifugal treating is vacantly added dropwise in the middle part of adsorbed film, 5min, DNA samples are placed at room temperature
Product put -20 DEG C of refrigerator preservations；
D, product PCR to be measured are expanded
PCR upstream and downstream primer, ROX reference dye ROX Reference are added in reagent SYBR Premix Ex Taq II
Dye, DNA sample and distilled water ddH2O, deformation process is 95 DEG C of pre-degeneration processing 30s, 95 DEG C of denaturation treatment 5s, 64 DEG C of annealing
34s is handled, deformation process is circulated 30-40 times；
E, PCR expand sensitivity and amplification efficiency detection
Sensitivity and the amplification efficiency of Cyt b and β-actin primers of the Cyt b primers amplification of product to be measured are detected, passes through step
Rapid B quantitative measurement standard curve models calculate detection meat products pig derived component content, realize and quantitatively detect meat products pig
Derived component.
Also include step F, quantitative measurement standard curve model to detect：Ox, sheep, chicken and duck sample press 1:1:1:1 mixing
Afterwards, pork sample is added in biased sample, pork sample adding proportion is 0,5,35,55 and 75%, reagent SYBR Premix
PCR upstream and downstream primer, ROX reference dye ROX Reference Dye, DNA sample and distilled water are added in Ex Taq II
ddH2O, adjustment DNA sample concentration is 50ng/μ L, deformation process be 95 DEG C of pre-degenerations processing 30s, 95 DEG C of denaturation treatment 5s,
64 DEG C of annealing 34s, deformation process is circulated 40 times, using β-actin as internal reference, the pork samples of different adding proportions for pair
According to using relative quantification method calculating DDCT methods and 2-DDCtValue, detecting step B curve model.
Deformation process is 95 DEG C of pre-degeneration processing 30s, 95 DEG C of denaturation treatment 5s, 64 DEG C of annealings in the step D
34s, deformation process is circulated 40 times.
TIANGEN tissue gene group DNA extraction kits, model DP304 are selected in the step C.
Beneficial effects of the present invention are：
1st, the PCR specific primers selection of pig DNA, the PCR primer of design specific amplification pig DNA is detection pig derived component
Precondition, therefore by analyses and comparison pig and other livestock and poultry mitochondrial cytochrome b genes DNA sequence dnas, with its variable region
For template design primer sequence, expanded through Real-time PCR, pig muscle sample DNA detects stronger fluorescence intensity, i.e.,
Ct values are low；Although ox, sheep, chicken and duck muscle samples DNA also detect that extremely weak fluorescence intensity, its Ct value ＞ 30, be higher than or
Close to the blank control of non-animal derived property composition.In theory, blank control and will not when being expanded without pig derived component sample DNA
Produce in fluorescence signal, but practical operation, often due to the reason such as instrument, reagent, primer and magnesium ion concentration, even if strict control
DNA profiling processed is not contaminated, amplification curve occurs after also being circulated 30, should be false positive, illustrates that designed primer has
Preferable species specificity.In the step D deformation process be 95 DEG C of pre-degenerations processing 30s, 95 DEG C of denaturation treatment 5s, 64 DEG C
34s is made annealing treatment, deformation process circulates 40 times, amplification curve is completely presented, is easy to testing result clearly to present.
2nd, in view of because the reasons such as food processing very likely have the problem of DNA extracted amounts are too low and right in real work
The requirement of halal food is, it is necessary to which detection method has very high sensitivity.Cooked meat product, the spirit of detection are detected in regular-PCR method
Sensitivity is only 5 %, and uses real-time PCR method, in detection beef or mutton product the test limit of doping pig derived component up to 1 ×
10-6ng/ μL.In this experiment, by pig DNA doubling dilution, its DNA detection limit is analyzed.When DNA concentration as little as 5 × 10- 5During ng/ μ L, amplification curve is still complete, clear, and Ct values are higher than blank control, illustrate that this method has higher sensitivity.
