CN108060242B - Dual digital PCR method for quantitative detection of African ostrich-derived components - Google Patents
Dual digital PCR method for quantitative detection of African ostrich-derived components Download PDFInfo
- Publication number
- CN108060242B CN108060242B CN201810149935.4A CN201810149935A CN108060242B CN 108060242 B CN108060242 B CN 108060242B CN 201810149935 A CN201810149935 A CN 201810149935A CN 108060242 B CN108060242 B CN 108060242B
- Authority
- CN
- China
- Prior art keywords
- digital pcr
- ostrich
- african
- african ostrich
- pcr reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Abstract
The invention provides a dual digital PCR method for quantitatively detecting African ostrich-derived components, which adopts a dual-channel detection method, utilizes a digital PCR system to simultaneously detect two fluorescent signals of African ostrich specific species genes and high-grade animal specific genes, respectively marks probes for detecting the African ostrich specific species genes and the high-grade animal specific gene sequences as FAM and VIC, and calculates the relative content of the African ostrich-derived components in the high-grade animal-derived components by the copy number of the African ostrich specific species genes and the high-grade animal specific gene sequences which are detected in the same PCR reaction system. The method can relatively quantify the copy number proportion of the African ostrich-derived components in the meat food to the total meat-derived components.
Description
Technical Field
The invention belongs to the field of molecular biological detection, and particularly relates to a dual digital PCR method for quantitatively detecting African ostrich source components.
Background
The horse meat wind wave in europe rolled in 2013 pushed animal-derived ingredients in food to the wind tip of the wind gap, and economic benefit-driven food adulteration (EMA) became a global food safety hotspot problem. Meat-based food products contain ingredients of meat, particularly non-edible meat, which are not identified in ingredient lists, and are one of the major types of EMA. The beef products of 16 countries of the European Union such as France, Germany and Italy in the horse meat storm contain unidentified horse meat components. Some beef and mutton products found by official investigation in south Africa include buffalo meat, donkey meat, and even kangaroo meat, long neck deer meat, zebra meat and the like. In China, the problem mutton is also discovered when the problem mutton is searched and treated, and the adulterated mutton relates to animal meat such as foxes, minks, camels, mice and the like.
Animals commonly known as ostriches, including african ostriches (Struthio camellus), australian ostriches (dromiaus novaehollandia) and large american ostriches (Rhea americana), belong to different subjects in terms of animal taxonomy. Among them, African ostriches are large-sized meat poultry which are widely bred in the world, and have high economic value. China is the country in Asia where the African ostriches are bred most at present, ostriches are bred in more than 20 provinces and cities in China, and the country also sets various policies such as tax collection for ostriches breeding. In 1996, China established the Chinese ostrich breeding and developing society and the ostrich breeding industrialization healthy development. The killed ostrich meat is used for meat product processing and export, the purchase price is more expensive than that of chicken and duck, and enterprises can sell and process meat products by filling the ostrich meat with the chicken and duck meat. In addition, Australian ostrich, also known as emu, is also used as an ostrich in China to develop industrial breeding, and the meat poultry breeding and food processing industries often treat emus as African ostrich in a significant proportion by mistake or by accident.
In order to ensure food safety and maintain fair and legal trade, a quantitative determination method of ostrich-derived components in food needs to be established, the ostrich-derived components in meat food are accurately detected, and accurate and reliable technical basis is provided for law enforcement supervision and related industry self-discipline.
At present, the ostrich-derived component detection in food is limited to molecular biological detection technologies such as real-time fluorescence PCR, common PCR, agarose gel electrophoresis and the like, and a quantitative detection method capable of meeting the industrial requirements is not available. The invention is urgently needed to be developed based on the reality.
Disclosure of Invention
The invention aims to provide a dual digital PCR method for quantitatively detecting African ostrich-derived components, aiming at overcoming the defects and shortcomings to be solved, and the dual digital PCR method for simultaneously detecting the African ostrich and the genome single copy of the higher animal-derived components can relatively quantify the copy number proportion of the African ostrich-derived components in the meat food to the total meat-derived components.
