CN108103210A - A kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false - Google Patents
A kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false Download PDFInfo
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Abstract
The invention discloses a kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false, while can be realized by once expanding to bird's nest and its adulterant white fungus and pigskin ingredient, quick, real-time qualitative detection, its cardinal principle contains the DNA of esculent swift for bird's nest, differentiates the presence or absence of bird's nest by detecting in bird's nest esculent swift cytb genes；Differentiate the presence or absence of adulterant and its specific species in bird's nest by detecting in pigskin in pig ATP synthase genes and white fungus white fungus α tubulin genes.Compared with substance real-time fluorescence PCR, not only high specificity, high sensitivity, while be greatly improved detection efficiency, save testing cost detect this method particularly suitable for the adulterated doping to bird's nest of largely entering a country and quality safety.
Description
Technical field
The invention belongs to technical field of biological chemistry detection, in particular to the triple glimmering in real time of the discriminating bird's nest true and false
Light PCR method.
Background technology
Bird's nest is to be mixed and condensed in built up nest with saliva and down etc. by esculent swift and a variety of swallow classes that belong to together, from ancient times with
Come be considered as a kind of rare Chinese medicine and rare food, it is a variety of potential that modern scientific research shows that the glycoprotein in bird's nest has
Pharmacological effect.Bird's nest main product is in Southeast Asian countries and regions, such as Indonesia, Malaysia, Thailand, Vietnam, Philippine
Deng, and China is then the consumption market of bird's nest maximum.In recent years, due to improvement of living standard and the enhancing of health care consciousness, swallow
The demand sustainable growth of nest.Contradiction between the strong market demand and limited bird's nest source expedites the emergence of and promotes bird's nest production
The generation of the adulterated doping phenomenon of product.
Possible adulterant has white fungus, pigskin etc. in bird's nest, and also establishing a variety of physics and chemistry currently for these adulterants examines
Survey method, such as SDS-PAGE, dielectrophoresis, Fourier transform infrared spectroscopy (FTIR).These methods are mainly based upon bird's nest
And some bases in adulterant, such as the progress such as protein, carbohydrate, however these into branch with the bird's nest place of production, gold
The differences of factors such as silk swallow source of species, time of picking and change, it is therefore not objective to the judgement of result.
The content of the invention
In view of the above-mentioned problems, the present invention is based on the real-time fluorescence PCR technologies of molecular biology, a kind of discriminating bird's nest is provided
Triple real time fluorescent PCR methods of the true and false, by once expanding while detecting esculent swift ingredient and two kinds of adulterants (pigskin and silver
Ear) ingredient, science can be made to testing result from gene level, is objectively judged, realize to the rapid screening of bird's nest sample and
Detection.This method has high specificity, high sensitivity compared with Standard PCR, effectively reduce PCR pollution rates, it is continuous it is quick, from
The features such as dynamicization degree is high；Compared with substance real-time fluorescence PCR, detection efficiency higher, testing cost decline to a great extent, available for pair
The rapid screening of a large amount of immigration bird's nest products and detection.
The cardinal principle of the present invention is the saliva that bird's nest is esculent swift, wherein the DNA containing esculent swift, by detecting bird's nest
Middle esculent swift cytb genes differentiate the presence or absence of bird's nest；And pigskin and white fungus are the common adulterants of bird's nest, pass through detection
In pigskin in pig ATP synthase genes and white fungus white fungus α-tubulin genes come differentiate in bird's nest the presence or absence of adulterant and
Its specific species, so as to achieve the purpose that quickly to differentiate the bird's nest true and false and adulterant contained therein.If bird's nest product to be checked is only
It detects esculent swift ingredient, and does not detect two kinds of adulterant pigskins and white fungus ingredient, then bird's nest product to be checked is certified products bird's nest；
If bird's nest product to be checked not only detects esculent swift ingredient, while also detects at least one adulterant ingredient (pigskin or silver
Ear), then bird's nest product to be checked is adulterated doping bird's nest；If bird's nest product to be checked does not detect esculent swift ingredient, and detects
At least one adulterant ingredient (pigskin or white fungus) does not detect above two adulterant ingredient, then bird's nest product to be checked is puppet
Product bird's nest.
A kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false, it is described compound using composite primer and probe solution
Primer and probe solution includes：
(1) it is used to expand esculent swift cytb genes：
First sense primer, sequence are as follows
5 '-CAGTAGACAACCCCACATTA-3 ',
First anti-sense primer, sequence are as follows
5 '-GTGGGGTGAGTATGATGATG-3 ',
First probe, sequence are as follows
FAM-5’-CCCGATTCTTCGCCCTACACTTCC-3’-TAMRA；
(2) it is used to expand white fungus α-tubulin genes：
Second sense primer, sequence are as follows
5 '-TGGTGGTCCTCTTGGCACTC-3 ',
Second anti-sense primer, sequence are as follows
5 '-CCACCAAGCGAGTGGGTAAT-3 ',
Second probe, sequence are as follows
CY5-5’-AGCCCTCGGCCTCCCGGCGGACG-3’-BHQ2；
(3) it is used to expand pig ATP synthase genes：
3rd sense primer, sequence are as follows
5’-AACCAGTAGCCCTAGCCGTA-3’；
3rd anti-sense primer, sequence are as follows
5’-TGAGTAGTGCTAATGTGGCCC-3’；
3rd probe, sequence are as follows
VIC-5’-TGACAGCCAACATTACAGCAGGG-3’-TAMRA。
Preferably, nucleic acid extraction kit extraction can be used in triple real time fluorescent PCR methods of the above-mentioned discriminating bird's nest true and false
Bird's nest product dna to be measured and using Eukaryotic 18s universal primers to carried DNA carry out standard PCR amplification.
Preferably, in triple real time fluorescent PCR methods of the above-mentioned discriminating bird's nest true and false, triple real-time fluorescence PCR reactants
It is to be：PCR Master Mix12.5 μ L, compound sense primer and each 1.5 μ L of compound anti-sense primer solution, combined probe solution
1.5 μ L, bird's nest product dna solution 5.0 μ L, ddH to be measured2O3.0 μ L, 25 μ L of total volume；
The compound sense primer solution is by 10 μM, the first sense primer solution of 0.5 μ L, and the of 12.5 μ Μ, 0.5 μ L
Two sense primer solution and 7.5 μ Μ, the 3rd sense primer solution of 0.5 μ L are prepared to obtain；
The compound anti-sense primer solution is by 10 μM, the first anti-sense primer solution of 0.5 μ L, and the of 12.5 μ Μ, 0.5 μ L
Two anti-sense primer solution and 7.5 μ Μ, the 3rd anti-sense primer solution of 0.5 μ L are prepared to obtain；
The combined probe solution is by 7.5 μM, the first probe solution of 0.5 μ L, 7.5 μ Μ, and the second probe of 0.5 μ L is molten
Liquid and 12.5 μ Μ, the 3rd probe solution of 0.5 μ L are prepared to obtain.
Preferably, in triple real time fluorescent PCR methods of the above-mentioned discriminating bird's nest true and false, amplification condition is：95 DEG C of pre-degenerations
10min, 95 DEG C of denaturation 15s, extends 1min, 35 Xun Huans after 60 DEG C of annealing.
Preferably, in triple real time fluorescent PCR methods of the above-mentioned discriminating bird's nest true and false, triple real-time fluorescence PCR expansions are carried out
Negative control and the blank control without DNA profiling are set up during increasing.