3rd, the animal derived materials PCR detection method predominantly qualitative detection set up, and the reality of meat contains in meat products
Adulterated amount is also the problem of consumer pays close attention in amount and adulterated food.Therefore, we use real-time fluorescence PCR relative quantification method
By making standard curve, the method for setting up detection pig derived component content.To mix meat sample known to pig derived component content
DNA is template, and linear regression utilizes regression analysis in mathematical statistics, complementary between two or more variable to determine
A kind of statistical analysis technique of quantitative relationship, with quite varied.In regression analysis, an independent variable and one are only included because becoming
Amount, and the relation of the two can use straight line approximate representation, this regression analysis is referred to as simple linear regression analysis.We are herein
What is done is exactly linear regression, that is, finds out pig derived component percentage composition (x) and 2-DDCtValue（y）Between regression relation.R2 is judgement
Coefficient, is the Judging index for judging linear regression straight line degree of fitting quality, square for being R（R calculation formula is as follows）.Linear phase
The index that coefficients R is linear correlation degree between two stochastic variables of measurement is closed, it is by karr Pearson（Karl
Pearson）Proposed in the 1880's, be widely used in the every field of science.R2 wherein in Fig. 4 and Fig. 5 is not by
Same variable is calculated, withβ-actinGene is internal reference, detectionCyt bGene, sets up the standard curve of quantitative analysis, knot
Fruit display 2-DDCtValue has significant linear relationship with mixing the content of pig derived component in meat sample（R² = 0.9914）.With pig source
Property component content different mixing meat sample DNA verified that detected value and actual content matching degree are high to standard curve, return
Yield 99.85-102.60%, in general allowed band.Select suitable reference gene to standardize target gene, eliminate sample
The influence to DNA extracted amounts, sampling amount such as product constitutional heterogenity, DNA extraction process, extraction batch, so that amplification curve
It is more reliable with testing result.In this experiment, Pork Tissue is mixed in proportion with other animal tissues, with 2-DDCtValue makes mark
Directrix curve, with exclude it is actually detected in because sample constitutes influence that is complicated and may being caused to amplification.
In existing food in pig derived component detection method, it is believed that Ct values are limited within 35 circulations, in the spy of detection
The opposite sex, sensitivity and false positive control aspect can reach preferable effect.The Ct values detection positive is defined to 30 however, also having
Circulation, similar, we, without in pig derived component muscle samples DNA test experiences, can often detect to ox, sheep, chicken and duck etc.
To faint fluorescence signal, although amplification cycles number or Ct values are higher than or close to blank control, but still there is more completely amplification bent
Line, can be to whether the accurate qualitative judgement containing pig derived component brings interference in real work；If with reference to quantitative analysis,
Target gene is standardized with the reference gene Ct values of empty species-specific amplification, and compared with the control group without pig derived component
（As shown in Table 3, 4）, then can exclude " false positive ", strengthen the reliability qualitatively judged.
Brief description of the drawings
Fig. 1 is different genes group DNA electrophoretograms；
Fig. 2 is pig, ox, sheep, chicken and duck DNA Real-time PCR amplification curves；
Fig. 3 is the Real-time PCR amplification curves after pig DNA is serially diluted；
Fig. 4 is that Cyt b and β-actin primers amplification efficiency analyzes schematic diagram；
Fig. 5 is quantitative measurement standard curve model schematic diagram；
Fig. 6 is Accuracy Verification PCR amplification curves；
The commercially available ham sausage sample P CR amplification curves of Fig. 7.