The purpose of the invention is realized by the following technical scheme:
a dual digital PCR method for quantitatively detecting African ostrich-derived components adopts a dual-channel detection method, a digital PCR system is utilized to simultaneously detect two fluorescent signals of African ostrich specific species genes and high-grade animal specific genes, probes for detecting the African ostrich specific species genes and the high-grade animal specific gene sequences are respectively marked as FAM and VIC, and the relative content of the African ostrich-derived components in the high-grade animal-derived components is calculated through the copy number of the African ostrich specific species genes and the high-grade animal specific gene sequences which are detected in the same PCR reaction system.
Preferably, the method of the invention comprises the steps of:
(1) extracting animal tissue genome DNA of meat products containing African ostrich-derived ingredients;
(2) preparing a digital PCR reaction system;
(3) carrying out digital PCR reaction;
(4) reading and analyzing the digital PCR reaction result;
(5) calculating the relative content of the African ostrich-derived components in the total meat components
The relative content of African ostrich derived components in total meat components
Wherein A is the specific gene copy number concentration of the African ostrich, and B is the specific gene copy number concentration of the higher animal;
preferably, the digital PCR reaction includes, but is not limited to, a microdroplet digital PCR reaction and a chip digital PCR reaction.
Preferably, in the step (1), the animal tissue genome DNA of the meat product is extracted by a kit method.
Preferably, the kit includes, but is not limited to, animal tissue Genomic DNA extraction kit (Kurabo quickGene DNA extraction kit DT-S), Wizard Genomic DNA purification kit (Promega, A1120), PSS nucleic acid automatic extractor and other DNA extraction methods.
Preferably, in the step (2), when the digital PCR reaction is a microdroplet digital PCR reaction, the microdroplet digital PCR reaction system is 20 μ L, and each component is as follows: 2 XddPCR TM10 mu L of premix liquid; 0.8. mu.L each of primers at a concentration of 10. mu. mol/. mu.L, 0.4. mu.L each of probes at a concentration of 10. mu. mol/. mu.L, 2. mu.L of DNA template, and water to 20. mu.L. Respectively adding a 20 mu L reaction system and 70 mu L droplet generating oil into a droplet generating clamping groove, covering a rubber mat, putting into a droplet generating instrument for droplet generation, transferring all generated droplets into a 96-well plate by using a single-channel electric pipetting gun after droplet generation is finished, sealing a membrane, and then placing into a thermal cycler for PCR reaction.
More preferably, in the step (3), the microdroplet digital PCR reaction conditions are: 95 ℃, 5min, 1 ℃/s; 49 cycles of 94 ℃, 15s, 1 ℃/s, 60 ℃, 1min, 1 ℃/s; at 98 ℃, 10min, 1 ℃/s; the reaction product was stored at 12 ℃.
More preferably, in step (4), the droplet digital PCR data is read as follows: after amplification, the 96-well plate was placed in a microdroplet analyzer to read the fluorescence signal and the experimental data was analyzed using QuantaSoft V1.3.2 software.
Preferably, in the step (2), when the digital PCR reaction is a chip digital PCR reaction, the chip digital PCR reaction system is 15 μ L, and each component is as follows:
7.5 mu L of premix; 0.6 muL of each primer with the concentration of 10 mumol/muL, 0.3 muL of each probe with the concentration of 10 mumol/muL, 1.5 muL of DNA template and 15 muL of water; and automatically loading the prepared 15-microliter reaction system into micropores on the chip through a chip loader, immediately covering the surface of the chip with sealing oil by using an oil sealing injector after the system is loaded, sealing the chip, and placing the sealed chip on a PCR system for amplification.
More preferably, in the step (3), the chip digital PCR reaction conditions are: 96 ℃ for 10 min; 49 cycles of 60 ℃, 2min, 98 ℃, 30 s; 60 ℃ for 2 min; the reaction product was stored at 10 ℃.
More preferably, in the step (4), the chip digital PCR data is read as follows: after the amplification is finished, after the chip is restored to the room temperature, the chip is placed in a chip analyzer to read and preliminarily analyze the chip result, and then the chip result is subjected to QuantStaudioTMThe experimental data were analyzed twice by 3D AnalysisSuiteTM Cloud Software.