Preferably, in triple real time fluorescent PCR methods of the above-mentioned discriminating bird's nest true and false, specific determination method is as follows：
A. as the amplification curve of esculent swift ingredient occurs in bird's nest product to be checked, and Ct values are no more than 35, and negative control with
Blank control then judges to contain bird's nest ingredient in the bird's nest product to be checked without amplification；As bird's nest product to be checked does not occur esculent swift
The amplification curve of ingredient, and negative control and blank control are also set up, then judge that the bird's nest product to be checked does not contain bird's nest ingredient；
B. as the amplification curve of white fungus ingredient occurs in bird's nest product to be checked, and Ct values are not more than 30, and negative control and sky
White control then judges to contain adulterant white fungus ingredient in the bird's nest product to be checked without amplification；As white fungus occurs in bird's nest product to be checked
The amplification curve but Ct values of ingredient are between 30-35, and negative control and blank control are also set up, then need to expand again, if knot
Fruit is still in this way, then judge that the bird's nest product to be checked does not contain adulterant white fungus ingredient；If Ct values are less than 30 after expanding again, feminine gender
Control and blank control are also set up, then judge that the bird's nest product to be checked contains adulterant white fungus ingredient；Such as bird's nest product to be checked not
There is the amplification curve of white fungus ingredient, and negative control and blank control are also set up, then judge that the bird's nest product to be checked does not contain
Adulterant white fungus ingredient；
C. as the amplification curve of pig ingredient occurs in bird's nest product to be checked, and Ct values are not more than 30, and negative control and blank
Control then judges to contain adulterant pigskin ingredient in the bird's nest product to be checked without amplification；As pig ingredient occurs in bird's nest product to be checked
Amplification curve but Ct values between 30-35, and negative control and blank control are also set up, then need to expand again, if result is still
In this way, then judge that the bird's nest product to be checked does not contain adulterant pigskin ingredient；If Ct values are less than 30 after expanding again, negative control
It is also set up with blank control, then judges that the bird's nest product to be checked contains adulterant pigskin ingredient；As bird's nest product to be checked does not occur
The amplification curve of pig ingredient, and negative control and blank control are also set up, then judge that the bird's nest product to be checked does not contain adulterant
Pigskin ingredient.
A kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false provided by the present invention are suitable for swallow of largely entering a country
The quick detection and examination of nest product, have following advantage：
1. specific good, high sensitivity, can effectively prevent to the false negative result of esculent swift composition detection and to adulterant
The false positive results of composition detection are suitble to the customary sampling observation detection in laboratory to use.
2. simple and quick, entire detection process only needs or so 4 hours.
3. the detection to three kinds of heterogeneities once can be achieved at the same time in operation, testing cost is substantially reduced, improves detection examination
Agent and the utilization rate of reaction system.
Description of the drawings
Fig. 1 is triple real-time fluorescent PCR amplification graphs of bird's nest and two kinds of adulterant (pigskin and white fungus) ingredients.
Fig. 2 is relative sensitivity curves figure of the triple real time fluorescent PCR methods to adulterant composition detection.
Specific embodiment
For those skilled in the art is made to more fully understand technical scheme, with reference to specific embodiment to this
The triple real time fluorescent PCR methods for inventing a kind of discriminating bird's nest true and false provided are described in detail.
Embodiment 1：
(1) synthesis of primer and probe：By Shanghai, handsome Bioisystech Co., Ltd synthesizes, specific primer and probe sequence
It is as follows：
A. expand the upstream and downstream primer of esculent swift cytb genes (210bp) and probe sequence is respectively：
Sense primer (YW-F)：5’-CAGTAGACAACCCCACATTA-3’；
Anti-sense primer (YW-R)：5’-GTGGGGTGAGTATGATGATG-3’；
Probe (YW-P)：FAM-5’-CCCGATTCTTCGCCCTACACTTCC-3’-TAMRA.
B. expand the upstream and downstream primer of white fungus α-tubulin genes (192bp) and probe sequence is respectively：
Sense primer (YE-F)：5’-TGGTGGTCCTCTTGGCACTC-3’；
Anti-sense primer (YE-R)：5’-CCACCAAGCGAGTGGGTAAT-3’；
Probe (YE-P)：CY5-5’-AGCCCTCGGCCTCCCGGCGGACG-3’-BHQ2.
C. expand the upstream and downstream primer of pig ATP synthase genes (93bp) and probe sequence is respectively：
Sense primer (ZP-F)：5’-AACCAGTAGCCCTAGCCGTA-3’；
Anti-sense primer (ZP-R)：5’-TGAGTAGTGCTAATGTGGCCC-3’；
Probe (ZP-P)：
VIC-5’-TGACAGCCAACATTACAGCAGGG-3’-TAMRA。
(2) extraction of bird's nest product dna to be checked：Carrying for DNA is carried out according to Nucleospin Kit nucleic acid extraction kits
It takes, is specially as follows：
The bird's nest sample to be checked of 200mg grind into powder is taken to be placed in 2.0mL centrifuge tubes, is separately added into 650 μ L lysates
With the Proteinase K Solution that 10 μ L concentration are 10mg/mL；12000rpm centrifuges 10min after 65 DEG C of oscillation incubation 1h, shifts supernatant
Into new centrifuge tube.Isometric C4 (binding buffer) and absolute ethyl alcohol are separately added into, is taken after the mixing that turns upside down
700 μ L are placed on DNA adsorption columns, and filtrate is discarded after 11000 × g centrifugations 1min, repeat this operation until in surplus solution
DNA is adsorbed successively.400 μ L CQW (wash buffer 1) is added to be discarded on adsorption column after 11000 × g centrifugations 1min
Screening；700 μ L CQW (wash buffer 2) is added to discard filtrate after 11000 × g centrifugations 1min on adsorption column；Add 200 μ
Adsorption column after 11000 × g centrifugations 2min is directly taken out and is transferred in new centrifuge tube on adsorption column by L CQW.Add 100 μ
L CE (elution buffer, 65 DEG C of preheatings) 11000 × g centrifugations 1min, gained mistake after 5min on adsorption column, is incubated at room temperature
Screening is DNA solution.