Embodiment
A kind of method that meat products pig derived component is quantitatively detected based on real-time fluorescence PCR, is comprised the following steps：
A, pig DNA PCR specific primers selection
PCR primer selection livestock and poultry mitochondrial cytochrome b-cytochrome b, the Cyt b genes DNA of specific amplification pig DNA
Sequence, using its variable region as template design primer sequence, Cyt b sense primers be 5 '-
CATGCGTATCACCACCATTATAT-3 ', anti-sense primer is 5 '-TGCCAAGCGGGTTGCT-3 ', the bp of primer size 100；With
β-actin are internal reference, universal amplification primer, and sense primer is 5 '-GATCGTGCGGGACATCAA-3 ', anti-sense primer is 5 '-
AGGAAGGAGGGCTGGAAGA G-3 ', the bp of primer size 180；
B, quantitative measurement standard curve model are set up
Ox, sheep, chicken and duck sample press 1:1:1:After 1 mixing, pork sample, pork sample adding proportion are added in biased sample
For 0,1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, in reagent SYBR Premix Ex Taq II
Add PCR upstream and downstream primer, ROX reference dye ROX Reference Dye, DNA sample and distilled water ddH2O, adjusts DNA
Sample concentration is 50ng/μ L, and deformation process is 95 DEG C of pre-degeneration processing 30s, 95 DEG C of denaturation treatment 5s, 64 DEG C of annealings
34s, deformation process is circulated 40 times, using β-actin as internal reference, and the pork sample of different adding proportions is control, using relatively fixed
Amount method calculates DDCT methods and 2-DDCtValue, draws y=3075.5x+160.83 --- y: 2-DDCtValue, x:Pig derived component hundred
Divide content；
C, product to be measured extracting genome DNA
Take 20mg tested animal tissue samples to be put into 2ml centrifuge tubes, protease K digesting time 4h is added, from TIANGEN groups
Genome DNA extracting reagent kit is knitted, 200 μ l buffer solution GB are added, mixing 15 seconds of turning upside down, 70 DEG C of placement 13min, rotating speed 12
000rpm centrifugal treating 2min, the solution of centrifugal treating is vacantly added dropwise in the middle part of adsorbed film, 5min, DNA samples are placed at room temperature
Product put -20 DEG C of refrigerator preservations；
D, product PCR to be measured are expanded
PCR upstream and downstream primer, ROX reference dye ROX Reference are added in reagent SYBR Premix Ex Taq II
Dye, DNA sample and distilled water ddH2O, deformation process is 95 DEG C of pre-degeneration processing 30s, 95 DEG C of denaturation treatment 5s, 64 DEG C of annealing
34s is handled, deformation process is circulated 30-40 times；
E, PCR expand sensitivity and amplification efficiency detection
Sensitivity and the amplification efficiency of Cyt b and β-actin primers of the Cyt b primers amplification of product to be measured are detected, passes through step
Rapid B quantitative measurement standard curve models calculate detection meat products pig derived component content, realize and quantitatively detect meat products pig
Derived component.
Also include step F, quantitative measurement standard curve model to detect：Ox, sheep, chicken and duck sample press 1:1:1:1 mixing
Afterwards, pork sample is added in biased sample, pork sample adding proportion is 0,5,35,55 and 75%, reagent SYBR Premix
PCR upstream and downstream primer, ROX reference dye ROX Reference Dye, DNA sample and distilled water are added in Ex Taq II
ddH2O, adjustment DNA sample concentration is 50ng/μ L, deformation process be 95 DEG C of pre-degenerations processing 30s, 95 DEG C of denaturation treatment 5s,
64 DEG C of annealing 34s, deformation process is circulated 40 times, using β-actin as internal reference, the pork samples of different adding proportions for pair
According to using relative quantification method calculating DDCT methods and 2-DDCtValue, detecting step B curve model.
It is preferred that the step D in deformation process be 95 DEG C of pre-degenerations processing 30s, 95 DEG C of denaturation treatment 5s, 64 DEG C of annealing
34s is handled, deformation process is circulated 40 times.TIANGEN tissue gene group DNA extraction kits, model are selected in the step C
DP304。
Commercially available part brand ham sausage is gathered with supermarket（Sample message is shown in Table 1）, DNA is extracted, by reaction described above
Condition carries out Real-time PCR detections, detects whether containing pig derived component, and calculates its content progress labor.
（1）The specificity of Real-time PCR reaction detection pig derived components
Pig, ox, sheep, chicken and the duck musculature DNA of extraction detect that band is clear, complete through 1% agarose gel electrophoresis（Figure
1）, available for PCR amplifications.
Optimization Real-time PCR reaction systems and under the conditions of, to pig, ox, sheep, chicken and duckCyt bGene enters
The Real-time PCR that gone are detected.As shown in Fig. 2 in Real-time PCR amplifications, pig muscle sample is after 16 circulate
Enter the exponential amplification phase, and ox, sheep, chicken and duck muscle samples are then appeared in after 30 circulations, its Ct value is all higher than 32, detection
The fluorescence intensity arrived is extremely weak, should be false positive, illustrates that designed primer has species specificity.
The PCR primer of design specific amplification pig DNA is to detect the precondition of pig derived component, therefore is compared by analyzing
To pig and other livestock and poultry mitochondrial cytochrome b genes DNA sequence dnas, using its variable region as template design primer sequence, warp
Real-time PCR are expanded, and pig muscle sample DNA detects stronger fluorescence intensity, i.e. Ct values are low；Although ox, sheep, chicken and
Duck muscle samples DNA also detects that extremely weak fluorescence intensity, but its Ct value ＞ 30, is higher than or close to the sky of non-animal derived property composition
White control.In theory, blank control and fluorescence signal will not be produced when being expanded without pig derived component sample DNA, but actual behaviour
In work, often due to the reason such as instrument, reagent, primer and magnesium ion concentration, even if strictly control DNA profiling is not contaminated,
There is amplification curve after being circulated 30, should be false positive, illustrate that designed primer has preferable species specificity.