More preferably, the African ostrich specific gene is the African ostrich transforming growth factor beta 3 gene.
More preferably, the nucleotide sequences of the primer and the probe of the African ostrich specific gene are as follows:
african ostrich specific gene-F: GCCACCCAAAGCCAAGAG (SEQ ID NO.1)
African ostrich specific gene-R: CCACAGGTGCTGGTGCTTG (SEQ ID NO.2)
Specific gene-P of African ostrich: FAM-CCACTCTCCTGCTCCCAAGATAACGTA-BHQ1(SEQ ID NO. 3).
More preferably, the higher animal-specific gene is a higher animal muscle growth inhibitory gene.
More preferably, the nucleotide sequences of the primers and probes for the higher animal-specific genes are as follows:
higher animal-specific gene-F: TTGTGCAAATCCTGAGACTCAT (SEQ ID NO.4)
Higher animal-specific gene-R: ATACCAGTGCCTGGGTTCAT (SEQ ID NO.5)
Higher animal-specific gene-P: VIC-ACGGTACCCCATGAAAGAAGGTATACTG-BHQ1(SEQ ID NO. 6).
The African ostrich (Struthio camellus) belongs to ostrich genus of ostrich family of ostriches of the class of birds, and is the only species in ostriches. Practical tests prove that the African ostrich specific primer probe designed by the invention can specifically detect the African ostrich.
The above-mentioned higher animal specific gene can effectively detect 27 kinds of higher animal components, and said components are pig, cattle, buffalo, yak, goat, sheep, horse, donkey, fox, racoon dog, fruit racoon dog, camel, cat, mink, deer, dog, rabbit, roe deer, mouse, chicken, duck, goose, pigeon, quail, turkey, African ostrich and partridge.
Digital PCR (dPCR) is a nucleic acid detection technique based on single molecule amplification. By partitioning conventional PCR reaction systems through different formats, a large number of partitioned amplification systems are created. After the separated PCR reaction systems are amplified, whether positive fluorescence signals are generated in each small reaction system is checked one by one. The average copy number in the micro-reaction obtained by Poisson distribution can be combined with the number of positive bright spots to obtain the total copy number of the target fragment in the system.
The method adopts a dual-channel detection method, utilizes a primer probe capable of quantifying the total meat component, the African ostrich specific species gene and the higher animal specific gene are both constantly copied in a genome, a digital PCR system can be used for simultaneously detecting two fluorescent signals, the probes for detecting the specific species gene and the higher animal specific gene sequence are respectively marked as FAM and VIC, the African ostrich specific species gene and the higher animal specific gene sequence copy number measured in the same PCR reaction system are used for quantifying the African ostrich derived component and the total meat component, and the relative content of the African ostrich specific species component in the higher animal component can be calculated.
Experimental results show that the relative qualitative detection Limit (LOD) of the African ostrich derived components in the total meat components is 0.01%, and the quantitative detection Limit (LOQ) is 0.1%. In addition, the method of the invention can effectively avoid system errors existing in different reaction systems and errors between parallels caused by sampling and DNA extraction by carrying out double PCR in the same PCR reaction system, and can save reagent and time cost.
Drawings
FIG. 1 is a 2D graph showing the relative qualitative detection limit of the copy number concentration of the African ostrich-derived component by ddPCR.
FIG. 2 is a 2D graph showing the relative qualitative detection limit of the copy number concentration of the ostrich-derived component in African by cdPCR.
FIG. 3 is an analysis chart of the data of the validation experiment for the relative quantitative detection limit of the copy number concentration of the African ostrich-derived component by ddPCR.
FIG. 4 is a 2D graph showing the results of a relatively quantitative detection limit verification experiment for copy number concentration of an African ostrich-derived component by cdPCR.
FIG. 5 is a graph showing the result 1D of actual sample detection by ddPCR.
FIG. 6 is a 2D graph showing the results of actual sample detection by cdPCR.
Detailed Description
The present invention will be described in further detail below with reference to specific examples and drawings, but the embodiments of the present invention are not limited thereto.