(3) purity and concentration for carrying DNA are measured：After appropriate DNA stostes is taken to add aqua sterilisa dilution certain multiple, nucleic acid is used
The DNA of protein analyzer determination sample, by OD260/OD280Ratio and concentration value judge the purity and content of DNA.
(4) determining for DNA mass is carried：Standard PCR expansion is carried out to carried DNA using Eukaryotic 18s universal primers
Increase, detailed process is：
Primer sequence：F：5’-TCTGCCCTATCAACTTTCGATGGTA-3’；
R：5’-AATTTGCGCGCCTGCTGCCTTCCTT-3’.
PCR reaction systems：2.5 μ L10 × buffer, 400mmol/L each 0.5 μ L of dNTPs, 10 μm of ol/L primers Fs, R,
5.0 μ LDNA templates, use ddH2O is mended to 25 μ L of total volume.
PCR response procedures：95 DEG C of pre-degeneration 10min；95 DEG C denaturation 30s, 60 DEG C annealing 30s, 72 DEG C extension 30s, 35
Xun Huan；72 DEG C of fully extension 10min after the completion of cycling.
As a result detect：PCR reaction products are separated using 2% Ago-Gel, dye gel with EB after electrophoresis,
PCR reaction results are observed by gel imaging system.
As a result judge：Sample DNA generates apparent amplification, and primer size is 137bp, and blank control does not expand
Band illustrates that carried DNA mass meets PCR reaction requirements, can carry out subsequent experimental；Otherwise, it is necessary to extract again.
(5) preparation (25 μ L of total volume) of triple real-time fluorescence PCR reaction systems：PCR is added in PCR reaction tubes
Master Mix12.5 μ L, each 1.5 μ L of upstream and downstream primer of mixing, probe 1.5 the μ L, 5.0 μ of bird's nest product dna to be checked of mixing
L, ddH2O3.0 μ L, vortex mixing.The upstream and downstream primer and probe wherein mixed is both needed to prepare in advance, the sense primer tool of mixing
Body preparation method is：By following 3 kinds of sense primers, 10 μM of YW-F, 12.5 μ Μ YE-F and 7.5 μ Μ ZP-F according to 1:1:1
(v/v/v) ratio is mixed, and the mixing that is vortexed, and 4 DEG C save backup；The specific preparation method of anti-sense primer of mixing is：It will
Following 3 kinds of anti-sense primers, 10 μM of YW-R, 12.5 μ Μ YE-R and 7.5 μ Μ ZP-R are according to 1:1:The ratio of 1 (v/v/v) carries out
Mixing, and the mixing that is vortexed, 4 DEG C save backup；The probe preparation method of mixing is：By following 3 kinds of probes, 7.5 μM of YW-P, 7.5 μ
Μ YE-P and 12.5 μ Μ ZP-P are according to 1:1:The ratio of 1 (v/v/v) is mixed, and the mixing that is vortexed, and 4 DEG C save backup.
(6) triple real-time fluorescent PCR amplifications are carried out：First, PCR pipe is put into real-time fluorescence PCR instrument；Secondly, under
Row condition completes the setting of amplification program：95 DEG C of pre-degeneration 10min, 95 DEG C of denaturation 15s, extend 1min after 60 DEG C of annealing, 35
Xun Huan；Finally, Start operation amplification programs are clicked on.