（2）The sensitivity of Real-time PCR amplification pig derived components and amplification efficiency
In order to detectCyt bThe sensitivity of primer amplification, is respectively 5 × 10 to concentration-4- 50ng/ μ L pig DNA is carried out
Real-time PCR react.As shown in figure 3, when reactant DNA concentration as little as 5 × 10-5During ng/ μ L, reaction is still clearly
Into exponential phase amplification, and earlier than blank control, therefore, this method detects lower bound up to 5 × 10-5 ng/ μ L.
In view of because the reasons such as food processing very likely have the problem of DNA extracted amounts are too low and to clear in real work
The requirement of true food is, it is necessary to which detection method has very high sensitivity.With regular-PCR method detect cooked meat product, detection it is sensitive
Degree is only 5 %, and uses real-time PCR method, in detection beef or mutton product the test limit of doping pig derived component up to 1 ×
10-6ng/ μL.In this experiment, by pig DNA doubling dilution, its DNA detection limit is analyzed.When DNA concentration as little as 5 × 10- 5During ng/ μ L, amplification curve is still complete, clear, and Ct values are higher than blank control, illustrate that this method has higher sensitivity.
Under the DNA diluted concentrations, determineCyt bWithβ-actinThe Ct values of primer amplification, with DCt（Ct β-actin -
Ct Cyt b ）It is abscissa mapping for the logarithm of ordinate, template DNA concentration（Fig. 4）, the absolute value of its slope is 0.0769, is connect
0 is bordering on, i.e.,Cyt bWithβ-actinThe amplification efficiency of primer is basically identical, can use 2-DDCtMethod is calculatedCyt bRelative quantity.
（3）The making of standard curve
Mixing meat sample DNA by 0-100% of pig derived component content is template, carries out Real-time PCR reactions.Withβ- actinFor internal reference, using without pig derived component sample as control, analysis pig derived component content withCyt bGene C t values, DCt
Value ,-DDCt and 2-DDCtCorrelation between value（Table 2）.As a result show, utilize 2-DDCtThe relative quantification value that method is calculated and pig source
The percentage composition of property composition is significantly correlated, and standard curve is simulated using Excel（Fig. 5）, regression formula is：y = 3075.5x
+ 160.83（y: 2-DDCtValue, x:Pig derived component percentage composition, R2=0.9914）.
The animal derived materials PCR detection method predominantly qualitative detection set up, and in meat products meat actual content
And adulterated amount is also the problem of consumer pays close attention in adulterated food.Therefore, we are logical using real-time fluorescence PCR relative quantification method
Making standard curve is crossed, the method for setting up detection pig derived component content.To mix meat sample DNA known to pig derived component content
For template, withβ-actinGene is internal reference, detectionCyt bGene, sets up the standard curve of quantitative analysis, as a result shows 2-DDCt
Value has significant linear relationship with mixing the content of pig derived component in meat sample（R² = 0.9914）.With pig derived component content
Different mixing meat sample DNA are verified that detected value is high with actual content matching degree to standard curve, and the rate of recovery reaches
99.85-102.60%, in general allowed band.Select suitable reference gene to standardize target gene, eliminate sample sets
Into inhomogeneity, DNA extraction process, the influence to DNA extracted amounts, sampling amount such as batch is extracted, so that amplification curve and inspection
Survey result more reliable.In this experiment, Pork Tissue is mixed in proportion with other animal tissues, with 2-DDCtIt is worth making standard bent
Line, with exclude it is actually detected in because sample constitutes influence that is complicated and may being caused to amplification.
In existing food in pig derived component detection method, it is believed that Ct values are limited within 35 circulations, in the spy of detection
The opposite sex, sensitivity and false positive control aspect can reach preferable effect.The Ct values detection positive is defined to 30 however, also having
Circulation, similar, we, without in pig derived component muscle samples DNA test experiences, can often detect to ox, sheep, chicken and duck etc.