Instruments and reagents
1. Instrument for measuring the position of a moving object
QX200TMDroplet Digital PCR system: comprises a thermal cycler (C1000 Touch)TMthermal cycler), droplet generator (droplet generator), droplet analyzer (droplet reader) and membrane sealer (PCR plate sealer)4 sections, purchased from Bio-rad, usa.
QuantStudioTM3D Digital PCR System: including a PCR system (Dual Flat Block)
PCR System 9700), Chip Loader (Digital Chip Loader) and Chip analyzer (Digital PCR Instrument)3 sections, available from Applied Biosystems by Life Technologies, USA.
The Nanodrop 1000 nucleic acid protein analyzer was purchased from Thermo Scientific, usa.
2. Reagent
ddPCR：ddPCRTMPremix (Super Mix for Probes, no dUTP), Droplet Generation Oil (Droplet Generation Oil), Droplet analysis Oil (Droplet Reader Oil), Droplet Generation card slot (Droplet Generator DG8 card), Droplet Generation card slot gel pad (Droplet Generator DG8 mask), and 96-well plate, available from Bio-Rad, usa.
cdPCR：
Premix (3D Digital PCR Master Mix v2), Chip Kit (3D Digital PCR 20K Chip Kit v2, including Chip, Chip lid, brush head, oil seal syringe), purchased from Applied Biosystems by Life Technologies, USA.
Both primers and probes were synthesized by Shanghai scintillation molecular Biotechnology, Inc.
The probes for detecting the African ostrich specific species genes and the higher animal specific gene sequences are as follows:
3. test sample
Meat products (e.g. food products) containing ingredients of African ostrich origin.
Detection method
The double digital PCR method for relatively quantitatively detecting the African ostrich components in the animal-derived food is carried out according to the following steps:
1. preparation of sample and extraction of DNA template: after 25-30 g of a sample (meat or meat food and the like) is cut into pieces, the pieces are crushed by using a tissue grinder under the conditions of 1800 rpm for 3 minutes. Weighing 20-50 mg of prepared sample in a 1.5mL centrifugal tube, and extracting sample DNA by a kit method, wherein the kit can be selected from the following components: DNA extraction methods such as animal tissue genome DNA extraction kit (Kurabo quickGene DNA extraction kit DT-S), Wizard Genomic DNA purification kit (Promega, A1120), and PSS nucleic acid automatic extractor.
2. Preparation and Dispersion of the reaction System
(1) The reaction system of ddPCR digital PCR is as follows:
the ddPCR (microdroplet digital PCR) reaction system was 20. mu.L, and the components were as follows: 2 XddPCR TM10 mu L of premix liquid; 0.8. mu.L each of primers at a concentration of 10. mu. mol/. mu.L, 0.4. mu.L each of probes at a concentration of 10. mu. mol/. mu.L, 2. mu.L of DNA template, and water to 20. mu.L.
Respectively adding a 20 mu L reaction system and 70 mu L microdroplet generating oil into a microdroplet generating clamping groove, covering a rubber mat, putting into a microdroplet generating instrument for microdroplet generation, transferring all generated microdroplets (about 40 mu L) into a 96-well plate by using a single-channel electric pipetting gun after the microdroplets are generated, sealing the membrane by using a membrane sealing instrument, and then putting into a thermal cycler for PCR reaction.
(2) The reaction system of the cdPCR is as follows:
the cdPCR (Chip digital PCR) reaction system is 15 mu L, and the components are as follows:
7.5 mu L of premix; 0.6. mu.L each of primers at a concentration of 10. mu. mol/. mu.L, 0.3. mu.L each of probes at a concentration of 10. mu. mol/. mu.L, 1.5. mu.L of DNA template, and 15. mu.L of water.
And automatically loading the prepared 15-microliter reaction system into micropores on the chip by using a chip loader, immediately covering the surface of the chip with sealing oil by using an oil sealing injector after the system is loaded, and sealing the chip. The sealed chip is placed on a PCR system for amplification.