(7) analysing amplified result：The analysis of software progress result, specific analytical method are carried using real-time fluorescence PCR instrument
For：
A. as the amplification curve of esculent swift ingredient occurs in bird's nest product to be checked, and Ct values are no more than 35, and negative control with
Blank control then judges to contain bird's nest ingredient in the bird's nest product to be checked without amplification；As bird's nest product to be checked does not occur esculent swift
The amplification curve of ingredient, and negative control and blank control are also set up, then judge that the bird's nest product to be checked does not contain bird's nest ingredient.
B. as the amplification curve of white fungus ingredient occurs in bird's nest product to be checked, and Ct values are not more than 30, and negative control and sky
White control then judges to contain adulterant white fungus ingredient in the bird's nest product to be checked without amplification；As white fungus occurs in bird's nest product to be checked
The amplification curve but Ct values of ingredient are between 30-35, and negative control and blank control are also set up, then need to expand again, if knot
Fruit is still in this way, then judge that the bird's nest product to be checked does not contain adulterant white fungus ingredient；If Ct values are less than 30 after expanding again, feminine gender
Control and blank control are also set up, then judge that the bird's nest product to be checked contains adulterant white fungus ingredient；Such as bird's nest product to be checked not
There is the amplification curve of white fungus ingredient, and negative control and blank control are also set up, then judge that the bird's nest product to be checked does not contain
Adulterant white fungus ingredient.
C. as the amplification curve of pig ingredient occurs in bird's nest product to be checked, and Ct values are not more than 30, and negative control and blank
Control then judges to contain adulterant pigskin ingredient in the bird's nest product to be checked without amplification；As pig ingredient occurs in bird's nest product to be checked
Amplification curve but Ct values between 30-35, and negative control and blank control are also set up, then need to expand again, if result is still
In this way, then judge that the bird's nest product to be checked does not contain adulterant pigskin ingredient；If Ct values are less than 30 after expanding again, negative control
It is also set up with blank control, then judges that the bird's nest product to be checked contains adulterant pigskin ingredient；As bird's nest product to be checked does not occur
The amplification curve of pig ingredient, and negative control and blank control are also set up, then judge that the bird's nest product to be checked does not contain adulterant
Pigskin ingredient.
Embodiment 2：The compositional optimization of upstream and downstream mix primer and probe
(1) respectively to sense primer (YW-F, YE-F and ZP-F), anti-sense primer (YW-R, YE-R and ZP-R) and probe
(YW-P, YE-P and ZP-P) sets 5 concentration gradients (2.5,5.0,7.5,10.0,12.5 μm of ol/L), horizontal according to 3 factor 5
Orthogonal Composite principle [L25(53)] primer or probe combinations of various concentration are set, then using similar in embodiment 1
Reaction system and amplification program carry out real-time fluorescent PCR amplification, according to the size of Ct values and the smoothness of amplification curve come true
Fixed optimal triple PCR reaction system.
(2) detailed process is that the concentration and probe concentration of fixed first esculent swift, white fungus and pig component system is 10 μM, then
25 groups of quadratic-orthogonal experiments are carried out to different primer concentrations；Next, immobilized primer concentration is optimal combination, to different spies
Pin concentration carries out 25 groups of quadratic-orthogonal experiments.
(3) the optimal primer concentration finally determined is combined as：The primer concentration difference of esculent swift, white fungus and pig component system
For 10 μM, 12.5 μM and 7.5 μM, the primer final concentration of three is respectively 200,250 and 150nmol/L at this time；Finally choose
Optimal concentration and probe concentration is combined as：The concentration and probe concentration of esculent swift, white fungus and pig component system distinguishes 7.5 μM, 7.5 μM and 12.5 μM,
The probe final concentration of three is respectively 150,150 and 250nmol/L at this time.
Embodiment 3：Method is to the relative sensitivity of adulterant composition detection
(1) preparation of sample：0.2g white fungus and 0.2g frying pigskin powder is taken to be added in 1.6g bird's nest powder respectively,
It is sufficiently mixed using tissue grinder and the bird's nest powder sample containing 10% white fungus and 10% pigskin ingredient uniformly, is made.It takes
It states that 0.2g contains 10% white fungus and the bird's nest sample of 10% pigskin ingredient is added in 1.8g bird's nest powder, is sufficiently mixed uniformly,
The bird's nest powder sample containing 1% white fungus and 1% pigskin ingredient is made；Be made successively in this way containing 0.1%,
0.01%th, the bird's nest powder sample of 0.001% white fungus and 0.1%, 0.01%, 0.001% pigskin ingredient.