To faint fluorescence signal, although amplification cycles number or Ct values are higher than or close to blank control, but still there is more completely amplification bent
Line, can be to whether the accurate qualitative judgement containing pig derived component brings interference in real work；If with reference to quantitative analysis,
Target gene is standardized with the reference gene Ct values of empty species-specific amplification, and compared with the control group without pig derived component
（As shown in Table 3, 4）, then can exclude " false positive ", strengthen the reliability qualitatively judged.
（4）Standard curve is verified
The mixing meat sample DNA that pig derived component content is respectively 0%, 5%, 35%, 55% and 75% is prepared, Real-time PCR are carried out
Reaction, will survey 2-DDCtValue brings in standard curve calculating pig derived component content into, with test stone curve and quantitative determination
Accuracy.From table 3, the pig derived component content and actual value matching degree of detection are higher, and the rate of recovery is 99.85-
102.60%。
（5）The commercially available ham sausage testing result in part
To the 6 kinds of commercially available ham sausage sample extraction DNA taken, its pig is detected using the Real-time PCR reaction systems of foundation
Derived component, testing result is as shown in Fig. 7 and table 4.It is 21.94 to be denoted as Cyt b genes Ct averages in the ham sausage of non-Islamic,
Its pig derived component content is 12.7%, and remaining sample Cyt b gene Ct average for being denoted as halal food is all higher than 30, is higher than
Or close to the control without pig derived component, no pig derived component is should be, quantitative analysis is shown as negative value.
Commercially available ham sausage and its markup information that table 1 is inspected by random samples
The pig derived component content detection the result of table 3
The commercially available ham sausage pig derived component testing result of table 4
Note：Pig derived component content detection result negative value person should be detection feminine gender, i.e., without pig derived component.
Claims (4)
1. a kind of method that meat products pig derived component is quantitatively detected based on real-time fluorescence PCR, it is characterised in that including following step
Suddenly：
A, pig DNA PCR specific primers selection
PCR primer selection livestock and poultry mitochondrial cytochrome b-cytochrome b, the Cyt b genes DNA of specific amplification pig DNA
Sequence, using its variable region as template design primer sequence, Cyt b sense primers be 5 '-
CATGCGTATCACCACCATTATAT-3 ', anti-sense primer is 5 '-TGCCAAGCGGGTTGCT-3 ', the bp of primer size 100；With
β-actin are internal reference, universal amplification primer, and sense primer is 5 '-GATCGTGCGGGACATCAA-3 ', anti-sense primer is 5 '-
AGGAAGGAGGGCTGGAAGA G-3 ', the bp of primer size 180；
B, quantitative measurement standard curve model are set up
Ox, sheep, chicken and duck sample press 1:1:1:After 1 mixing, pork sample, pork sample adding proportion are added in biased sample
For 0,1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100%, in reagent SYBR Premix Ex Taq II
Add PCR upstream and downstream primer, ROX reference dye ROX Reference Dye, DNA sample and distilled water ddH2O, adjusts DNA
Sample concentration is 50ng/μ L, and deformation process is 95 DEG C of pre-degeneration processing 30s, 95 DEG C of denaturation treatment 5s, 64 DEG C of annealings
34s, deformation process is circulated 40 times, using β-actin as internal reference, and the pork sample of different adding proportions is control, using relatively fixed
Amount method calculates DDCT methods and 2-DDCtValue, draws y=3075.5x+160.83 --- y: 2-DDCtValue, x:Pig derived component hundred
Divide content；
C, product to be measured extracting genome DNA
Take 20mg tested animal tissue samples to be put into 2ml centrifuge tubes, protease K digesting time 4h is added, from TIANGEN groups
Genome DNA extracting reagent kit is knitted, 200 μ l buffer solution GB are added, mixing 15 seconds of turning upside down, 70 DEG C of placement 13min, rotating speed 12
000rpm centrifugal treating 2min, the solution of centrifugal treating is vacantly added dropwise in the middle part of adsorbed film, 5min, DNA samples are placed at room temperature
Product put -20 DEG C of refrigerator preservations；
D, product PCR to be measured are expanded
PCR upstream and downstream primer, ROX reference dye ROX Reference are added in reagent SYBR Premix Ex Taq II
Dye, DNA sample and distilled water ddH2O, deformation process is 95 DEG C of pre-degeneration processing 30s, 95 DEG C of denaturation treatment 5s, 64 DEG C of annealing
34s is handled, deformation process is circulated 30-40 times；
E, PCR expand sensitivity and amplification efficiency detection
Sensitivity and the amplification efficiency of Cyt b and β-actin primers of the Cyt b primers amplification of product to be measured are detected, passes through step
Rapid B quantitative measurement standard curve models calculate detection meat products pig derived component content, realize and quantitatively detect meat products pig
Derived component.