3. Digital PCR reaction procedure
ddPCR reaction conditions: 95 ℃ for 5min (1 ℃/s); 49 cycles of 94 ℃, 15s (1 ℃/s), 60 ℃, 1min (1 ℃/s); the reaction product was stored at 98 ℃ for 10min (1 ℃/s) and 12 ℃.
cdPCR reaction conditions: 96 ℃ for 10 min; 49 cycles of 60 ℃, 2min, 98 ℃, 30 s; 2min at 60 ℃; the reaction product was stored at 10 ℃.
4. Fluorescence signal reading and analysis
The fluorescence reading in the standard adopts FAM or VIC double-channel fluorescence detection.
ddPCR data reading: after amplification, the 96-well plate was placed in a microdroplet analyzer to read the fluorescence signal and the experimental data was analyzed using QuantaSoft V1.3.2 software.
cdPCR data read: after the amplification is finished, after the chip is restored to the room temperature, the chip is placed in a chip analyzer to read and preliminarily analyze the chip result, and then the chip result is subjected to QuantStaudioTMThe experimental data were analyzed twice by 3D AnalysisSuiteTM Cloud Software.
After the fluorescence collection is finished, determining a fluorescence threshold value according to the reaction heat point diagram, and distinguishing a negative point from a positive point.
5. Calculation of results
Calculation of relative content of African ostrich-derived components in total meat components
The relative content of African ostrich derived components in total meat components
A-African ostrich specific gene copy number concentration
B-higher animal-specific Gene copy number concentration
6. Quality control
(1) Quality control of sample testing
a. Calculation of relative standard deviation between sample parallel
Two parallel sample digital PCR reactions are set, and under the condition that the copy number concentration of the detection result is greater than the quantitative detection limit and the quantity of positive reactions is lower than 80% of the total reaction quantity, the relative standard deviation calculation formula is as follows:
wherein X1And X2The copy number concentration of the African ostrich specific/higher animal specific gene content of two parallel samples, and X is the average value of the copy number concentration of two groups of parallel groups. The Relative Standard Deviation (RSD) value of the copy number concentration of the two parallel samples is required to be less than 25%, and the average value measured by the two parallel samples is used as the species specificity/higher animal specificity gene content of the sample for subsequent analysis.
b. Control of effective microreaction number
The total number of effective micro-reactions generated during the segmentation of the digital PCR system must not be less than 60% (i.e., 12000) of the theoretical number of platforms; the number of positive systems must not exceed 80% of the total number of systems.
c. Quality control of blank control
The theoretical detection result of the digital PCR blank control should be zero. However, in actual testing, a very small number of positive coefficients were allowed to occur. The positive microreaction coefficient in the blank should be less than 0.03% of the actual effective value.
If one of the above quality control conditions is not satisfied, the test result should be discarded and the digital PCR test should be performed again.
(2) Confirmation of performance index
a. Verification of absolute quantitation limits
The absolute quantitative detection limit of the method on the components of the African ostriches and higher animals is 6 copies/mu L. The positive samples with copy number concentration of 6 copies/. mu.L are subjected to digital PCR quantitative detection, 3 replicates are arranged at each concentration, and the RSD value of the parallel detection result of each concentration is calculated. And the RSD is less than or equal to 25 percent and is used as a judgment basis of effective quantitative data, and the absolute quantitative detection lower limit is the lowest copy number concentration when the RSD of the detection result is less than or equal to 25 percent.
b. Relative quantitative detection low limit and recovery rate
The relative quantitative detection limit of the method for the proportion of the African ostriches in the components of the higher animals is 1 percent. And (3) carrying out digital PCR quantitative detection on positive samples/standard substances with known relative copy number concentration, setting 2 parallels for each concentration, and calculating the RSD value of each relative copy number concentration parallel detection result and the deviation of the measured relative copy number concentration from a theoretical value. The RSD is less than or equal to 25 percent and the deviation is less than or equal to +/-10 percent to serve as the judgment basis of effective quantitative data.
Example 1 verification of the relative qualitative detection limit of the copy number concentration of African ostrich-derived ingredients
Supplying a sample book: the African ostrich genome DNA is mixed into a test DNA sample with the African ostrich genome DNA copy percentage of 0.01 percent according to the copy percentage by taking the genome DNA of pigs, cows, sheep and chickens as a substrate. 3 parallel ddPCR and cdPCR experiments were performed, respectively, and the data obtained are shown in FIGS. 1 and 2. The results showed that both ddPCR and cdPCR could be detected when the content of the African ostrich-derived component was 0.01%.