(2) Nucleospin kits are used, after the extraction each sample of DNA extraction process described in embodiment 1 DNA,
Triple real-time fluorescence PCRs are carried out according to the reaction system that is optimized in embodiment 2, each sample Parallel testing 10 times.
(3) figure it is seen that method can effectively detect that content is only 0.1% white fungus and pigskin ingredient simultaneously, and
Repetitive rate is 100% (10/10), while showing that method can be used for white fungus in bird's nest product and pigskin adulterant ingredient,
Effectively detection.
It is understood that the principle that embodiment of above is intended to be merely illustrative of the present and the exemplary implementation that uses
Mode, however the present invention is not limited thereto.For those skilled in the art, the essence of the present invention is not being departed from
In the case of refreshing and essence, it can make a variety of changes and improve, these changes and improvements are also considered as protection scope of the present invention.
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Sequence table
<110>Shanxi university of TCM
<120>A kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false
<141> 2018-01-29
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tctgccctat caactttcga tggta 25
<210> 11
<211> 25
<212> DNA
<213>Artificial synthesized (artificial)
<400> 11
aatttgcgcg cctgctgcct tcctt 25
Claims (6)
1. a kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false, using composite primer and probe solution, feature exists
In the composite primer and probe solution include：
(1) it is used to expand esculent swift cytb genes：
First sense primer, sequence are as follows
5 '-CAGTAGACAACCCCACATTA-3 ',
First anti-sense primer, sequence are as follows
5 '-GTGGGGTGAGTATGATGATG-3 ',
First probe, sequence are as follows
FAM-5’-CCCGATTCTTCGCCCTACACTTCC-3’-TAMRA；
(2) it is used to expand white fungus α-tubulin genes：
Second sense primer, sequence are as follows
5 '-TGGTGGTCCTCTTGGCACTC-3 ',
Second anti-sense primer, sequence are as follows
5 '-CCACCAAGCGAGTGGGTAAT-3 ',
Second probe, sequence are as follows
CY5-5’-AGCCCTCGGCCTCCCGGCGGACG-3’-BHQ2；
(3) it is used to expand pig ATP synthase genes：
3rd sense primer, sequence are as follows
5’-AACCAGTAGCCCTAGCCGTA-3’；
3rd anti-sense primer, sequence are as follows
5’-TGAGTAGTGCTAATGTGGCCC-3’；
3rd probe, sequence are as follows
VIC-5’-TGACAGCCAACATTACAGCAGGG-3’-TAMRA。
2. a kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false according to claim 1, which is characterized in that adopt
Extract bird's nest product dna to be measured with nucleic acid extraction kit, and using Eukaryotic 18s universal primers to carried DNA into
Row standard PCR amplification.
3. a kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false according to claim 2, which is characterized in that three
Real-time fluorescence PCR reaction system is again：PCR Master Mix12.5 μ L, compound sense primer and compound anti-sense primer solution are each
1.5 μ L, combined probe solution 1.5 μ L, bird's nest product dna solution 5.0 μ L, ddH to be measured2O3.0 μ L, 25 μ L of total volume；
The compound sense primer solution is by 10 μM, the first sense primer solution of 0.5 μ L, 12.5 μ Μ, 0.5 μ L second on
Trip primer solution and 7.5 μ Μ, the 3rd sense primer solution of 0.5 μ L are prepared to obtain；
The compound anti-sense primer solution is by 10 μM, the first anti-sense primer solution of 0.5 μ L, 12.5 μ Μ, 0.5 μ L second under
Trip primer solution and 7.5 μ Μ, the 3rd anti-sense primer solution of 0.5 μ L are prepared to obtain；
The combined probe solution is by 7.5 μM, the first probe solution of 0.5 μ L, 7.5 μ Μ, the second probe solution of 0.5 μ L and
12.5 μ Μ, the 3rd probe solution of 0.5 μ L are prepared to obtain.
4. a kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false according to claim 3, which is characterized in that expand
Increasing condition is：95 DEG C of pre-degenerations 10min, 95 DEG C of denaturation 15s extend 1min, 35 Xun Huans after 60 DEG C of annealing.