2. a kind of method that meat products pig derived component is quantitatively detected based on real-time fluorescence PCR according to claim 1, it is special
Levy and be that also including step F, quantitative measurement standard curve model detects：Ox, sheep, chicken and duck sample press 1:1:1:After 1 mixing,
Pork sample is added in biased sample, pork sample adding proportion is 0,5,35,55 and 75%, reagent SYBR Premix Ex
PCR upstream and downstream primer, ROX reference dye ROX Reference Dye, DNA sample and distilled water are added in Taq II
ddH2O, adjustment DNA sample concentration is 50ng/μ L, deformation process be 95 DEG C of pre-degenerations processing 30s, 95 DEG C of denaturation treatment 5s,
64 DEG C of annealing 34s, deformation process is circulated 40 times, using β-actin as internal reference, the pork samples of different adding proportions for pair
According to using relative quantification method calculating DDCT methods and 2-DDCtValue, detecting step B curve model.
3. a kind of method that meat products pig derived component is quantitatively detected based on real-time fluorescence PCR according to claim 1, it is special
Levy and be in the step D that deformation process is that 95 DEG C of pre-degenerations handle 30s, 95 DEG C of denaturation treatment 5s, 64 DEG C of annealing 34s,
Deformation process is circulated 40 times.
4. a kind of method that meat products pig derived component is quantitatively detected based on real-time fluorescence PCR according to claim 1, it is special
Levy and be in the step C to select TIANGEN tissue gene group DNA extraction kits, model DP304.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106987647A (en) * | 2017-05-25 | 2017-07-28 | 上海瑞丰农业科技有限公司 | A kind of RPA primers, kit and detection method for detecting pig derived component |
| CN109251984A (en) * | 2018-10-10 | 2019-01-22 | 珠海出入境检验检疫局检验检疫技术中心 | The method and kit of pork content ratio in identification mixing meat sample |
| CN110904245A (en) * | 2019-12-23 | 2020-03-24 | 中南民族大学 | TaqMan fluorescent quantitative PCR method for identifying pork components by using CACA gene and application thereof |
| CN112322792A (en) * | 2020-11-30 | 2021-02-05 | 天津市农业科学院 | Pig source reference gene and application thereof in internal control nucleic acid test strip |
| CN113088531A (en) * | 2021-03-25 | 2021-07-09 | 四川省食品药品检验检测院(四川省药品质量研究所、四川省医疗器械检测中心) | Bovine-derived component quantitative analysis standard plasmid, preparation and detection method and application |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106987647A (en) * | 2017-05-25 | 2017-07-28 | 上海瑞丰农业科技有限公司 | A kind of RPA primers, kit and detection method for detecting pig derived component |
| CN109251984A (en) * | 2018-10-10 | 2019-01-22 | 珠海出入境检验检疫局检验检疫技术中心 | The method and kit of pork content ratio in identification mixing meat sample |
| CN110904245A (en) * | 2019-12-23 | 2020-03-24 | 中南民族大学 | TaqMan fluorescent quantitative PCR method for identifying pork components by using CACA gene and application thereof |
| CN110904245B (en) * | 2019-12-23 | 2023-08-15 | 中南民族大学 | TaqMan fluorescent quantitative PCR method for identifying pork components by utilizing CACA genes and application thereof |
| CN112322792A (en) * | 2020-11-30 | 2021-02-05 | 天津市农业科学院 | Pig source reference gene and application thereof in internal control nucleic acid test strip |
| CN113088531A (en) * | 2021-03-25 | 2021-07-09 | 四川省食品药品检验检测院(四川省药品质量研究所、四川省医疗器械检测中心) | Bovine-derived component quantitative analysis standard plasmid, preparation and detection method and application |
| CN113088531B (en) * | 2021-03-25 | 2023-10-17 | 四川省药品检验研究院(四川省医疗器械检测中心) | Bovine-derived ingredient quantitative analysis standard plasmid, preparation and detection methods and application |
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