Example 2 verification of the relative quantitative detection limit of the copy number concentration of African ostrich-derived components
Supplying a sample book: in order to verify the quantitative detection limit of the method, the genomic DNA of pigs, cows, sheep and chickens is taken as a substrate, and the genomic DNA of African ostriches with copy percentage of 0.1%, 1%, 10% and 100% is doped in the substrate. 3 parallel ddPCR and cdPCR experiments were performed, respectively, and the results are shown in FIGS. 3 and 4.
For African ostrich genome DNA samples with copy percentage ratios of 0.1%, 1%, 10% and 100%, the detection results on the ddPCR platform are 0.098%, 1.062%, 9.697% and 101.34%, the RSD value among the three parallel is 1.03-5.34%, and the recovery rate is 96.97-101.34%; the detection results on the cdPCR platform are respectively 0.099%, 0.979%, 10.64% and 104.08%, the RSD value between three parallels is 3.08-14.11%, and the recovery rate is 97.91-106.43%.
EXAMPLE 3 actual sample detection capability
Supplying a sample book: takes mixed raw meat of pig, cattle, sheep and chicken as a substrate, and African ostrich meat with the mass percentage of 1 percent, 10 percent, 50 percent and 100 percent is mixed into the substrateBy using
The mixture was mixed by a tube mill to prepare 10g each of mixed meat samples containing various animal-derived components. 3 replicates of each sample, 30mg of each, were weighed and subjected to animal tissue genomic DNA extraction, and 1 replicate of ddPCR and cdPCR, respectively, was run on each sample. The results are shown in FIGS. 5 and 6.
In FIG. 5, E03, F03 and G03 are samples of African ostriches at 1% by mass, C04, D04 and E04 are samples of African ostriches at 10% by mass, A05, B05 and C05 are samples of African ostriches at 50% by mass, and D05, E05 and F05 are samples of African ostriches at 100% by mass.
For the samples of the African ostriches with the meat mass percentages of 1%, 10%, 50% and 100%, the detection results on the ddPCR platform are 0.9293%, 8.46%, 48.42% and 101.32%, the RSD value among the three parallel samples is between 0.94% and 10.97%, and the recovery rate is between 84.59% and 101.32%; the detection results on the cdPCR platform are 0.809%, 8.501%, 44.22% and 101.39% respectively, the RSD value between three parallels is 0.46-5.99%, and the recovery rate is 80.98-101.39%.
Sequence listing
<110> inspection and quarantine technology center of Guangdong entry-exit inspection and quarantine bureau
<120> double digital PCR method for quantitative detection of African ostrich-derived components
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gccacccaaa gccaagag 18
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccacaggtgc tggtgcttg 19
<210> 3
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ccactctcct gctcccaaga taacgta 27
<210> 4
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ttgtgcaaat cctgagactc at 22
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ataccagtgc ctgggttcat 20
<210> 6
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
acggtacccc atgaaagaag gtatactg 28
Claims (5)
1. A dual digital PCR method for quantitatively detecting African ostrich-derived components is characterized in that: the method comprises the following steps of (1) simultaneously detecting two fluorescence signals of a African ostrich specific species gene and a high-grade animal specific gene by using a dual-channel detection method and using a digital PCR system, respectively marking probes for detecting the African ostrich specific species gene and the high-grade animal specific gene sequence as FAM and VIC, and calculating the relative content of the African ostrich-derived components in the high-grade animal-derived components by using the copy number of the African ostrich specific species gene and the high-grade animal specific gene sequence measured in the same PCR reaction system;
the method comprises the following steps:
(1) extracting animal tissue genome DNA of meat products containing African ostrich-derived ingredients;
(2) preparing a digital PCR reaction system;
(3) carrying out digital PCR reaction;
(4) reading and analyzing the digital PCR reaction result;
(5) calculating the relative content of the African ostrich-derived components in the total meat components,
relative content X =of African ostrich-derived component in total meat component
，
Wherein A is the specific gene copy number concentration of the African ostrich, and B is the specific gene copy number concentration of the higher animal;
the nucleotide sequences of the primer and the probe of the African ostrich specific gene are shown as SEQ ID number 1, SEQ ID number 2 and SEQ ID number 3, and the nucleotide sequences of the primer and the probe of the higher animal specific gene are shown as SEQ ID number 4, SEQ ID number 5 and SEQ ID number 6.