5. a kind of triple real time fluorescent PCR methods of discriminating bird's nest true and false according to claim 1 or 4, which is characterized in that
It carries out setting up negative control and the blank control without DNA profiling during triple real-time fluorescent PCR amplifications.
6. a kind of triple real time fluorescent PCR methods for differentiating the bird's nest true and false according to claim 5, which is characterized in that sentence
It is as follows to determine method：
A. as the amplification curve of esculent swift ingredient occurs in bird's nest product to be checked, and Ct values are not more than 35, and negative control and blank
Control then judges to contain bird's nest ingredient in the bird's nest product to be checked without amplification；As bird's nest product to be checked does not occur esculent swift ingredient
Amplification curve, and negative control and blank control are also set up, then judge that the bird's nest product to be checked does not contain bird's nest ingredient；
B. as the amplification curve of white fungus ingredient occurs in bird's nest product to be checked, and Ct values are not more than 30, and negative control and blank pair
According to no amplification, then judge to contain adulterant white fungus ingredient in the bird's nest product to be checked；As white fungus ingredient occurs in bird's nest product to be checked
Amplification curve but Ct values between 30-35, and negative control and blank control are also set up, then need to expand again, if result is still
In this way, then judge that the bird's nest product to be checked does not contain adulterant white fungus ingredient；If Ct values are less than 30 after expanding again, negative control
It is also set up with blank control, then judges that the bird's nest product to be checked contains adulterant white fungus ingredient；As bird's nest product to be checked does not occur
The amplification curve of white fungus ingredient, and negative control and blank control are also set up, then it is adulterated to judge that the bird's nest product to be checked does not contain
Object white fungus ingredient；
C. as the amplification curve of pig ingredient occurs in bird's nest product to be checked, and Ct values are not more than 30, and negative control and blank control
Without amplification, then judge to contain adulterant pigskin ingredient in the bird's nest product to be checked；As the expansion of pig ingredient occurs in bird's nest product to be checked
Increase curve but Ct values between 30-35, and negative control and blank control are also set up, then need to expand again, if result still in this way,
Then judge that the bird's nest product to be checked does not contain adulterant pigskin ingredient；If Ct values are less than 30 after expanding again, negative control and sky
White control is also set up, then judges that the bird's nest product to be checked contains adulterant pigskin ingredient；As bird's nest product to be checked do not occur pig into
The amplification curve divided, and negative control and blank control are also set up, then judge that the bird's nest product to be checked does not contain adulterant pigskin
Ingredient.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023092861A1 (en) * | 2021-11-25 | 2023-06-01 | 百年同康药业集团有限公司 | Lamp-lfd primer set and kit for identifying origin of edible bird's nest, and detection method therefor and use thereof |
| WO2023092860A1 (en) * | 2021-11-25 | 2023-06-01 | 百年同康药业集团有限公司 | Arms-pcr primer set, kit and detection method for identifying gene of cubilose, and use of arms-pcr primer set |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586397A (en) * | 2015-06-19 | 2016-05-18 | 英格尔检测技术服务（上海）有限公司 | Method for conducting real-time fluorescence identification on edible bird's nest product through PCR |
-
2018
- 2018-01-29 CN CN201810082383.XA patent/CN108103210A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586397A (en) * | 2015-06-19 | 2016-05-18 | 英格尔检测技术服务（上海）有限公司 | Method for conducting real-time fluorescence identification on edible bird's nest product through PCR |
Non-Patent Citations (2)
| Title |
|---|
| LILI GUO: "Authentication of Edible Bird’s nests by TaqMan-based real-time PCR", 《FOOD CONTROL》 * |
| 何国林: "TaqMan实时荧光定量PCR鉴定燕窝方法的建立", 《生物技术通讯》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023092861A1 (en) * | 2021-11-25 | 2023-06-01 | 百年同康药业集团有限公司 | Lamp-lfd primer set and kit for identifying origin of edible bird's nest, and detection method therefor and use thereof |
| WO2023092860A1 (en) * | 2021-11-25 | 2023-06-01 | 百年同康药业集团有限公司 | Arms-pcr primer set, kit and detection method for identifying gene of cubilose, and use of arms-pcr primer set |
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Application publication date: 20180601 |