2. The method of claim 1, wherein in step (1), the animal tissue genomic DNA of the meat product is extracted by a kit method.
3. The method of claim 1, wherein the digital PCR reaction is any one of a microdroplet digital PCR reaction and a chip digital PCR reaction.
4. The method according to claim 3, wherein in the step (2), when the digital PCR reaction is a microdroplet digital PCR reaction, the microdroplet digital PCR reaction system is 20 μ L, and each component is as follows: 2 XddPCR premix 10. mu.L; 0.8. mu.L each of primers at a concentration of 10. mu. mol/. mu.L, 0.4. mu.L each of probes at a concentration of 10. mu. mol/. mu.L, 2. mu.L of DNA template, and water to 20. mu.L.
5. The method of claim 4, wherein in step (3), the microdroplet digital PCR reaction conditions are: 95 ℃, 5min, 1 ℃/s; 49 cycles of 94 ℃, 15s, 1 ℃/s, 60 ℃, 1min, 1 ℃/s; at 98 ℃, 10min, 1 ℃/s; the reaction product was stored at 12 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810149935.4A CN108060242B (en) | 2018-02-13 | 2018-02-13 | Dual digital PCR method for quantitative detection of African ostrich-derived components |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810149935.4A CN108060242B (en) | 2018-02-13 | 2018-02-13 | Dual digital PCR method for quantitative detection of African ostrich-derived components |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108060242A CN108060242A (en) | 2018-05-22 |
| CN108060242B true CN108060242B (en) | 2021-07-16 |
Family
ID=62134529
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810149935.4A Active CN108060242B (en) | 2018-02-13 | 2018-02-13 | Dual digital PCR method for quantitative detection of African ostrich-derived components |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108060242B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913789B (en) * | 2018-08-13 | 2019-08-06 | 石家庄海关技术中心 | Primer, probe, detection kit and detection method for ostrich derived component in quantitative detection meat products |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001084903A2 (en) * | 2000-05-09 | 2001-11-15 | Centre National De La Recherche Scientifique | Method for detecting and identifying the presence of biological substances derived from birds, and oligonucleotides therefor |
| CN102758002A (en) * | 2011-04-29 | 2012-10-31 | 宗卉 | Method of using gene chip technology to identify and detect 16 animal-derived compositions |
| CN104531890A (en) * | 2015-01-16 | 2015-04-22 | 北京市食品安全监控和风险评估中心 | Kit for detecting ostrich-derived component in food and application of kit |
| CN106676189A (en) * | 2017-02-23 | 2017-05-17 | 珠海出入境检验检疫局检验检疫技术中心 | Quantitative detection method of bovine-derived and porcine-derived components based on droplet digital PCR (polymerase chain reaction) as well as primer, probe and kit |
-
2018
- 2018-02-13 CN CN201810149935.4A patent/CN108060242B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001084903A2 (en) * | 2000-05-09 | 2001-11-15 | Centre National De La Recherche Scientifique | Method for detecting and identifying the presence of biological substances derived from birds, and oligonucleotides therefor |
| CN102758002A (en) * | 2011-04-29 | 2012-10-31 | 宗卉 | Method of using gene chip technology to identify and detect 16 animal-derived compositions |
| CN104531890A (en) * | 2015-01-16 | 2015-04-22 | 北京市食品安全监控和风险评估中心 | Kit for detecting ostrich-derived component in food and application of kit |
| CN106676189A (en) * | 2017-02-23 | 2017-05-17 | 珠海出入境检验检疫局检验检疫技术中心 | Quantitative detection method of bovine-derived and porcine-derived components based on droplet digital PCR (polymerase chain reaction) as well as primer, probe and kit |
Non-Patent Citations (5)
| Title |
|---|
| Detection and quantification of beef and pork materials in meat products by duplex droplet digital PCR;Yicun Cai等;《PLoS One》;20170803;第12卷(第8期);第1页摘要，第3页2.2，第8页3.6 * |
| PREDICTED: Struthio camelus australis transforming growth factor, beta 3 (TGFB3), mRNA;GenBank;《GenBank》;20141015;第1页Origin部分 * |
| Quantitative analysis of pork and chicken products by droplet digital PCR;Yicun Cai等;《Biomed Res Int.》;20140827;第1页摘要，第2页2.3、2.5 * |
| Sus scrofa isolate TJ Tabasco breed Duroc chromosome 15, Sscrofa11.1, whole genome shotgun sequence;GenBank;《GenBank》;20180112;第1-3页 * |
| 肉制品中鸵鸟源性成分的实时荧光PCR检测;齐春萌等;《中国食品学报》;20160902(第7期);第246页摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108060242A (en) | 2018-05-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108060241B (en) | Double-digital PCR method for quantitatively detecting pigeon-derived components | |
| CN105648046B (en) | Method for identifying sheep, goat, mink, nutria and duck meat at one time | |
| CN104099405A (en) | PCR (polymerase chain reaction) detection method and kit for one-time identification of mutton, chicken, duck and pork | |
| CN102776289B (en) | Reagent kit for distinguishing four components including pork, beef, mutton and chicken in food at same time and application thereof | |
| CN108085374B (en) | Dual digital PCR method for quantitatively detecting turkey-derived components | |
| Fontanesi | Meat authenticity and traceability | |
| CN108060242B (en) | Dual digital PCR method for quantitative detection of African ostrich-derived components | |
| CN107974492A (en) | A kind of animal derived materials detection method and its application | |
| CN109852705B (en) | Method and kit for rapidly detecting horse-derived components in food | |
| CN107012247B (en) | Real-time fluorescence PCR detection method for detecting goat-derived ingredients in food and feed by using single-copy nuclear gene | |
| CN104962650B (en) | A kind of synchronous PCR method and kit for differentiating animal derived materials | |
| CN108300770B (en) | Dual digital PCR method for quantitative detection of camel-derived components | |
| CN108300771B (en) | Dual-digital PCR method for quantitatively detecting fox-derived components | |
| CN113493826A (en) | Animal-derived component detection method and application thereof | |
| CN110343766A (en) | A method of based on calf-derived Cyclospora in QPCR quantitative detection livestock meat | |
| CN114752690A (en) | Method for rapidly identifying duck-origin components in meat products based on MIRA technology | |
| CN116875702B (en) | Primer group for identifying common animal species, kit and application thereof | |
| CN109825613B (en) | Method and kit for rapidly detecting duck-origin components in food | |
| CN109825610B (en) | Kit for rapidly detecting horse-derived components in food and application thereof | |
| CN106811534A (en) | A kind of detection primer for disposably detecting various meat derived components, method and kit | |
| CN114836523B (en) | Real-time fluorescence PCR detection method for detecting turkey-derived components in foods and feeds by utilizing single copy nuclear genes | |
| CN111440881B (en) | Primer group, kit and detection method for detecting pork and application | |
| CN104894263A (en) | Multiple-species-component real-time fluorescence PCR (polymerase chain reaction) combined detection method | |
| CN116411086B (en) | Murine component RPA nucleic acid isothermal amplification primer probe group, kit, detection method and application thereof | |
| CN109897902B (en) | Rapid detection test method and kit for donkey-derived components in food |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 510623 block B, Guangdong check tower, 66 Huacheng Road, Zhujiang New Town, Guangzhou, Guangdong. Applicant after: Guangzhou Customs Technology Center Address before: 510623 block B, Guangdong check tower, 66 Huacheng Road, Zhujiang New Town, Guangzhou, Guangdong. Applicant before: INSPECTION & QUARANTINE TECHNOLOGY CENTER OF GUANGDONG ENTRY-EXIT INSPECTION & QUARANTINE BUREAU |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |